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Attorney Docket No.: 00.22US 



PATENT 



IN THE UNITED STATES PATENT AND TRADEMARK OFFICE 

In re Application of: Maes, et al. 



For: Cholesterol Sulfate and Amino Sugar Compositions for Enhancement of Stratum Corneum Function 

APPELLANT'S BRIEF PURSUANT TO 37 CFR 41.37 

Assistant Commissioner of Patents and Trademarks 
Attention: Board of Patent Appeals and Interferences 
Washington, D.C. 20231 
Dear Sir: 

Appellants hereby appeal to the Board of Patent Appeals and Interferences from the final rejection of 
claims 1 and 3 to 20 in the present application in the decision of April 12, 2006. A notice of appeal was filed on 
August 10, 2006. Please see the table of contents below. 

REAL PARTY IN INTEREST 2 

RELATED APPEALS AND INTERFERENCES 3 

STATUS OF CLAIMS 4 

STATUS OF AMENDMENTS 5 

SUMMARY OF THE CLAIMED SUBJECT MATTER 6 

GROUNDS OF REJECTION TO BE REVIEWED ON APPEAL 7 

ARGUMENTS 8 

1) Disclosure of lipid vesicle bilayers, starting materials and components in Ribier et al. (U.S. Patent No. 5,650,166, 
hereinafter Ribier- 166) would not be known and understood by one of ordinary skill in the art as an integral mixture of 
cholesterol sulfate and an exfoliant 8 

• Claims 1, and 3 to 9 8 

2) The claims of the present invention are non-obvious in view of Ribier- 166 1 1 

• Claims 13 to 20 11 

3) The claims of the present invention are non-obvious over Ribier et. al. (U.S. Patent No. 5,925,364, hereinafter Ribier- 
364) in view of Sebag et al. (U.S. Patent No. 5,41 1,742, hereinafter Sebag) 13 

• Claims 1,3, 4, 6 to 9, 11 and 18 13 

4) The present invention is non-obvious over Ribier- 166 in view of Subbiah (U.S. Patent No. 6,150,381, Subbiah) and 
Ichinose et al. (U.S. Patent No. 5,702,691, Ichinose) and the term "integral mixture" is supported by the present 
specification 18 

• Claims 10 to 12 and 20 18 

5) Conclusion 20 

CLAIMS APPENDIX 21 

EVIDENCE APPENDIX 24 

RELATED PROCEEDINGS APPENDIX 25 



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REAL PARTY IN INTEREST 

The name of the real party in interest in this appeal is Color Access, Inc., the assignee of the application. 



2 



RELATED APPEALS AND INTERFERENCES 

There are no other appeals or interferences relating to the instant application that would directly affect, be 
directly affected by, or have a bearing of any kind on the Board's decision in this appeal that are known to 
Appellants. 



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STATUS OF CLAIMS 

Claims 1, and 3 to 20 remain pending and rejected in the application. All pending claims, a copy of which is 
attached hereto, are included in this appeal as of the submission of a Response to a non-final Office Action dated 
August 18, 2005 submitted on January 11, 2006. 



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STATUS OF AMENDMENTS 

In response to the Office Actions of April 12, 2006, September 10, 2004, March 23, 2004, May 6, 2003, 
May 1, 2002 and in a Preliminary Amendment submitted with a Request for Continued Examination on April 12, 
2002 no amendments were made to Claims 1 and 3 to 20. In Appellants' response to the Office Action of July 5, 
2001, Claims 12 and 20 were amended. Claim 18 was amended in a response to the Final Office Action of 
December 12, 2001; and, Claims 1, 13, 16, and 19 were amended in a Preliminary Amendment submitted with a 
Request for Continued Examination on October 1, 2002. Claims 1, 2, 4, 7, and 10 were amended in Appellants' 
response to the first Office Action of June 3, 2002. Amendments were made to claims 1, 16, 18 and 19, and Claim 
2 was canceled in a response to an Office Action dated November 5, 2002. Further amendments were made in a 
Preliminary Amendment submitted with a Request for Continued Examination on January 5, 2004 to Claims 1,13, 
16, and 19; and still further amendments were made in a Preliminary Amendment submitted with a Request for 
Continued Examination on May 9, 2005 to claims 1, 13, 16, and 19. Final amendments were made in Appellants' 
response to the Office Action of August 18, 2005 to Claims 1, 13, 16, and 19. All pending claims, a copy of which 
is attached hereto, are included in this appeal. 



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SUMMARY OF THE CLAIMED SUBJECT MATTER 

The present invention relates to compositions containing an integral mixture of cholesterol sulfate and an 
amino sugar for the enhancement of the protective barrier of the skin and repair of the barrier if it has been damaged 
by chronological aging or other environmental factors. This is described in the present specification at page 1, lines 
5 to 7; page 3, lines 5 to 1 1; and page 4, lines 1 1 to 24. 

None of the dependent claims that are separately argued include a means plus function or a step plus 
function. 



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GROUNDS OF REJECTION TO BE REVIEWED ON APPEAL 

The outstanding issues center around one main question which is whether or not one of ordinary skill in the 
art would know and understand the teaching of bilayers, starting materials and components of lipid vesicles in two 
Ribier et al. references, (U.S. Patent Nos. 5,650 5 166 ? and 5,925,364), to be a teaching of an integral mixture of an 
exfoliant and cholesterol sulfate in the present invention. This main issue applies to the following four issues 
raised by the Examiner. 

1) whether one of ordinary skill in the art would know and understand the disclosure of lipid vesicles and their 
bilayers, starting materials and components in Ribier et al (U.S. Patent No. 5,650,166, hereinafter Ribier-166) 
when read as a whole to be a disclosure of an integral mixture of an exfoliant and cholesterol sulfate in the present 
invention such that it anticipates Claims 1 and 3 to 9; 

2) whether Claims 1, 3, 4, 6 to 9, 1 1 and 18 are obvious over Ribier et. al. (U.S. Patent No. 5,925,364, hereinafter 
Ribier-364 in view of Sebag et al. (U.S. Patent No. 5,41 1,742, hereinafter Sebag) based on teachings in Ribier-364 
similar to Ribier-166 of bilayers, starting materials and components of lipid vesicles such that one of ordinary skill 
in the art would know and understand them in combination with the teaching of cholesteryl phosphate in Sebag to 
be an integral mixture of cholesterol sulfate and an exfoliant of the present invention; 

3) whether Claims 13 to 20 are obvious over Ribier-166 again based on teachings of bilayers, starting materials and 
components of lipid vesicles because one of ordinary skill in the art would know and understand them in Ribier- 
166 alone to be an integral mixture of cholesterol sulfate and an exfoliant of the present invention; and 

4) whether Claims 10 to 12 and 20 are obvious over Ribier-166 in view of Subbiah (U.S. Patent No. 6,150,381, 
Subbiah) and Ichinose et al. (U.S. Patent No. 5,702,691, Ichinose) based on teachings of bilayers, starting materials 
and components of lipid vesicles because one of ordinary skill in the art would know and understand them in 
Ribier-166 in combination with Subbiah and Ichinose to be an integral mixture of cholesterol sulfate and an 
exfoliant of the present invention. 

Each of the four sets of rejections outlined above is addressed in the Arguments section below. 



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ARGUMENTS 



1) Disclosure of lipid vesicle bilayers, starting materials and components in Ribier et al. (U.S. Patent 
No. 5,650,166, hereinafter Ribier-166) would not be known and understood by one of ordinary skill in the 
art as an integral mixture of cholesterol sulfate and an exfoliant. 

• Claims 1, and 3 to 9 

With respect to the anticipation rejection the Examiner finds the following in the Office Action of April 
12, 2006, page 3. 

Ribier et al. discloses a moisturizing composition for the treatment of surface and deep layers 
of the skin clear comprising the instant ingredients such as cholesterol sulfate in the salt of 
alkali metal (including potassium)(see col. 3, lines 64-67), N-acetylglucosamine (see col. 5, 
lines 59-67), the particular sterol, cholesterol (see col. 3 line 60 and col. 6, lines 47-49), fatty 
acids, including linoleic acid (see col. 6, lines 44-46.) Ribier et al. further teaches the use of 
plant extracts (see col. 7, lines 5-8.) The compositions of Ribier et al. may be an emulsion, gel, 
lotion, and ointment form (see col. 7, lines 10-14.) 

Thus the disclosure of Ribier et al. anticipates claims 1 and 3-9. . . . 

Appellants have argued that the arrangement of the components in Ribier et al. (U.S. Patent No. 
5,650,166, hereinafter referred to "Ribier-166") are not as an integral mixture, and therefore, Ribier-166 fails 
to disclose an element of the present claims. Anticipation requires identity of invention: the claimed invention, 
as described in appropriately construed claims, must be the same as that of the reference, in order to 
anticipate. Continental Can Co. USA, Inc. v. Monsanto Co., 948 F.2d 1264, 1267, 20 USPQ2d 1746, 1748 
(Fed. Cir. 1991). See also In re Spada, 911 F.2d 705, 708, 15 USPQ2d 1655, 1657 (Fed. Cir. 1990) ("the 
reference must describe the applicant's claimed invention sufficiently to have placed a person of ordinary skill 
in the field of the invention in possession of it"). Therefore, Appellants assert that it is relevant whether 
features set forth in the present claims are present in Ribier-166. This is because the present invention is 
based on the arrangement of the ingredients as an integral mixture. The elements in Ribier-166 are not 
arranged as they are in the present invention and this issue has not been addressed. 

The arrangement in Ribier-166 is not an "integral mixture" as one of ordinary skill in the art would 
understand it. Two ingredients that are separated from one another, as they are in the Ribier-166 by virtue of 
the vesicle formation, cannot be a mixture or be integral with one another because they are not actually 
combined. The Examiner has admitted in the Final Office Action of April 14, 2006 that a mixture can be 
interpreted by one of ordinary skill in the art as being integral with and notes that Appellants have accepted 
this plain meaning as well. However, Appellants assert that the present claims are limited in a way that one of 
ordinary skill in the art would understand that the present invention is an integral mixture separate and distinct 
from the separate lipid bilayers of Ribier-166. There is no integration (or mixture) where there is separation. 
Therefore, Ribier-166 does not anticipate the claims of the present invention because it fails to disclose an 



8 



integral mixture. In deciding the issue of anticipation, two steps must be taken: first, the elements of the 
claims must be identified to determine their meaning in light of the specification; and second, the 
corresponding elements disclosed in the allegedly anticipating reference must be identified. Lindemann 
Maschinenfabrik GMBG v. Am. Hoist and Derrick Co. et al> 221 USPQ 481, 485; Cf. Slimfold Mfg. Co. v. 
Kinkead Indus., Inc., 810 F.2d 1113, 1 1 16, 1 USPQ2d 1563, 1566 (Fed. Cir. 1987). The second step has not 
been taken and cannot be taken because an integral mixture is not found in Ribier-166. 

A. Claims Are Limited to an Integral Mixture in a Vehicle Supported by the Specification 
The Examiner has noted at page 7 of the Office Action of August 18, 2005 that the present claims are 
not limited to "the act of mixing produce a mixture" [sic] with respect to the novelty rejection over Ribier- 
166. Further at page 7 of the August 18, 2005 Office Action, the Examiner questions what orderly manner is 
used to form discrete layers of a vesicle dispersed in the aqueous phase. Thus, Appellants believe that the 
Examiner acknowledged that Ribier-166 discloses discrete layers of a vesicle dispersed in the aqueous phase. 
However, according to the Examiner at page 8 of the August 18, 2005 Office Action, at the time the claims 
only recited a composition comprising a mixture of effective amounts of cholesterol sulfate or salts, integral 
with or mixed with an exfoliant in a vehicle . The Examiner again found that the features that Appellants 
argued and asserted were not in the rejected claims. In response, Appellants asserted that the claims 
previously, and as amended in the Appellants Response of January 11, 2006 to further enhance the clarity, 
were directed to the very claim limitation, an integral mixture in a vehicle, which is missing from Ribier-166. 
To advance prosecution, the claims were amended to precisely state "an integral mixture." Specifically, 
Claim 1 reads as follows. 

1. A composition for topical application to the skin comprising an integral mixture of 
cholesterol sulfate or salts thereof present in an amount between 0,05 to about 5.00 percent, 
integral with an exfoliant present in an amount between 0.1 to about 10.0 percent by weight of 
the composition in a cosmetically or pharmaceutically acceptable vehicle. 

Appellants assert that regardless of the method used to achieve their formation, the materials found in 
Ribier-166 discrete layers are not integral with each other, and therefore, no anticipation can be found. Based 
on a review of Ribier-166 as a whole, one of ordinary skill in the art would expect to make vesicles with 
discrete layers and not the integral mixture of components of the present invention. Because the product of 
the present invention is different than the alleged product taught by Ribier-166, the present claims are 
adequately directed to limitations that are not disclosed by Ribier-166. 

The Examiner also raises at page 13, of the Final Office Action of April 12, 2006 and at page 3, of the 
Office Action of March 23, 2004 an issue that the integral mixture is not defined in the present specification 
to exclude compositions having vesicles or two or more phases. Instead, the Examiner notes that the present 



9 



specification refers to "integrated results" mentioned on page 4, lines 22 to 24, of the present specification in 
the sense that effects provided by each component are not canceled out. Therefore, the Examiner finds that 
there is no support in the specification under 35 U.S.C. §112, first paragraph , for an "integral mixture" that 
excludes compositions having vesicles of two or more phases. However, the Examiner does find that the term 
"integral mixture" would be subject to its ordinary plain meaning. Appellants respectfully traverse this 
rejection because the integrated result is only rendered by the integral mixture. The result is inextricably 
bound to the mixture because it is only by virtue of the mixture that the result is achieved. Since the plain 
meaning of the term integral mixture is according to the Examiner applicable, and since the mixture is like the 
result - integral, Appellants request that the rejection for lack of support in the specification under 35 U.S.C. 
112, first paragraph be withdrawn. 

B. Present invention is distinct from Ribier-166 

The Examiner has argued at page 7 of the August 18, 2005 Office Action that the Ribier-166 
compositions are a mixture of the ingredients in the present invention such as cholesterol sulfate in the salt of 
alkali metal (including potassium), N-acetylglucosamine, the particular sterol, cholesterol, and fatty acids, 
including linoleic acid. However, this is simply not the case. The Ribier-166 compositions are vesicles 
containing materials that would not be recognized by one of ordinary skill in the art as an integral mixture. 
In the present invention, an integral mixture of ingredients is in a cosmetic or pharmaceutical vehicle. Unlike 
the present invention, the Ribier-166 compositions are discrete layers in an aqueous phase. This is taught in 
Ribier-166 at column 1, line 65 to column 2, line 13, and column 1, lines 24 to 30, where it is explained that 
the Ribier-166 compositions are two types of lipid vesicles and that lipid vesicles are understood by one of 
ordinary skill in the art to be particles formed of a membrane of concentric lamellae where the lamellae 
contain bimolecular layers of amphiphilic lipids encapsulating an aqueous phase. Thus, the Ribier-166 end 
product of a vesicle is indeed different than the present invention. Appellants have repeatedly asserted that 
the method and/or steps for achieving the end result is irrelevant as long as the arrangement of the ingredients 
in the final product of the present invention are distinct from what is described in Ribier-166. It is not 
necessary to know how or why the elements in the ' 166 reference end up being arranged as they are. What is 
at issue here is that the Ribier-166 elements are not arranged as an integral mixture in a cosmetic or 
pharmaceutical vehicle, and therefore, Ribier-166 fails to anticipate the present invention. The elements in 
Ribier-166 are not arranged as they are in the claims of the present invention and this has not been addressed. 

Contrary to the Examiner's assertion, the arrangement in Ribier-166 is not an "integral mixture" in a 
vehicle as one of ordinary skill in the art would understand it. Two ingredients that are separated from one 
another, as they are in the Ribier-166 by virtue of the vesicle formation, are not integral with one another. The 
Examiner has previously admitted in the Office Action of September 10, 2004, page 2, that a mixture can be 



10 



interpreted by one of ordinary skill in the art as being integral with. Therefore, Appellants assert that one of 
ordinary skill in the art would understand that the present invention is an integral mixture distinct from the 
separate lipid bilayers of Ribier-166. Although the steps to preparing the Ribier-166 lipid bilayers involve a 
stirring step, there is no disclosure that the ingredients simply stirred are added to a cosmetic or 
pharmaceutical vehicle. Rather, Ribier-166 discloses at column 8, lines 7 to 14, and column 8, lines 15 to 22, 
stirring to form vesicles that can then be added to a fatty phase. The vesicles formed in Ribier-166 are defined 
at column 1, lines 23 to 29 and include bimolecular layers . Additional hydration and/or dialysis treatment 
steps are also disclosed in Ribier-166 at column 7, lines 42 to 61. The separate layers of Ribier-166 vesicles 
are contrary and opposite to the integral mixture of the present invention. Therefore, Ribier-166 does not 
anticipate the claims of the present invention because it fails to disclose an integral mixture of cholesterol 
sulphate integral with an exfoliant in a vehicle, and Appellants request withdrawal of this rejection. 

2) The claims of the present invention are non-obvious in view of Ribier-166 

• Claims 13 to 20 

The Examiner notes in the Final Office Action of September 10, 2004 at page 9, that notwithstanding the 
fact that Ribier-166 fails to disclose the amount of each of the ingredient components of the present invention, one 
of ordinary skill in the art would know to use the amounts of the ingredients taught in Ribier-166. Therefore, 
according to the Examiner Claims 13 to 20 of the present invention are obvious. Appellants respectfully traverse 
this line of reasoning because Ribier-166, as discussed above with respect to the novelty rejection, fails to teach or 
suggest a mixture of the ingredients such that they are integral with one another. 

The limitations of the claims sufficiently describe an integral mixture of components which one of 
ordinary skill in the art would recognize as being distinct and separate from the same components physically 
located in separate bilayers of a liposome (or vesicle) as they are in Ribier-166. Appellants have not found that this 
point has been addressed. "A proper analysis under §103 requires, inter alia, consideration of two factors: (1) 
whether the prior art would have suggested to those of ordinary skill in the art that they should make the claimed 
composition or device, or carry out the claimed process; and (2) whether the prior art would also have revealed that 
in so making or carrying out [the claimed process], those of ordinary skill would have a reasonable expectation of 
success." In re Vaeck, 20 USPQ2d 1438, 1442 (CAFC 1991); see in re Dow Chemical Co., 5 USPQ2d 1529, 1531 
(Fed. Cir. 1988). These two factors have not been met in the present case. 

First, there is no teaching or suggestion in the prior to make an integral mixture of the pertinent 
components in Ribier-166. The teaching in Ribier-166 of the starting materials and components physically located 
in separate bilayers of a liposome is contrary and opposite to the integral mixture of the same components of the 
present invention. In an integral mixture, the components are not separated; but rather, are integrated. Since 



11 



Ribier-166 only teaches the components in a state of separation, namely present in separate bilayers of the 
liposome, the integral mixture of the present invention is not taught or suggested by Ribier-166. 

The second factor of an obviousness analysis is likewise not met because Ribier-166 fails to reveal that 
making the composition of the present invention, namely the integral mixture of the components, would be 
expected by one of ordinary skill in the art to have reasonable success. This factor is linked to the first factor 
because as long as there is no teaching or suggestion in Ribier-166 to make the mixture of the present invention, 
there likewise, cannot be a reasonable expectation of success to do what is not taught or suggested. But beyond 
this, the teachings of Ribier-166 are aimed at treating two different layers of the skin at the same time. Thus, the 
components of Ribier-166 compositions start out separated in the composition and the components remain 
separated as they are directed to two different areas of the skin. There is never a deliberate mixing or integration of 
the components of Ribier-166 compositions in a vehicle of a composition. This is illustrated by the teaching at 
column 1, lines 11 to 14, where Ribier-166 compositions are described as comprising at least one active agent 
conveyed via at least two distinct types of lipid vesicles. Additional support is found at column 2, lines 19 to 21, of 
Ribier-166 wherein it is taught that the alleged invention involves two different agents to act in different areas of 
the skin. The different agents act in different areas due to the different Ribier-166 lipid vesicles containing them. 
The different Ribier-166 vesicles are classified based on the different types of action (see column 2, lines 34 to 41 
of Ribier-166.) Every aspect of Ribier-166 compositions relates to being separate and distinct. Thus, Ribier-166 
does not teach, suggest, nor motivate one of ordinary skill in the art to make the compositions of the present 
invention having integrally mixed components. 

The contrast between the integral mixture of two ingredients in a vehicle in the present invention and 
the teachings of Ribier-166 is worthy of note. Ribier-166 only discloses the use of cholesterol sulfate to form 
lipid vesicles that can be used to encapsulate NADG. Thus, the two ingredients in Ribier-166 reference are 
separated by the discrete layers of the vesicle that are formed. The ingredients are separated by virtue of their 
being present in separate and discrete layers of the lipid vesicle. The vesicle is similar to a nut in a shell. Like 
NADG and cholesterol sulfate in Ribier-166, the nut and the shell are not mixed, integrally or otherwise. 

Cholesterol sulfate in Ribier-166 is part of the bilayer of the liposome and resembles the shell of a 
nut. Inside of the shell lies the nut, which resembles NADG inside of the bilayers of the liposome. The 
important feature of this analogy is that the shell and the nut are not mixed just as NADG when it is 
encapsulated by a lipid bilayer containing cholesterol sulfate is not mixed with cholesterol sulfate. To further 
support the analogy Appellants submit herewith a copy of Abraham, et al., "Interaction between corneocytes 
and stratum comeum lipid liposomes in vitro," Biochimica et Biophysica Acta, 1021, pp. 119-25 (1990). In 
the article, the authors provide pictures of the liposomes which appear as bubbles in Figures 1 and 3, pp. 121 
to 123. Even in looking at the liposome as a bubble or a balloon, it can be seen that cholesterol sulfate is used 
to make the thin layered wall of the bubble or balloon. For decorative purposes some translucent balloons are 



12 



made to hold items inside. This is similar to NADG held inside of a liposome. In each case, a balloon or a 
nut in a shell, the outer wall is not mixed with the content held inside. Likewise, inside of a liposome, NADG 
is not mixed with cholesterol sulfate as taught in Ribier-166. Therefore, the arrangement of the components 
taught in Ribier-166 is not the arrangement of the present invention, and thus, the present invention is not 
obvious in light of Ribier-166. 

Cholesterol sulfate is taught at column 3, lines 57 to 67, of Ribier-166 as being an additive to the lipid 
membrane of the vesicle. Further, one of the actives contained within the lipid vesicle with deep down action 
are taught at column 5, lines 59 to 67, to include inter alia, NADG. Thus, there is no mixing of these 
ingredients as they are in the present invention by adding them in combination to a vehicle. Unlike the present 
invention, the Ribier-166 vesicles contain these two ingredients in separate and distinct layers and the vehicle 
containing them in separate layers is added to a medium as taught at column 8, lines 32 to 35. This is in 
contrast to adding the ingredients themselves directly to a vehicle. This is especially the case for N- 
acetylglucosamine (NADG) as Ribier-166 fails to teach or suggest adding the NADG directly to a vehicle 
because it teaches that the NADG is contained within the lipid vesicle. Therefore, even though the vesicle is 
added to a vehicle, because the NADG is inside the vesicle, there is no teaching in Ribier-166 where NADG, 
per se, is added directly to the vehicle. An integral mixture of NADG with cholesterol sulfate was not in the 
possession of one of ordinary skill in the art as Ribier-166 merely suggests that cholesterol sulfate is useful as 
a lipid bilayer of a vesicle holding NADG inside of it. Accordingly, the present invention is not obvious in 
view of Ribier-166 and Appellants request that this rejection be withdrawn. 

3) The claims of the present invention are non-obvious over Ribier et. al. (U.S. Patent No. 5,925,364, 
hereinafter Ribier-364) in view of Sebag et al. (U.S. Patent No. 5,411,742, hereinafter Sebag) 

• Claims 1, 3, 4, 6 to 9, 11 and 18 

With respect to the rejection of these claims, the Examiner notes at pages 5 to 6 of the April 12, 2006 

Final Office Action the following. 

One having ordinary skill in the art at the time the invention was made would have been motivated to 
employ the particular fatty acid, linoleic acid, and cholesterol in the composition of Ribier et al, since fatty 
acids broadly and the particular fatty acid, linoleic acid, and known to be useful in a cosmetic or 
dermatological composition for treating skin based on the prior art. Moreover, cholesterol is well known 
to be used as a cosmetic or dermatological composition for treating skin according to Sebag. Therefore, 
one of ordinary skill in the art would have reasonably expected that combining the composition of Ribier 
et al and the composition of Sebag known to be useful for the same purpose, treating skin, in a 
composition to be administered would improve the therapeutic effect for treating skin, in a composition to 
be administered would improve the therapeutic effect for treating skin. 

As noted above, Appellants have asserted that the act of mixing the starting materials and components 
of Ribier-166 can produce two different results, namely, one being an integral mixture and the other being a 



13 



vesicle with discrete layers. Therefore, the process is irrelevant. The same is true for Ribier-364 because its 
teachings are similar to that of Ribier-166. Thus, the arguments above apply to Ribier-364 as well. What is at 
issue in the present application is that the results of mixing are different, and that the claims are directed to 
features that are not present in a vesicle. Appellants respectfully traverse the line of reasoning presented by 
the Examiner in the Final Office Action of April 12, 2006 at page 12 with respect to Ribier-166 and Ribier- 
364 because they each fail to teach or suggest a unit (the composition) based on a dictionary definition of the 
term "integral" in the Merriam- Webster Online Dictionary (without submission by the Examiner of the 
definition on a Notice of References cited or a copy of the definition provided with the office action). At page 
12, of the Final Office Action of April 12, 2006, the Examiner notes in a parenthetical "(formed as a unit with 
another part <a seat with integral headrest.)" The Examiner goes on to note that the prior art teaches and/or 
suggests such a composition, because cholesterol sulfate is combined with an exfoliant in a single cosmetic 
composition (a single unit). However, the issue is what is occurring throughout and within the unit (the 
composition). 

The comparison that probes the issue is at the level of cholesterol sulfate and the exfoliant. In the 
present invention, these two components are an integral mixture in the composition, and this is in stark 
contrast to the Ribier et al. references where cholesterol sulfate and N-acetylglucosamine are separate and 
distinct and in no way are integrated throughout and within the unit (the vesicle or the composition). While it 
is true that the cholesterol sulfate and another component are confined within a unitary space, i.e., the vesicle 
or composition, this does not lead to the conclusion that the cholesterol sulfate is a unit or unified with the 
components in the other layers of the vesicle. This is what separates Ribier-166 and Ribier-364 from the 
present invention and one of ordinary skill in the art would readily know and understand this difference. 

The cholesterol sulfate in one layer of the Ribier-364 vesicle is not integrally mixed with the N- 
acetylglucosamine of any of the other layers of the vesicle. Rather, the cholesterol sulfate layer is separated 
from the N-acetylglucosmine layer of the vesicle. No mixing of cholesterol sulfate in one layer of the Ribier- 
364 vesicle occurs with the other N-acetylglucosamine layer of the Ribier-364 vesicle. However, unlike the 
Ribier references, the present invention is an integral mixture of cholesterol sulfate and an exfoliant 
throughout and within the composition that produces the integrated result of a balanced nurturing of the skin 
barrier. Therefore, the integral mixture of the present invention is not taught or suggested by the Ribier 
references. At best, Ribier-364 or Ribier-166 could be interpreted as teaching how to bring cholesterol 
sulfate and N-acetylglucosamine in close proximity to one another in separate bilayers, but not as a mixture 
and certainly not as an integral mixture. Sebag fails to remedy the defect of Ribier-364. 



14 



A. Sebag teaching of cholesteryl phosphate in a vesicle is not an integral mixture 
Both the Sebag and Ribier-364 preparations fail to produce a mixture because the ionic lipids swell 
under the action of mixing to form discrete layers of a lipid vesicle which separates its contents from the other 
ingredients in the composition, namely the outside media (e.g., the aqueous phase). The Examiner notes at 
page 4 of the Final Office Action of September 10, 2004 and the Final Office Action of April 12, 2006 at page 
5 that Ribier-364 fails to disclose linoleic acid and cholesterol. However, these compounds are not disclosed 
in the present claims, and it is not clear why this is pointed out. Specifically, one of ordinary skill in the art 
would not substitute cholesterol with cholesterol sulfate or cholesteryl phosphate with cholesterol sulfate. In 
support of this assertion, Appellants submitted a declaration by Philip Wesley Wertz ("the Wertz 
Declaration") associated with Application No. 08/865,821 for its pertinence to the distinct nature of 
cholesterol and cholesterol sulfate. In paragraph 5, of the Wertz Declaration, the declarant makes the 
following statement. 

One of ordinary skill in the art recognizes that cholesteryl esters, cholesterol, and cholesteryl sulphate 
and phosphate are distinct compounds and treats each of these compounds as non-equivalent 
compounds due to their distinct distribution in the stratum corneum as well as their different fimctions 
in the stratum corneum. 

Therefore, the Examiner's reliance on the teaching of cholesterol (specifically cholesteryl phosphate ) in 
Sebag at column 2, line 34 is misplaced. It would not have been obvious nor does the combination of linoleic 
acid, cholesterol and exfoliant in the cited references make the present invention. 

Appellants pointed out in their Response of June 23, 2004 at page 6, lines 5 to 7, and in their 
Preliminary Amendment accompanying a Request for Continued Examination submitted on May 9, 2005 that 
the cited references, Ribier-364 and Sebag, teach that discrete layers of a lipid vesicle are formed by mixing. 
If an inventor takes steps that the prior art suggests cannot be made, it is probative of non-obviousness. 
Yamanouchi Pharm. Co. v. Danbury Pharmacol Inc., 21 F. Supp. 2d 366, 374 n. 15, 48 USPQ2d 1741, 1748 
n. 15 (S.D. N.Y. 1998), aff'd, 231 F.3d 1339, 56 USPQ2d 1641 (Fed. Cir. 2000). Thus, based on the cited 
references, one of ordinary skill in the art would expect to make vesicles with discrete layers by mixing and 
not the mixture of the present invention. Because the result of the present invention is different than the result 
taught by the cited references, the claims are adequately directed to limitations that distinguish these results. It 
is not permissible to pick and choose only so much of any given reference as will support a given position and 
ignore the reference in its totality." Lubrizol Corp, V. Exxon Corp., 986 F. supp. 302, 322, 7 USPQ2d 1513, 
1527 (N.D. Ohio 1988). Specifically, and consistent with Ribier-166 and Ribier-364, it is indicated in Sebag 
at column 1, lines 38 to 54, that ionic lipids are capable of swelling in an aqueous solution to form a lamellar 
phase, and after stirring, to form vesicles dispersed in the aqueous solution. The Ribier-364 preparation does 
not produce a mixture because the ionic lipids swell under the action of mixing to form discrete layers of a 



15 



lipid vesicle which separates its contents from the other ingredients in the composition, namely the outside 
media (e.g., the aqueous phase). Therefore, there is no teaching or suggestion of a mixture like that of the 
present invention in Ribier-364 and Sebag, alone or in combination with one another. 

As previously discussed in Appellants Response of June 23, 2004, neither Ribier-364 nor Sebag 
teaches or suggests that the act of mixing produces an integral mixture. To the contrary, the act of mixing in 
Ribier-364 and Sebag would be understood by one of ordinary skill in the art to cause the ionic lipid to swell 
and arrange itself in an orderly manner to form discrete layers of a vesicle dispersed in the aqueous phase. 
Thus, the ionic lipid used with other materials to make the vesicle is not integrally mixed with the content of 
the aqueous phase; but, rather is used to form a discrete entities present in the outside media (i.e., the aqueous 
phase). It is also not mixed as an end product. As previously discussed, the vesicle holds active agents within 
and keeps the actives separate from media outside of its walls. Creating a vesicle is akin to encapsulation 
where the actives inside and the materials used to encapsulate are not mixed with the outside media. 
Therefore, the combination of Ribier-364 and Sebag fails to teach or suggest the integral mixture of the 
present invention. 

B. Unexpected Results 

Even if the interpretation of one of ordinary skill in the art were that a lipid vesicle containing 
cholesterol sulfate in the membrane layer and NADG encapsulated therein was equivalent to the integral 
mixture of the present invention, Appellants assert that it would be rebutted by the surprising results of the 
present invention. The Examiner asserts in the Final Office Action of September 10, 2004, page 1 1, that the 
Example in the present specification provides no clear and convincing evidence of nonobviousness or 
unexpected results since it is not a direct comparison between the present invention and the cited prior art 
references. Appellants note that the standard is not a clear and convincing evidence of nonobviousness but, 
rather a clear and convincing showing of obviousness. All evidence of nonobviousness, however, must be 
considered. In re Soni, 44 USPQ2d 1684, 1687 (Fed. Cir. 1995). Unexpected results must be sufficient to 
overcome a clear and convincing showing of obviousness. Richardson-Vicks Inc. v. The Upjohn Co., 
44 USPQ2d 1181, 1 188 (CAFC 1997). As previously noted, a clear and convincing showing of obviousness 
has not been made. However, even if such a showing were made, comparative test results are not the only 
evidence that can be presented to overcome a clear and convincing showing of obviousness. The unexpected 
result in the present invention lies in the fact that two opposite acting agents are combined and do not cancel 
out their activity. 

As Appellants have pointed out above and in previous responses, the systems in the cited prior art and 
that of the present invention are not the same, and there is no reason to believe that the integral mixture of the 
ingredients directly in a vehicle would necessitate a comparison with a lipid vesicle as these are two 



16 



completely different systems and different arrangements of the components. To support this fact, Appellants 
submitted a copy of an article, Bouwstra et al, "Cholesterol sulfate and calcium affect stratum corneum lipid 
organization over a wide temperature range" Journal of Lipid Research, vol. 40, 2303-3212 (Dec. 1999). In 
the article, the authors note at page 2, lines 4 to 6, that reduced levels of cholesterol sulfate in stratum 
corneum contribute to desquamation, thus indicating that the presence of cholesterol sulfate would maintain 
the integrity of the stratum corneum and prevent desquamation. Therefore, Appellants maintain that one of 
ordinary skill in the art would expect a combination of cholesterol sulfate and an exfoliant to have no effect on 
the surface on the skin because while the exfoliant would contribute to desquamation, the presence of 
cholesterol sulfate would act to prevent desquamation. As previously mentioned, this benefit cannot even be 
addressed with the combination of Ribier-364 and Sebag because these two materials form lipid vesicles, and 
therefore, are not in fact mixed. Rather, they are separated such that one, the cholesterol sulfate, is part of a 
protective membrane that encases the other, the NADG. The whole point of the lipid vesicles/lamellar 
systems of the cited references is to protect and prevent the active inside from being exposed to anything else. 
Thus, a comparison of this kind would be futile. 

While Appellants refrain from speculating on a specific method for forming separate lipid layers, 
Appellants note that it is indicated in Sebag at column 1, lines 38 to 54, that ionic amphiphilic lipids possess 
the property of forming mesomorphic phases and are capable of swelling in an aqueous solution to form a 
lamellar phase. Thus, after stirring in the presence of an aqueous solution, Sebag amphiphilic lipids form 
vesicles dispersed in the aqueous solution. Further, in the Summary of the Invention at column 2, lines 19 to 
23, in Ribier-364, the alleged invention is described as being oily globules that are coated and dispersed in an 
aqueous phase. In other words, Ribier-364 oily globules are encapsulated, and thus, the contents of the ionic 
lipids in the lamellar phase are not mixed with, but rather, are separated from the aqueous phase. Further, the 
teaching of cholesterol sulphate in Ribier-364 is with respect to the coating for the oily globule as taught at 
column 2, lines 37 to 41, wherein it states that "each oily globule is individually coated with a . . . layer 
obtained from ... at least one ionic amphiphilic lipid." An alkali metal salt of cholesterol sulphate is 
provided as an exemplary ionic amphiphilic lipid at column 3, lines 43 to 55 of Ribier-364. As the alkali 
metal salt of cholesterol sulphate is part of the coating of the oily globule, there is no integral mixture with an 
exfoliant formed by the teachings of Ribier-364. The potential exfoliants cited by the Examiner include 
salicylic acid, at column 15, line 6 of Ribier-364 and keratolytic agents at column 14, line 49 of Ribier-364. 
However, in both of these examples they are lipophilic actives contained inside the Ribier-364 oily globule. 
Ribier-364 lipophilic actives are, therefore, not integrally mixed with the coating on the oily globule. Thus, 
there is no teaching or suggestion in Ribier-364 or Sebag, alone or combined, of an integral mixture like that 
of the present invention and Appellants request that the Examiner's rejection of the claims based on this 
combination of references be withdrawn. 



17 



4) The present invention is non-obvious over Ribier-166 in view of Subbiah (U.S. Patent No. 6,150,381, 
Subbiah) and Ichinose et al. (U.S. Patent No. 5,702,691, Ichinose) and the term "integral mixture" is 
supported by the present specification. 

• Claims 10 to 12 and 20 

These claims are rejected by the Examiner for the following reasons. 

It would have been obvious to one of ordinary skill in the art at the time the invention was 
made to have modified the composition of Ribier et al. by the addition of sclareolide for its 
anti-acne properties as taught by Subbiah and by the addition of white birch extract for its anti- 
inflammatory properties as taught by Ichinose. The motivation for modification comes from 
the benefit of such properties in formulating cosmetic compositions. The missing ingredients 
have art-recognized suitability for the intended purpose of formulation cosmetic compositions. 

Appellants respectfully traverse the line of reasoning presented by the Examiner in the Final Office 
Action of April 12, 2004 because Ribier-166 fails to teach or suggest a unit (the composition) as presented 
above with respect to Ribier-364. The line of reasoning is also traversed for the reasons presented above for 
the previous rejections based on Ribier-166. As noted above, the present claims are directed to an integral 
mixture of cholesterol sulfate in certain amounts with an exfoliant in certain amounts in a cosmetic or 
pharmaceutical vehicle. Subbiah teaches sclareolide-like compounds for treating disorders caused by 
microbials such as, for example, bacteria, and one specific disorder is acne. As disclosed in Subbiah, topical 
formulations containing sclareolide are generally prepared by admixing sclareolide in water and at least one 
organic solvent. However, this does not remedy the defect of Ribier-166 discussed above. 

Since Ribier-166 teaches lipid vesicles encapsulating water soluble actives, the combination of these 
references at most suggests that sclareolide could be incorporated within the aqueous phase of the Ribier-166 
lipid vesicles (i.e., sclareolide could be encapsulated). Because lipid vesicles are not integral mixtures, the 
combination of Ribier-166 and Subbiah fails to teach or suggest the present invention. Finally, Ichinose 
teaches flavanonol derivatives in hair nourishing and hair growth products and is cited by the Examiner for its 
teaching of the anti-inflammatory properties of white birch extract. However, like that of Subbiah, the 
teachings of Ichinose do nothing to remedy the defect of Ribier -166. In order to make out a prima facie case 
of obviousness, it must be shown that there is a suggestion or a teaching to one of ordinary skill in the art to 
make the combination of cited references or a reasonable expectation of success. In re Vaeck, 20 USPQ2d 
1438 (Fed. Cir. 1991). Essentially, none of the cited references alone or in combination teach or suggest an 
integral mixture of cholesterol sulfate and an exfoliant in a cosmetic or pharmaceutical vehicle as an end 
product. 



18 



The integrated result further supports Appellants' argument above regarding the §102 and §103 
rejections with respect to the Ribier references. With the Ribier references, producing an integrated result is 
not an issue because the cholesterol sulfate and N-acetylglucosamine remain in separate layers of a vesicle 
presumably in order for them to exhibit their individual activities without concern for canceling each other out 
with respect to their activity. However, the surprising result achieved with the present invention is the fact 
that the cholesterol sulfate and exfoliant do not need to be separated. In the present invention, they can be 
combined in an integral mixture with a vehicle and provide a benefit to the barrier of the skin surface. One of 
ordinary skill in the art as noted in the present specification at page 4, lines 5 to 24, would have expected the 
two components having opposing activities to cancel each other out in a mixture and not produce an integrated 
result. But, to the contrary, it has been unexpectedly found with the present invention that the two opposing 
components do not cancel each other out. This would never have been found based on the separated 
components of the Ribier references. 

Subbiah teaches a method for treating microbial infections with a sclareolide-like compound, and 
therefore, fails to provide any teaching of a cholesterol sulfate and an exfoliant in an integral mixture. And, 
Ichinose teaches a flavanolol derivative for treating hair, and like the other secondary reference fails to teach 
or suggest a cholesterol sulfate and an exfoliant in an integral mixture like that of the present invention. The 
arrangement within Ribier- 166 compositions is not an "integral mixture" in a vehicle as one of ordinary skill 
in the art would understand its plain meaning. Two ingredients that are separated from one another, as they 
are in Ribier- 166 by virtue of the vesicle formation, are not integral with one another. 

Even if the interpretation of one of ordinary skill in the art were that a lipid vesicle containing 
cholesterol sulfate in the membrane layer and NADG encapsulated therein was equivalent to the integral 
mixture of the present invention, Appellants assert that it would be rebutted by the surprising results of the 
present invention as discussed above. As Appellants have pointed out above, the vesicles in Ribier-166 and 
the integral mixtures of the present invention are not the same, and there is no reason to believe that the 
integral mixture of the ingredients directly in a vehicle would necessitate a comparison with a lipid vesicle as 
these are two completely different systems and different arrangements of the components. Therefore, 
Appellants maintain that one of ordinary skill in the art would not know and understand an integral mixture of 
cholesterol sulfate and an exfoliant based on the teachings of Ribier-166 in combination with Subbiah and 
Ichinose. Therefore, Appellants request that this rejection be withdrawn. 

Appellants acknowledged the provisional double patent rejection made by the Examiner. However, in 
light of the arguments set forth above, Appellants will make a terminal disclaimer to obviate the rejection over 
U.S. Application No. 10/424,616, if necessary, in the event that allowable subject matter is indicated. 



19 



5) 



Conclusion 



To recapitulate, the present invention is based on the finding that two ingredients, the cholesterol sulfate 
and the exfoliant, although they have opposing activities , when added as an integral mixture to a pharmaceutical 
or cosmetic vehicle, do not neutralize one another's activities, but rather their activity occurs in tandem, and can 
improve or maintain a healthy skin barrier. Of the five cited references, Ribier-166, Ribier-364, Subbiah, Sebag, 
and Ichinose, none of them disclose, teach or suggest an integral mixture of cholesterol sulfate and an exfoliant 
for an effect on the barrier repair of the skin surface. Ribier-166, Ribier-364 and Subbiah consistently teach 
how to make a vesicle having separate bilayers, and that a cholesterol or derivative may be used as part of the 
formation of the bilayer. There is no integration (or mixture) where there is separation. These three references 
also consistently teach that actives, such as N-acetylglucosamine (an exfoliant) can be an active inside of the 
bilayers. This however is not a disclosure, teaching or suggestion of an integral mixture of cholesterol sulfate 
and an exfoliant. Neither Sebag or Ichinose remedies the defect of the other three references in that they do not 
disclose, teach or suggest cholesterol sulfate in an integral mixture with an exfoliant. Due to the opposing 
activities of cholesterol sulfate and an exfoliant, this combination, based on the cited references is not in 
possession of one of ordinary skill in the art. Therefore, Appellants request that the rejections of the Examiner 
for the present application be withdrawn. 



Respectfully submitted, 





Dorene M. Price, Reg. No. 43,018 

Estee Lauder Companies 

155 Pinelawn Road 

Suite 345 South 

Melville, NY 11747 

(631)414-6087 



20 



CLAIMS APPENDIX 



1. A composition for topical application to the skin comprising an integral mixture of cholesterol sulfate or salts 
thereof present in an amount between 0.05 to about 5.00 percent, integral with an exfoliant present in an amount 
between 0.1 to about 10.0 percent by weight of the composition in a cosmetically or pharmaceutical^ 
acceptable vehicle. 

2 (canceled). 

3. The composition of claim 1 wherein the composition contains a salt of cholesterol sulfate. 

4. The composition of claim 3 wherein the salt is potassium. 

5. The composition of claim 1 wherein the exfoliant is an amino sugar selected from the group consisting of N- 
acetyl-D-glucosamine, N-acetylgalactosamine, and a combination thereof. 

6. The composition of claim 1 further comprising at least one fatty acid selected from the group consisting of 
butyric acid, caproic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, palmitic acid, 
stearic acid, linoleic acid and oleic acid. 

7. The composition of claim 6 wherein said fatty acid is linoleic acid. 

8. The composition of claim 1 further comprising cholesterol. 

9. The composition of claim 1 further comprising both linoleic acid and cholesterol. 

10. The composition of claim 1 further comprising sclareolide. 

1 1 . The composition of claim 1 further comprising a protease inhibitor selected from the group consisting of 
white birch extract, silver birch extract, Boswellia extract, bearberry extract, Centella asiatica extract, and 
Pygeum africanum extract. 

12. The composition of claim 1 further comprising both sclareolide and white birch extract. 



21 



13. A cosmetic or pharmaceutical formulation for topical application of a composition to the skin, the 
formulation containing a mixture comprising an integral mixture of cholesterol sulfate or salts thereof in an 
amount from about 0.05 to about 5.00 percent, and from about 0.1 to about 10.0 percent by weight of an amino 
sugar selected from the group consisting of N-acetyl-D-glucosamine, N-acetylgalactosamine, and a combination 
thereof by weight of the composition being integral with one another in a cosmetically or pharmaceutically 
acceptable vehicle. 

14. The formulation of claim 13 further comprising both cholesterol and a fatty acid selected from the group 
consisting of butyric acid, caproic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, 
palmitic acid, stearic acid, linoleic acid and oleic acid. 

15. The formulation of claim 14 wherein the fatty acid is linoleic acid present in an amount less than 1 percent 
and the cholesterol is present in an amount less than 1 percent. 

16. A method for improving or maintaining a healthy skin barrier which comprises adding an effective amount 
of an integral mixture to a cosmetically or pharmaceutically acceptable vehicle wherein said integral_mixture 
comprises cholesterol sulfate or salts thereof in an amount from about 0.05 to about 5.00 percent by weight of 
the composition, and from about 0.1 to 10.0 percent by weight of the composition an amino sugar selected from 
the group consisting of N-acetyl-D-glucosamine, N-acetylgalactosamine, and a combination thereof, and 
applying said vehicle containing said mixture to the skin. 

17. The method of claim 16 in which the mixture comprises from about 0.1 to about 2.0 percent cholesterol 
sulfate. 

18. The method of claim 16 in which the composition comprises about 0.04 to about 1 .0 percent cholesterol 
sulfate. 

19. A method of treating or reducing damage to the skin, wherein the damage is associated with a reduction or 
loss of skin barrier function, which comprises adding an effective amount of an integral mixture to a 
cosmetically or pharmaceutically acceptable vehicle wherein said mixture comprises cholesterol sulfate or salts 
thereof in an amount from about 0.05 to about 5.00 percent by weight of the composition, and about 0.1 to about 
10.0 percent of an amino sugar selected from the group consisting of N-acetyl-D-glucosamine, N- 



22 



acetylgalactosamine, and a combination thereof by weight of the composition, and applying said vehicle 
containing said mixture to the skin. 

20. The method of claim 19 further comprising cholesterol sulfate or salts thereof in an amount of about 0.1 
to 2.0 percent, about 0.5 to 8.0 percent of N-acetyl-D-glucosamine, cholesterol in an amount of about 0.2 to 1.0 
percent, linoleic acid in an amount of about 0.2 to 1 .0 percent by weight of the composition, sclareolide in an 
amount of about 0.001 to about 1.000 percent, and white birch extract in an amount of about 0.001 to about 
1.000 percent. 



23 



EVIDENCE APPENDIX 



1 . The Wertz Declaration - Appellants submitted the Wertz Declaration with a Response to an Office Action 
dated August 18, 2005 submitted on January 11, 2006. In the Examiner's Office Action of April 12, 2006, the 
Examiner fails to expressly state that Appellants' Wertz Declaration is entered. It is noted by the Examiner that 
the Wertz Declaration is provided by Appellants (Applicants). Based on the consideration given to the Wertz 
Declaration in the Office Action of April 12, 2005, Appellants believe that the Wertz Declaration was entered. 

2. Bouwstra et al.« "Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide 
temperature range". Journal of Lipid Research, vol. 40. 2303-3212 (Dec. 1999) - Information Disclosure 
Statement (IDS) initially submitted with Appellants' Request for Continued Examination submitted on 
January 5, 2004, secondarily with the Request for Continued Examination submitted on May 9, 2005, and 
thirdly with the Response to an Office Action dated August 18, 2005 submitted on January 11, 2006. IDS 
initialed by the Examiner indicating consideration of citation with Office Action of March 23, 2004. 
Appellants believe that this document has been entered and considered. 

3. Abraham et al., "Interaction between corneocytes and stratum corneum lipid liposomes in vitro," 
Biochimica et Biophvsica Acta, 1021, pp. 119 25 (1990) - Information Disclosure Statement (IDS) initially 
submitted with Appellants' Request for Continued Examination submitted on January 5, 2004. IDS initialed 
by the Examiner indicating consideration of citation with Office Action of March 23, 2004. Appellants 
believe that this document has been entered and considered. 



24 



RELATED PROCEEDINGS APPENDIX 

NONE 



25 



Attorney Docket No.: 96.27US 



PATENT 



IN THE UNITED STATES PATENT AND TRADEMARK OFFICE 
In re Application of: Pelle et al. 



I, Philip Wesley Wertz, declare and say that: 

1. I am a citizen of the United States and I reside at 1412 Laurel Street, Iowa City, LA 
52240. 

2. I am a professor at the University of Iowa, College of Dentistry located in Iowa City, 
IA, and I have an A.B. degree in Biochemistry from Rutgers University, New Brunswick, 
NJ and a Ph.D. degree in Biochemistry from the University of Wisconsin, Madison, WL A 
copy of my curriculum vitae is attached hereto. 

3. For over 20 years, I have taught, published and conducted research in the field of 
skin lipids. I have published numerous book chapters and articles related to skin lipids, 
including, in particular, the subject of the structure and function of lipids in the skin. I am 
very familiar with the scientific principles and issues related to the different structures and 
physiological roles of lipids in the skin and related to skin lipid research. 

4. I have read U.S. Patent No. Zysman, U.S. Pat. No. 5,618,523 (hereinafter referred to as 
"the '523 reference"') entitled "Ceramides, Process for Their Preparation and Their 
Applications in the Cosmetic and Dermopharmaceutical Fields" which relates to ceramides 
having a particular structure and compounds containing the ceramide. One of the 
compositions can be an aqueous dispersion of lipid spherules composed of organized 
molecular layers made of the ceramide and another lipid compound. The other lipid 
compound can be cholesterol, or cholesteryl sulphate or phosphate. I understand that the 



Serial No.: 08/865,821 

Filed: May 30, 1997 

For: Lipid Mix for Lip Product 



Group Art Unit: 1615 
Examiner: Faulkner, Diedra 



DECLARATION UNDER 37 GFJL §1.132 



Examiner in the present application has asserted that "cholesteryl esters are encompassed by 
the generic term, cholesterol" in this reference. 

5 . In my opinion, the Examiner' s statement is incorrect. One of ordinary skill in the art 
recognizes that cholesteryl esters, cholesterol, and cholesteryl sulphate and phosphate are 
distinct compounds and treats each of these compounds as non-equivalent compounds due 
to their distinct distribution in the stratum corneum as well as their different functions in the 
stratum corneum. The c 523 reference does not make a generic disclosure of cholesterol 
derivatives and the c 523 reference does not disclose cholesteryl esters. More specifically, as 
applied directly to the Examiner's statement, it is my opinion that the mere recitation of the 
term "cholesterol" or "cholesteryl sulphate and phosphate" does not encompass cholesteryl 
esters. 

6. Cholesterol is a major constituent of the stratum corneum, and it is believed to play 
a major role in the stratum corneum by contributing to the epidermal permeability barrier 
function and mediating desquamation of the skin. Moreover, cholesterol is a major 
component of the intercellular membrane system in the cornified layer of the epidermis. 
This intercellular membrane system provides the permeability barrier of the skin. In 
contrast, the cholesterol esters found in the cornified layer are not membrane forming lipids, 
and at a physiologic temperature they are liquid. The cholesterol esters in the cornified layer 
are thought to separate into isolated liquid phase pockets, and therefore, it is thought that 
cholesterol esters do not function as components of the intercellular membrane system. 

7. Those skilled in the art also do not consider cholesterol and cholesteryl esters to be 
interchangeable. More specifically, because of the different functions of the compounds in 
the skin, one skilled in the art, on reading about a particular use for one of these compounds, 
would not assume that one of the other compounds would also function for the same 
purpose. This is evidenced by the separate treatment, both structurally and functionally, of 
cholesterol and cholesteryl esters in the scientific literature, especially literature pertaining 
to dermatology. 

8. With respect to biochemistry and biology, one of ordinary skill in the physiological 
and/or dermatological arts recognizes that cholesterol, and cholesteryl sulphate and 



9627DEC.DOC 



2 



phosphate, as disclosed in the 4 523 reference, are each individual compounds having 
individual qualities and functions in a physiological and/or dermatological system. 
Therefore, one of ordinary skill in the art would not interpret or imply that cholesterol, as it 
is disclosed in the 4 523 reference, includes cholesteryl ester nor would one of ordinary skill 
in the art be led to substitute cholesterol with cholesteryl esters, 

9. In my opinion, based on the compositions taught in the '523 reference, it would not 
have been obvious to one of ordinary skill in the art to replace the cholesterol in the 
compositions of the '523 reference with the cholesteryl ester in the lipid mixture of the 
present invention. 

10. The undersigned declares further that all statements made herein of his own 
knowledge are true and that all statements made on information and belief are believed to 
be true; and further that these statements were made with the knowledge that willful false 
statements and the like so made are punishable by fine or imprisonment, or both, under 
Section 1001 of Title 18 of the United States Code and that such willful false statements 
may jeopardize the validity of the application or any patents issuing thereon. 





9627DEC.DOC 



3 



FACULTY PROFESSIONAL BIOGRAPHY 
College of Dentistry 
University of Iowa 



Personal Data 

1 . Name Philip Wesley Wertz 
3 . Birth Date 10/25/49 



5. Present Rank 



Professor 



Date of preparation 



12/12/98 



173-42-0860 



2. Social Security # _ 

4. Department Oral Pathology. Radiology & 
Medicine 

6. Date appointed to present rank 7/93 

Mo./Yr. 



Higher Education , formal programs (most recent first) 

Date Awarded Degree Major 
(Mo./Yr.) 



1976 
1971 



Ph.D. 
A.B. 



Biochemistry 
Biochemistry 



Institution 



Dates Attended 



University of Wisconsin 1971-1976 
Rutgers University 1967-1971 



Professional Employment : Appointments (most recent first) 
Mo./Yr. Rank/Title 



Institution 



1993- 
present 

1990- 
1993 

1984- 
1990 



1981- 
1984 



1979- 
1981 

1976- 
1979 



Professor 

Associate Professor 
Assoc. Research Sci. 

Assist. Research Sci. 

Assoc. of the Laboratory 
Postdoctoral Fellow 



University of Iowa College of Dentistry 
Dows Institute for Dental Research 

University of Iowa College of Dentistry and 
Dows Institute for Dental Research 

University of Iowa College of Medicine 
Department of Dermatology University of Iowa Iowa 
City, Iowa 

University of Iowa College of Medicine 
Department of Dermatology University of Iowa Iowa 
City, Iowa 

McArdle Laboratory for Cancer Research 
University of Wisconsin 

McArdle Laboratory for Cancer Research 
University of Wisconsin 



Faculty Professional Biography -2- Philip Wesley Wertz 

IV. Certification and Licensure (Eligibility, stage of completion) 

Board Date 



V. Honors and Awards (most recent first) 

Certificate of appreciation for lecture delivered at Catholic University Graduate School, Seoul, Korea 
(1996) 

Certificate of appreciation for presentation at the annual meeting of the Cosmetic Chemists Society 
(1994) 

Certificate of appreciation from NIH for participation in minority apprenticeship program (1992) 
A.B. with highest honors (1971) 
Phi Beta Kappa (1970) 



VI. Professional Appointments (consultantships, editorships, review panels, etc.; most recent first) 

Year Title 

Reviewer for: 

Biochimica et Biophysica Acta 
Canadian Journal of Microbiology 
Chemistry and Phisics of Lipids 
Journal of Investigative Dermatology 
Journal of Lipid Research 
Journal of Nutritional Biochemistry 
Lipids 

and others 

1992- Director of the Research Seminar Series 

1992 External Reviewer for: 

Advisory Board for Research 

College of Dentistry 

The University of Illinois at Chicago 

1991 Consultant for: 

Functions of Lipids in the Skin. 
Program Director: Donald T. Downing 
Department of Dermatology 
University of Iowa College of Medicine 
Program Project Grant Application — not funded 



Faculty Professional Biography 



-3- 



Philip Wesley Wertz 



1 99 1- Ceramide Metabolism & Transport in Keratinocyte Cultures 

Principal Investigator: Kathi Madison 
Department of Dermatology 
University of Iowa College of Medicine 
RO-1 application — active 

1 991- Effect of Nicotine on Inflammatory Mediators 

Principal Investigator: Georgia Johnson 
Department of Periodontics & Dows Institute 
University of Iowa College of Dentistry 
R-29 application ~ active 

1991- Effect of Nicotine on Inflammatory Mediators 

Principal Investigator: Georgia Johnson 
Department of Periodontics & Dows Institute 
University of Iowa College of Dentistry 
Funded by Smokeless Tobacco Council - completed 

1 987 External Grant Reviewer for NIH 

General Medicine A Study Section 

VII. Committees (include chair and officer positions; most recent first) 

Year Committee Organization 



National : 

1995 Session Chairman, Presenter and Workshop Co-chairman at the Gordon 

Conference on the Barrier Function of the Skin 



1987 Session Co-chairman; Gordon Conference; 

Epithelial Differentiation and 
Keratinaization: The Cell Periphery. 

University : 

1996-1998 Ethics and Scientific Misconduct Commitee 

1992-1993 Biological Sciences Committee 

(Advisory Committee to the Vice 
President for Research) Pew Proposal 
Review Subcommittee (1992-1993) 



1997-1998 



Biological Sciences Committee 
(Convener) 



Faculty Professional Biography -4- Philip Wesley Wertz 

1991-1997 Committee B (Human Subjects) 

1997- Committee A 

1997-1998 Library Committee 

Collegiate : 
1998 - 
1998 
1996 

1991-1994 
1991- 



VIIL Professional Memberships (include offices held; most recent first) 



Year Organization 

1 992- Secretary/Treasurer of Iowa Chapter of AADR 
1993 

1992- ASBMB Congressional Correspondent 

1991- American Association for Dental Research 

1989- American Society for Biochemistry and Molecular Biology 

1981- American Oil Chemists Society 

1981- Society for Investigative Dermatology 

1997- American Association of Pharmaceutical Scientists 

1998- Controlled Release Society 



IX. Areas of Research 

Lipids of skin and oral epithelium - structures, functions and metabolism 
Epithelial permeability and drug delivery 
Biochemistry of carcinogenesis 
Essential fatty acids 

X. Current Projects 



Senior Faculty Peer Review Committee 
Periodontics Department Self Study Review Committee 
Basic Science Subcommittee of the Curriculum Committee 
Research Committee 
Student Dental Research Committee 
Special Advisory Subcommittee on Design and 
Conduct of Retrospective and Prospective 
Clinical Studies (1992- )- 



I 



Faculty Professional Biography -5- Philip Wesley Wertz 

Compositions and structures of lipids from oral mucosa and skin 

Isolation and characterization of membrane coating granules 

Lipid alteration in hyperplasia 

Biochemical basis for oral mucosal drug delivery 

Preparation of liposomes for drug delivery 

Comparative enzymology of skin and oral epithelium. 

Quantitative morphology of oral epithelia 

Role of alcohol dehydrogenase in oral carcinogenesis 

XI. Financial Resources (Grants and Contracts; include funded, pending, and approved but not funded 
applications; list most recent first in each category) 

Role on Project 

Tide (eg: PL Co-PL consultant) Dates Amount 

Federal: 

A Tissue Engineered Skin Model Contractor/Investigator 
(ROD 

Source: NIH 
Status: Pending 

Principal Investigator: Bozena Michniak 
Salary Support for PWW: 1 1% 
Comment: This is an ROl 
Submitted from the University 
of South Carolina. 

Local Effects of Alcohol, Aldehyde Investigator 
And Tobacco Carcinogens in the 
Pathogenesis of Oral Cancer 
Source: NIH/NIDR 
Status: Pending 

Principal Investigator: Christopher Squier 
Salary Support for PWW: 10% 
Comment: 

This is a component of a PPG. 

Production of Carcinogens by Investigator 
Oral Candida Strains 
Source: NIH/NIDR 
Status: Pending 

Principal Investigator: David Soil 
Salary Support for PWW: 5% 
Comment: 

This is a component of a PPG. 



02/01/00- 
01/31/03 



08/01/99- 
03/31/04 



08/01/99- 
03/31/04 



Stratum Corneum Lipid 
Disks: A Novel Skin Model 



Contractor/Collaborator 



1-/01/99- 
09/30/02 



Faculty Professional Biography -6- Philip Wesley Wertz 

Source: NIH 
Status: Pending 

Principal Investigator: Bozena B . 
Michniak 

Salary Support for PWW: 25% 
Comment: ThisisanROl 
submitted from the University 
of South Carolina. The major 
focus is a series of studies on 
mechanisms of penetration 
enhancer action using lipid 
disks prepared at UI. Some EPR 
spectra and TEM work will also 
be done at Iowa, 

Tissue Engineering, Biomimetics * Investigator 08/01/98- 
And Medical Implant Science 07/01/03 
Source: NIH 
Status: Active 

Principal Investigator: Jackie 
Bickenbach 

Salary Support for PWW: 5% 
Comment: Lipid composition will be 
one of several markers used to judge 
epithelial differentiation in several 
bioengineered systems. 

Biological Basis For Oral Investigator 
Mucosal Drug Delivery. 
Source: NIH/NIDR 
Status: Active 

Principal Investigator: Christopher 
A. Squier 

Salary Support for PWW: 20% 
Comment: The proposed 
experiments would identify the 
physiochemical parameters required 
for effective drug delivery through 
the oral mucosa and would identify 
permeability enhancers for use in 
oral mucosal drug delivery systems. 

Effects Of Alcohol On Oral Principal Investigator 07/01/95- $70,835 

Mucosal Permeability. 06/31/97 

Source: NIH/NIDR 

Status: Approved but not funded 



07/01/95- $474,624 
06/31/98 



Faculty Professional Biography 

Principal Investigator; Philip W. Wertz 
Salary Support for PWW: 15% 
Comment: The effects of alcohol on 
oral mucosal permeability to 
the tobacco carcinogen NNN 
would be examined. These 
studies could provide a 
mechanistic explanation 
for the association of alcohol 
and tobacco use with oral cancer. 



Faculty Professional Biography 



-8- 



Philip Wesley Wertz 



Transmucosal Peptide Delivery. Investigator 

Source: MH/NIDR 

Status: Approved but not funded 

Principal Investigator: William Abraham 

Salary Support for PWW: 5% 

Comment: This is a Small Business 

Technology Transfer application. 

If funded we would collaborate with 

Cygnus Therapeutics Inc of Redwood CA 

on the development of trans oral 

mucosal peptide delivery systems. 

Lipids And The Permeability Principal Investigator 

Barrier of Oral Epithelium. 
Source: NM-NIDR 
Status: Active 

Salary Support for PWW: 30% 
Comment: This grant would support 
studies on the formation, structure, 
permeability barriers. 
It would also include studies on 
the perturbation of barrier 
function by essential fatty acid 
deficiency. 

Epithelial Permeability; The Investigator 
Effects Of Age and Alcohol. 
Source: NIH-NIDR 
Status: Completed 

Principal Investigator: Christopher A. Squier 
Salary Support for PWW: 20% 
Comment: This grant supports 
work on the structure, lipid 
composition and permeability of 
human epithelia and the effects of 
aging on these parameters. Also, the 
effects of alcohol consumption 
on oral epithelial properties and 
carcinogenesis will be studied. 



07/01795- $100,000 
06/31/97 



09/01/93- $403,504 
08/31/97 



07/01/92- $328,611 
06/30/95 



Faculty Professional Biography 



-9- 



Philip Wesley Wertz 



Structure And Function of 
Polar Lipids In Human Skin. 



Investigator 



12/01/83- 
07/01/90 



$725,000 



Source: NIH-NIADDK 

Principal Investigator: Donald T. Downing 

Salary support for PWW: 60% 

Comment: PWW was involved in all 

phases of planning and execution of this 

project. This included 

extraction, isolation and analysis 

of epidermal lipids, synthesis of 

novel lipids, planning of metabolic 

experiments and formulation and 

testing of topical treatments. 

Evaluation Of Pathogenic Investigator 04/15/81- $1,341,000 

Factors In Acne Vulgaris. 07/01/90 

Source: NIH-NIADDK 

Principal Investigator: John S.Strauss 

Salary support for PWW: 40% 

Comment: As an Inversigator 

on this project, PWW was 

specifically responsible for isolation 

and analysis of polar lipids from 

normal skin and from patients with 

acne and scaling dermatoses. 

College of Dentistry : 
none 



Source: Cellegy Pharmaceuticals 
Status: Active 

Comment: This project is linked 
To a US Patent application on 
Mucoadhesive liposomes. 
($30,000) 

Lipid Dermatophysiology. Principal Investigator 10/30/91- 

Source: ConvaTec - 12/3 1/94 

Bristol-Myers Squibb 

Status: Completed ($56,900 in account) 

Comment: This project 

focuses on the effects of 

sebaceous lipids, synthetic 

lipids and liposomes on 



Other: 



Mucoadhesive Liposomes 



Principal Investigator 



08/30/98- 
04/30/99 



J 



Faculty Professional Biography -10- Philip Wesley Wertz 

properties of the skin surface 
including permeability and 
adhesive performance. It is 
renewable on an annual basis 
at $35,000 per year. 

Ceramides & Ceramidases of Skin Principal Investigator 01/01/95- 

12/31/95 

Source: Gist-brocades 
Status: Completed ($1,788 remaining) 
Comment: This project involved several 
experiments in which the penetration of 
ceramides into stratum corneum was 
monitored, and the activity of ceramidase 
in epidermal homogenates was assayed. 
(Total costs - $5,000) 

Lipid Analytical Service Principal Investigator 11/15/90 - 

Source: Various Agencies 
Status: Active on an intermittant basis 
($40,000 currently in account) 
This is a service with 
prospects of long term 
continuity and accumulation 
of small amounts of money in 
exchange for occasional advise 
or analytical services. 

Roles of Lipids In Desquamation. Co-Principal Investigator 
Source: The Procter & Gamble 
Company 

Co-Principal Investigators: Donald T. 
Downing & Philip W. Wertz 
Comment: This contract was renewed 
on an annual basis at $75,000 per year. 

Roles Of Lipids In Cosmetic Investigator 
Properties of The Skin. 
Source: Richardson Vicks Inc. 
Principal Investigator: John S. Strauss 
Comment: PWW was 
an investigator on this contract, 
which provided partial salary 
support during 1981-1983 and 
supplemented funds available 
through federal grants. The 



07/01/86- $300,00 
07/01/90 



4/15/81- $200,000 
6/30/86 



f 



Faculty Professional Biography - 1 1 - Philip Wesley Wertz 

contract was renewed on an 
annual basis. It started at 
$25,000 per year, and ended 
at $75,000 per year. 

XII. Bibliography (attach sections in the following order, with authors in sequence; most recent first; list 

work that is published or "in press". Submitted work may also be listed; include journal title and date of 
submission; manuscripts in progress should not be included) 

1. Books and/or Chapters 

Wertz PW, Michniak BB; Epidermal lipid metabolism and barrier function of stratum corneum. In: 
BIOMEDICAL MODULATIONS OF SKIN REACTIONS IN DERMAL AND 
TRANSDERMAL DRUG DELIVERY, JJ Wille, A. Kydonieus (Eds), CRC Press, Boca 
Raton, 1999, in press 

Michniak BB, Wertz PW: Ceramides and lipids. In: HANDBOOK OF COSMETIC SCIENCE 
AND TECHNOLOGY, HI Maibach, A Barel, M Paye (Eds.), Marcel Dekker, New York, 
1999, in press 

Wertz PW: Integral lipids of hair and stratum corneum. In Hair: Biology And Structure, H. Zahn 
and P. Jolles, Eds., Birkhauser Verlag, Basel,, 1996, pp 227-238, 

Wertz PW: Skin lipids in health and disease. In Phospholipids: Characterization, Metabolism and 
Novel Biological Applications, G. Cevc, F. Paltauf, Eds., AOCS Press, Champaign, IL, pp 
141-160, 1996. 

Wertz PW: Percutaneous absorption: Role of lipids, In Dermatotoxicology - 5th Edition, HX 
Maibach and F.N. Marzulli, Eds., Taylor and Francis, Washington DC, pp 29-32, 1996 



Wertz PW, Hoogstraate AJ, Squier CA: Biochemical basis of the permeability barrier in skin and 
oral mucosa. In Oral Mucosa Drug Delivery, M J. Rathbone and J. Hadgraft, Eds., Marcel 
Dekker, Inc., New York, pp 27-49, 1996 

Squier CA, Wertz PW: Structure and function of the oral mucosa and implications for drug 

delivery. In Oral Mucosal Drug Delivery, MJ Rathbone, Ed., AAI Inc, Wilmington, pp 1-26, 
1996 

Wertz PW, Squier CA: Biochemical Basis of the Permeability Barrier in Skin and Oral Mucosa. In 
Oral Mucosal Drug Delivery, MJ Rathbone, Ed., AAI Inc, Wilmington, pp 1-26, 1996 

Squier C A, Wertz PW, Williams DM, Cruchley AT: " Permeability of oral mucosa and skin with age. 
In The Effect Of Aging In Oral Mucosa And Skin, CA. Squier and M.W. Hill, Eds., CRC 
Press, Boca Raton, pp 91-98, 1994. 



Faculty Professional Biography 



-12- 



Philip Wesley Wertz 



Wertz PW, Karthigasan J, Hill MW: Effects of Aging on Epithelial Metabolism. In The Effect of 
Aging In Oral Mucosa And Skin, C.A. Squier and M.W. Hill, Eds., CRC Press, Boca Raton, 
pp 107-112, 1994. 

Downing DT, Stewart ME, Wertz PW, Strauss JS: Lipids of the epidermis and the sebaceous 

glands. In Dermatology In General Medicine, 4th edition, TB Fitzpatrick, AZ Eisen, K Wolff, 
KF Austen (eds.) McGraw-Hill, Inc., New York, 210-211, 1993. 

Wertz PW, Downing DT: Biology and biochemistry of the epidermis. In Biochemistry And 

Physiology Of The Skin, 2nd edition, LA Goldfsmith (Ed.) Oxford University Press, New 
York, 1992. 

Wertz PW: Liposome dermatics - chemical aspects of the skin lipid approach. In: Liposome 

Dermatics, O. Braun-Falco, HC Korting, HI Maibach (Eds.), Springer- Verlag, Berline, 1992, 
pp 38-43. 

Wertz PW, Swartzendruber DC, Downing DT: The role of the corneocyte lipid envelope in the 
structure and function of the epidermal barrier. In Proceedings Of The Conference On 
Prediction Of Percutaneous Penetration, RC Scott, J Hadgraft, RH Guy, J McDougal (Eds.), 
IBCTechnical Services Ltd., London, 1990, pp. 405-411. 

Downing DT, Stewart ME, Wertz PW, Strauss JS: The effect of sebum on epidermal lipid 
composition. In Acne And Related Disorders, R Marks, G Plewig (Eds.) Martin Dunitz, 
London, 1989. 

Wertz PW, Downing DT: Stratum corneum: biological and biochemical considerations. In 
Transdermal Delivery Systems, J Hadgraft, RH Guy (Eds.), Marcel Dekker, NY, 1988, 

Wertz PW: Lipids of keratinized tissues. In Biology Of The Integument, Vol. 2. J Bereiter-Hahn, 
GAMatoltsy, KS Richards (Eds.), Springer-Verlag, Berlin, 1986. 

Wertz PW: Effects of phorbol esters on phospholipid metabolism in vitro. In Arachidonic Acid 
Metabolism And Tumor Promotion, Vol. 3. TJ Slaga, SM Fisher (Eds.), Martinas Nijhoff 
Publishing, Boston, 1985. 

2. Journal Articles (include initial and final page numbers) 

Di Nardo A, Wertz PW, Giannetti A, Seidenari S: Ceramide and cholesterol composition of the skin 
of patients with atopic dermatitis. Acta Derm Venereol (Stockh) 78:27-30, 1998 

Madison KC, Sando GN, Howard EJ, True CA, Gilbert D, Swartzendruber DC, Wertz PW: 
Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme 
transport, and the golgi. J Invest Dermatol Symp Proc 3:80-86, 1998 

Kuempel D, Swartzendruber DC, Squier CA, Wertz PW: In vitro reconstitution of stratum 
corneum lipid lamellae. Biochim Biophys Acta 1372:135-140, 1998 



Faculty Professional Biography 



-13- 



Philip Wesley Wertz 



Wertz PW, van den Bergh BAT. The Physical, chemical and functional properties of lipids in the 
skin and other biotogical barriers. Chem Phys Lipids 91 :85-96, 1998 

van den Bergh BAI, Bouwstra JA, Junginger HE, Wertz PW: Interaction of flexible liquid-state 
niosomes with hairless mouse skin, assessed by electron microscopy, in vivo, and by 
diffusion studies in vitro. Proceed Int Symp Control Rel Bioact Mater 25:18-19, 1998 

Law S, Fotos PG, Wertz PW: Skin surface lipids inhibit adherence of Candida albicans to stratum 
corneum. Dermatology 195:220-223, 1997 

Whittle S, Swartzendruber DC, Kremer M, Squier CA, Wertz PW: Lipids of hamster cheek pouch 
epithelium. Lipids 32:961-964, 1997 

Hoogstraate AJ, Wertz PW, Squier CA, Bos-van Geest A, Abraham W, Garrison MD, Verhoef JC, 
Junginger HE, Bodde HE: Effects of the penetration enhancer glycodeoxycholate on the lipid 
integrity in porcine buccal epithelium in vitro. Euro J Pharm Sci 5:189-198, 1997 

Atar PS, Wertz PW, McArthur M, Imakado S, Bickenbach JR, Roop DR: Inhibition of retinoid, 
signaling in transgenic mice alters lipid processing and disrupts epidermal barrier function. 
Molecular Endocrin 11:792-800, 1997 

Squier CA, Kremer M, Wertz PW: Continuous flow mucosal cells for measuring the in vitro 
permeability of small tissue samples. J Pharm Sci, 86:82-84, 1997 

Di Nardo A, Sugino K, Ademola J, Wertz PW, Maibach HI: Rote of ceramides in proclivity to 
toluene and xylene-induced skin irritation. Dermatosen 44:1 19-125, 1996 

Di Nardo A, Sugino K, Ademola J, Wertz PW, Maibach HI: Sodium lauryl sulfate (SLS) induced 
irritant contact dermatitis: a correlation study between ceramides and in vivo parameters of 
irritation. Contact Dermatitis. 35:86-91,1996 

Law S, Wertz PW, Swartzendruber DC, Squier C A: Regional variation in content, composition 
and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and 
transmission electron microscopy. Archs oral Biol 40:1085-1091, 1995 

Swartzendruber DC, Manganaro A, Madison KC, Kremer M, Wertz PW, Squier CA: Organization 
of the intercellular spaces of porcine epidermal and palatal stratum corneum: a Quantitative 
study employing ruthenium tetroxide. Cell & Tissue Res 279:271-276, 1995. 

Swartzendruber DC, Burnett IH, Wertz PW, Madison KC, Squier C A: Osmium tetroxide and 

ruthenium tetroxide are complementary reagents for the preparation of epidermal samples for 
transmission electron microscopy. J Invest Dermatol, 104:417-420, 1995. 

Vargas K, Wertz PW, Drake D, Morrow B, Squier C A, Soil DR: Differences in adhesion of the 
switch phenotypes of Candida albicans strain 3 153 A to buccal epithelium and stratum 
corneum. Infection and Immunity, 62:1328-1335, 1994. 



Faculty Professional Biography 



-14- 



Philip Wesley Wertz 



Johnson GK, Poore TK, Squier CA, Wertz PW, Reinhardt RA, Vincent SD: Prostaglandin E2 and 
interleukin-1 levels in smokeless tobacco-induced oral mucosal lesions. J Periodont Res, 29: 
430-438, 1994. 

Abraham W, Wertz PW, Potts RO, Garrison MD: Investigation of phase transition in oral mucosa 
by fluorescent spectroscopy. Proc Internat Symposium Control Release Bioact Mater, 
21:557-558, 1994. 

Steen Law SL, Squier CA, Wertz PW: Free sphingosine in oral epithelium. Comp Biochem 
Physiol, 110B:511-513, 1994. 

Chang F, Wertz PW, Squier CA: Localization of p-glucosidase activity within keratinizing 
epithelia. Comp Biochem Physiol, 105A:251-253, 1993. 

Chang F, Swartzendruber DC, Wertz PW, Squier CA: Covalently bound lipids in keratinizing 
epithelia. Biochim Biophys Acta 1150:98-102,1993. 

Abrams K, Harvell J, Shriner D, Wertz P, Maibach H, Maibach HI, Rehfeld SJ: Effect of organic 
solvents on in vitro human skin water barrier function. J Invest Dermatol 101 :609-613, 1993. 

Di Nardo A, Sugino K, Wertz PW, Ademola J, Maibach HI: Cutaneous reactions to the organic 
solvents toluene and xylene: determination of surface lipids and instrumental determination of 
inflammatory reaction. Determatologia Oggi 2:17-21, 1993. 

Wertz PW, Kremer M, Squier CA: Comparison of lipids from epidermal and palatal stratum 
corneum. J Invest Dermatol, 98:375-378, 1992. 

Squier CA, Cox P, Wertz PW: Lipid content and water permeability of skin and oral mucosa. J 
Invest Dermatol, 96:123-126, 1991. 

Chang F, Wertz PW, Squier CA: Comparison of glycosidase activities in epidermis, palatal 
epithelium and buccal epithelium. Comp Biochem Physiol, 100B:137-139, 1991. 

Squier CA, Wertz PW, Cox P: Thin-layer chromatographic analyses of lipids in different layers of 
porcine epidermis and oral epithelia. Arch Oral Biol, 36:647-653, 1991. 

Wertz PW, Downing DT: Metabolism of topically applied fatty acid methyl esters in BALB/c 
mouse epidermis. Journal of Dermatologic Science 1:33-38, 1990. 

Wertz PW, Downing DT: Free sphingosine in human epidermis. J Invest Dermatol 94: 159-161, 
1990. 

Bortz JT, Wertz PW, Downing DT: Composition of cerumen lipids. J Amer Acad Dermatol, 
23:845-849, 1990. 

Wertz PW, Downing DT: Ceramidase activity in porcine epidermis. FEBS Letters, 268: 1 10-1 12, 
1990. 



Faculty Professional Biography 



-15- 



Philip Wesley Wertz 



Wertz PW, Downing DT: Metabolism of linoleic acid in porcine epidermis. J Lipid Res 31:1839- 
1844, 1990. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Sphingolipid metabolism in 
organotypic mouse keratinocyte cultures. J Invest Dermatol, 95:657-664, 1990. 

Wertz PW, Madison KC, Downing DT: Covalently bound lipids of human stratum corneum. J 
Invest Dermatol 91:109-111, 1989. 

Takamura T, Wertz PW, Sato K: Free fatty acids and sterols of human eccrine sweat. Brit J 
Dermatol 120:43-47, 1989. 

Swartzendruber DC, Wertz PW, Kitko DJ, Madison KC, Downing DT: Molecular models of the 
intercellular lipid lamellae in mammalian stratum corneum. J Invest Dermatol 92:251-257, 
1989. 

Wertz PW, Downing DT: Integral lipids of mammalian hair. Comp Biochem Biophys 92B:759- 
761, 1989. 

Wertz PW, Downing DT: w-Hydroxyacid derivatives in the epidermis of several mammalian 
species. Comp Biochem Physiol 93B:265-269, 1989. 

Wertz PW, Downing DT: b-Glucosidase activity in porcine epidermis. Biochim Biophys Acta 
1001:115-119,1989. 

Wertz PW, Downing DT: Free sphingosine in porcine epidermis. Biochim Biophys Acta 
1002:213-217, 1989. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Murine keratinocyte cultures grown 
at the air/medium interface synthesize stratum corneum lipids and "recycle" linoleate during 
differentiation. J Invest Dermatol 93:10-17, 1989. 

Wertz PW. Swartzendruber DC, Kitko DJ, Madison KC, Downing DT: The role of the comeocyte 
lipid envelopes in cohesion of the stratum corneum. J Invest Dermatol 93:169-172, 1989. 

Bortz JT, Wertz PW, Downing DT: On the origin of alkanes found in human skin surface lipids. J 
Invest Dermatol 93:723-727, 1989. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Lamellar granule extrusion and 
stratum comeum intercellular lamellae in murine keratinocyte cultures. J Invest Dermatol 
90:110-116,1988. 

Hedberg CL, Wertz PW, Downing DT: The nonpolar lipids of pig epidermis. J Invest Dermatol 
90:225-229, 1988. 



Faculty Professional Biography 



-16- 



Philip Wesley Wertz 



Abraham W, Wertz PW, Downing DT; Fusion patterns of liposomes formed from stratum corneum 
lipids. J Invest Dermatol 90:259-262, 1988. 

Perisho KM, Wertz PW, Madison KC, Stewart ME, Downing DT: Fatty acids of acylceramides 
from comedones and from the skin surface of acne patients and control subjects. J Invest 
Dermato 90:350-353, 1988. 

Abraham W, Wertz PW, Downing DT: Effect of epidermal acylglucosylceramides and 

acylceramides on the morphology of liposomes prepared from stratum corneum lipids. 
Biochim Biophys Acta 939:403-408, 1988. 

Hedberg CL, Wertz PW, Downing DT: The time course of lipid biosynthesis in pig epidermis. J 
Invest Dermatol 91:169-174, 1988. 

Wertz PW, Downing DT: Hydroxyacid derivatives in human epidermis. Lipids 23:415-418, 1988. 

Wertz PW, Downing DT: Integral lipids of human hair. Lipids 23:878-881, 1988. 

Swartzendruber DC, Kitko DJ, Wertz PW, Downing DT: Isolation of corneocyte envelopes from 
porcine epidermis. Arch Dermatol Res 280:424-429, 1988. 

Wertz PW, Downing DT: Covalently bound o-hydroxyacylsphingosine in the stratum corneum. 
Biochim Biophys Acta 917:108-1 1 1, 1987. 

Abraham W, Wertz PW, Landmann L, Downing DT: Stratum corneum lipid liposomes: calcium- 
induced transformation into lamellar sheets. J Invest Dermatol 88:212- 214, 1987. 

Abraham W, Wertz PW, Burken RR> Downing DT: Glucosylsterol and acylglucosylsterol of snake 
epidermis. J Lipid Res 28:446-449, 1987. 

Wix MA, Wertz PW, Downing DT: Polar lipid composition of mammalian hair. Comp Biochem 
Biophys 86B:671-673, 1987. 

Swartzendruber DC, Wertz PW, Madison KC, Downing DT: Evidence that the corneocyte has a 
chemically bound lipid envelope. J Invest Dermatol 88:709-713, 1987. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Presence of intact intercellular 
lamellae in the upper layers of the stratum corneum. J Invest Dermatol 88:714-718, 1987. 

Wertz PW, Swartzendruber DC, Madison KC, Downing DT. The composition and morphology of 
epidermal cyst lipids. J Invest Dermatol 89:419-425, 1987. 

Melton JL, Wertz PW, Swartzendruber DC, Downing DT: Effects of essential fatty acid deficiency 
on 0-acylsphingolipids and transepidermal water loss in young pigs. Biochim Biophys Acta 
921:191-197, 1987. 



Faculty Professional Biography 



-17- 



Philip Wesley Wertz 



Ranasinghe AW, Wertz PW, Downing DT, Mackenzie IC: Lipid composition of cohesive and 
desquamated corneocytes from mouse ear skin, J Invest Dermatol 86:187-190, 1986. 

Wertz PW, Cox PS, Squier CA, Downing DT: Lipids of epidermis and keratinized and non- 
keratinized oral epithelia. Comp Biochem Physiol 83B: 529-531, 1986. 

Wertz PW, Downing DT: Linoleate content of epidermal acylglucosylceramide in newborn, 
growing, and mature mice. Biochim Biophys Acta 876:469-473, 1986. 

Wertz PW, Downing DT: Covalent attachment of co-hydroxyacid derivatives to epidermal 

macromolecules: a prelimnary characterization. Biochem Biophys Res Comm 137-992-997, 
1986. 

Wertz PW, Stover PM, Downing DT: A survey of polar and nonpolar lipids from epidermis and 
epidermal appendages of the chicken f Gallus domesticus Y Gomp Biochem Physiol 84B:203- 
206, 1986. 

Wertz PW, Stover PM, Abraham W, Downing DT: Lipids of chicken epidermis. J Lipid Res 
27:427-435, 1986. 

Madison KC, Wertz PW, Strauss JS, Downing DT: Lipid composition of cultured murine 
keratinocytes. J Invest Dermatol 87:253-259, 1986. 

Squier CA, Cox PS, Wertz PW, Downing DT: The lipid composition of porcine epidermis and oral 
epithelium. Archs Oral Biol 31:741-747, 1986. 

Merrill AH Jr., Wang E, and Wertz PW: Differences in long chain (sphingoid) base composition of 
sphingomyelin from rats bearing Morris hepatoma 7777. Lipids 21 .529-530, 1986. 

Downing DT, Wertz PW, Stewart ME: The role of sebum and epidermal lipids in the cosmetic 
properties of the skin. Int J Cosmetic Sci 8:115-123, 1986. 

Wertz PW, Abraham W, Landmann L, Downing DT: Preparation of liposomes from stratum 
corneum lipids. J Invest Dermatol 87:582-584, 1986. 

Stewart ME, GrahekMO, Cambier LS, Wertz PW, Downing DT: Dilutional effect of increased 
sebaceous gland activity on the proportion of linoleic acid in sebaceous wax esters and in 
epidermal acylceramides. J Invest Dermatol 87:733- 736, 1986. 

Downing DT, Stewart ME, Wertz PW, Strauss JS: Essential fatty acids and acne. J Amer Acad 
Dermatol 14:221-225,1986. 

Wertz PW, Abraham W, Cho ES, Downing DT: Linoleate-rich O-acylsphingolipids of mammalian 
epidermis: structures and effects of essential fatty acid deficiency. Prog Lipid Res 25:383- 
389, 1986. 



Faculty Professional Biography - 1 8- Philip Wesley Wertz 

Burken RR, Wertz PW, Downing DT: The effects of lipids on transepidermal water permeation in 
snakes. Comp Biochem Physiol 81 A:2I3-216, 1985. 

Burken RR, Wertz PW, Downing DT: A survey of polar and nonpolar lipids extracted from snake 
skin. Comp Biochem Physiol 81B:315-318, 1985. 

Wertz PW, Miethke MC, Long S A, Strauss JS, Downing DT: The composition of the ceramides 
from human stratum corneum and from comedones. J Invest Dermatol 84:410-412, 1985. 

Long S A, Wertz PW, Strauss JS, Downing DT: Human stratum corneum polar lipids and 
desquamation. Arch Dermatol Res 277:284-287, 1985. 

Abraham W, Wertz PW, Downing DT: Linoleate-rich acylglucosylceramides from, pig epidermis; 
structure determination by proton magnetic resonance. J Lipid Res 26:761-766, 1985. 

Schoephoerster RT, Wertz PW, Madison KC, Downing DT: A survey of polar and nonpolar lipids 
of mouse organs. Comp Biochem Physiol 82B:229-232, 1985. 

Wertz PW, Abraham W, Stover PM, Downing DT: Uropygiols: confirmation of structure by 
proton magnetic resonance. J Lipid Res 26:1333-1337, 1985. 

Wertz PW, Downing DT, Freinkel RK, Traczyk TN: Sphingolipids of the stratum corneum and 
lamellar granules of fetal rat epidermis. J Invest Dermatol 83:193- 195, 1984. 

Frost ML, Colton SW 6th, Wertz PW, Downing DT: Structures of the dienoic lactones of horse 
sebum. Comp Biochem Physiol 78B:549-552, 1984. 

Wertz PW, Downing DT: Cholesteryl sulfate: the major polar lipid from horse hoof. J Lipid Res 
25:1320-1323, 1984. 

Landmann L, Wertz PW, Downing DT: Acylglucosylceramide causes flattening and stacking of 
liposomes: an analogy for assembly of the epidermal permeability barrier. Biochim Biophys 
Acta 778:412-418, 1984. 

Wertz PW Colton SW, VI, Downing DT: Comparison of the hydroxyacids from the epidermis and 
the sebaceous glands of the horse. Comp Biochem Physiol 75B:217- 220, 1983. 

Wertz PW, Downing DT: Acylglucosylceramides of pig epidermis: structure determination. J 
Lipid Res 24:753-758, 1983. 

Wertz PW, Downing DT: Ceramides of pig epidermis: structure determination. J Lipid Res 
24:759-765, 1983. 

Wertz PW, Cho ES, Downing DT: Effects of essential fatty acid deficiency on the epidermal 
sphingolipids of the rat. Biochim Biophys Acta 753:350-355, 1983. 



Faculty Professional Biography 



-19- 



Philip Wesley Wertz 



WertzPW, Downing DT: Glucosylceramides of pig epidermis: structure determination. J Lipid 
Res24:1135-1139, 1983. 

Downing DT, Stewart ME, Wertz PW, Colton SW 6th, Strauss JS: Skin lipids. Comp Biochem 
Physiol 76B: 673-678, 1983. 

Wertz PW, and Mueller GC: A possible role for protein alkylation in phorbol ester action. 
Carcinogenesis 7:499-51 1, 1982. 

Birkby CS, Wertz PW, Downing DT: The polar lipids from keratinized tissues of some 
vertebrates. Comp Biochem Physiol 73B:239-242, 1982. 

Wertz PW- Downing DT: Glycolipids in mammalian epidermis: structure and function in the water 
barrier. Science 217:1261-1262, 1982. 

Mueller GC, Wertz PW, Kwong CH, Anderson KA, and Wrighton SA: Dissection of the early 
molecular events in the activation of lymphocytes by 12-0- tetradecanoylphorbol- 13 -acetate. 
Jerusalem Symposia on Quantum Chemistry and Biochemistry 13:319-333, 1981. 

Wertz PW, Garver JC, and Anderson L: The anatomy of a complex mutarotation. J Amer Chem 
Soc 103:3916-1922, 1981. 

WertzPW and Mueller GC: Inhibition of 1 2-0-tetradecanoylphorobol- 13 -acetate accelerated 
phospolipid metabolism by 5,8,1 1,14-eicosatetraynoic acid. Cancer Res 40:776-781, 1980. 



Verma AK, Slaga TJ, Wertz PW, Mueller GC, and Boutwell RK: Inhibition of skin tumor 

promotion by retinoic acid and its metabolite 5,6-epoxyretinoic acid. Cancer Res 40:2367- 
2371, 1980. 

WertzPW, Kensler TW, Mueller GC, Verma AK, and Boutwell RK: 5,6-Epoxyretinoic acid 

opposes the effects of 12-0-tetradecanoylphorbol- 13 -acetate in bovine lymphocytes. Nature 
277:227-229, 1979. 

Kensler TW, Wertz PW, and Mueller GC: Inhibition of phorbol ester accelerated amino acid 
transport in bovine lymphocytes. Biochim Biophys Acta 585:43-52, 1979. 

Wertz PW, and Mueller GC: Rapid stimulation of phospholipid metabolism in bovine lymphocytes 
by tumor-promoting phorbol esters. Cancer Res 38:2900-2904, 1978. 

Wertz PW, Kensler TW, and Mueller GC: Inhibition of phorbol ester action in lymphocytes by 5,6- 
epoxy-p-ionone. Biochem Biophys Res Commun 83: 138-143, 1978. 

3. Reviews 

WertzPW: Sebum. In: DRUGS VS. COSMETICS: COSMECEUTICALS?, HI Maibach, P Eisner 
(Eds.), Marcel Dekker, New York, 1999, in press 



Faculty Professional Biography -20- Philip Wesley Wertz 

Hoogstraate AJ, Garza J, Wertz PW: Barrier lipids from epidermis and oral epithelium. Recent 
Develop Lipids Res, 1999, in press 

Hoogstraate AJ, Wertz PW: Drug delivery via the buccal mucosa. Pharm Sci Tech Today 1:309- 
316, 1998 

Wertz PW: The nature of the epidermal barrier: biochemical aspects. Advan Drug Delivery Rev 
18:283-294, 1996 

Book Review (Lipids — Molecular Organization, Physical Functions And Technical Applications 
Kare Larsson, The Oily Press, Dendee, Scotland, 1994) for Chemistry and Physics of Lipids, 
in press, 1994. 

Soil DR, Morrow B, Srikantha T, Vargas K, Wertz PW: The developmental and molecular biology 
of switching in Candida albicans . Oral Surgery, Oral Medicine and Oral Pathology, 78:194- 
201, 1994. 

Wertz PW, Swartzendruber DC, Squier CA: Regional variation in the structure and permeability of 
oral mucosa and skin. Advanced Drug Delivery Reviews, 12:1-12, 1993. 

Squier CA, Wertz PW: Permeability and the pathophysiology of oral mucosa. Advanced Drug 
Delivery Reviews, 12:13-24, 1993. 

Wertz PW: Epidermal lipids. Seminars in Dermatology, 11:106-113, 1992. 

Book review (Interfacial Phenomena In Biological Systems M. Bender, Ed., Marcel Dekker Inc., 
New York, 1991) for Inform - a publication of the American Oil Chemists' Society. Inform 
3:835,1992. 

Wertz PW, Squier CA: Cellular and molecular basis of permeability barrier function in oral 
epithelium. Crit Rev Ther Drug Carrier Sys, 8:237-269, 1991 . 

Wertz PW, Swartzendruber DC, Abraham A, Madison KC, Downing DT: Essential fatty acids and 
epidermal integrity. Arch Dermatol 123:1381-1384, 1987. 

Downing DT, Stewart ME, Wertz PW, Colton SW 6th, Abraham W, Strauss JS: Skin lipids: An 
update. J Invest Dermatol 88:2s-6s, 1987. 

4. Abstracts 

Gilbert D, MA KC, Sando GN, Squier CA, Wertz PW: p-Glucocerebrosidase in lamellar 
granules. J Dent Res, 78:719, 1999 

Squire D, Squier CA, Wertz PW: Effects of protease inhibitors on desquamation from buccal 
mucosa. J Dent Res, 78: 536, 1999 

Garza J, Madison KC, Sando GN, Wertz PW: N-Butyldeoxygalactonojirimycin inhibits glycolipid 
and ceramide synthesis and permeability barrier formation. J Dent Res, 78:719, 1999 



Faculty Professional Biography 



-21- 



Philip Wesley Wertz 



Ahmad M, Hoogstraate AJ, Squier CA, Wertz PW: Effects of potential permeability enhancers 
on buccal epithelial integrity. J Dent Res, 78:719, 1999 

Howard M, Swartzendruber DC, Squier CA, Wertz PW: In vitro reconstitution of epidermal and 
palatal stratum corneum lipid lamellae. J Dent Res, 536, 1999 

Guo R, Swartzendruber DC, Squier CA, Wertz: Quantitative morphology of the intercellular 
spaces of superficial buccal epithelium. J Dent Res, 78:719, 1999 

Kremer MJ, Denel S, Kas SH r Wertz PW, Squier C A: Oral mucosal drug delivery: Chitosanm as 
vehicle and permeabilizer. J Dent Res, 78:719, 1999 

Zantua JB, Kremer MJ, Lafoon JE, Squier CA, Wertz PW: Role of mucin glycoproteins in the 
permeability of oral mucosa. J Dent Res, 78:719, 1999 

Wade TL, Vargas MA, Wertz PW: Effect of preparation and acid conditioning on dentin collagen. 
J Dent Res 77:177, 1998 

Squire D, Squier CA, Wertz PW: Effects of SDS and a protease inhibitor on desquamation. J 
Dent Res 77:293, 1998 

Gilbert D, Swartzendruber DC, Squier CA, Wertz PW: Lipid composition of isolated lamellar 
granules. J Dent Res 77:293, 1998 

Ahmad M, Hoogstraate AJ, Krema C, Squier CA, Wertz PW: Size limit and pathway for glucan 
diffusion across buccal mucosa. J Dent Res 77:293, 1998 

Whittle S, Quinn J, Squier CA, Wertz PW: Sphingomyelinase activity in epidermis and oral 
epithelium. J Dent Res 77:293, 1998 

Garza J, Swartzendruber DC, Vincent S, Squier CA, Wertz PW: Membrane structures in human 
buccal epithelium. J Dent Res 77:293, 1998 

Kuempel D, Swartzendruber DC, Squier CA, Wertz PW: In vitro reconstitution of stratum 
corneum lipid lamellae. J Dent Res 77:294, 1998 

Kremer MJ, Squier CA, Wertz PW: Absorption and release of triamcinolone in oral mucosa. J 
Dent Res 76:361, 1997 

Kuempel D, Swartzendruber, DC, Kremer M, Wertz PW, Squier CA: Reconstruction of epithelial 
membrane systems. J Dent Res 76:361, 1997 

Lund TM, Wertz PW, Drake DR. Development of an in vitro Candida albicans oral infection 
model. J Dent Res 76:121, 1997 



Faculty Professional Biography 



-22- 



Philip Wesley Wertz 



Jarpa P, Johnson GK, Squier CA, Wertz PW: Effects of smokeless tobacco products on pH. J Dent 
Res 76:448, 1997 

Grau M, Donly KJ, Wertz PW: In vitro degradation of a guided tissue regeneration matrix barrier. 
J Dent Res 76:444, 1997 

Squire D, Wertz PW, Squier CA: A model system for studies of desquamation from buccal 
epithelium. J Dent Res 76:362, 1997 

Du X, Kremer M, Wertz PW, Squier C A: Oral epithelial flux and reservoir function following brief 
exposure to ethanol. J Dent Res 76:362, 1997 

Whittle S, Du X, Squier CA, Wertz PW: Effects of hyperplasia on stratum corneum barrier function 
and lipids. J Dent Res 76:362, 1 997 

Lafoon JE, Squier CA, Wertz PW, Barrieshi K, Kremer MJ: Role of vascularized rete pegs in 
permeability of oral mucosa. J Dent Res 76:362, 1997 

Ortale K, Hall E, Squier CA, Wertz PW: Patterns of DNA fragmentation during epithelial 
differentiation. J Dent Res 76:362, 1997 

Ortale K, Fromm R, Wertz PW, Squier C A: Adherence of positively charged liposomes to buccal 
mucosa, J Dent Res 75:330, 1996 

Whittle S, Squier CA, Kremer M, Swartzendruber DC, Johnson GK, Wertz PW: Alteration of oral 
epithelial lipids in hyperplasia, J Dent Res 75:260,1996 

Lund T, Drake DR, Wertz PW: Liposomal modification of mucosal surfaces and adherence of 
Candida albicans, J Dent Res 75:1 14, 1996 

Alves J, Barrieshi K, Walton R, Wilcox L, Wertz PW, Drake DR: Endotoxin penetration from 
mixed culture through obturated post-prepared canals, J Dent Res 75:373, 1996 

Kremer M, Squier CA, Wertz PW: Mucosal permeability is best predicted by flow through perfusion 
chambers, J Dent Res 75:330, 1996 

Gell G, Drake DR, Wertz PW: Peridex and lauric acid exhibit a synergistic effect on Streptococcus 
mutans. Annual meeting of the American Association for Dental Research, J Dent Res 74:76, 
1995 

Ortale K, Wertz PW, Squier CA: Preparation of liposomes from buccal barrier lipids. Annual 
meeting of the American Association for Dental Research, J Dent Res 74:59, 1995 

Steen Law SL, Wertz PW, Squier CA: Regional variation in content and composition of epithelial 
barrier lipids. Annual meeting of the American Association for Dental Research J Dent Res, 
74:62, 1995 



Faculty Professional Biography 



-23- 



Philip Wesley Wertz 



Bayati P, Wertz PW, Swartzend ruber DC, Squier CA: Relationship of orientation to electron 
microscopic images of lamellar granules. Annual meeting of the American Association for 
Dental Research, J Dent Res 74:62, 1995 

Bickenbach JR, Attar PS, Imakado S, Rothnagel JA, Wertz PW, Roop DR: Dominant negative 
retinoic acid receptor produces loss of epidermal barrier function in transgenic mice. J Invest 
Dermatol, in press, 1995 

Quinn J, Cox P, Squier C A, Wertz PW: Epithelial sphingomyelin and sphingomyelinase. Annual 
meeting of the American Association for Dental Research, J Dent Res 73:108, 1994 

Steen Law SL, Squier CA, Wertz PW: Free sphingosine in oral epithelium. Annual meeting of the 
American Association for Dental Research, J Dent Res 73:108, 1994 

Williams DM, Cruchley AT, Wertz PW, Squier CA: The lipid composition of human oral 

epithelium. Annual meeting of the American Association for Dental Research, J Dent Res 
73:168, 1994 

Bayati P, Chang F, Swartzendruber DC, Madison KC, Johnson GK, Squier C A, Wertz PW: 
Organization of human epithelial permeability barriers. Annual meeting of the American 
Association for Dental Research, J Dent Res 73:168, 1994 

Johnson GK, Poore TK, Wertz PW, Organ CC, Reinhardt RA: IL-1 and PGE2 levels in smokeless 
tobacco-induced lesions. Annual meeting of the American Association for Dental Research, J 
Dent Res 73:359, 1994 

Cannon CL, Neal PJ, Kulilus J, Klausner M, Swartzendruber DC, Squier CA, Kremer MJ, Wertz 
PW: Lipid and ultrastructural characterization of a new epidermal model shows good 
correspondence to normal human epidermis. Annual meeting of the Society for Investigative 
Dermatology, J Invest Dermatol 102:600, 1994 

Swartzendruber DC, Manganaro A, Ahonkhai A, Wertz PW, Squier CA: Organization of epithelial 
permeability barriers. Annual meeting of the American Association for Dental Research, J 
Dent Res 72:166, 1993. 

Manganaro A, Wertz PW, Squier CA: Enhancement of buccal permeability by oleic acid. Annual 
meeting of the American Association for Dental Research, J Dent Res 72:103, 1993. 

Chang F, Swartzendruber DC, Wertz PW, Squier CA: Covalently bound lipids in oral epithelia. 
Annual meeting of the American Association for Dental Research, J Dent Res 72:165, 1993. 

Kremer M, Hu L, Wertz PW, Squier CA: Method for isolation of the buccal permeability barrier. 
Annual meeting of the American Association for Dental Research, J Dent Res 72:166, 1993. 

Gell G, Drake D, Wertz PW: Antimicrobial effects of epithelial lipids. Annual meeting of the 
American Association for Dental Research, J Dent Res 72:399, 1993. 



Faculty Professional Biography 



-24- 



Philip Wesley Wertz 



Wertz PW, Squier CA: Glycosylceramides of buccal epithelium - structure determination. Annual 
meeting of the American Association for Dental Research, J Dent Res 72:166, 1993. 

Squier CA, Kremer M, Wertz PW, Squier SU: Penetration of ovalbumin across nonkeratinized oral 
epithelia. Annual meeting of the American Association for Dental Research, J Dent Res 
72:166, 1993. 

Law SS, Fotos PG, Wertz PW: Effects of lipids on adherence of Candida albicans to a keratinized 
epithelial surface. Annual meeting of the American Association for Dental Research, J Dent 
Res 72:300, 1993. 

Di Nardo A, Sugino K, Wertz PW, Ademola J, Maibach HI: Irritation induced by organic solvents: 
An in vivo study of the effects of toluene and xylene in man. Third Congress of the European 
Academy of Dermatology and Venereology, Denmark, 1993 

Di Nardo A, Sugino K, Wertz PW, Ademola J, Maibach HI: Evaluation with noninvasive methods 
of irritation by xylene and toluene and analysis of superficial lipids. 68th National Congress of 
the Itallian Society of Dermatology and Venereology, Pisa, 1993 

Kremer MJ, Shade WB II, Holbrook NS, Wertz PW, Squier CA: Morphologic Location of the 
Buccal Permeability Barrier. Annual meeting of the American Association for Dental 
Research, 1992. J Dent Res 71:213, 1992. 

Wertz PW, Kremer M, Squier CA: A Comparison of Lipids From Epidermal and Palatal Stratum 
Corneum. Annual meeting of the American Association for Dental Research, J Dent Res 
71:214, 1992. 

Chang F, Wertz PW, Squier CA: Distribution of b-Glucosidase in Keratinizing Epithelia. Annual 
meeting of the American Association for Dental Research, J Dent Res 71:214, 1992. 

Kremer MJ, Shade WB II, Holbrook NS, Wertz PW, Squier C A: Morphologic Location of the 
Buccal Permeability Barrier. Annual meeting of the American Association for Dental 
Research, 1992. J Dent Res 71:213, 1992. 

Wertz PW, Kremer M, Squier CA: A Comparison of Lipids from Epidermal and Palatal Stratum 
Corneum. Annual meeting of the American Association for Dental Research, J Dent Res 
71:214, 1992, 

Chang F, Wertz PW, Squier CA: Distribution of b-Glucosidase in Keratinizing Epithelia. Annual 
meeting of the American Association for Dental Research, J Dent Res 71 :214, 1992. 

Squier CA, Wertz PW, Laffoon J, Kremer M: Lipid Lamellae and the Permeability Barrier in Oral 
Epithelium. Annual meeting of the International Association for Dental Research, 1992. 

Abrams K, Harvell J, Shriner D, Wertz P, Maibach HI, Rehfeld SJ: The effect of various solvent 
systems on in vitro human skin barrier function. Annual meeting of the Society for 
Investigative Dermatology, 1992. J Invest Dermatol 98:641, 1992. 



Faculty Professional Biography 



-25- 



Philip Wesley Wertz 



Chang F, Squier CA, Wertz PW: Glycosidases in skin and oral mucosa. Annual meeting of the 
International Association for Dental Research, 1991. J Dent Res 70:513, 1991. 

Wertz PW, Downing DT: The time course of linoleate metabolism in pig epidermis in vivo. 

Annual meeting of the Society for Investigative Dermatology, 1990. Clin Res 38:603 A, 1990. 

Wertz PW, Downing DT : Epidermal ceramide hydrolase. Annual meeting of the Society for 
Investigative Dermatology, 1990. Clin Res 38:640A, 1990. 

Swartzendruber DC, Boysen DP, Wertz PW, Downing DT: The ultrastructure of lipid lamellae in 
avian epidermis. Annual meeting of the Society for Investigative Dermatology, 1990. Clin 
Res 38:640A, 1990. 

Madison KC, Swatzendruber DC, Wertz PW, Downing DT: Characterization of the ceramides of 
mouse epidermis and organotypic murine keratinocyte cultures. Annual meeting of the 
Society for Investigative Dermatology, 1990. Clin Res 38:685A, 1990. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: De novo synthesis of ceramides in 
organotypic mouse epidermal cultures. Annual meeting of the American Federation for 
Clinical Research, 1989. Clin Res 37:353A, 1989. 

Bortz JT, Wertz PW, Downing DT: On the originin of paraffin hydrocarbons found in skin surface 
lipids. Annual meeting of the Society for Investigative Dermatology, 1989. Clin Res 
37:617A, 1989. 

Hedberg CL, Wertz PW, Downing DT: Glucose is not a substrate for epidermal lipid biosynthesis 
in vivo. Annual meeting of the Society for Investigative Dermatology, 1989. Clin Res 
37:675A, 1989. 

Swartzendruber DC, Wertz PW, Madison KC, Downing DT: Modelling the molecular architecture 
of stratum corneum lipid lamellae. Annua! meeting of the Society for Investigative 
Dermatology, 1989. Clin Res 37.680A, 1989. 

Golden G, Francoeur ML, Potts RO, Abraham W, Wertz PW, Downing DT: Cedramides provide 
for the "solid-like" properties of stratum corneum intercellular lipids. Annual meeting of the 
Society for Investigative Dermatology, 1989. Clin Res 37:61 7A, 1989. 

Wertz PW, Downing DT: Free sphingosine in porcine epidermis. Annual meeting of the Society 
for Investigative Dermatology, 1989. Clin Res 37:622A, 1989. 

Downing DT, Stewart ME, Wertz PW, Strauss JS: The effect of sebum on epidermal lipid 
composition. International symposium on acne and related disorders, 1988. 

Downing DT, Stewart ME, Wertz PW, Strauss JS: Diet and acne. International symposium on 
acne and related disorders, 1988. 



Faculty Professional Biography 



-26- 



Philip Wesley Wertz 



Wertz PW, Downing DT: Integral lipids of human hair. Annual meeting of the Society for 
Investigative Dermatology, 1988. Clin Res 3 6:703 A, 1988. 

Abraham W, Wertz PW, Downing DT: Preparation of liposomes and lamellar sheets from the total 
stratum corneum extracts. Annual meeting of the Society for Investigative Dermatology, 

1988. Clin Res 36:629 A, 1988. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Lipid composition and metabolism in 
murine keratinocyte cultures grown at the air/medium interface. Annual meeting of the 
Society for Investigative Dermatology, 1988. Clin Res 36:670A, 1988. 

Wertz PW, Downing DT: Hydroxyacid derivatives in mammalian epidermis. Joint Meeting of the 
American Society for Biochemistry and Molecular Biology and the American Society for Cell 
Biology, 1989. J Cell Biol 107:361a, 1988. 

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Morphologic and biochemical lipid 
markers of terminal differentiation in cultured keratinocytes. Joint Meeting of the American 
Society for Biochemistry and Molecular Biology and the American Society for Cell Biology, 

1989. J Cell Biol 107:361a, 1988. 

Abraham PW, Wertz PW, Downing DT: Transformation of stratum corneum lipid liposomes into 
lamellar sheets. Joint Meeting of the American Society for Biochemistry and Molecular 
Biology and the American Society for Cell Biology, 1989. J Cell Biol 107:356a, 1988. 

Downing DT, Swartzendruber DC, Wertz PW, Madison KC: Molecular models of the extracellular 
membranes in mammalian stratum corneum. Joint Meeting of the American Society for 
Biochemistry and Molelcular biology and the Amrican Society for Cell Biology, 1989. J Cell 
Biol 107:356a, 1988. 

Swartzendruber DC, Madison KC, Wertz PW, Downing DT: Transmission electron microscopy 
(TEM) visualization of stratum corneum lipid lamellae after ruthenium tetroxide fixation. 
Joint Meeting fo the American Society for Biochemistry and Molecular Biology and the 
American Society for Cell Biology, 1989. J Cell Biol 107:357a, 1988. 

Abraham W, Wertz PW, Landmann L, Downing DT: O-Acylglucosylceramides and O- 

acylceramides cause aggregation and stacking of stratum corneum lipid liposomes. Annual 
meeting of the Society for Investigative Dermatology, 1987. Clin Res 35:665A, 1987. 

Downing DT, Swartzendruber DC, Madison KC, Wertz PW: The corneocyte plasma membrane is 
a chemically bound lipid envelope. Annual meeting of the Society for Investigative 
Dermatology, 1987. Clin Res 35:680A, 1987. 

Swartzenbdruber DC, Wertz PW, Madison KC, Downing DT: Demonstration of intercellular 
lamellae in intact and desquamated stratum corneum. Annual meeting of the Society for 
Investigative Dermatology, 1987. Clin Res 35:720A, 1987. 



Faculty Professional Biography 



-27- 



Philip Wesley Wertz 



Wertz PW, Melton JC, Swartzendruber DC, Downing DT; Effects of essential fatty acid deficiency 
on the structure and function of epidermal lipids. Annual meeting of the Society for 
Investigative Dermatology, 1987. Clin Res 35:724A, 1987. 

Abraham W, Wertz PW, Landmann L, Downing DT: Preparation of liposomes and lamellar sheets 
from stratum corneum lipids. Annual meeting of the Society for Investigative Dermatology, 
1986. Clin Res 34:733A, 1986. 

Burken RR, Wertz PW, Abraham W, Downing DT: The occurrence of "molecular rivets" in 
epidermis from different orders of vertebrates. Annual meeting of the Society for 
Investigative Dermatology, 1986. Clin Res 34:741 A, 1986. 

Wertz PW, Downing DT: Lirioleic acid in epidermal acylglucosylceramide from newborn, growing 
and mature mice. Annual meeting of the Society for Investigative Dermatology, 1985. Clin 
Res34:787A, 1986. 

Madison KC, Wertz PW, Downing DT, Strauss JS: Lipid composition of cultured murine 

keratinocytes. Annual meeting of the Society for Investigative Dermatology, 1986. Clin Res 
34:765A, 1986. 

Squier CA, Cox P, Lesch CA, Wertz PW: Identification of lipids in successive epithelial strata of 
skin and oral mucosa. International Association for Dental Research meeting, 1985. 

Stewart ME, Wertz PW, Grahek MO, Downing DT: Relationship between sebum secretion rates 
and the concentration of linoleate in sebum and epidermal lipids. Annual meeting of the 
Society for Investigative Dermatology, 1985. Clin Res 33:684A, 1985. 

Ranasinghe AW, Wertz PW, Mackenzie IC, Downing DT, Strauss JS: The role of lipids in 

corneocyte cohesion and desquamation in mouse ear skin. Annual meeting of the Society for 
Investigative Dermatology, 1985. Clin Res 33:677 A, 1985. 

Abraham W, Wertz PW, Downing DT: Linoleate-rich polar lipids of epidermis: structure 

determination by proton magnetic resonance. Annual meeting of the Society for Investigative 
Dermatology, 1985. Clin Res 33:621A, 1985. 

Long SA, Wertz PW, Strauss JS, Downing DT: Human stratum corneum polar lipids before and 
after desquamation. Annual meeting of the Society for Investigative Dermatology, 1983. Clin 
Res 32:599 A, 1984. 

Landmann L, Wertz PW, Downing DT: Acylglucosylceramide causes flattening and stacking of 
liposomes: an analogy for assembly of the epidermal permeability barrier. Annual meeting of 
the Society for Investigative Dermatology, 1984. Clin Res 32:596A, 1984. 

Wertz PW, Downing DT: The role of glycolipids in assembly of extracellular membranes in the 
skin. Annual meeting of the Iowa Academy of Science, 1984. 



Faculty Professional Biography 



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Philip Wesley Wertz 



Landmann L, Wertz PW, Downing DT: Acylglucosylceramide and the epidermal permeability 
barrier: a liposome reconstitution study. Annual meeting of the European Anatomical 
Society, 1984. Acta Anatomica 120:241-242, 1984. 

Cox PS, Wertz PW, Downing DT, Squier CA: Changes in lipid composition between consecutive 
sections through pig epidermis. Annual meeting of the British Society for Investigative 
Dermatology, 1984. 

Downing DT, Stewart ME, Wertz PW, Colton SW 6th: Analysis of waxes from vertebrate skin. 
Annual meeting of the American Oil Chemists Society, 1984. J Amer Oil Chem Soc 61:666, 
1984. 

Clancey CJ, Van Orden DE, Zlatnik FJ, Wertz PW, Dowining DT: Quantitation of amniotic fluid 
phospholipids: A novel determination of disaturated lecithins (SPC). Annual meeting of the 
Society for Gynecologic Investigation, 1983, 

Wertz PW, Downing DT: Epidermal sphingolipids: strucutres and possible functions in the 

.epidermal water barrier. Annual meeting of the Society for Investigative Dermatology, 1983. 
Clin Res 3 1:608 A, 1983. 

Wertz PW, Downing DT: Studies on the role of glycolipids in the assembly and maintenance of the 
extracellular membranes in the skin of terrestrial vertebrates. Twelfth International Congress 
of Biochemistry, 1982. 

Wertz PW, Downing DT: The structures and physiological role of lipids in the epidermal barrier to 
percutaneous water loss. Annual scientific meeting of the Society of Cosmetic Chemists, 
1982. 

Wertz PW, Mueller GC: A role for protein alkylation in phorbol ester action. Annual meeting of 
the American Association for Cancer Research, 1981. Proc Amer Assoc Cancer Res 22:137, 
1981. 

Wertz PW, Mueller GC: Activation of CTP:phosphorylcholine cytidyl transferase by 12-0- 
tetradecanoylphorbol- 13 -acetate (TP A) in bovine lymphocytes. Annual meeting of the 
American Association for Cancer Research, 1 980. Proc Amer Assoc Cancer Res 21 :51 1, 
1980. 

Wertz PW, Mueller GC: 5,8, 1 1 , 1 4-Eicosatetraynoic acid (ETY A) inhibits the biological action of 
12-0-tetradecanoylphorbol-l 3-acetate (TP A) in bovine lymphocytes. Annual meeting of the 
American Association for Cancer Research, 1979. Proc Amer Assoc Cancer Res 20:949, 
1979. 

Mueller GC, Kensler TW, Wertz PW, Kwong CH: Retinoic acid inhibition of phorbol ester 

mediated comitogenesis, phospholipid metabolism and capping in bovine lymphocytes. Cold 
Spring Harbor Symposium on Phorbol Esters, 1978. 



Faculty Professional Biography 



-29- 



Philip Wesley Wertz 



Wertz PW, Mueller GC: 12-0-TetradecanoylphorboM3-acetate stimulates phosphatidyl choline 
metabolism in bovine lymphocytes. Annual meeting of the American Society of Biological 
Chemists, 1978. Fed Proc 37:1494, 1978. 

Wertz PW: A computer assisted kinetic analysis of the tautomerization and complex mutarotation 
of D-galactose. Doctoral dissertation, University of Wisconsin, Madison (1976). Dissertation 
Abstracts International 37:4450-B, 1977. 

Anderson L, Wertz PW: Correlation between the complex mutarotation of an aldose and the 

kinetics of its tautomerization. Centennial meeting of the American Chemical Society, 1976. 

5. Invited External Presentations and Lectures 

Presented an invited lecture titled "Regional variation in lipids and barrier function in keratinizing 
epithelia" at the conference on Perspectives on Percutaneous Penetration in Leiden, September, 
1998. 

Seminars at the University of Modena, University of Leiden, University of South Carolina, Medical 
University of South Carolina, University of California at San Francisco and University of Iowa 
College of Dentistry and College of Medicine, 1998. 

Invited speaker at the Gordon Conference on the Barrier Function of Mammalian Skin, New 
Hampshire, 1997. 

Invited speaker at the World Congress of Dermatology, Sydney, Australia, 1997, 

Presented an invited lecture titled "Skin lipids in health and disease" at Catholic University, Seoul, 
Korea, August, 1996 

Presented an invited lecture titled "Effects of lipids on the cosmetic properties of the skin" at Pacific 
Research and Development Center, Seoul, Korea, August, 1996 

Presented an invited lecture titled "Lipids and the epidermal permeability barrier" at the annual 
meeting of the Korean Society for Investigation of the Barrier Function of the Stratum Corneum, 
Seoul, Korea, August, 1 996 

Presented an invited lecture titled "Chemistry and composition of lipids from the skin surface, 
stratum corneum and hair" at Amway Research Center, Grand Rapids, MI, October 1995 

Presented an invited lecture titled "Lipids and the epidermal permeability barrier" at ALZA 
Corporatiuon, Palo Alto, CA, September 1995 

Invited speaker at the Gordon Conference on Barrier Function of Mammalian Skin. Also co- 
directed an afternoon workshop on the ROLES OF LIPrDS IN BARRIER FUNCTION. 

Presented an invited lecture titled "Lipids and the epidermal permeability barrier" at the annual 
scientific meeting of the Society of Cosmetic Chemists, New York, 8-9 December, 1994. 



Faculty Professional Biography 



-30- 



Philip Wesley Wertz 



Presented an invited lecture titled "Antimicrobial lipids from the skin surface" at ConvaTec, a 
subsidiary of Bristol-Myers Squibb, Princeton, NJ, December 1994. 

Served as co-chairman at the Dental Student Research Conference, 1990, 1992 & 1994. 

Presented an invited lecture titled "Epidermal lipids - opportunities for transdermal drug delivery 
and improved skin adhesives" at ConvaTec, a subsidiary of Bristol-Myers Squibb, Princeton, NJ, 
October 1993. 

Presented an invited lecture titled "Skin lipids in health and disease" at The 6th International 
Colloquium on Phospholipids, Hamburg, Germany, 24-27 October, 1993. 

Presented an invited lecture titled "The epidermal permeability barrier — biochemical and 
ultrastructural considerations" Second Annual New Technologies Workshop: Skin Toxicology And 
Pharmacology — In Vitro And Clinical Techniques, Microbiological Associates, Inc., Bethesda, 
MD, 9-10 September 1993. 

Presented an invited lecture titled "Roles of lipids in formation and function of the epidermal 
permeability barrier and the influence of skin surface lipids on adherence of Candida albicans" at the 
ConvaTec Skin Care Symposium, Princeton, NJ, 14 May 1993 

Presented an invited lecture titled "Composition, structure and organization of lipids in stratum 
corneum" at The Winter Workshop (The Physical Organization of Stratum Corneum Lipid) held at 
the University of British Columbia, Vancouver, Canada, February 28 - March 2, 1993 

Served as a judge of the Max Smith Award competition at the 1992 annual meeting of the Iowa 
Chapter of the American Association for Dental Research 

Speaker at conference on the effects of aging on oral mucosa and skin held in Iowa City, May 1992. 

Presented an invited lecture titled "Lipid nature of the epidermal permeability barrier" at ConvaTec, 
a subsidiary of Bristol-Myers Squibb, Princeton, NJ, 1991. 

Speaker and session chairman at International Conference on Liposome Dermatics in Bad 
Griesbach, Germany, October 1991. 

Presented invited lecture titled "Essential fatty acids and epidermal integrity" at the 36th Annual 
Symposium on the Biology of the Skin sponsored by the Cutaneous Biology Foundation, Inc., 
Salishan Lodge, Oregon, 1986. 

Presentations on epidermal lipids at Vicks Research Center, Shelton, CT, in 1982, 1983 and 1984, 

Presented and invited lecture title "Strucutre and function of the polar lipids of epidermis" at the 
Gordon Conference on Epithelial Differentiation and Keratinization, 1983. 



Faculty Professional Biography 



-31- 



Philip Wesley Wertz 



Presented invited lecture titled "The structure and physiological role of lipids in the epidermal 
barrier to percutaneous water loss" at the annual meeting of the Society of Cosmetic Chemists, 
New York. 1982. 

6. Other: Exhibits, films, tapes, special presentations 

XIIL Student Mentoring : 

1. Graduate Theses Directed : 

Degree Year . Candidate Title . . 



M.S. 1993 Sandra Law 
(Co-Directed with Pete Fotos) 

M;S. 1992 Albert Manganaro 



M.S. 



1997 Patricio Jarpa 



Adherence of Candida albicans to porcine stratum 
corneum in vitro. 

Effects of permeabilizers on penetration of propranolol 
through buccal epithelium in vitro. 

Variations of pH, nicotine levels and mutagenicity of 
smokeless tobacco products from U.S., India and 
Venezuela 



Faculty Professional Biography -32- 
I have served or am serving on the thesis committees of: 



Philip Wesley Wertz 



Deborah Vezeau 
David Rolf 
Thomas Poore 
Riaz Ali 
Bruce Ringdahl 
Hudda Hamaddi 

Mike Huberman 

Joseli Alves 

Deena Kuempel 

Earnest Lam 

Kaaren Vargas 

Heather Reid 

Xiobing Du 

2. Other Graduate Committee Service : 

Degree Year Candidate Title 



3. Predoctoral Student Research Mentorship : 

Student Year Awards Received bv Student 



Bennedicte van den Bergh 1998 
(Dutch Ph.D. student) 



Jennifer Garza 


1997- 


Dows Award 
NIDR Award 


Matt Howard 


1998- 


NIDR Award 


Manal Ahmad 


1997 - 


DRA 


Rebecca Guo 


1998- 


Dows Award 


Shannon Li 


1998 - 


DRA 


Kevin Ortale 


1994-1997 


Max Smith Award 
NIDR Awards 


Janet Hoogstraate 


1994 





(Dutch PhD student) 



Sara Whittle 1995-1998 



NIDR Awards 



Faculty Professional Biography 



-33- 



Philip Wesley Wertz 



Trace Lund 1994-1997 DRA 
(David Drake Co-Mentor) 

MattGrau 1996 Director's Award 

(Kevin Donly Co-Mentor) 

DougSquier 1995- Dows Award 

(predental 1995, dental student 1996- ) DRA 

Delon Gilbert 1996-1997 
(Predental, dental student 1997-) 



Keith Slaymaker 1996-1998 
(Predental) 

Matt Abraham 1996 
(Predental) 

P. Bayati 1993-1995 Director's Award 

Dows Award 



Howard Hughs Fellowship 
Dows Award 
AADS Gies Award 



Ajanet Mclain 
(SSTP) 

GGell 

Candace Wright 
(SSTP) 

Aima Ahonkai 
(SSTP) 

Felicia Chang 

Niki Holbrook 
(SSTP) 



1994 



1993-1995 



1992 



1992 



1991-1994 
1992 



Dows Award 



Dows Awards 



Harry Fang 1992 

(local high school student; Science Research 91:86) 

Nate Stevens 1991 
(Predental) 



William B Shade II 1991 
(SSTP, CA Squier Co-Mentor) 



Faculty Professional Biography 



-34- 



Philip Wesley Wertz 



Karen N. Ekpenyong 1991 
(SSTP, CA Squier Co-Mentor) 

Sofia F. Betancourt 1991 
(SSTP, David Drake Co-Mentor) 

Jennifer Haake 1991 
(local high school student; Science Research 91:86) 

Jeffrey T Bortz 1990 
(Medical Student) 



Christopher L Hedberg 
(Medical Student) 

Kathy M Perisho 
(Medical Student) 

Mark A Wix 
(Medical Student) 

Jeffrey C Melton 
(Medical Student) 

Russel R Burken 
(Medical Student) 

Susan Rubes 
(Medical Student) 

Paul M Stover 
(Medical Student) 

Saundrett Gibbs 
(Medical Student) 

Dan Johnson 
(Medical Student) 

Marianne C Miethke 
(Medical Student) 

Sherri Long 



1989 



1988 



1987 



1987 



1983-1984 



1986 



1986 



1985 



1985 



1985 



1985 



Best Basic Science Presentation 
Best Clinical Presentation 
Best Presentation in Dermatology 
All at the 1986 Medical Student 
Research Symposium at Galviston 



Faculty Professional Biography 



-35- 



Philip Wesley Wertz 



Richard T Schoephoerster 1985 
(Undergraduate) 

Mark L Frost 1984 
(Medical Student) 

Craig Birkby 1982 
(Medical Student) 



XIV. Continuing Education (sponsored by the University; presented during the past five years most recent 
first) 

Presentations qualifying for continuing education credit at: 
Dermatology meetings & symposia 20 
Clinical research meeting 1 
Seminars 2 

XVI. Teaching Activities (Include the past five years; most recent first; provide narrative describing 
responsibility in each course) 

1 . Classroom . Seminar , or Teaching Laboratory 

Year Course Title and No. Mo. registered length of course 

1996- Systemic Disease Manifestations 75 16 wks 

86:155 

facilitator for problem based learning 

1993- Pathophysiology of Skin & 9 16 wks 

Mucosa 112:251 

1992-95 Dental Biochemistry 

99:161 70 16 wks 

1991- Dental Science Research 

Methodology 151:210 31 8 wks 

Course Director 



1991- Research Seminar Series 15 16 wks 100% 

151:200 
Director 

1991- Pathophysiology of Salivary 12 16 wks 10% 



Faculty Professional Biography 



-36- 



Philip Wesley Wertz 



Glands 112:254 



1990-91 



Dental Science Research 



25 



8wks 10% 



Methodology 112:210 



1982-90 



Basic Science for Dermatology 
College of Medicine 



12 



16 wks 25% 



1982-90 



Research in Dermatology 



6 



16 wks 35% 



62:4 



2. Clinical Teaching (undergraduate, graduate and direct patient care) 



Students supervised: 

2 local high school students 
7 SSTP students 

6 University of Iowa undergraduates 
14 medical students 
14 dental students 

3 College of Dentistry MS students 

1 high school science teacher 

2 postdoctoral associates 
2 Dutch PhD students 

see pages 25-32 for details. 



Year 



Where Occurred 



weeks/year 



hrs/wk 



Attorney Docket No.: 00.22US 

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE 



First Inventor: MAES.etal. 

Serial No.: 09/773,351 Group Art Unit: 1617 

Filed: January 31, 2001 Examiner: JIANG, Shaojia A. 



For: Cholesterol Sulfate and Amino Sugar Compositions for Enhancement of Stratum Corneum Function 



Transmittal of Second Supplemental 
Information Disclosure Statement under 37 C.F.R. 1.97(c) 



Commissioner for Patents 

P,0. Box 1450 

Alexandria, VA 22313-1450 



Sir: 

In accordance with 37 C.F.R. 1.56, 1.97 and 1.98, Applicant submits herewith references which 
he believes may be material to the examination of this application and with respect to which there may be 
a duty to disclose in accordance with 37 C.F.R. 1 .56. 



While the references may be "material" under 37 C.F.R. 1 .56, it is not intended to constitute an 
admission that the references are "prior art" unless specifically designated as such. 



The filing of this Information Disclosure Statement (IDS) shall not be construed as a 
representation that no other material references exist or that a search has been conducted. 



These references are listed on the enclosed Form PTO/SB/08A (substitute for Form 1449/PTO) 
which is in accordance with the requirements of M.P.E.P. 609. Copies of required English-language 
abstracts and English-language publications, if any, are enclosed for each foreign language reference to 
satisfy the requirement regarding a concise explanation of foreign language references. 



1) JP11-005742, 01/12/1999, Shiseido Co. Ltd. (Abstract) 

2) US 6,589,945 B1, 07/08/2003, Wachter, etal. (Counterpart of WO 98/17244) 

3) US 5,989,568, 11/23/1999, Breton, et aL (Counterpart of JP08-231342 A) 

4) WO88/01274 , 02/25/1988, Broadbent (Counterpart of J P Patent Publication JP02-503554 A) 

5) JP2001 -002551, 01/09/2001, Kanebo Ltd. (Abstract) 

6) JP59-013708, 01/24/1984, Shiseido Co. Ltd. (Abstract) 



It is respectfully requested that these references be considered by the Patent and Trademark 
Office in its examination of the above-identified application and be made of record therein. The Examiner 
is also invited to contact the undersigned if there are any questions concerning this paper or the attached 
references. 



This IDS is being submitted after the first Office Action on the merits of this application, but prior 
to Final Rejection under 37 C.F.R. 1.113, or a Notice of Allowance under 37 C.F.R. 1.311. A fee of 
$180.00 under 37 C.F.R. 1.17(p) is paid with this submission. If any other fee should be required for the 
submission of this IDS, please charge Deposit Account No. 05-1320; a duplicate copy of this sheet is 
enclosed. 



Respectfully submitted, 



Date:U<U^UuJ l0,ZDQl I 





Dorene M. Price (Reg. No. 43,108) 
Estee Lauder Companies 
155 Pinelawn Road 
Suite 345 South 
Melville, NY 11747 



Attorney Docket No.: 00.22US 



IN THE UNITED STATES PATENT AND TRADEMARK OFFICE 



First Inventor: MAES, et al. 

Serial No.: 09/773,351 Group Art Unit: 1617 

Filed: January 31, 2001 Examiner: JIANG, Shaojia A. 



For: Cholesterol Sulfate and Amino Sugar Compositions for Enhancement of Stratum Corneum Function 



Transmittal of Second Supplemental 
Information Disclosure Statement under 37 C.F.R. 1.97(c) 



Commissioner for Patents 
P.O. Box 1450 
Alexandria, VA 22313-1450 



Sir: 

In accordance with 37 C.F.R. 1 .56, 1 .97 and 1 .98, Applicant submits herewith references which 
he believes may be material to the examination of this application and with respect to which there may be 
a duty to disclose in accordance with 37 C.F.R. 1 .56. 



While the references may be "material" under 37 C.F.R. 1 .56, it is not intended to constitute an 
admission that the references are "prior art" unless specifically designated as such. 



The filing of this Information Disclosure Statement (IDS) shall not be construed as a 
representation that no other material references exist or that a search has been conducted. 



These references are listed on the enclosed Form PTO/SB/08A (substitute for Form 1449/PTO) 
which is in accordance with the requirements of M.P.E.P. 609. Copies of required English-language 
abstracts and English-language publications, if any, are enclosed for each foreign language reference to 
satisfy the requirement regarding a concise explanation of foreign language references. 



1) JP11-005742, 01/12/1999, Shiseido Co. Ltd. (Abstract) 

2) US 6,589,945 B1, 07/08/2003, Wachter, et al. (Counterpart of WO 98/17244) 

3) US 5,989,568, 1 1/23/1999, Breton, et al. (Counterpart of JP08-231342 A) 

4) WO88/01274 , 02/25/1988, Broadbent (Counterpart of JP Patent Publication JP02-503554 A) 

5) JP2001-002551, 01/09/2001, Kanebo Ltd. (Abstract) 

6) JP59-013708, 01/24/1984, Shiseido Co. Ltd. (Abstract) 



It is respectfully requested that these references be considered by the Patent and Trademark 
Office in its examination of the above-identified application and be made of record therein. The Examiner 
is also invited to contact the undersigned if there are any questions concerning this paper or the attached 
references. 



This IDS is being submitted after the first Office Action on the merits of this application, but prior 
to Final Rejection under 37 C.F.R. 1 .1 13, or a Notice of Allowance under 37 C.F.R. 1 .31 1 . A fee of 
$180.00 under 37 C.F.R. 1.1 7(p) is paid with this submission. If any other fee should be required for the 
submission of this IDS, please charge Deposit Account No. 05-1320; a duplicate copy of this sheet is 
enclosed. 



Respectfully submitted, 



Date: ^UuLA>oJL 10/ SS DL 1^£UjJ^l 
7] 0 Dorene I 




Dorene M. Price (Reg. No. 43,108) 
Estee Lauder Companies 
155 Pinelawn Road 
Suite 345 South 
Melville, NY 11747 



PTO/SB/OBA (07-05) 
Approved for use through 07/31/2006. OMB 0651-0031 
U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE 
Under the Paperwork Reduction Act of 1995. no persons are required to respond to a collection of information unless it contains a valid OMB control numbe r. 



Substitute for form 1449/PTO 



INFORMATION DISCLOSURE 
STATEMENT BY APPLICANT 

(Use as many sheets as necessary) 

V Sheet 



Complete if Known 



Application Number 



Filing Date 



January 31,2001 



First Named Inventor 



Art Unit 



Examiner Name 



09/773,351 



MAES, et al. 



1617 



JIANG, Shaojia A. 



1 



of 



Attorney Docket Number 



00.22US 



U. S. PATENT DOCUMENTS 


Examiner 
Initials* 


Cite 
No. 1 - 


Document Numhpr 
Number-Kind Code 2 r * ^ 


Publication Date 
MM-DD-YYYY 


Namp nf Patentpp nr 

I^ICllllW vl 1 DLwl ILwu Ul 

Applicant of Cited Document 


Pages, Columns, Lines, Where 
Relevant Passages or Relevant 
Figures Appear 






US- 












US- 












US- 












US- 












US- 












US- 












us- 












us- 












us~ 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 









FOREIGN PATENT DOCUMENTS 


Examiner 
Initials* 


Cite 
No. 1 


Foreign Patent Document 


Publication 
Date 
MM-DD-YYYY 


Name of Patentee or 
Applicant of Cited Document 


Pages, Columns, Lines, 
Where Relevant Passages 
Or Relevant Figures Appear 


T 6 


Country Code 3 'Number 4 "Kind Code 5 {if known) 




















JP1 1-005742 (Abstract) 


01-12-1999 


Shiseido Co. Ltd. 










US 6,589,945 B1 - 


07-08-2003 


Wachter, et al. 










(CntrprtW098/17244) 












US 5,989,568 -- 


11-23-1999 


Breton, et al. 










(CntrprtJP08-231342 A) 









Examiner 




Date 




Signature 




Considered 





considered. Include copy of this form with next communication to applicant. Applicant's unique citation designation number (optional). 2 See Kinds Codes of 
USPTO Patent Documents at www.uspto.Qov or MPEP 901,04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For 
Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by 
the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language 
Translation is attached. 

This collection of information is required by 37 CFR 1.97 and 1.98. The information is required to obtain or retain a benefit by the public which is to file (and by the 
USPTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1.14. This collection is estimated to take 2 hours to complete, 
including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments 
on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent 
and Trademark Office, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND 
TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450. 

If you need assistance in completing the form, call 1-800-PTO-9199 (1-800-786-9199) and select option 2. 



PTO/SB/08A (07-05) 
Approved for use through 07/31/2006. OMB 0651-0031 
U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE 
Under the Paperwork Reduction Act of 1 995, no persons are required to respond to a collection of information unless it contains a valid OMB control numb er. 



Substitute for form 1449/PTO 



INFORMATION DISCLOSURE 
STATEMENT BY APPLICANT 

(Use as many sheets as necessary) 



V Sheet 



Complete if Known 



Application Number 



Filing Date 



January 31 , 2001 



First Named Inventor 



Art Unit 



Examiner Name 



09/773,351 



MAES.etal. 



1617 



JIANG, Shaojia A. 



-2L 



Attorney Docket Number 



00.20US 



U. S. PATENT DOCUMENTS 


Examiner 
Initials* 


Cite 
No. 1 


HnfMimont Mnmhar 
UUOUIIICIIL INUIIIUCl 

Number-Kind Cofe ZOfkf,own) 


Pnhliratinn Date 

MM-DD-YYYY 


INdlllc Ul rdlcMLct? Ul 

Applicant of Cited Document 


Relevant Passages or Relevant 
Figures Appear 






US- 












US- 












US- 












US- 












US- 












US- 












US- 












US- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 












us- 









FOREIGN PATENT DOCU 


WIENTS 


Examiner 
Initials* 


Cite 
No. 1 


Foreign Patent Document 


Publication 
Date 
MM-DD-YYYY 


Name of Patentee or 
Applicant of Cited Document 


Pages, Columns, Lines, 
Where Relevant Passages 
Or Relevant Figures Appear 


T* 


Country Codo 3 ~Number 4 "Kind Code 5 (if known) 




















WO88/01274 


02-25-1988 


Broadbent 










(Cntrprt JP02-503554 A) 














JP2001-002551 (Abstract) 


01-09-2001 


Kanebo Ltd. 










JP59-013708 (Abstract) 


01-24-1984 


Shiseido Co. Ltd. 





















Examiner 




Date 




Signature 

*CVA»J11ICD 




Considered 





considered. Include copy of this form with next communication to applicant. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of 
USPTO Patent Documents at www.usDto.QOv or MPEP 901 .04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For 
Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by 
the appropriate symbols as indicated on the document under WIPO Standard ST. 16 if possible. 6 Applicant is to place a check mark here if English language 
Translation is attached. 

This collection of information is required by 37 CFR 1 .97 and 1 .98. The information is required to obtain or retain a benefit by the public which is to file (and by the 
USPTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1.14. This collection is estimated to take 2 hours to complete, 
including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments 
on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent 
and Trademark Office, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND 
TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450, 

If you need assistance in completing the form, call 1-B00-PTO-9199 (1-800-786-9199) and select option 2. 



English abstract of reference A \ 

PATENT ABSTRACTS OF JAPAN 



(1 1 publication number : 



11-005742 



(43)Date of publication of application : 12.01.1999 



(51)IntCI. 



A61K 31/575 
A61K 7/00 
A61K 7/48 




(21 Application number : 09-21 8288 (7 1 Applicant : SHISEIDO CO LTD 
(22)Date of filing: 30.07.1997 (72)Inventor : SATO JUNKO 



(30)Priority 

Priority number : 091 1 7508 Priority date : 21 .04.1 997 Priority country : JP 



(54) EXTERNAL PREPARATION CONTAINING CHOLESTEROL SULFATE 

(57)Abstract 

PROBLEM TO BE SOLVED: To obtain the subject dermal external preparation for 
controlling decomposition of desmosome in the skin by including a cholesterol sulfate. 
SOLUTION: This external composition is obtained by including a cholesterol sulfate 
(cholesterol 3-sulfate ester) derived from a living body or partially or totally 
synthesized at approximately 0.005 to 20 wt.%, based on the whole composition, 
preferably 0.5 to 5 wt.%, for lotion, cream or the like, and further including a diluent or 
aid (alcohol, water, chelating agent, urea, surfactant or the like) which is commonly 
used for cosmetics and external medicines. 



[Date of sending the examiner's decision 
of rejection] 

[Kind of final disposal of application other 
than the examiner's decision of rejection 
or application converted registration] 



DENDA MITSUHIRO 
KOYAMA JUNICHI 




LEGAL STATUS 



[Date of request for examination] 



29.05.2002 



[Date of final disposal for application] 

[Patent number] 

[Date of registration] 

[Number of appeal against examiner's 
decision of rejection] 

[Date of requesting appeal against 
examiner's decision of rejection] 
[Date of extinction of right] 



Copyright (C); 1998,2003 Japan Patent Office 



CORRECTED 
-VERSION* 



PCT 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 



Counterpart of reference M 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 4 ; 
C07J 31/00, A61K 31/575 



Al 



(11) International Publication Number: WO 88/ 01274 
(43) International Publication Date: 25 February 1988 (25.02.88) 



(21) International Application Number: PCT/AUS7/0028 1 

(22) International Filing Date: 21 August 1987 (21.08.87) 

(31) Priority Application Number: PH 7614 

(32) Priority Date: 21 August 1986 (21.08.86) 

(33) Priority Country: AU 



(71) Applicant (for all designated States except US): 

BROADBENT, James, Meredyth IAU/AU]; Suite 2, 
227 Burwood Road, Hawthorn, VIC 3122 (AU). 

(71X72) Applicant and Inventor: KOSUGE, Yoshiki 
[JP/JP]; 3-4-18, Kamiashiarai, Shizuoka (JP), 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): KOSUGE, Takuo 
[JP/JP1; 3-4-18, Kamiashiarai, Shizuoka (JP). TSUJI, 
Kuniro [JP/JP]; 2-11-17, Kamiashiarai, Shfeuoka (JP). 
ISHIDA, Hiioshi riP/JP]; 200-16, Sena, Shizuoka (JP). 



(74) Agents: SLATTERY, John, Michael et al.; Davies & 
Collison, 1 Little Collins Street, Melbourne, VIC 
3000 (AU). 



(81) Designated States: AT (European patent), AU, BE (Eu- 
. ropean patent), CH (European patent), DE (Euro 
pean patent), DK, FR (European patent), GB (Euro- 
pean patent), IT (European patent), JP, KR, LU (Eu- 
ropean patent), NL (European patent), SE (European 
patent), US. 



Published 

With international search report, 



(54) Title: ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES 




♦(Referred .to in PCT Gafcette No.05/1990, Section II) 

r 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 



appl 


[cations under the PCT. 










AT 


Austrni 


ES 


Spam 


MO 


Madagascar 


Att 


Australia 


FT 


Finland 


ML 


Malt 


BB 


Barbados 


FR 


France 


MR 


Mauritania 


BE 


Bcfeum 


GA 


Gabon 


MW 


Malawi 


BF 


Burkina Fasaa 


GB 


United Kingdom 


NL 


Netherlands 


BG 


Bulgaria. 


EU 


Hungary 


NO 


Norway 


BJ 


Benin 


tr 


Italy 


BO 


Romania: 


BR 


Brazil 


jp 


Japan 


SD 


Sudan 


CA 


Canada 


KP 


Democratic People's RenubBc 


SE 


Sweden 


CF 


Central African Republic 




of Korea 


SN 


Senega! 


CG 


Congo 


KR 


RcpublbqfKorea 


su 


Soviet Union 


CH 


Switzerland 


U 


Liechtenstein 


TO 


Chad 


CM 


Cameroon 


LK 


Sri Lanka 


TG 


Ta*o 


DC 


Germany, Federal Rcpubfcof 


UJ 


Luxembourg 


US 


United States of America 


Wt 


Denmark. 


MC 


Monaco 







per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 4 : 
C07J 31/00, A61K 31/575 



Al 



(11) International Publication Number: WO 88/ 01274 

(43) International Publication Date: 25 February 1988 (25.02.88) 



(21) International Application Number: PCT/AU87/0028 1 

(22) International Filing Date: 21 August 1987 (2 L08.87) 

(31) Priority Application Number: PH 7614 

(32) Priority Date: 21 August 1986 (21.08.86) 

(33) Priority Country: AU 



(71) Applicant (for all designated States except US): 

BROADBENT, James, Meredyth [AU/AU]; Suite 2, 
227 Burwood Road, Hawthorn, VIC 3122 (AU). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) ; KOSUGE, Yoshiki 
[JP/JPJ; KOSUGE, Takuo [JP/JPJ; 3-4-18, Kamiashia- 
rai, Shizuoka (JP). TSUJI, Kuniro [JP/JP]; 2-11-17, 
Kamiashiarai, Shizuoka (JP). ISHIDA, Hitoshi [JP/ 
JP]; 200-16, Sena, Shizuoka (JP). 



(74) Agents: SLATTERY, John, Michael et al.: Davies & 
Collison, 1 Little Collins Street, Melbourne, VIC 
3000 (AU). 

(81) Designated States: AT (European patent), AU, BE (Eu- 
ropean patent), CH (European patent), DE (Euro- 
pean patent), DK, FR (European patent), GB (Euro- 
pean patent), IT (European patent), JP, KR, LU (Eu- 
ropean patent), NL (European patent), SE (European 
patent), US. 



Published 

With international search report. 



(54) Title: ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES 




(57) Abstract 

A compound of general formula (I), in substantially pure form, wherein A is a cation. A method for preparation is 
also disclosed, together with compositions and methods of use thereof. 



FOR THE PURPOSES Of INFORMATION ONLY 



Codes used to identifyStatespar^t0thePCToathefi:oatpagesofpamp Metspublishdnginteniationalappli- 
cations under the PCX 



AT Austria " 

All Australia 

BB Barbados 

BE Belgium 

BG Bulgaria. 

BJ Benin 

BR Brazil 

CF Central African Republic 

CG Congo 

CH Switzerland 

CM Camecooa 

DE Germany Federal Republic of 

DK Denmark 

Ft Finland 



FR France 

GA Gabon 

GB United Kingdbm 

HU Hungary 

IT Italy 

JP Japan 

KP Democratic Feople's Republic 

of Korea 
ER Republic of Korea 
LI Liechtenstein 
LK Sri Lanka 
LU Luxembourg 
MC Monaco 
MG 



ML Mali 

MR Mauritania 

MW Malawi 

NL Netherlands 

NO Norway 

RO Romania 

SD Sudan 

SE Sweden 

. SN Senegal 

SU Sonet Union 

TD Chad 

TG Togo 

US United States of America 



WO 88/01274 



1 



PCT/AU87/00281 



"ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES" 

This invention relates to the 
identification, isolation and preparation of an active 
principle by extraction from natural tissues, and in 
particular it relates to the identification, isolation 

5 and preparation of such an active principle by 

extraction from particular tissues of sharks- 

In Japan, a preparation known as "deep-sea 
shark liver oil" has been used as a folk remedy for a 
long time. It is an oil prepared from shark's liver 

10 and is. normally capsulated in soft capsules* The 

liver oil is said to be effective in treatment of many 
kinds of diseases, especially those which are related 
to the liver, such as hepatitis, nephritis, diabetes, 
etc. As well, when- used externally, it is widely 

15 recognised that the liver oil is effective in 

treatment of scalds, burns or other types of skin 
trouble, and also is ideal as an ingredient for 
cosmetics . 

The present inventors have been studying 
20 this material for many years, and recently have 
discovered the unexpected fact that an active 
substance exists in the aqueous component of shark's 
liver rather than the oil soluble component* This 
fact was recognised from a comparison of the practical 
25 use of the liver oil and a powder produced from the 



WO 88/01274 PCT/AU87/00281 

2 

aqueous component of the liver by evaporation of the 
water. In comparative tests of a dosage of 94)0mg of 
the Liver oil per day and 60mg of the powder per day, 
the latter gave a better clinical result than the 
5 former- Furthermore , where the liver oil was 

thoroughly washed with water r the resulting oil showed 
almost no effect. These facts indicate that the 
active substance of deep-sea shark liver is not 
oil-soluble as previously believed, but is 
10 water-soluble. 

According to the present invention, there is 
provided an active principle which is isolated from an 
aqueous extract of the liver and/or gallbladder of a 
shark. 

15 in a first aspect of the invention, there is 

provided a compound of the general formula I, in 
substantially pure form r 



OH 



25 




4U wherein A is a cation, such as a sodium, potassium, 
calcium or ammonium ion, or an organic amine. 

In other aspects, this invention provides a 
method for the preparation of a compound of general 
formula I in. substantially pure form, together with 

35 



compositions for pharmaceutical, dietary or cosmetic 
purposes which comprise such a compound. 

By using activity assays which are described 
in detail below, it has been shown that the active 
principle is water-soluble and does not exist in the 
oil-soluble component of shark's liver. These assays 
have been used in a series of tests to ascertain 
whether the active principle exists only in the liver. 
All parts of the shark's body, such as the bones, 
meat, gallbladder, ovary, alimentary canal, etc., have 
been investigated, and it has been found that the 
gallbladder showed the same activities as liver in the 
assays. This result indicates that the active 
principle exists only in liver and gallbladder. 

In general terms, the two bioassays referred 
to herein and used to identify sources of the active 
principle and to assess the degree of purity of an 
extract, are designed to identify characteristic 
pharmacological activities of the substance. In 
particular, the bioassays, designated as (A) and (B) 
are based on the following activities: 

(A) The active principle prevents liver trouble in 
mice caused by carbon tetrachloride. 

(B) The active principle increases the respiration 
rate in mice when a toxic substances such as 
nicotine is administered. 

The present invention also provides a method 
for preparing an active principle as described above, 
which comprises the steps of preparing an aqueous 
extract of the liver and/or gallbladder of a shark, 
and isolating the active principle from the aqueous 
extract. 

The following description sets out general 
procedures for isolation of the active principle from 



WO88/01274 - PCT/AU87/00281 

4 

the aqueous extract of the liver and/or gallbladder of 
a shark r involving the steps of extraction with polar 
organic solvents/ adsorption on suitable adsorbents 
and/or chromatography techniques. 
5 In order to determine whether the active 

principle is soluble in polar organic solvents, such 
as methanol t ethanol, acetone r etc, r the powder 
obtained by freeze-drying of shark's bile was 
. extracted with polar organic solvent f then the £A) and 
10 (B) assays were applied to both the soluble part and 

the insoluble part* Activity was seen only in the 
assays on the soluble portion, thus establishing that 
active principle is soluble in polar organic solvents. 

In testing to determine whether the active 
15 principle can be isolated utilising adsorbents, many 

adsorbents were examined and it was found that the 
active principle can be adsorbed by ion exchange 
resins of basic anion exchange type r or by synthetic 
adsorbents such as XAD f Sep-pak cl8' f etc, or 

20 charcoal. This absorption test was performed by 

extracting shark f s liver and/ or gallbladder with 
water. Each adsorbent under test was added to the 
extract and left to stand overnight. The mixture was 
then filtered and each filtrate tested for activity by 
25 the (A) and (B) assays. The results indicate that the 

active substance is adsorbed by those adsorbents 
mentioned above. The active principle may be 
recovered from the adsorbent resins by extraction with 
acid, alkali or salts, and front the synthetic 
30 adsorbents and charcoal by extraction with polar 

organic solvents. 

Further purification of the active principle 
is achieved by chromatography r for example in a silica 
column, Sephadex LH-20 column, or by preparative TLC 



WO 88/01274 PCT/AU87/00281 

5 

(thin layer chromatography) or HPLC (high performance 
liquid chromatography) , etc. Each method gave 
satisfactory results, but HPLC gave the best 
purification. The active principle as isolated by 

5 HPLC was quite pure because it gave very sharp single 

peak and also gave a single spot of approximate 
representative Rf value of 0.36 on TLC. The active 
principle in its purified form is a white powder of 
melting point of 140 °C„ 

10 Testing of the purified active principle by 

vanillin sulfuric acid gave a purple colour, 
indicating that it contains bile acid or bile alcohol 
in its structure. It has already been found that the 
bile of sharks contains a bile alcohol named scymnol. 

15 After partial acetylation of the active principle with 

acetic anhydride, followed by treatment of the crude 
product with dry dioxan-trichloroacetic acid for 
several days, scymnol was identified from the reaction 
mixture. The result indicated that the active 

20 principle is a scymnol derivative. It was the first 

isolation of the pure scymnol derivative contained in 
bile of shark, as the active principle. 

A preferred procedure for isolation of the 
active principle from the lyophilized bile of 

25 Rhizoprinodon acutus (obtained by homogenization and 

f reeze-drying of gall-bladders) , is set out in the 
following chart: 



30 



35 



WO 88/01274 



PCT/AU87/00281 



6 

Lyophilized bile pf Khizoprionodon acutus 
extracted with 1, n-Hexane (100mlx3) 
2. MeOH {100mlx3) 

Fraction I (MeOE-extraCt) 
1* dissolved in 1^0 

2. Amberlite XAD-2 cvc. , eluting with 
i. H 2 0 (400ml) 
ii- MeOH (400ml) 

Fraction II (MeOH-eluate) 

1. dissolved in CECl 3 -MeOH(l:l) 

2. Sephadex LH-20 c-c« eluted with 

i. CECl 3 -MeOH(l:l) (300ml) 

ii. MeOH • (500ml) 

Fraction III 

HPLC; YMC-Pack A-324 (ODS) 

Colorless powder (compound I) 

As set out above r in this procedure the 
lyapphilized material is deflated with n-hexane, and 
then extracted with methanol. The concentrate thus 
obtained is applied to an Amberlite XftD-2 column in 

5 batches, using H 2 0, and ethanol as eluents. As the 

ethanol eluate contains the active principle (as 
determined by color reagent) , this fraction is 
successively subjected to gel filtration on Sephadex 
LH-*20 with chloroform-methanol and methanol* The 

10 active principle is so effectively contained in the 

methanol eluate that its final purification is 
achieved by successive application of HPLC with a 
reverse phase column. 



WO 88/01274 PCT/AU87/00281 

7 

It has been suggested that scymnol might be 
in the form of a sulphate ester, but no positive 
information has been published about the position of 
attachment of the sulphate ester, because scymnol has 

5 six hydroxyl groups where the sulphate ester group 

might be attached. The present scymnol derivative has 
never been isolated as a pure substance. The active 
powder as purified by HPLC was subjected to elementary 
analyses. Results were anal: calcd for C 27 H 51 0gNS, 

10 C;57.34, H;9.02, N;2.47, S;5.66. Found C;57.23, 

H;8.92, N;2.45, S;5.30. These results suggested that 
the active compound has ammonium sulphate ester in the 
structure. Nuclear magnetic resonance spectroscopy of 
the active powder showed the following properties. 

15 -"-H-NMRdn d 4 -MeOH) 6 (ppm) : 

4.22(dd, 1H, J=4.5 and 10.0Hz), 

4.11(dd, 1H, J=10.0 and 16.7Hz), 

4.00(bs, 1H) , 3.80(d, 1H, J=1.2Hz), 

3.60-3.80 (ia, 4H) , 3.30-3.45(m, 1H) , 0.72(3, 3H) . 

20 

72.9(d) , 
48.4(d) , 
41.0(d) , 
35.8(t) , 
28.8 (t) , 
13.1(g) . 

J ' J C-NMR spectrum shows that the active 
compound has 27 carbon atoms made up of three methyl, 
11 methylene, 11 methine and two tertiary carbons. 
The signals at low field (0.72-2.35) in 1 H-NMR 

spectrum suggest that it seems to be a coprostane 

13 

derivative. At the higher field in C-NMR spectrum, 
signals at 74.1(d), 72.9(d), 71.3(d) and 69.1(d) are 



C-NMR(i 
71.3(d) , 
47.8(d) , 
40.4 (t), 
25 33.3(t), 
27.9(d), 



d 4 -MeOH) 
69.1(d) , 
47.5(s) , 
37.0 (d) , 
32.1(t) , 
24.3(t) , 



6 (ppm) : 
66.7 (t) , 
43.1(d) , 
36.5 (t) , 
31.2 (t) , 
23.2(g) , 



74.1(d) , 
61.2(t) , 
43.0 (d) , 
35.9(s) , 
29.6 (t) , 
18.1(g) , 



35 



WO 88/01274 



PCT/AU87/00281 



20 



25 



30 



8 

assignable to the metfaine carbon with hydroxy! group ♦ 
And the two signals at 66. 7 (t) and 61.2 (t> are 
ascribable to the O-substituted methylene carbon. 
2D0COSY0NHR spectra and C-H-shift-COSY relationship 
indicate that these two carbons attach to a methine 
carbon and one of them with low chemical shift (66.7) 
has two unequivalent protons at 4.22 (dd) snd 
4*14(dd)ppra in the ^H-NMR spectrum , which indicates 
that the active compound has the partial moiety of 
HOCE 2 -CH-GH 2 OR in the molecule* From the results of 
elementary analyses, R is -SOjHH^. 



From these NMR spectra and elementary 
analyses, the powder is characterised as 3a , 7a, 12a , 
15 245, 26 -pentahydroxycoprostane-27 -ammonium sulphate 

ester. The ammonium ion in the structure possibly 
came from the phosphate ammonium buffer used as mobile 
phase in HPLC, by replacement of a sodium ion. To 
verify this point, an active powder purified by XAD-2 
and then by column chromatography on Sepadex LH-20 was 
subjected to atomic absorption spectrophotometry for 
sodium and to elementary analysis for nitrogen. The 
results were, calcd. for C^E^OgSNa, Na;4,03, N;0.00, 
found Na?3.57, N;0.02. The stereochemistry of the 
C-24 position in the structure was determined as 24R 
by X-ray crystallographic analysis of scymnol and the 
specific rotation of sodium scymnol sulphate is 
positive. Accordingly, it is concluded that the 
active principle isolated from shark is 24R-(+)-3a, 
7a r 12a,24 , 26 -pentahydroxycoprostane-27 -sodium sulphate 
ester. 

The sodium or ammonium ion in the sulphate 
ester is easily replaced by other metal ions such as 
potassium, calcium, etc., or by organic amine cations 

35 



WO 88/01274 



PCT/AU87/00281 



such as amino acids , etc., by means of well known 
procedures* 

The following Tables illustrate the activity 
of the aqueous extracts of this invention: 

TABLE I 



Dosage 



Bioassay (A) Bioassay (B) 



(Units) 



(Seconds) 



Oil-soluble part of 

shark's liver 500mg 

Water-soluble part 

of shark's liver 50ntg 

Control 



13,800 

9,500 
13,000 



21 

15 
22 



TABLE II 



Aqueous Extract of 
Shark's Gallbladder , 
Purified by: 



- Bioassay (A) Bioassay (B) 

Dosage (Units) (Seconds) 



Charcoal adsorption 


5mg 






15 


XAD-2 adsorption 


lmg 






16 


Anion-exchange resin 
adsorption 


0 . 5mg 


8 


,200 


14 


Purified active principle 


0 .15mg 


9 


,600 


15 


Control 




14 


,000 


22 



Standard bioassays referred to in the above 
description were performed as follows: 
Bioassay (A) 

Biological test for protective activity against 



WO 88/01274 



PCT/AU87/00281 



10 

carbon tetrachloride (cci^) -induced liver lesions in 
mice. 



Kale Stdiddy mice (weight 30-35g) were used in 
5 groups of 5 animals. Samples of test materials were 

administered orally 7 days at a suitable daily dose 
and Q^lml of 5% CCl^ in olive oil was. orally 
administered at 24hrs after "the last sample 
administration^ Blood was obtained from the orbital 
10 sinus at 24hrs after the CCl^ administration. Serum 

was obtained by centrifugation (3,000 rpnu, lOmin) and 
glutamic pyruvic transaminase (GPT) activity was 
measured, by Reitman-Frankel-Momose method. Activity 
was expressed as a comparison of X3FT values between 
15 the sample-administered groups and controls. 

Bioassay (&) 

Effect on respiration in nicotine administration 
to mice. 

Kale Stdrddy mice (weight 20-22g) were used in 
groups. of 5 animals. Nicotine tartrate (3mg) was 
injected subcutaneously* Samples of test materials 
were orally administered 3hrs before nicotine 
administration. The time taken for 30 respirations 
was counted 5 minutes after nicotine administration. 
Activity was expressed as -a comparison of the counted 
time between the sample- administered groups and 
controls; 

The present invention also provides a 
pharmaceutical composition comprising an active 
substance as described above, together with a 
pharmaceutical^ acceptable carrier or diluent 
therefor. By way of example, the active substance can 
be formulated as stable tablets after being mixed as a 

35 



20 



25 



30 



WO 88/01274 



PCT/AU87/00281 



11 

powder with a known carrier or bulking agent. 
Alternatively, the active substance can be 
incorporated into a lotion or cream base for topical 
application. In yet another alternative, the active 

5 substance can for example be filled in soft gelatin 

capsules, if desired after being admixed with shark 1 s 
liver oil. Such pharmaceutical compositions may be 
used, for example, for the protection of the liver or 
activation of liver function in the treatment of 

10 diseases or conditions affecting the liver such as 

hepatitis, nephritis, diabetes, etc.. Such 
compositions may also be used for the activation of 
regeneration of skin tissue, for example, in the 
treatment of dermatitis, trauma or acne. 

15 Clinical tests which have been performed 

using compositions containing the active substance 
have specifically demonstrated its activity in 
restoration of the liver function, and in the 
treatment of seborrhea. 

20 in a further aspect of this invention, there 

is provided a dietary or health food composition which 
comprises the active principle described herein, 
together with one or more appropriate base or carrier 
materials. Such a composition may, for example, be 

25 useful in the treatment of a hangover. 

In another aspect, the present invention 
provides a cosmetic composition comprising the active 
principle as described above, together with a cosmetic 
base material. 

30 The compositions of the present invention 

may also incorporate known pharmaceuticals or other 
active ingredients, for example, antibiotics or other 
antibacterial substances. 



WO 88/01274 PCT/AU87/00281 

12 

Further details of this invention will be 
apparent front the following Examples which illustrate 
the invention without: limiting it in any way. 

5 EXAMPLE 1 - Preparation of Crude Active Principle 

28 Og of a mixture of liver and gallbladder 
isolated from 4kg of shark was homogenised in 300ml of 
water, and the mixture was centrifuged at 12 f 000 rpm 
for 30 minutes to obtain a clear aqueous layer. 50g 

10 of ion exchange resin of basic anion exchange type was 

added to the aqueous . layer and the mixture was left to 
stand overnight. The resin was removed by filtration 
and washed with water. The resin was then extracted 
with 200ml of 0*5% sodium chloride solution. lOOg of 

15 X&D2 was added to the extracted solution. XAD2 was 

removed by filtration and washed with water. XAD2 was 
extracted with 200ml of ethanol. From the extract, 
ethanoi was removed by distillation to obtain 45mg of 
crude active powder. 



20 



25 



30 



EXAMPLE 2 — Silica gel column chromatography 

10 Og of crude active compound, obtained by 
adsorption on a XAD-2 column was subjected to 
chromatography on. a. silica gel column, using MeOH- 
CHC1 3 -H 2 0 (30:70:6) as solvent-, to afford white powder 
(40g), 

EXAMPLE 3 r Thin layer chromatography (TLC) 

Crude active compound was subjected to TLC on a 
precoated silica gel 60 thin layer plate (Merck) , 
using the system (parts by volume) : 
U-BuOH(85)-AcOH(10)-H 2 O(5> and MeOH(40) -CHC1 3 (60) ~ 
1^0 (10)* The active principle showed as a single spot 



35 



WO 88/01274 PCT/AU87/00281 



on TLC, and was visualized by spraying with vanillin 
sulfuric acid reagent. 

EXAMPLE 4 - High performance liquid chromatography 

5 (HPLC) 

Final purification of crude active powder was 
achieved by successive application of preparative HPLC 
with a reverse phase column. 31g of the active 
compound in the form of white powder, mp.l40°, was 

10 obtained from lOOg of XAD-2 purified sample. The 

approximate representative retention time of the 
active compound was 16 minute. The conditions for 
HPLC were as follows; column: YMC-Pack A-324 (ODS) ; 
flow rate: 20ml/min.; mobile phase: CH 3 CN-0.02N 

15 phosphate ammonium buff er (pH 7.45) (8:2); detector: 

refractive index. 



EXAMPLE 5 - Column chromatography on Sephadex LH-20 
Crude active compound (lOOg) obtained by 

20 adsorption on a XAD-2 column was subjected to gel 

filtration on Sephadex LH-20 column, using 
MeOH-CHCl 3 (l:l) and then MeOH as eluents, to afford 
white powder (45g) from the MeOH fraction. 
Rechromatography on the same column afforded 30g of 

25 almost pure white powder. 



EXAMPLE 6 

Gall-bladders (65g) , obtained from 5 sharks of 
the species Rhizoprionodon acutus (ca 8Kg weight) , 
30 were homogenized and then f reeze-dried. This material 

(10.25g) was used as a source of the active principle, 
sodium scymnol sulphate. After defatting the material 
with refluxing n-hexane (100ml x3) , it was extracted 
with methanol (100ml x3) under reflux for Ih. The 



35 



WO 88/01274 PCT/AU87/00281 

14 

concentrate (3.67gl was dissolved in HjO (80ml) f and 
applied to an Amberlite XAD-2 column (3.0 x 16.0cm) ^ 
The column was eluted with (400ml) and then with 

ethanol (400ml). Then, the ethanol eluate (l-95g) was 

5 applied to Sephadex LH-20 column (3.0 x 32.0cm), 

chloroform and methanol (1:1). After elution with 
chloroform and methanol (200ml) , the column was 
developed with methanol in batches of 50ml. 
Concentration of the methanol eluate containing the 

10 sodium salt gave a white gum (1.06g) . Purification of 

this material (120mg) by HPLC yielded 85.6mg of sodium 
scymnol sulphate as white powder. The conditions for 
HPLC were as follows: column/ YMC-Pack A-324(ODS) 
10x300mm; flow rate, 2ml/min? mobile phase, 35% 

15 CH 3 CN-0.1N Sodium Phosphate Buffer (pH 6.43); 

detector, Refractive Index, Sodium scymnol sulphate 

25 

has the following physical data: White powder? t a l D 

21. 75(0. 5c, in MeOH) ; Anal.: Calcd. for C^H^O^SNa 
: C^56.82 H;8.30 ' S;5.62 Na?4.03. Found: C;56.99 
20 H?8.79 S;5.62 Na;4.23. SIMS mass (m/e) : 

654t S27 H 47?° 9^ HNtC 2 H 6°>2 1 ' 574 tC 27 H 47 Q 6 -WC^O) 2 . 

IRv 55™* 5 3420, 2950, 1470, 1380 , 1230, 1070, 980, 
max « 

910,- 810. H-NMR (in CDjOD) ; 6 (ppm) : 
. 4.22(lE,dd,J=4.5, 10.0Hz), 4-11 (lH,dd,J=6.6, 10.0Hz), 

25 4.00 (1H, broad) , 3.80(1H, m) , 3 .80-3 .62 (3H, m) , 

3.45-3.30 (1H, m), 2.35-2.15(2H, m.) , 2.05-1.02 (23H, 
m) , 1.02(3H, d, J-6.2Hz) , 0.92(3H, s) , 0.72(3H, 
s) . 13 C-NMR (inC& 3 OD) ; «(ppm) : 74.1(d) 72.9 (d), 
71.4(d) , 69.1(d) , 66.7(t), 61.2(t>, 48.3(d), 47.8(d), 

30 47.5(s) r 43.1(d), 43.0(d), 41.0(d) , 40.3(t) f 37.0(d), 

36.5(t), 35.9 (s) , 35.8(H), 33.3(fc) , 32.1(t), 31.2(t), 
29. 5 (t), 28. 8 (t), 27.9(d), 24. 3 (t), 23. 2 (q), 18.1(q), 
13.1 (q). 



35 



WO 88/01274 



15 



PCT/AU87/00281 



EXAMPLE 7 

Trials have been conducted using the active 
principle of this invention in an antiseborrheous 
lotion applied topically by 40 male and female 
patients affected by long established (72) years 
facial hyper seborrhea . The trials were conducted as 
double blind trials with 20 patients, applying a 
placebo and 20 patients applying the lotion containng 
the active principle. 

In these trials, the treatment was applied 
three times daily (morning, midday and evening) over a 
period of 20 days, and an evaluation of seborrhea 
(Seborrhea Index) made at days 0, (prior to 
treatment), 10 and 21, (at end of treatment). 

The results showed a significantly greater 
improvement in the seborrhea for patients using the 
lotion containing the active principle than for 
patients using the placebo. It was also observed that 
this improvement was shown in both male and female 
patients. 



25 



30 



35 



WO 88/01274 



PCT/AU87/0Q281 



16 

EXAMPLE 8 - Compositions 
1. Cold cream 



Spenaacetti 6 . Og 

Beeswax 6.0g 

Carbopol 934 10 *0g 

Sodium Carbonate 4*75g 

Rose water 5* 0ml 

Rose oil . . 0 .02ml 

Expressed almond oil 56*0g 

Active principle 0*05g 

Distilled water 20«0g 

2. Tonic 

Ethanol 30 ml 

Active principle 20mg 

Flavour q*s» 



Distilled water - sufficient quantity to 

make 100ml 



WO 88/01274 



PCT/AU87/00281 



17 

CLAIMS : 

1 . A compound of the general f oraula I , in 

substantially pure form, 




therein A is a cation. 

2. A compound according to claim l f wherein the 
cation is a sodium, potassium, calcium or ammonium 
ion, or an organic amine . 

3 . A method for the preparation of a compound 
of the general formula I as defined in claim 1, in 
substantially pure form, which comprises the steps of 
preparing an aqueous extract of the liver and/or 
gall-bladder of a shark, and isolating the said 
compound from said aqueous extract. 

4 „ A method according to claim 3 , wherein said 

step of isolation from the aqueous extract comprises 
at least one step selected from solvent extraction, 
adsorption and chromatography . 



WO 88/01274 



PCT/AU87/00281 



18 

5. A pharmaceutical composition;, comprising a 
compound of the general formula I as defined in claim 
i f together with a phanaaceutically acceptable carrier 
or diluent therefor. 

6 . A pharmaceutical composition according to 
claim 5/ in the form of a tablet/ capsule, lotion or 
cream* - ' 

7. Use of a compound of the general formula I 
as defined in claim 1, for the protection of the liver 
or actxvation of liver function in the treatment of 
diseases or conditions affecting the liver. 

8. A composition for the treatment of the skin 
comprising a compound of the general formula I as 
defined in claim 1 , together ^rith a topically 
acceptable carrier or diluent therefor. 

9. A composition according to claim 8 , further 
comprising an antibiotic or other antibacterial 
substance* 

10 ♦ A cosmetic composition comprising a compound 

of the general formula I as defined in claim 1, 
together with a cosmetic base material. 



11.. Use of a compound of the general formula I 

as defined in claim 1, for the treatment of the skin. 



INTERNATIONAL SEARCH REPORT 

InltrnMiontl A»sli<ilian No PCT/AU 87/00281 



1. CLASSIFICATION 0* SUtJKCT MATTtft M W«*l< CUtti'eiiio* ivno«-i iaot|. -<%di;*:a •♦'» 1 


A<eo*«iA4 i« fn«irn«uon«l *ata<« CfMitfcwon (If CI of (a 4otrt k*Uo«tt CU«*/f<aci*A aAd IfC 


Int. CI. 


C07J 31/00, A61K 31/575 


II. miQ* StAACMIO 


MiMmwm Oa«um««ititiOfl 5aar<ft»o * 




CUttrieitiofl Svmaata 


IPC 


C07J 31/00 


US CI. 


260/397.2 




0«<vAtf rtittaA 5««reh«4 man Mtmmvm OocuflHAtatiOA 




to tho tittm wtt aye* OocumoAti iff tfttfudad ut tno ftolda Soarcftad • 


AU : IPC 


as above 



Ht DOCUMENTS COHStOIWCO TQ It MUVAHT* 
Cauoory * > GUtoon ol QocwAiaM. " «Hn indicatiOA. *ftart aooroprUta. ol u»a *tlt*«nt oumn '* j IWaVaM w CU«* No. " 



A U$,A, 4296109 (LAURENT et al) 20 October 1981 (1) 
(20.10.81) 

A US.A, 3994878 (PARTRIDGE, Jr. et al) 30 November (1) 
1976 (30.11.76) 



* Spoctat cattoonaa of ciiM documoAU: 
"A* docufftaAt d#A<u«i4 tno 9«n«r«i tuto ol tfto Ort w*H<h »* not 
cOAaitfo<td M So of OdtttfuU' ro«ovwK« 

•f tartar docvfflOAt ftut pyoitaftoo) on olw IM mt««nau«n«t 
fthnf ««to 

t" daeyinoAt wench may m<o« doubt* oa onaMv eloimUt or 

wiuctl l« CltH tO OttlOhtA t«« pUOltCltiO* 04t0 Ol tAOtnO* 
CltOttOA Of OtAOf aOO«l44 cm too ui tooofio*! 

•O* docymoftt rottfnnfl to «a oral dtacJoay/o. woo, oimmioa or 
otftor (Mini 

"f documaftt owftttihtd pcto* to tAo lAtorftatiOAol ftbna <«to »wt 
(atar wan ifto onarity data ctumtd 



"T" utor doeu«am stittMAfd 4ftor ttio lAtarAattorui Alma data 
or oftontf ««• 4no *** »* «Altt«f <**tn t«a wowwort ovi 
citad to uMoutaftd tna pnfloolo or tnoorf uAdortf*** ,n * 

"X* documant ol oarticutar ralovoAco: mo ctumtd iavoama 
<«m«o( to cofttiotrod Aotot of €«m«l so conaioorad to 
•rtvOlvo an iavomivo *tao 

-T- docurnoM ol poiticutor ro»o*a«ca; iao dotmod irwdAtiOA 
c«Aaot oo eoAaidarad l« «*ot#o 4A i*»oAtwo aiao waoa 
do«wnoAt to ctfflotrtH «wt* oao or moro otMr awcn doc«* 
mama. w«« <omo«iotio« aoiflf osmoyo to 4 soroon t**H 
ia Ifto art* 

-4- oocwAMAt mambt/ al tno M«a patMt lamtlr 



Oata ol tna Actual Comotauon ol ma IntorflatiOfMt Saarcft 

17 November 1987 (17.11.87) 


1 Qato ol Uatfutg ol tma lotorfioUOAOl SooreA Kaaart 


Intarnauonal S«arct\ir*9 Autnortlv 

Australian Patent Office 




AwmadMd Offlctr 

J - s - HANS0N 



fefm PCTjl3Ar2lO fs«cona tJ«nu«or tMSt 



ANNEX TO TOE TKTERNM^QE&L SESRCII.EEPORP (3T 



PgTEBMftjTICN^L APELICATION SO, FCT/ATI 87/00281 



This Annex lists t±e known h A" publication level patent fargily 
members relating to the patent documents cited ia the above-nientioned 
international search report* The Australian Patent Office is in no way liable 
for these particulars which are merely given. for the purpose of information* 



Patent Docment 

Cited in Search Patent Family mribars 



Report 



US 4296109 



AU 51423/79 
DK 4048/79 
GB 2034715 
DE 2932166 



CA 1127630 
EP 10056 
JP 55051100 



DE 2843690 
ES 484706 
SU 818489 



US 3994878 



AT 7513/76 

AT 5465/79 

CH 634337 

FR 2407941 

GB 1564808 

H 1068692 

CH 626096 



AT 5464/79 

EE 847131 

DE 2645527 

GB 1564806 

GB' 1564809 

JP 52046061 

CH 626097 



AT 5463/79 

CH 628907 

FR 2351998 

GB 1564807 

GB" 1564810 

NL 7611155 

US 4038272 



END CF ANNEX 



English abstract of reference {£> 

PATENT ABSTRACTS OF JAPAN 



(11)Publication number : 



2001-002551 



(43)Date of publication of application : 09.04,2001 




(51)Int.CI. 



A61K 7/48 
A61K 7/00 




(21 Application number : 11-172415 
(22)Date of filing: 18.06.1999 



ODApplicant : KANEBO LTD 
(72)Inventor : SAKAI SHINGO 



SAYO TETSUYA 
YASUDA SUESHIGE 
INOUE SHINTARO 




(54) AGENT FOR INCREASING HYALURONIC ACID CONTENT OF HORNY LAYER 

(57)Abstract: 

PROBLEM TO BE SOLVED: To obtain the subject agent which can increase the 
hyaluronic acid content of the horny layer to optimize the water environment and 
hardness of the horny layer, allows to expect an effect for preventing rough skins and 
wrinkles, is stable in preparations, and is useful for skin cosmetics, by including 
N-acetylglucosamine. 

SOLUTION: This agent for increasing the hyaluronic acid content of a horny layer 
comprises N-acetylglucosamine. The agent is preferably added to a skin cosmetic 
preferably in an amount of 0.001 to 10%, especially preferably 0.01 to 5%, based on the 
total amount of the cosmetic composition. The agent may preferably further be 
compounded with a polyhydric alcohol such as 1,3-butylene glycol, a vitamin 
derivative such as an ascorbic acid phosphoric acid ester salt, an anionic surfactant 
such as N-stearoyhL-glutamic acid salt, and so on. 

LEGAL STATUS 

[Date of request for examination] 

[Date of sending the examiner's decision 
of rejection] 

[Kind of final disposal of application other 
than the examiner s decision of rejection 



or application converted registration] 

[Date of final disposal for application] 

[Patent number] 

[Date of registration] 

[Number of appeal against examiner s 

decision of rejection] 

[Date of requesting appeal against 
examiner's decision of rejection] 

[Date of extinction of right] 



Copyright (C); 1998,2003 Japan Patent Office 



English abstract of reference Ab 

PATENT ABSTRACTS OF JAPAN 



(1 DPublication number : 59-013708 
(43)Date of publication of application : 24.01 .1984 



(SDlnLOL A61K 7/00 



(21) Application number : 57-122729 (71)Applicant : SHISEIDO CO LTD 

(22) Date of filing : 1 4.07.1 982 (72)Inventor : SHIMADA TADAHIRO 

TOYODA HIDEKAZU 

(54) COSMETIC 
(57)Abstract: 

PURPOSE: To provide a cosmetic containing amino sugars, N-acetylamino sugars or 
their salts existing in the crust of insects and crustaceans, and giving smoothness and 
moist feeling to the skin, and luster and finishing effect to the hair. 
CONSTITUTION: The cosmetic contains 0.1W5.0wt% of one or more compounds 
selected from N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucosamine, 
D-galactosamine, their hydrochloride, sulfate, etc. The cosmetic has moistening 
effect, emmolient effect and skin activating effect, and gives smooth feeling, 
springiness and luster to the skin. It is also effective to moisten and soften the hair, 
and improves the combing property of the hair. The above compound can be obtained 
by decomposing the chitin of the crust of insects and crustaceans such as crab, 
mucopolysaccharide and sugar proteins, etc. of animals and vegetables, etc. with acid, 
alkali, enzyme, etc. 



LEGAL STATUS 

[Date of request for examination] 

[Date of sending the examiner's decision 
of rejection] 

[Kind of final disposal of application other 
than the examiner's decision of rejection 
or application converted registration] 

[Date of final disposal for application] 

[Patent number] 



[Date of registration] 

[Number of appeal against examiner's 
decision of rejection] 

[Date of requesting appeal against 
examiners decision of rejection] 

[Date of extinction of right] 



Copyright (C); 1998,2003 Japan Patent Office 



- bouwstra et ai. 4U 1 12): ZJUJ 



Page 1 of 18 



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Journal of Lipid Research, Vol. 40, 2303-2312, December 1999 
Copyright © 1999 by Lipid Research, Inc. 



Original Article 



Cholesterol sulfate and calcium affect 
stratum corneum lipid organization 
over a wide temperature range 

J. A. Bouwstra*, G. S. Gooris a , F. E. R. Dubbelaar a , and M. 



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Ponec 0 

a Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA 
Leiden, The Netherlands 

b Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands 
Correspondence to: J. A. Bouwstra 



# ABSTRACT 



^TOP 

■ ABSTRACT 
^ INTRODUCTION 
^MATERIALS AND METHODS 



^RESULTS 

^ DISCUSSION 

^REFERENCES 



The main diffusion barrier for drugs penetrating through the skin is 
located in the intercellular lipid matrix in the upper layer of the 
skin, the stratum corneum (SC). The main lipid classes in the SC 
are ceramides (CER), free fatty acids (FFA) and cholesterol 
(CHOL). The lipids in SC are organized into two lamellar phases 
with periodicities of approximately 13 and 6 nm, respectively. Similar lipid organization has been 
found with equimolar CHOL:CER:FFA mixtures in SAXD studies performed at room temperature. 
However, one may conclude that the phase behavior of the mixtures is similar to that in SC only 
when the lipid organization of the lipid mixtures resembles that in SC over a wide temperature range. 
Therefore, in the present study, the organization of the lipid mixtures has been studied in a 
temperature range between 20° and 95°C. From these experiments it appeared that at elevated 
temperatures in equimolar CHOL:CER:FFA mixtures a new prominent 4.3 nm phase is formed 
between 35;-55°C, which is absent or only weakly formed in intact human and pig SC, respectively. 
As it has been suggested that gradients of pH and cholesterol sulfate exist in the SC and that Ca is 
present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca on the lipid phase 
behavior has been investigated with lipid mixtures. Both an increase in pH from 5 (pH at the skin 
surface) to 7.4 (pH at the SC;-stratum granulosum interface) and the presence of cholesterol sulfate 
promote the formation of the 13 nm lamellar phase. Furthermore, cholesterol sulfate reduces the 
amount of CHOL that is present in crystalline domains, causes a shift in the formation of the 4.3 nm 
phase to higher temperatures, and makes this phase less prominent at higher temperatures. 



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The finding that Ca counteracts the effects of cholesterol sulfate indicates the importance of a 
proper balance of minor SC components for appropriate SC lipid organization. In addition, when the 
findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is required to 
dissolve cholesterol in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in 
cholesterol sulfate content in the superficial layers of the SC is expected to destabilize the lipid 
lamellar phases, which might facilitate the desquamation process. — Bouwstra, J. A., G. S. Gooris, 
F. E. R. Dubbelaar, and M. Ponec. Cholesterol sulfate and calcium affect stratum corneum lipid 
organization over a wide temperature range. J. Lipid Res. 1999. 40: 2303;-2312. 

Supplementary key words: stratum corneum, ceramides, phase behavior, X-ray diffraction 



INTRODUCTION 



^ TQP 

^ABSTRACT 



INTRODUCTION 
-MATERIALS AND METHODS 



- RESULTS 
DISCUSSION 
-REFERENCES 



One of the main functions of the skin is to act as a barrier for 
undesired exogenous substances. However, for topical application 
of drugs, this barrier forms the main problem. The skin barrier is 
located in the upper layer of the skin, the stratum corneum (SC) 
that is composed of keratin-rich cells surrounded by hydrophobic 
crystalline lipid lamellar domains. It has frequently been suggested that SC lipids play a dominant 
role in proper functioning of the skin barrier, as topically applied substances have often to pass the 
SC lipid regions that form a very dense structure, Therefore, a detailed knowledge about the SC lipid 
organization is of great importance. The main lipid classes present in the SC are ceramides (CER), 
cholesterol (CHOL), and free fatty acids (FFA). The ceramide fraction is composed of at least six 
ceramides, referred to as CER 1, CER 2 ... CER 6 (1). The FFA and CER both vary in hydrocarbon 
chain length, the main population of FFA and CER having an acyl chain length between 22 and 26 C 
atoms. 



In diseased skin the barrier function is frequently impaired. There, a deviation in lipid composition 
has often been found. This includes i) a significant change in CER profile in atopic dermatitis and 
psoriatic scales (2) (3), ii) reduced FFA/CHOL and FFA/CER ratios in lamellar ichthyosis patients 
(4); and Hi) 3- to 4-fold elevated levels of CS0 4 in recessive X-linked ichthyosis patients (5). It 

remains to be established whether the impaired barrier function can always be linked to a modulated 
lipid composition. In order to delineate the link between the barrier function and lipid composition, 
the role that individual lipid classes play in SC lipid phase behavior should be examined. Such studies 
with native tissue are hampered by the low availability of the required material from the diseased 
skin. In order to mimic diseased SC, one can consider to modulate lipid composition in SC isolated 
from normal skin. However this approach is not feasible, as it is impossible to selectively extract 
certain lipid classes from the SC. For this reason another approach, like the use of lipid mixtures 
isolated from SC, should be chosen. 

Using X-ray techniques it has been established that in the SC the lipids are organized into two 
crystalline lamellar phases with periodicities of approximately 6 and 13 nm (6) (7) (8). A similar 



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Page 3 of 18 



organization has been observed using equimolar mixtures of CHOL, FFA, and CER isolated from pig 
SC, except that in these mixtures a larger proportion of CHOL appears in separate crystalline domains 
than observed in SC. These observations have been made in experiments performed at room 
temperature (9). In this study we have observed that in CHOL:CER mixtures, the phase behavior at 
room temperature is similar to that of intact SC. However, when the temperature-induced changes in 
phase behavior were examined, we observed that between 35° and 55°C a new phase with a 
periodicity varying between 4.3 and 4.5 nm was formed, which remained dominantly present with 
further increase of the temperature (9). In human SC this phase has not been encountered (8). The 
presence of such a phase at elevated temperatures indicates a lower stability of the lamellar phases in 
the lipid mixtures than in the SC. These observations illustrate the relevance for measuring 
temperature-induced phase changes, as differences in phase behavior at elevated temperature indicate 
that differences in lipid organization also exist at room temperature. Sometimes this is difficult to 
trace when performing lipid organization studies only at room temperature. 

Not only the major SC lipids, CHOL, CER and FFA, but also minor fractions, like cholesterol sulfate 
(CS0 4 ) may affect SC lipid organization. The CS0 4 level throughout the SC does not remain 

constant, but reaches very low levels in the superficial SC layers (10) (11). In addition, a pH gradient 

94- 94- 

exists in the SC (12) (13) (14) and Ca also displays a characteristic gradient in the epidermis. Ca 
level increases from the basal layers to the granulosum layer and declines again in the SC (15) (16) 
07). 

One of the methods to investigate the SC lipid organization is X-ray diffraction. However, X-ray 
diffraction is a bulk method that provides only information on overall lipid organization in the SC and 

94- 

does not allow study of the effects of local changes in pH and CS0 4 and Ca contents on lipid 

organization in native SC. A systematic study can be performed with lipid mixtures prepared from 
isolated SC lipids. This allows experimental modulation in lipid composition to mimic the situation 
either at the stratum granulosum(SG);-SC interface (pH 7.4, high Ca 2+ and high CS0 4 content) or in 

9+ 9-1- 

the superficial SC layers (pH 5;-6, low Ca and low CS0 4 content). Because a drop in Ca 
concentration already occurs in the lowest SC layers, but CS0 4 content decreases in the most 
superficial SC layer, the effect of CS0 4 in the absence of Ca on the SC lipid organization is also of 
interest. In our present studies we chose to incorporate either 2% m/m or 10% m/m CS0 4 into 
equimolar CHOL:CER:FFA mixtures, to approximate the CS0 4 levels observed in normal and in 

9-1- 

recessive X-linked ichthyosis skin, respectively (5). Furthermore, as substantial amounts of Ca are 
also present in superficial SC layers in psoriatic skin (18) and in skin covered for several weeks by a 
plaster after bone fracture (M. Ponec, unpublished results), we decided to study also the effect of 
Ca at pH 5. Because at present no information is available on the local Ca concentrations in the 
SC, we have chosen a concentration of 2 mmol Ca , similar to that used to study the effect of Ca 
on phospholipid membrane systems (19) (20) (21) (22) (23) being used as model systems for cell 
membranes. 



t MATERIALS AND METHODS 



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Isolation of stratum corneum from pig skin 

Fresh pig skin was obtained from a slaughter house at the C.D.I. in 
Lelystad in the Netherlands and SC was isolated from the skin as 
described before (9). 

Extraction, separation, and identification of lipids from stratum corneum 

Epidermal lipids were extracted using the method of Bligh and Dyer (24). The extracted lipids were 
applied on a silicagel 60 (Merck) column with a diameter of 2 cm and a length of 33 cm. The various 
lipid classes were eluted sequentially using various solvent mixtures as published recently (9). The 
lipid composition of the collected fractions was established by one-dimensional high performance 
thin-layer chromatography, as described before (25) . For quantification, authentic standards (Sigma) 
were run in parallel. The quantification was performed after charring using a photodensitometer with 
automatic peak integration (Desaga, Germany). Isolated CER fractions 1 to 6 were mixed to achieve 
the same ratio as found in the pig SC. 

Preparation of lipid mixtures 

The lipids were mixed in various molar ratios, using a mean CER molar weight of 700. For 
calculation of the mean ceramide molecular weight, the data on the ceramide composition and alkyl 
chain length distributions (26) were used. Mixtures were prepared from either i) CHOL, CER and 
FFA or ii) CHOL, CER, FFA and CS0 4 or Hi) CHOL, CER, FFA and CS0 4 in the presence of 2 mM 

CaCl 2 . For the FFA mixture, we chose long-chain free fatty acids in a molar ratio according to Wertz 

and Downing (1). The following fatty acids were included in the lipid mixtures: C16:0, C18:0, C22:0, 
C24:0, and C26:0 in a molar ratio of 1 :3:42:37:7, respectively. The mixtures were prepared at either a 
pH of 7.4, which is the pH close to the stratum corneunr-stratum granulosum interface, or a pH 5, 
which is the pH of the skin surface (9) (10) (11). To achieve a pH of 7.4, a HEPES buffer (10 mmol, 
Na + based) was used. To prepare mixtures at a pH of 5, we used a citrate buffer (10 mmol, Na + 
based). 

Small angle X-ray diffraction (SAXD) 

All measurements were carried out at the Synchrotron Radiation Source at Daresbury Laboratory 
using station 8.2. The samples were put in a special designed sample holder with two mica windows. 
A detailed description of the equipment has been given elsewhere (7). The experimental conditions 
were similar as described before (9). The scattered intensities were measured as a function of 8, the 
scattering angle. Calibration of the detector was carried out with rattail and cholesterol. From the 
scattering angle the scattering vector (Q) was calculated Q = 4 n (sin #)/A, in which A is the 
wavelength being 0.154 nm at the sample position. 

The diffraction pattern of a lamellar is characterized by a series of peaks at equal interpeak distance, 
Q n = n Qp in which Q n is the position of the nth order peak and Q 1 is the position of the 1 st order 

peak. The periodicity can directly be calculated from the position of the peaks d = n 2 w/Q - The 

diffraction pattern of CHOL is characterized by two peaks at Q = 1.87 and 3.74 nm" 1 . 

The lipid phase behavior was also measured as a function of temperature. The temperature was 



^TOP 

^ ABSTRACT 
^ INTRODUCTION 
- MATERIALS AND METHODS 
^RESULTS 
^ DISCUSSION 
^REFERENCES 



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Page 5 of 18 



increased at a heating rate of 2°C/min between 25° and 95°C. Data collection was carried out 
continuously. Each minute a new diffraction curve was collected. In such a way each successive set 
of diffraction data reflects the mean phase behavior during a temperature rise of 2°C. 



RESULTS 



Phase behavior at room temperature 

Mixtures prepared at pH 5. CHOL:CER:FFA mixture. The diffraction 
pattern of the equimolar CHOL:CER:FFA mixture is presented in 
( Figure 1 ) A. The spacings and corresponding periodicities are 
summarized in Table 1 . As can be noticed from Table 1 and Fi gure 
la, this mixture formed two lamellar phases with periodicities of 13.0 and 5.4 nm, respectively. In 
addition, 3.36 and 1 .69 nm diffraction peaks were observed that could be attributed to CHOL that 
separates as a phase in crystalline domains. The phase behavior of the equimolar CHOL:CER:FFA 
mixture was similar to that published recently (9). 



^TOP 

^ ABSTRACT 
^ INTRODUCTION 
^ MATERIALS AND METHODS 
- RESULTS 
^ DISCUSSION 
^REFERENCES 




Figure 1. The effect of CS0 4 and Ca 2+ on the phase behavior 

of equimolar CHOL:CER:FFA mixtures. The arabic numbers 
indicate the diffraction orders of the long periodicity phase 
(repeat distance between 12 and 13 nm). The roman numbers 
indicate the diffraction orders of the short periodicity phase 
(repeat distance between 5.3 and 5.5 nm). The diffraction 
patterns of the CHOL:CER:FFA:CS0 4 mixtures in molar 

ratios of A: (a) 1:1:1:0, (b)l:l:l:0.06 and (c) 1:1:1:0.3 atpH 
5;B: (a) 1:1:1:0.06 and(b) 1:1:1:0.3 in the presence of 2 

mmol Ca 2+ at pH 5 ; C: (a) 1 : 1 : 1 :0, (b) 1 : 1 : 1 :0.06 and (c) 
1 : 1 : 1 :0.3 at pH 7.4. At curve (a) the shoulder indicated by an 
arrow refers to the first order diffraction peak of the short 
periodicity phase; D: (a) 1 : 1 : 1 :0.06 and (b) 1 : 1 : 1 :0.3 in the 

presence of 2 mmol in the presence of 2 mmol Ca 2+ at pH 
7.4. 



cm 



4 



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Page 6 of 18 




View larger version (95K): 
[in this window] 
[in a new windowl 



Table 1. Effect of CSO A and Ca 2+ o*/ r*#E Xi^d oF/m>*<aTtoi/ a* £$fe|ioArf 
View this table: TTT ^ , T1 _„ A A „ 4 
[in this window] «HOA«EP»A ^Xr&P^r «r rfl = 

T in a new windowl 



CHOL:CER:FFA:CS0 4 mixture. In order to study the effect of CS0 4 on the lipid phase behavior 

either 2% m/m or 10% m/m CS0 4 has been incorporated into the equimolar CHOL:CER:FFA 

mixture. Inspection of Table 1 and Figure la revealed that lipids in CHOL:CER:FFA:CS0 4 mixture 

in a molar ratio of 1 : 1 : 1 :0.06 (2% m/m) formed two lamellar phases with periodicities of 12.8 and 5 A 
nm, respectively, similar to that seen in the absence of CS0 4 . Furthermore, the intensity of peaks 

attributed to crystalline CHOL decreased. A further increase in CS0 4 content to 10% m/m (molar 

ratio: 1:1:1 :0.3) induced a pronounced change in the lamellar phase behavior: the diffraction peaks 
attributed to the 5.4 nm phase and the peaks assigned to crystalline CHOL disappeared (Figure la) . 

CHOL:CER:FFA:CS0 4 and Ca z . The diffraction curve and corresponding spacings are depicted in 

Figure lb and Table 1 5 respectively. Addition of 2 mmol CaCl 2 to a CHOL:CER:FFA:CS0 4 mixture 

in a molar ratio of 1:1:1:0.06 did not affect the peak intensities attributed to the 5.4 and 13.0 nm 
lamellar phases, but slightly increased the intensity of the CHOL reflections. This indicates that at 
low CS0 4 the presence of Ca 2+ decreased the CHOL solubility in the lamellar phases. In contrast to 

that, addition of Ca 2+ to 1:1:1 :0.3 CHOL:CER:FFA:CS0 4 mixture did not result in the reappearance 

of the CHOL reflections. However, a reappearance of the 5.4 nm peak was observed. 

The results at pH 5 demonstrate that CS0 4 increases the solubility of CHOL in the lamellar phases 



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Page 7 of 18 



and when present at high levels it promotes the formation of the long periodicity phase, while Ca 
counterbalances partly the lipid phase changes induced by CS0 4 . 

Mixtures prepared at pH 7.4. CHOL:CER:FFA mixtures. The phase behavior of the equimolar 
CHOL:CER:FFA mixtures prepared at a pH of 7.4 is shown in Figure lc and the corresponding 
spacings .are presented in Table 2 . From Figure lc and Table 2 it is obvious that at pH 7.4 only one 
dominant lamellar phase was present, with a periodicity of 12.6 nm, which is in good agreement with 
our recent findings (27). The 6.2 nm peak (2nd order peak of the 12.5 nm phase) was asymmetric 
with a shoulder on its right-hand side, indicating the presence of an additional peak at a slightly 
shorter spacing (see arrow in Figure lc ). This shoulder is most likely the first order diffraction peak 
of the 5;-6 nm phase, which is prominently present at a pH 5 (see above). In addition, two diffraction 
peaks at 3.37 and 1.68 nm were observed that could be attributed to crystalline cholesterol. 



Table 2. Effect of CSO A and Ca oi/ r*2€ Xtwd o^Tm^.Ca^o;/ a? Q3§tu,okaP 

\^iew this table* 

[ in this window] *HOA«EP»A |i*rfl«r or *rH =F>*- 
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CHOL:CER:FFA:CS0 4 mixture. Incorporation of 2% m/m CS0 4 into the equimolar 

CHOL:CER:FFA mixture caused a change of the 6.2 nm asymmetric peak into a symmetric one (2nd 
order peak of the 12.4 nm phase) and a slight reduction of the intensities of the CHOL reflections 
(Figure lb). From these observations it is obvious that in the presence of 2% m/m CS0 4 the 

formation of the 5;-6 nm phase was suppressed and the solubility of CHOL in the lamellar phases 
was increased. An increase in CS0 4 content to 10% m/m increased the solubility of CHOL further. 

No further changes in the 12.5 nm lamellar phase were observed. 

CHOL:CER:FFA:CS0 4 and Ca 2+ . As shown in Table 2 and Figure Id , addition of 2 mM Ca 2+ to the 
1:1:1 :0.06 mixture of CHOL:CER:FFA:CS0 4 induced a reappearance of a 5.4 nm shoulder at the 

9-1- 

right-hand side of the 6.2 nm diffraction peak. Furthermore, in the presence of Ca , the intensities of 

9-1- 

the peaks attributed to crystalline CHOL increased. Addition of Ca to the 1:1:1:0.3 mixture of 
CHOL:CER:FFA:CS0 4 also caused a reappearance of the 5.4 nm shoulder, but no CHOL reflections 

9+ 

were observed. The above results indicate that 2 mmol Ca balances, at least partly, the changes in 
lipid phase behavior induced by CS0 4 . 

The results at pH 7.4 obviously show that CS0 4 increases the solubility of CHOL and promotes the 

formation of the long periodicity phase, while Ca opposes the effect similar to that observed at pH 
5. 

Temperature-induced changes in the SC lipid phase behavior of mixtures prepared at pH 5 

CHOL:CER:FFA mixtures. The phase behavior of the CHOL:CER:FFA mixtures has been followed 
as a function of temperature between 25° and 95°C ( Figure 2 A). Each curve represents the lipid 



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phase behavior during a 2°C rise in temperature. The phase behavior at room temperature is similar to 
that shown in Figure la . Two lamellar phases are present with periodicities 13.0 and 5.4 nm, 
respectively. The peaks attributed to the 5.4 and 13.0 nm phases start to decrease in intensity at 
approximately 55°C and disappear at about 63°C. At approximately 35;-39°C a new 4.3 nm peak is 
formed. A further temperature rise leads to a strong increase in the intensity of this peak and to a 
small shift in position to a spacing of 4.4 nm. Simultaneously with the increase in intensity of the 4.3 
nm peak between 39° and 63°C, a 2.2 nm peak appears. This peak can most likely be attributed to the 
same phase, as it follows the positional and intensity changes of the 4.4 nm peak. At around 60°C the 
4.4 nm peak transforms into a doublet that turns to a singlet again at 71°C. A further increase in 
temperature reduces the 4.4 nm and 2.2 nm peak intensity, but at 95°C the reflections are still present. 
The CHOL reflections start to decrease in intensity at approximately 37°C and disappear at around 
55°C. 




fan's hm its*** 



1 



.2+ 



on 



Figure 2. Temperature-dependent effect of CS0 4 and Ca 

the lipid phase behavior of CHOL:CER:FFA mixtures. The 
mixtures were prepared at pH 5. The arabic numbers indicate 
the diffraction orders of the long periodicity lamellar phase 
(repeat distance varies between 12.8 and 13 nm). The roman 
numbers refer to the diffraction peaks attributed to the short 
periodicity phase (repeat distance 5.4 nm). Each diffraction 
curve represents the scattered x-rays during a temperature rise 
of 2°C. A: Equimolar CHOL:CER:FFA mixture. The arrow 
indicates the formation of a new 4.3 nm phase at elevated 
temperatures. B: CHOL:CER:FFA:CS0 4 mixture in a molar 

ratio of 1:1:1:0.06. C: CHOL:CER:FFA:CS0 4 mixture with 

addition of 2 mmol CaCl 2 . Note the arrow indicating the 2nd 

order reflection of the 4.4 nm phase. 




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CHOL:CER:FFA:CS0 4 mixture. As depicted in Figure 2b, no changes in the lipid phase behavior 

have been noticed in the 1:1:1:0.06 CHOL:CER:FFA:CS0 4 mixture until the temperature reaches 

approximately 45°C. Between 50° and 55°C the CHOL diffraction peaks gradually reduced in 
intensity and subsequently disappeared at about 55°C. At about 60°C the intensity of the diffraction 
peaks attributed to the 5.4 and 12.8 nm lamellar phases started to decrease. In contrast to the 
observations made with the CHOL:CER:FFA mixture, no new 4.3 nm peak appeared at 35;-39°C, 
but the intensity of the 4.4 nm diffraction peak (3rd order of the 12.8 nm lamellar phase) started to 
increase at around 45°C. It is not clear whether in this temperature region it is caused by the 
formation of a new phase with a peak position at exactly the same spacing as the already existing 
peak or that the intensity increase is caused by a change in the lipid organization of the 12.8 nm 
phase. A further rise in temperature caused a further increase in the 4.4 nm peak intensity. A 
maximum peak intensity was reached at 61°C. The observation that the diffraction peaks attributed to 
the 5.4 and 12.8 nm phases disappear at 65°, while the 4.4 nm phase was still present, is indicative for 
the formation of a new 4.3 nm phase. The 4.4 nm peak disappeared at approximately 83°C. 

As depicted in Figure2c, the presence of 2 mmol Ca had a profound effect on the phase behavior 
of the 1:1:1:0.06 CHOL:CER:FFA:CS0 4 mixtures at elevated temperature. Most substantial is the 

increase in 4.4 nm peak intensity that starts already at around 40°C. A further increase in temperature 
increased the peak spacing and peak intensity gradually. The peak intensity reached its maximum at 
approximately 67°C. Above this temperature the peak intensity and spacing decreased. At 95°C the 
peak (4.2 nm spacing) was still present. Because the 5.4 and 13 nm phases disappeared at 70°C, it is 
obvious that a new phase appeared at high temperature with a 2nd order peak at 2.15 nm (see arrow). 



Our studies reveal that the presence of CS0 4 is required to mimic SC lipid phase behavior over a 

v 



9+ 

wide temperature range and that Ca acts partly towards the CS0 4 effect not only at room 



temperature, but also at elevated temperatures. 



t DISCUSSION 



^ INTRODUCTION 
^MATERIALS AND METHODS 



^RESULTS 
- DISCUSSION 
^REFERENCES 



During epidermal differentiation, characteristic changes in lipid 

composition occur, consistent with the formation of a waterproof 

barrier. These changes include a progressive depletion of 

phospholipids and glucosphingolipids accompanied by enrichment 
in CER, CHOL, FFA, and small amounts of other polar (e.g., 

CS0 4 ) and nonpolar lipid species (cholesteryl esters and triglycerides). The relative amounts of 

CHOL, CER and FFA remain unchanged throughout the whole SC (M. Ponec and A. Weerheim, 
unpublished results). There is increasing evidence that within the SC also no significant change in 



^TOP 

^ABSTRACT 



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CER composition occurs (28 ). 

The SC intercellular lipid bilayers originate from lamellar bodies that are abundantly present in the 
SG and are extruded to intercellular space at the SG/SC interface. After extrusion, the content of 

0-4- 

lamellar bodies is reorganized into lamellae. It has been suggested that the presence ofCa ZT IS 
required for the formation of intercellular lamellae (29). The Ca 2+ concentration is high at the SG/SC 
interface and in the first SC layers. Subsequently, it drops to very low levels in the upper SC. Not 

94- 

only the Ca levels but also the pH and CS0 4 content decrease in the direction of the skin surface 
(ID (.12) (13) (14) (30) (31). 

The previous (27) and present studies have shown that the solubility of cholesterol in equimolar 
CHOL:CER:FFA mixtures increases in the presence of CS0 4 ( Figure 3). Most likely the increased 

solubility of CHOL in the lamellar phases can be ascribed to the presence of the charged sulfate 
group. Due to the electric repulsion forces, the charged sulfate group increases the interfacial area per 
lipid molecule and thus reduces the lattice density and/or increases the chain mobility in the bilayers. 
This is in agreement with our recent findings (27) which showed that in the absence of CS0 4 the 

CHOL:CER:FFA mixtures form an orthorhombic phase, while upon addition of CS0 4 a liquid lateral 

packing appears. An increased fluidity has indeed been found in recessive X-linked ichthyosis by 
electron spin resonance (32). Studies of Kitson et al. (33) revealed that intercalation of CS0 4 in 

sphingomyelin bilayers increased the transition temperature of the lamellar to reversed hexagonal 
phase. They explained this phenomenon by an increase in the interfacial area. A stabilization of the 
membrane structure by CS0 4 has also been found in other phospholipid systems (21) (34). 




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Figure 3. A schematic presentation of the changes in the lipid 
phase behavior in equimolar CHOL:CER:FFA mixtures by 

changes of pH, Ca (2 mmol), and CS0 4 content. The long 

periodicity phase, repeat distance of which varied between 
12.4 and 13 nm> was always prominently present. The intensity 
of the 1st order diffraction peak attributed to the short 
periodicity phase (repeat distance varied between 5 and 6) 
varied strongly. This peak, indicative for the presence of the 
short periodicity phase, has been categorized in the following 
way: strong, weak and not present. 



The formation of the long periodicity phase is also promoted by an increase in pH from 5 to 7.4. This 
pH change also increases the repulsion forces between the lipids and therefore is expected to reduce 
the lattice density and increase the chain mobility as well. These observations suggest a more general 
mechanism, namely that a reduction in lattice density or an increase in chain mobility promotes the 



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formation of the 12;— 13 nm phase. This hypothesis is supported by at least two other recent 
observations. In SC isolated from reconstructed human epidermis and from murine skin, lattice 
density is reduced: in the former a hexagonal lateral packing is present (35), while in the latter a 
liquid phase coexists with an orthorhombic lateral packing (6). In both SC samples the 12;— 13 nm 
lamellar phase dominates. 

It has often been suggested that CS0 4 plays an important role in the desquamation process. Recently 

it has been shown that CS0 4 inhibits the enzymatic activity of enzymes responsible for desmosomal 

degradation (36). A drop in the CS0 4 content in the superficial SC layers (10) (1 1) will activate these 

enzymes and facilitate the desquamation process. However, not only the desmosomes, but also the 
lipid bilayers are responsible for the intercellular cohesion in the SC. The results of the present study 
demonstrate that a drop in the CS0 4 content will affect the SC lipid phase behavior. As observed 

from the measurements at elevated temperature (see below), the reduction in CS0 4 content decreases 

the stability of the bilayers and reduces the fraction of lipids arranged in a liquid lateral packing (27) . 
The absence of this fluid phase is expected to reduce the elasticity of the lipid phases (37) and might 
even prevent the lamellae from following the contours of the corneocyte surfaces. This will decrease 
the cohesion between the cells and promote the desquamation process. Therefore, our data suggest 
that CS0 4 plays an important role in a proper functioning of the SC. In deeper SC layers CSO^ 

increases the lipid mobility and, in the superficial layers, the decrease in CS0 4 levels promotes the 

desquamation process not only due to the increased desmosomal degradation (36 ), but also by 
destabilizing the lipid lamellar phases. 

Changes induced by CSO^ can be partly counterbalanced by the presence of 2 mmol Ca (Figure 3). 

Namely, while 2% CS0 4 increases the solubility of CHOL in the lamellar phases, Ca promotes 

formation of separate crystalline CHOL domains in the presence of 2% m/m CSO^. Furthermore, 

while the 5-6 nm reflection at pH 7.4 disappears in the presence of 2 or 10% m/m CS0 4 , in the 

presence of Ca reappearance of the 5-6 nm reflection occurs. At pH 5 the same events are observed 
with 10% m/m CS0 4 . These findings indicate that in the presence of 2 mmol Ca the formation of 

the 5;-6 nm phase and the reformation of crystalline domains of CHOL is facilitated. However, in 
mixtures containing 10% m/m CS0 4 , most probably the CS0 4 content is increased to such high 

levels that the formation of CHOL crystals is prevented. In phospholipid systems Ca stimulates 
dehydration of phospholipid head groups and induces crystallization. This has been demonstrated for 
cardiolipin, phosphatidylserine, phosphatidylglycerol, and phosphatidylcholine (19) (20) (21 ) (22) 
(23). Because hardly any water is present between the bilayers in CER:CHOL:FFA mixtures it is 
unlikely that a similar mechanism is involved. Most likely Ca is able to reduce the surface charge 
density induced by either CS0 4 or dissociated FFA (the pKa values of the fatty acids are 

approximately 6;-6.5 (38). This results in a reduction of the intermolecular electrostatic repulsion and 

9+ 

an increase in lattice density. Whether Ca links two opposing CS0 4 lipids as suggested in a 
previous paper (32) remains to be established. Consequently, when extrapolating these findings to the 



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situation in intact SC, changes induced by CS0 4 might be partly counterbalanced by Ca . In normal 

9+ 7 -J- 

skin this occurs only in the lowest SC layers where Ca is present. In higher SC layers no Ca 

could be detected (17). In contrast to this, CS0 4 levels remain unchanged up to the superficial SC cell 

layers. The observation that in about 50% of the human SC samples a small amount of CHOL phase 
separates (J. A. Bouwstra, G. S. Gooris, A. Weerheim, and M. Ponec, unpublished results) indicates 
that the level of CS0 4 is probably such that the SC lamellae are partially or completely saturated with 

CHOL. At the most superficial SC layer the CS0 4 level drops, which might induce a crystallization 

of CHOL and decrease the cohesion between the lipid lamellae. 

At 10% m/m CS0 4 levels, only the 12;— 13 nm phase is present. One can expect that in recessive x- 

linked ichthyosis skin, in which the CS0 4 level is increased from 3.4% w/w to 1 1 .2% w/w (5), a 

change in the lipid phase behavior occurs. In a previous study (27) it has been shown that at low 
CS0 4 levels a fraction of lipids is arranged in a liquid lateral packing. One can speculate that a further 

increase in CS0 4 levels, as observed in recessive x-linked ichthyosis SC, might reduce the lattice 

density further and consequently might increase the permeability of the SC, which is in agreement 
with previous findings (32 ). The consequences of the presence of only the 12;— 13 nm phase for the 
skin barrier function is yet unknown. 

Phase behavior at elevated temperatures 

Although it has been observed that at room temperature the CHOL:CER and CHOL:CER:FFA 
mixtures mimic the SC lipid organization quite closely, one may conclude that the phase behavior of 
the mixtures is similar to that in the SC only when the phase behavior of the lipid mixtures resembles 
that of SC over a wide temperature range. Furthermore, it has often been demonstrated that the lipid 
phase behavior is more sensitive to compositional changes at increased temperature than at room 
temperature (39). In a previous paper (9) we have reported that at increased temperature an additional 
phase, indicated by 4.3 nm spacing, is formed in CHOL:CER mixtures, which is more prominently 
present when the temperature is increased. This 4.3 nm phase has hardly ever been observed in SC 
derived from healthy persons and is only weakly present in pig SC ( Figure 4 A and B), but it has 
been observed in a pilot study in psoriatic scales (J. A. Bouwstra, unpublished results). From the 
experiments presented here it became clear that in the presence of only 2% m/m CS0 4 the appearance 

of the 4.3 nm phase shifts to higher temperatures and that the intensity of the corresponding 
diffraction peak reduces remarkably. This finding demonstrates that the SC lipid phase behavior is 
mimicked more closely in the presence of CS0 4 than in its absence. In addition, the 5.4 and 12.8 nm 

lamellar phases remain stable over a larger temperature range and the phases are less sensitive for 
temperature-induced changes. This clearly indicates that CS0 4 increases the stability of these phases. 

7+ 

The situation is different when Ca is also present. There the intensity of the 4.4 nm peak starts to 

7+ 

increase at lower temperature, indicating that Ca destabilizes the lamellar phases and counteracts 
the effect of CS0 4 . Therefore an increase in Ca level, such as present in psoriatic scales (1 8), might 

lead to destabilization of the SC lipid organization. 



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Figure 4. Temperature-induced changes in the diffraction 
pattern of pig and human SC. The arabic numbers indicate the 
diffraction orders of the 13 nm lamellar phase. The peak 
indicated by an asterisk is attributed to the 2nd order 
diffraction peak of the 13 nm phase and the 1st order of the 6 
nm phase. Each diffraction curve represents the scattered X- 
rays during a temperature rise of 2°C. A: Human stratum 
corneum. Note the appearance of a new phase at around 50°C 
(see arrow). B: Pig stratum corneum. No new phase has been 
formed at elevated temperatures. 



To elucidate whether the stabilization of the lamellar phases by CS0 4 is caused by the introduction of 

the negative charge in the lamellae or whether it is due to the increased solubility of CHOL, we 
performed an additional experiment in which the CHOL content in CHOL:CER:FFA mixture (pH = 
5) was reduced to such a level that no crystalline CHOL was present. In the 0.6:1:1 CHOL:CER:FFA 
mixture no separated crystalline domains of CHOL were detected, see Figure 5 and increase in 
temperature up to 60°C did not affect the lipid phase behavior. At this temperature the diffraction 
peaks attributed to the 5.4 and 12.8 nm phases start to reduce in intensity and finally disappear 
between 60° and 70°C. In the same temperature region, a very broad diffraction peak is observed that 
disappears at approximately 75°C. No increase in the intensity of the 4.4 nm peak has been observed 
in this mixture. When extrapolating these results to 1:1:1:0.06 CHOL:CER:FFA:CS0 4 mixture, it 

became clear that in the diffraction pattern of this mixture the reduction in the 4.4 nm peak intensity 
at elevated temperatures is not caused by the presence of an additional negative charge introduced by 
CS0 4 , but can be ascribed to the decrease in crystalline CHOL that separates as a phase at room 

temperature. This obviously demonstrates that the increased stability of the lamellar phases induced 
by CS0 4 is based on its capacity to enhance the solubility of CHOL in the lamellar phases. 



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Figure 5. Temperature-induced changes in diffraction patterns 
of the CHOL:CER:FFA mixture prepared in a molar ratio of 
0.6:1:1 at pH 5. Each curve represents the phase behavior 
during a temperature rise of 2°C. The arabic numbers indicate 
the diffraction orders of the 12.8 nm. The roman numbers 
indicate the diffraction orders of the 5.4 nm lamellar phase. 



In conclusion, despite a low CS0 4 level present in SC of normal skin, CS0 4 might be important in 

stabilizing the lamellar phases in the deeper layers of the SC. Furthermore, its absence in the 
superficial layers of SC might destabilize the lipid lamellae and therefore facilitate the desquamation 
process. 

Manuscript received May 24, 1999; and in revised form August 31, 1999 Abbreviations: CHOL, 
cholesterol; CER, ceramides; FFA, free fatty acids; CS0 4 , cholesterol sulfate; A, wave length; 9, 

scattering angle; d, periodicity; SC, stratum corneum 



REFERENCES 

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Physiology, Biochemistry and Molecular Biology in the 
Skin. 2nd Edition. L. A. Goldsmith, editor. Oxford 
University Press, Oxford. 205;-236. 



^TOP 

^ ABSTRACT 
^ INTRODUCTION 
^MATERIALS AND METHODS 



^ RESULTS 
^ DISCUSSION 
• REFERENCES 



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26. Wertz, P. W., Downing, D. T. 1983. Ceramides of pig stratum epidermis, structure 
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31. Ohman, H., Vahlquist, A. 1998. The pH gradient over the stratum corneum differs in X-linked 
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32. Rehfeld, S. J., Plachy, W. Z., Williams, M. L., Elias, P. M. 1988. Calorimetric and electron 
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Invest. Dermatol. 91:499-505 [Abstractl . 

33. Kitson, N., Monck, M., Thewalt, J., Cullis, P. 1992. The influence of CS0 4 on phase behavior 
and hydrocarbon order in model membranes. Biochim. Biophys. Acta. 1111: 127-1 33 [Medline! . 

34. Epand, R. M., Bottega, R. ? Robinson, K. 1990. Cholesteryl phosphate and cholesteryl 
pyrophosphate inhibit formation of the hexagonal phase. Chem. Phys. Lipids. 55:49-53. 



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35. Bouwstra, J. A., Gooris, G. S., Weerheim, A. ? Kempenaar, J. A., Ponec, M. 1995. 
Characterization of stratum corneum structure in reconstructed epidermis by X-ray diffraction. 
1 Lipid Res. 36:496-504[Abstract]. 

36. Sato, J. ? M. Denda, J. Nakanishi, J. Nomura, and J. Koyama. 1998. Cholesterol sulfate inhibits 
proteases that are involved in desquamation of stratum corneum. J. Invest Dermatol 111: 
189;-93. 

37. Forslind, B. 1994. A domain mosaic model of the skin barrier. Acta Derm. Venereol. 74:1-6 
[Medline] . 

38. Lieckfeldt, R., Villalain, J., J-C. Gomez-Fernandez,, Lee, G. 1996. Apparant pKa of the fatty 
acids within ordered mixtures of model human stratum corneum lipids. Pharm. Res. 12:1614- 
1617. 

39. Sheddon, J. M. 1990. Structure of inverted hexagonal(H n ) phase, and non-lamellar phase 
transitions of lipids. Biochim. Biophys. Acta. 1031:l-69JMedline]. 



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Biochimica et Biophysica Acta, 1021 (1990) 119-125 
Elsevier 



119 



BBAMEM 74706 

Interaction between corneocytes and stratum corneum lipid 

liposomes in vitro 

William Abraham and Donald T. Downing 

Marshall Dermatology Research Laboratories* Department of Dermatology, University of Iowa College of Medicine, 

Iowa City, IA (U.S. A.) 

(Received 2 August 1989) 

Key words: Stratum corneum; Liposome; Corneocyte; Cell envelope 

Small unilamellar vesicles were made from a mixture of epidermal ceramides (45%), cholesterol (35%), free fatty acids 
(15%) and cholesteryl sulfate (5%). Isolated corneocytes prepared from pig epidermis were added to the liposomes and 
the interaction between corneocytes and liposomes was studied by (1) thin-section electron microscopy and (2) 
monitoring the release of aqueous contents of the vesicles by following the fluorescence intensity of carboxyfluorescein 
entrapped in the vesicles. The vesicles adsorbed readily onto the corneocytes and slowly transformed into lamellar 
sheets. Enhanced fluorescence intensity indicated a corneocyte-induced membrane fusion process that resulted in the 
release of aqueous contents of the vesicles. The results suggest a cohesive role for the corneocyte cell envelope, which 
consists of a monomolecular layer of lipids covalently bound to the outside of a cross-linked protein envelope. This may 
be one of the major factors in the reassembly of extruded membranous disks into lamellar sheets which occurs during 
the final stages of epidermal differentiation. 



Introduction 

The stratum corneum (SC) of the mammalian epider- 
mis consists of keratinized cells embedded in an ex- 
tracellular matrix of multiple lipid lamellae [1]. These 
intercellular membranous sheets of the SC constitute 
the epidermal water barrier [2] and are known to 
originate from the lamellar bodies found in the cells of 
the granular layer of the epidermis [3,4]. The lamellar 
bodies contain stacks of membranous disks, which ap- 
pear to be flattened vesicles [4]. After their discharge 
into the intercellular space, the disks are reassembled to 
form the intercellular lamellar sheets of the SC. During 
this process, the lipids undergo drastic biochemical 
changes as the cells progress from the granular layer to 
the horny layer, and unlike other biolpgical membranes, 
the SC lipid lamellae have been shown to be virtually . 
devoid of phospholipids [5]. Recently,^ we. have demon- 
strated the ability of relatively nonrpolar lipid mixtures 
(ceramides, cholesterol, free fatty acids arid cholesteryl 
sulfate) of the SC to form liposomes in- vitro [6] arid 

Abbreviations: CF f carboxyfluoresceiri; SC, stratum', corneum; SUV, 
small unilamellar vesicle; TMA, trimcthylarninei -. 

Correspondence: W. Abraham, 270 Medical l^bpratones/University 
of Iowa College of Medicine, Iowa . City; Iowa ; 52242; U.S.A. . ■ 



investigated some of the factors that cause the fusion of 
these vesicles into lamellar sheets [7-9]. 

Interactions of liposomes with a range of biological 
membranes and cell types have been widely studied, in 
cell biology to provide models for membrane-mediated 
biophysical processes such as fusion and cell-cell ad- . 
hesion [i0— 13]. The recent discovery In our laboratory 
of a chemically bound lipid envelope surrounding 
corneocytes [14] suggested a cohesive interaction be- 
tween the lipid envelope and the intercellular lipid 
lamellae. In the present study we investigated: whether 
the interaction between corneocytes and SC lipid lipo- 
somes may have !a role in promoting transformation of 
the extruded lamellar disks into the intercellular lamel- 
lae of the stratum corneum. . 

Materials and Methods 

Lipids. Epidermal ceramides were isolated by pre- 
parative thiri-layer. chromatography from total lipid ex- 
tracts of pig epideVrmYas. described previously [15].; A 
mixture of \f ree fatty : aeids- was prepared by; combining 
carnauba ^ax' fatty acids [16] with palnijtic.aQid -to a 
ratioof 8 : 2 by weight, forming a mixture close to: the 
distribution of free, .fatty acids in stratum corneum [17]. 
Cholesterol was obtained from. Sigma Chemical Co. (St. 
Louis, M(j). Cholesteryl sulfate, was prepared by reac- 
tion oft cholesterol with excess chlorosulfonic acid in 
pyridine arid purified chromatographically. 



0OO5-2736/90/S03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division) 



120 



Corneocytes. Corneocytes were prepared from pig SC 
as described previously [18]. Pieces of epidermis were 
obtained from freshly killed pigs by heating the skin to 
60-65 °C for 1 min. The epidermis was then peeled off 
and digested in 0.5% trypsin (Sigma Chemical Co., St. 
Louis, MO, Type III) in phosphate-buffered. saline (PBS) 
at pH 7.5 at 4°C overnight. After rinsing in distilled 
water, the tissue pieces were treated with fresh 
trypsin/PBS solution for 2 h at room temperature. 
Pieces of SC thus obtained were washed in distilled 
water and treated with a detergent solution containing S 
mM A^tf-dimethyldodecylamine. oxide (Procter and 
Gamble, Cincinnati, OH) and 2 mM sodium dodecyl 
sulfate (Mallinckrodt, Paris, KY) in PBS at 45 °C for 24 
h, SC sheets disintegrated into a mixture of individual 
corneocytes and empty corneocyte envelopes that were 
separated by centrifugation in cesium chloride solution 
of density 1.28 [18], The corneocytes were washed in 
ethanol to remove the detergents, resuspended in 1 mM 
sodium azide and stored at 4° C until further use. 

Preparation of liposomes. Appropriate volumes of 
solutions of individual lipids in chloroform/methanol 
(2 : 1, by vol.) were combined to obtain a mixture con- 
taining. 45% '.by weight of ceramides, 35% cholesterol, 
15% free fatty acids and 5% cholesteryl sulfate. 
Liposomes were prepared from these lipid mixtures in 
an aqueous buffer containing 10O mM NaCl, 5 mM 
Tris, 1 mM NaN 3 and 1 mM EDTA by sonication, as 
described elsewhere [6]. The total amount of lipid in the 
suspension was 5 mg/ml. The final pH of the disper- 
sions was adjusted to 7.5 by dialysis. In the fusion assay 
experiments Hposomes were prepared in a buffer con- 
taining 100 mM carboxyfluorescein (CF), 5 mM tri- 
methylamJne (TiA A) and 0.5 mlvi BDTA. Vesicles were 
separated from noneneapsulated.CF by gel filtration on 
Sephadex G-50. The elution buffer contained 5 mM 
TMA and 0,5 ."nj^ EDTA and was at pH ?, same as 
that of the liposome suspension. The final pH of the 
eluted suspensions were adjusted, to pH T.5 by dialysis. 

Interaction of liposomes with corneocytes) JC^rneocytes 
were added to the liposomes as ^.a suspension "in Tris 
buffer at 37°C to a final cbnwntration 61 lO'mg^ml, 
Dry weight of the corneocytes was de^r^ried after 
drying .an aliquot of ' the aqueous cell ' suspension.. The 
corneocytes were kept in suspension 1 by bubbling r niiro- 
gen at the rate of - 0.1 ml per min .: AU the suspensions 
were maintained at 37?C until : analysis by electron 
microscopy,. For .fusion assay experiments, corneocytes 
were ,added as a -suspension in, ;TMA buffer ancb. in- 
cubated %t31%iqz 1 h in the cell used for fluorescence 
measurement and the corheoeytes^ere kept in suspend 
sibn by .stirring: The final eonCentFattort of the;$uspen- 
sion was :1.5;!mg.of lipid and- l:mgiydf ■ corneocytes : ^per 
ml. - .■■ - - / >' :■ V ■ ■> •' 

Electron microscopy, Control liposorxies were analyzed, 
by freeze-fractiire electron microfscbpy as described pre-: 



viously [6]. The corneocyte-containing dispersions; t We 
analyzed by thin-section electron microscopy T'h#l2: 
and 1 week after the addition of corneocytgi&$ 
dispersions were centrifuged in a microfuge at lO.06j| 
and the wet pellets were fixed in 0.2% Ru0 4 in piji; 
late buffer for 15 min at room temperature. TheS 
pellets werepreembedded in agar> dehydrated in " 
acetones, embedded in Spurr's resin and sect^. 
Silver-gold sections were stained with uranyl Jit 
and lead citrate and examined in a Hitachi -til 
electron microscope operating at 75 kV. yf:£ 
Fusion Assay. The release of vesicle contents :f 
external medium induced by the corneocytes w^ 
sured by monitoring fluorescence intensity of qu- 
fluorescein (CF), encapsulated initially at a higj" 
quenching concentration in the vesicles. Whenvt 1 ^ 
trapped CF is released to the outside mediutt^ 
diluted and the fluorescence is directly propo£h<| 
the concentration of CF [19,20]. Fluorescence $r 
sured in an Aminco-Bowman fluorometer using& 
tion and emission wavelengths of 492 nm and:!" 
respectively. Fluorescence was measured befbrj| 
■after the disruption of the vesicles by the ad&fogi 
Triton XrlOO (0.2% v/v). CF-containing ves^l^S 
incubated at 37°C with corneocytes for 1 h and-f^S 
cence was measured before and after the ad^itiSn 
Triton. The vesicles were also incubated at ' ij^jSk 
the supernatant buffer from the corneocyte susjW 
for 1 h and fluorescence was measured before/ait 
the addition of Triton. Relative fluorescence inti 
were corrected for dilution after the addition^ 
suspensions and TX-10G. CF concentrations.^;? 
termined from calibration curve generated frbirj[g 
cence intensity measurements of CF solutions.^, 
in TMA buffer at pH 7.5. 

Results 

Fig! 1 shows a freeze-fracture micrograph . qj§f 
liposomes at pH 7.5. These liposomes were,-s '~ l 
ilamellar and ranged in size from 20 to 200? v 
liposomes remained stable for several weeksr jtg 
and 2B show the thiri-section electron micjj 
(TEMs) of corneocytes prepared from pig;^i 
Fig. 2A shows isolated corneocytes as well ,'k$f 
contact with each other. Fig. 2B shows two'cdf^ 
in contact with each other. The cell enveloped 
a protein envelope seen as a broad electrqn^dens 
outside of this is an electron-lucent band 'obti^ 
to the covalently linked lipids [14]. Corneocyt^ 
tact are seen to be separated by two elector 
bands, representing their respective lipid eifiv^t 
contact with each other.' Figs. 3A-3D sho'w^W 
of corneocytes that were added to the lipos : 6^V« 
sion.. Fig. 3A shows small unilamellar vesiej^> 
adsorbed on to the corneocyte lipid envelope^ 



121 




Fig. 1. Freeze-fracture electron micrograph of control liposomes. 
Arrowhead indicates the direction of shadowing. Bar - 200 nm. 



3B shows regions of multiple lamellae similar to those 
seen in the intercellular region of natural SC. Fig. 3C 
shows two corneocytes with their intercellular region 
filled with multiple lamellae formed from the added 
liposomes. There were regions where the SUVs were 
adsorbed onto fragments of cell envelope, as seen in 
Fig. 3D. There were some isolated SUVs that remained 
stable and unadsorbed even after a week of incubation 
with corneocytes at 37 ° C (not shown). 

Release of carboxyfluorescein from SC lipid vesicles 
under various conditions is shown in Table I. Fluores- 
cence obtained after lysis of the vesicles with Triton 
X-100 was taken as the value for 100% release. Initial 
fluorescence of the vesicles was taken as 0% release, 
which was 50 to 60% of the maximal value, indicating a 
high initial release of CF from these vesicles. The high 
initial release of the aqueous contents is due to the 
history of the vesicles. It was found necessary to use a 
high pH (> 9) during sonication to facilitate the disper- 
sion of these relatively nonpolar lipids in the presence 
of large quantity (100 mM) of CF. The vesicles were 
then dialyzed to pH 7.5 after the removal of nonen- 
capsulated CF. Amount of CF encapsulated at pH 9 
and 7.5 were found to be 3.77 and 1.28 nmol/>mol of 



I if} 



122 



f 




123 





Fig. 3. Thin-section electron micrographs of comeocytes added to (he liposomes. Incubation time was 1 h. (A) The liposomes are seen adsorbed 
onto the comeocytes. Bar -200 nm. (B) Liposomes are. seen at different stages of transformation. Bar = 100 nm. Inset shows multiple lamellae 
formed from the adsorbed liposomes (arrowheads). Bar —"50 nm. (Q Arrows show the intercellular region filled with multiple lamellae formed from 
the adsorbed liposomes. Arrowhead shows remnants of a partially- formed lamella. from the adsorbed liposome. Bar —100 nm. (D) Liposomes are 

seen adsorbed- onto the^xtenor of empty cell envelopes; Bar « 500 nm. - 



lipid, respectively, as shown in Table I. Incubation of 
the vesicles with supernatant buffer from .the comeo- 
cytes suspension resulted in some release of entrapped 
CF (10-12%) while incubation with comeocytes under 
similar conditions resulted in enhanced release lof CF 
(35-40%) as shown in Table I. . 

Discussion 

Liposomes formed from SC lipids were extremely 
stable at pH 7.5. The high curvature of these .SfcTVs, the 
repulsive hydration forces, and the. electrostatic fbrces 
of the charged headgroups (of the partially ionized fatty 
acids and the completely ioni^-.^;pl^ter^l:/stilfate)' 
apparently are sufficient to prevent them fr^in undergo- 
ing any fusion or aggregation; ■ >^ h- - y . 



The corrieocyte cell envelope has been thought of as 
a cross-linked protein envelope beneath the horny cell 
'plasma membrane* [21,22], These are usually seen' as in 
Fig: 2B, respectively, as a broad electron-dense band 
and a narrow electron-lucent band in TEMs. Recently, 
the electron-lucent band has been shown to consist of a 
monomolecular layer of lipids, predominantly made up 
of (^hydroxyacylsphingosines, that are covalently bound 
to the protein envelope [14]. These corneocyte lipid 
envelopes have been proposed to. have a cohesive role in 
regions of celUcell contact, especially in SC sheets that 
have been depleted of the intercellular lamellae by 
extensive extraction with chloroform/methanol [14], as 
well as in that was heated above, the 75 ° C transition, 
temperature k>f the SG lipids (unpublished observations). 
The , corneocyte ; lipid envelopes from contiguous 



■■5?T vi -Qi ■ 



1-T-, 



124 

TABLE I 

Release'of carboxyfluorescein from SC liposomes at 37°C 





CF released (nmol/>mol of lipid) 




during 


after lysis 




incubation - 


with Triton * 




with cells/ 






supernatant * 




Vesicles atpH7.5 




1.28±0.19 


Vesicles at pH 9.0 




3.77 c 


Vesicles incubated for 1 h 


0.44±0,06 


U3±0.18 


with corneocytes at pH 73 




1.45±0.18 


Vesicles incubated for 1 h with 


0.16 ±0.03 


supernatant (buffer) at pH 7.5 







• Calculated as the difference between .the amount of CF released 
from the vesicles after 1 h of incubation with cells/supernatant and 
the amount of CF released before the addition of cells/supernatant 
(initial release). 

b Calculated as the difference between the amount of CF released 
after the addition of Triton and the amount of CF released before 
the addition of cells/supernatant and Triton (initial release). 

c Mean of two measurements, n « 4 for rest of the data* 



corneocytes are seen as two lucent bands in regions 
where isolated corneocytes have come in contact with 
each other (Fig. 2B), supporting a cohesive role for the 
corneocyte lipid envelope. 

When corneocytes were added to the liposomes, the 
vesicles adsorbed onto the surface of the corneoeytes as 
seen in Fig. 3A. The liposomes, remained adsorbed even 
after the washing 1 and rinsing procedures during sample 
preparation for electron microscopy, indicating the sta- 
bility of the adsorption. Stable adsorption of phos- 
pholipid liposomes to different cells has been demon- 
strated by a variety of techniques [10]. Such adsorption 
may be mediated either by biochemical forces (surface 
receptors, antibodies, etc.), by purely physical forces 
(electrostatic, hydration, . hydrophobic, eta) or by a 
combination of both types of forces. 

SC has until recently been thought to be devoid of 
surface receptors such as lectins: 123,24]. Recently, a 40 
kD a -glycoprotein has-been isolated arid has been 
localized to the corneocyte lipid envelope: [25]; fcrysket 
al. have suggested that this endogenous lectin could 
play a major, role in the cell -cohesion in SC [26]. 
However, the same authors have shown that the mem- 
brane glycoproteins are solubilized by the jrionionic 
detergent used in the corneocyt^ preparation [2i5}: Thus, 
in corneocytes prepared by 

is reasonable to assume that iifcis. purely a physic^ force 
that causes the cell-ceH;c^^ a^p^ 
tion of SC lipid liposomes ;qrii tJ^ llurl^e ; of ^ 
corneocytes, ■;■ : ))''\? 'f.H? - : ""}'\ "; : 

The amount of CF Tele^ed'^fter- trie incubation of 
vesicles with corneocytes is^^ahr^tf caused 
by the incubation of the vesicIesl^itH the buffer' done 
suggesting a ceil.induced pwe^s: wliich result^ ft W 




enhanced release of the aqueous contents. 



of vesicles onto the corneocytes would not reiidfS' 



vesicles leaky. Enhanced release of CF from^^p! 
treated with corneocytes suggests a fusion proc^^j- 
renders the vesicles more leaky, presumably^^i 
from the collapse of the vesicles during or afte^? 
fusion process. CF release data along wiffijj&sfi 
pearance of lamellae on corneocytes treated'"^*"' 
lipid liposomes, as seen in Figs. 3B and 3C,sui 
formation of these lamellae from the adsorbed 
somes by a membrane fusion process. While 'iwpjj; 
the adsorbed liposomes transform to lamellae wittffi^- 
first hour of incubation, this transformation ap'^^^ 
be an inefficient process judging by the large fliijpgf^ 
liposomes that remained adsorbed on the cois^^; 
even a week after the addition of corneocyt^^j 
liposomes. This is not very surprising in the 'abSS|| 
fusogens that are known to promote aggre^a(^ 
fusion of SC lipid liposomes [7-91, whic^^ 
calcium [7]^ epidermal acylccramides and ac ^ 
ceramides [8,9], low pH ( < 6.5) and drying 
ence of large number of adsorbed liposqj 
corneocyte surfaces covered with lamellae i 
independence of the relative amounts of cdrrj 
surface with adsorbed liposomes versus cell sur$< 
lamellae suggests an alternative mechanism pE| 
tion of lamellar structures by direct transfort^fH| 
unadsorbed vesicles occurring concomitant t6 : ap^l 
pendent of adsorption process. We do not^^^j> 
experimental evidence to distinguish betwee|||| 
mechanisms of formation of the lamellae. -"v^L 

The adsorption of SC lipid liposomes b 
cytes and their transformation into lamellar^ 
the absence of any of these known fusogens iridfe 
independent role for the corneocyte lipid e$B 
these processes, Two plausible mechanisms c$B| 
tulated for the transformation of SUVs into|||j 
sheets on the basis of the appearance of these s||| 
TEM. After initial adsorption of the liposo)^ 
could be a transfer of lipids from the fiposc#g|| 
corneocyte lipid envelope. Such a transfer; patj 
tated through transient intermediary ;stn&| 
mixed micelles that can be formed betwee^ 
ionized fatty acids and other lipid caraKW 
through a direct transfer of lipids at tbc^p 
adsorption. This would lead to incorporatio^W 
the Vesicle biiayer into the lipid envelope/ Thi||| 
be accompanied by fusion of the adsorb^" 
with each other while they are immobiliz 
tion and are in close proximity with each 
would lead to the collapse of one layer 
liposomes and their transformation into a b$| 
emt to the cell envelope, seen as two eleq^| 
bands, as in Fig, 3B. * 
In regions where multilamellar structft^S 
attached to the corneocyte lipid envelope (£« 



125 



3C), the fusion mechanism may involve flattening of the 
adsorbed and/or unadsorbed liposomes and their sub- 
sequent edge-to-edge fusion to form multilamellar struc- 
tures. Corneocytes, which are flat and elongated, with 
the molecularly smooth lipid envelope on their surface 
would act as a template for the initial flattening and for 
the subsequent transformation of flattened vesicles into 
extended lamellar structures. It should be informative to 
see if SC lipid liposomes are adsorbed and/or lamellar 
structures are formed on delipidized corneocytes. How- 
ever, the alkaline hydrolysis procedure used for the 
removal of the ester-linked cj-hydroxyceramides re- 
sulted in completely delipidized corneocytes in which 
the protein envelopes were partially separated from the 
cell contents [14], These delipidized corneocytes dis- 
integrated easily and could not be used for any further 
study. 

The observations made in the present study suggest a 
cohesive role for the corneocyte lipid envelope in the 
transformation of SC lipid liposomes into lamellar 
sheets. This role may be one of the major factors in 
reassembly of the extruded membranous disks into 
lamellar sheets which occurs in the intercellular space 
between the upper granular layer and the horny layer 
during the final stages of epidermal differentiation [3,28]. 

Acknowledgements 

This study was supported in part by a grant from the 
United States Public Health Service (AR 32374). We 
would like to thank the staff of the Central Electron 
Microscopy Facility at the University of Iowa for their 
. assistance and provision of facilities and Fusako Sato 
■ for her assistance with fluorescence measurements. 

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