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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY {PCT) 



(51) International Patent Classification 6 : 
A61K 31/715 



Al 



(11) International Publication Number: WO 97/49412 

(43) International Publication Date: 31 December 1997 (31.12.97) 



(21) International Application Number: PCIYEP97/03238 

(22) International Filing Date: 20 June 1997 (20.06.97) 



(30) Priority Data: 

PD96A00OI63 



21 June 1996 (21.06.96) 



IT 



(71) Applicant (for alt designated Slates except US): FIDIA S.P.A. 

[IT/IT); Via Ponte della Fabbrica, 3/A, 1-35031 Abano 
Terme (IT). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): BELINI, Davide [IT/IT]; 
Via Po, 34, 1-35036 Montegrotto Terme (IT). PAPARELLA, 
Annamaria (IT/IT]; Via Principe Amedeo, 320, 1-70100 Ban 
(IT). O 'REGAN, Michael [IE/IT]; Via San Antonio, 5, I- 
35037 Teolo (IT). CALLEGARO, Lanfranco [IT/IT]; Via 
Monte Grappa. 6, 1-360I6 Thiene (IT). 

(74) Agents: GUTMANN, Ernest et al.; Ernest Gutmann-Yves 
Plasseraud S.A., 3. rue Chauveau-Lagarde, F-75008 Paris 
(FR). 



(81) Designated States: AL. AM. AT, AU. AZ, BA. BB. BG, BR, 
BY, CA, CH, CN, CU. CZ, DE. DK, EE, ES, FI. GB, GE, 
GH, HU, IL, IS, JP, KE, KG. KP, KR, KZ. LC. LK, LR, 
LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, 
PL. PT, RO. RU, SD, SE, SG, SI, SK t SL. TJ, TM. TR, 
TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent <GH, 
KE. LS, MW. SD. SZ. UG. ZW), Eurasian patent (AM, AZ. 
BY. KG, KZ, MD, RU, TJ, TM). European patent (AT, BE, 
CH, DE, DK, ES. Fl. FR, GB. GR. IE. IT, LU. MC, NL, 
PT. SE). OAPI patent (BF, BJ, CF. CG. CI. CM. GA, GN 
ML. MR. NE. SN, TD, TG). 



Published 

With international search report. 
Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: 



AUTOCROSS-LINKED HYALURONIC ACID AND RELATED PHARMACEUTICAL COMPOSITIONS FOR THE 
TREATMENT OF ARTHROPATHIES 



(57) Abstract 



The present invention relates to compositions containing an autocross- linked form of hyaluronic acid alone or as a first component in 
mixture with a second component noncross-1 inked hyaluronic acid, and possibly also in combination with another pharmacologically active 
substance. These compositions can be used in the treatment of arthropathies due to their unique viscoelastic properties. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AL Albania 

AM Armenia 

AT Austria 

AU Australia 

AZ Azerbaijan 

BA Bosnia and Herzegovina 

BB Barbados 

BE Belgium 

BF Burkina Faso 

DC Bulgaria 

BJ Benin 

BR Brazil 

BY Belarus 

CA Canada 

CF Central African Republic 

CG Congo 

CH Switzerland 

CI CNe d'l voire 

CM Cameroon 

CN China 

CU Cuba 

CZ Czech Republic 

DE Germany 

DK Denmark 

EE Estonia 



ES 


Spain 


LS 


Lesotho 


SI 


n 


Finland 


LT 


Lithuania 


SK 


KR 


France 


LU 


Luxembourg 


SN 


GA 


Gabon 


LV 


Latvia 


SZ 


GB 


United Kingdom 


MC 


Monaco 


TD 


CE 


Georgia 


MD 


Republic of Moldova 


TC 


Gil 


Ghana 


MG 


Madagascar 


TJ 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


GR 


Greece 




Republic of Macedonia 


TR 


1IU 


Hungary 


ML 


Mali 


TT 


IE 


Ireland 


MN 


Mongolia 


UA 


1L 


Israel 


MR 


Mauritania 


uc 


IS 


Iceland 


MW 


Malawi 


us 


IT 


Italy 


MX 


Mexico 


uz 


JP 


Japan 


NE 


Niger 


VN 


KE 


Kenya 


NL 


Netherlands 


YU 


KG 


Kyrgyzsian 


NO 


Norway 


ZW 


KP 


Democratic People's 


NZ 


New Zealand 






Republic of Korea 


PL 


Poland 




KR 


Republic of Korea 


FT 


Portugal 




KZ 


Kazakstan 


RO 


Romania 




LC 


Saint Lucia 


RU 


Russian Federation 




LI 


Liechtenstein 


SO 


Sudan 




LK 


Sri Lanka 


SE 


Sweden 




LR 


Liberia 


SC 


Singapore 





Slovenia 

Slovakia 

Senegal 

Swaziland 

Chad 

Togo 

Tajikistan 

Turkmenistan 

Turkey 

Trinidad and Tobago 

Ukraine 

Uganda 

United States of America 

Uzbekistan 

Viet Nam 

Yugoslavia 

Zimbabwe 



WO 97/49412 



1 



PCT/EP97/03238 



AUTOCROSS -LINKED HYALURONIC ACID AND 
RELATED PHARMACEUTICAL COMPOSITIONS FOR 
THE TREATMENT OF ARTHROPATHIES 

SUMMARY OF THE INVENTION 

The present invention relates to compositions contain- 
ing an autocross- linked form of hyaluronic acid alone or as 
a first component in mixture with a second component 
noncross- linked hyaluronic acid, and possibly also in com- 
bination with another pharmacologically active substance. 
These compositions can be used in the treatment of 
arthropathies due to their unique viscoelastic properties. 

BACKGROUN D OF THE INVENTION 

Hyaluronic acid (HA) is a naturally occurring 
polysaccharide of the glycosaminoglycan family which is 
present in particularly high concentration in the cartilage 
and synovial fluid of articular joints. It has been shown 
that synovial fluid acts as a viscous liquid at low shear 
(corresponding to the slowly moving joint) but shows an 
elastic behaviour at high shear (corresponding to the 
rapidly moving joint) (Balazs E.A., Univ. of Michigan, Med. 
Ctr. J. (Special Arthritis Issue), December 1968, 255). In 
patients with arthropathies such as osteoarthritis and 
rheumatoid, arthritis, the viscoelastic properties of 
synovial fluid are compromised and this has been 
demonstrated to reflect a decrease of the viscoelastic 
contribution given by the HA component {Kobayashi Y . et al, 



CONFIRMATION COPY 



WO 97/49412 PCT/EP97/03238 



Biorheology, 1994, 31, 235-244). This is clearly evident 
from Fig. l which shows the rheological profiles of 
synovial fluid from the joints of healthy volunteers and 
osteoarthritic donors ("The Rheological and Biological 
Function of Hyaluronic Acid," E. A. Balazs, D. A. Gibbs, in 
Chemistry and Molecular Biology of the Intercellular 
Matrix, ed. by E. A. Balazs, Academic Press, 1970). In 
normal synovial fluid, unlike in the case of 
osteoarthritis, viscoelasticity values are high and G' and 
G" cross each other. The existence of this crossover point 
is linked not only to the concentration of HA (2-4 mg/ml) , 
but also, and above all, to its high molecular weight 
(about 4-5 million). In osteoarthritic subjects, on the 
other hand, there is both degradation of the hyaluronic 
acid, with consequent lowering of its molecular weight, and 
a decrease in its concentration (1-2 mg/ml) . 

Administration of highly purified, exogenous HA by 
intraarticular injection has been shown to be effective in 
the treatment of osteoarthritis. This is due not only to 
the unique viscoelastic properties of HA but also to its 
potential pharmacological properties. In fact, the 
commercial HA-based products which are currently marketed 
for the treatment of osteoarthritis by intra-articular 
injection reflect two schools of thought concerning the 
mode of action of HA in the treatment of these pathologies. 
There is strong evidence that unmodified HA exhibits 
pharmacological activity in addition to . provoking a 
transient re -establishment of the viscoelastic properties 
of the synovial fluid (G. Abatangelo and M. 0' Regan, Eur. 
J. Rheumatol. Inflamm. , 1995, 15, 1:9-16; P. Ghosh, Clin 
Exp. Rheumatol., 1993, 12, 1-8; R.K. Strachan et al , An. 
Rheum. Dis., 1990, 49:949-952). On the other hand, 
manufacturers of chemically cross-linked HA derivatives 
promote the hypothesis that these derivatives act by solely 
mechanical means (E.A. Balazs and J.L. Denlinger, J. 
Rheumatology, 1993, vol. 20, supplement 39: 3-9). 



WO 97/49412 PCT/EP97/03238 

3 

Other forms of arthropathy besides osteoarthritis may 
result from modification of the viscoelastic properties of 
the synovial fluid of the articular joints which may occur 
as a result of particular mechanical or surgical operations 
carried out on the joint such as immobilization following 
joint distortion or fracture repair and joint arthroscopy. 
In the treatment of the functional consequences of these 
interventions, the lubricating potential of HA or 
derivatives thereof may be more pertinent than the long- 
term pharmacological effects of the compounds. In addition. 
HA is known to have a rapid turnover in the joint (Brown 
T.J. et al, Exp. Physiol., 1991, 76, 125-134; Praser J.R.E. 
et al., Semin. Arthritis Rheum. , 1993, 22 (Suppl. l) , 9-i7 ; 
Laurent U.B.G. et al . , Matrix, 1992, 12, 130-6). Therefore, 
a further objective of the formulations described in the 
present invention is to increase the residence time of 
exogenous HA which is injected into the joints for 
treatment of arthropathies. 

OBJECTS OF THE INVENTION 

Therefore, it is an object of the present invention to 
provide new hyaluronic acid (HA)- and/or autocross- linked 
polysaccharide (ACP) -based compositions, possibly together 
with a suitable pharmaceutical excipient or carrier and/or 
drug for intraarticular use, which compositions exhibit 
appropriate viscoelastic properties for the treatment of 
arthropathies . 

It is another object of the present invention to 
provide compositions which act as reservoirs of native HA. 

Another object of the present invention is to provide 
a method for the treatment of arthropathies by delivering 
an HA- and/or ACP-based composition which exhibits 
appropriate viscoelastic properties and residence time 
within the joint and which is administered in an effective 
amount to a patient in need thereof. 



WO 97/49412 



PCT/EP97/03238 



4 

The foregoing objects and others are accomplished in. 
accordance with the present invention by providing one of 
the following combinations: 

1. an autocross -linked form of hyaluronic acid 
alone; 

2. or an autocross- linked form of hyaluronic acid as 
a first component in mixtures containing, as a second 
component, hyaluronic acid and/or a pharmaceutically active 
drug for intraarticular use. 

BRIEF DESCRIPTION OF DRAWINGS 

The invention is further illustrated in the accompany- 
ing drawings wherein: 

Figure 1 shows viscoelastic spectra of human synovial 
fluid from young healthy donors, elderly healthy donors, 
and osteoarthritic donors; 

Figures 2A-2D show viscoelastic spectra for different 
relative proportions of ACP/HA mixtures in phosphate 
buffer, Cp-1% w/w, T=25°C (G' { ° ) ;G'\ { • ) ; i?* , <*) ; 

Figure 3 shows storage modulus (G')<°) and loss 
modulus (G") (•) as a function of ACP content {%) . Frequency 
=0.72 rad/s (corresponding to the movement of the joint at 
normal walking pace) , T=25°C; 

Figure 4 shows a comparison of the dynamic viscosities 
of formulations of ACP/HA at various ratios; 

Figure 5 shows viscoelastic spectrum of a formulation 
of ACP/HA, 100/0; ACP 20%, 0.5% H 2 0; 

Figure 6 shows viscoelastic spectrum of a formulation 
of ACP/HA, 75/25; ACP 20%, 0.5% H 2 0; 

Figure 7 shows viscoelastic spectrum of a formulation 
of ACP/HA, 50/50; ACP 5%, 0.5% H 2 0; 

Figure 8 shows viscoelastic spectrum of a formulation 
of ACP/HA, 50/50; ACP 20%, 0.5% H 2 0; 

Figure 9 shows a comparison of the dynamic viscosities 
of formulations of ACP/HA, 100/0; ACP 20%, 10%, and 5%, 
0.5% H 2 0; 



WO 97/49412 



PCT/EP97/03238 



5 

Figure 10 shows a comparison of the dynamic 
viscosities of formulations of ACP/HA, 50/50; ACP 20%, 10%, 
and 5%, 0.5% H 2 0; 

Figure 11 shows a comparison of the dynamic 
viscosities of formulations of ACP/HA, 40/60; ACP 20% and 
5%, 0.5% H 2 0; 

Figure 12 shows viscoelastic spectrum of synovial 
fluid from a non-osteoarthritic horse; 

Figure 13 shows viscoelastic spectrum of ACP/HA, 
100/0; ACP 10%, 0.5% H 2 0; 

Figure 14 shows viscoelastic spectrum of synovial 
fluid from a non-osteoarthritic horse with the addition of 
ACP 100/0, 10% 0.5% H 2 0, 3.3 mg/ml; 

Figure 15 shows viscoelastic spectrum of synovial 
fluid from a non-osteoarthritic horse with the addition of 
ACP 100/0, 10% 0.5% H 2 0, 5.5 mg/ml; 

Figure 16 shows a comparison of the dynamic 
viscosities of ACP, Synvisc, Fermentech, Artz and 
Hyalgan. 

DETAILED DESCRIPTION OF THE INVENTION 

It is an aim of the present invention to provide a new 
formulation able to improve the viscoelasticity and 
synovial residence time of exogenous HA which is injected 
into the joints for the treatment of arthropathies. 

This formulation consists of autocross- linked HA, 
alone or as a first component in mixtures containing as a 
second component hyaluronic acid. In these formulations, 
the autocross -linked polysaccharide (ACP) is obtained 
through an autocross-linking process that leads to the 
formation of intra- and inter- chain -ester bonds without 
introducing any foreign bridge between the polymer chains 
as described in EP 0341745 Bl . 

The ACP component can be synthesized from HA having a 
molecular weight in the range from 50 kDa to 5,000 kDa and 
must have a pharmaceutical grade purity and a level of 



WO 97/49412 



PCT/EP97/03238 



6 

cross-linking which ranges from 1% to 30% with respect to 
the carboxyl groups of the polymer. 

Preferred examples of the ACP component include: ACP 
5, ACP 10, ACP 15 and ACP 20 where the numbers 5, 10, 15 
and 20 reflect the nominal level of cross- linking based on 
the stoichiometry of the chemical reaction. 

These autocross- linked HA derivatives can, therefore, 
be used to advantage in the preparation of suspensions for 
the treatment of arthropathies due to their improved 
viscoelasticity with respect to native HA, which is 
released upon degradation of these autocross-linked 
derivatives. ACP HA derivatives therefore constitute ideal 
viscoelastic materials in addition to being a reservoir of 
native HA which is slowly released upon degradation 
resulting in a prolongation of the contact time of native 
HA with the joint tissues. The safety of these autocross- 
linked HA derivatives is also potentially better than HA 
derivatives produced by alternative cross- linking reactions 
since the native HA which is released by the degradation of 
ACP is metabolised by physiological metabolic pathways. 

Moreover, taking into account the gel -like behaviour 
of ACP's in aqueous media (Mensitieri et al . , Abstract, 
"12th European Conference on Biomaterials M Porto Portugal, 
Sept. 10-13, 1995), it is possible by blending HA with its 
ACP derivatives to obtain a wide range of systems which may 
combine viscoelastic and reservoir properties. 

The non- ideal rheological properties of ACP alone may 
be compensated for by preparing pharmaceutical compositions 
which are composed of mixtures of ACP and unmodified HA at 
varying ratios of the two components according to the 
condition of the patient and to the joint to be treated. 

The relative proportions of the ACP and HA employed in 
the formulations of the present invention generally include 
ACP/HA in relative amounts of about 95:05 to about 05:95. 
Preferred ratios for the ACP/HA formulations include ACP/HA 
at ratios of about 75:25 to about 25:75. 



WO 97/49412 



PCT/EP97/03238 



7 

The ACP/HA formulations of the present invention may 
be made into pharmaceutical compositions and may be 
combined with appropriate pharmaceutical^ active drugs, 
such as anaesthetics, antibiotics, steroidal and non- 
steroidal antiinflammatory drugs, hormonal -type antiinflam- 
matory agents, such as somatostatina, epitheliotrophic 
vitamins, cytokines such as IL-1 and IL-6, cytokine 
receptors, growth factors such as FGF and acceptable 
excipients. Moreover, it is possible to use pharmaceutical 
compositions starting from mixtures of ACP and HA wherein 
the HA is salified with silver, copper, zinc and calcium 
salts. These pharmaceutical compositions may be formulated 
into preparations in semi-solid or liquid forms for 
intraarticular use. 

The total quantity of HA, either in the form of ACP or 
HA, is in the range of 3-50 mg. A suitable dosage is that 
which contains a total amount of HA contained in the 
pharmaceutical compositions, either in the form of ACP or 
HA, of 20 mg in a final volume of 2 ml of suitable 
pharmaceutical excipient. 

1. Autocross- linked Polysaccharide (ACP) Product 

The ACP derivatives utilized in the present 
composition are autocross -linked derivatives of hyaluronic 
acid hyaluronic acid. In these derivatives, all or a part 
of the carboxyl groups of the hyaluronic acid are 
esterified with hydroxyl groups of the same molecule and/or 
of different hyaluronic acid (HA) molecules, thus forming 
lactone or intermolecular ester bonds. These "inner" esters 
of HA, in which there is no intervention by OH groups of 
other alcohols, can also be defined as "auto-crossl inked 
polysaccharides", since the formation of a mono- or 
polymolecular cross- link is the consequence of the above- 
mentioned internal esterif ication . The adjective 
"cross-linked" refers to the crosswise connections between 
the carboxyls and hydroxyls of the polysaccharide 
molecules . 



WO 97/49412 



PCT/EP97/03238 



8 

The inner esters can be total or partial, depending on 
whether all or only part of the carboxy functions are 
esterified in the above manner. In the partial inner 
esters, further carboxy functions can be either totally or 
partially esterified with monovalent or polyvalent 
alcohols, thus forming "external" ester groups, and in the 
partial esters of both these ester groups, the non- 
esterified carboxy functions may be free or salified with 
metals or organic bases. 

The inner esters used in the present invention can be 
prepared by the method described in EP 0 341 74 5 Bl which 
involves the activation of the carboxy groups by the 
addition of substances capable of inducing such activation. 
The unstable intermediate products obtained from the 
activation reaction separate spontaneously, either after 
the addition of catalysts and/or following a rise in 
temperature, forming the above-mentioned inner ester bonds 
with hydroxyls of the same or other HA molecule. According 
to the degree of inner esterif ication desired, either all 
or an aliquot part of the carboxy functions are activated 
(the aliquot part being obtained by using an excess of 
activating substances or by suitable dosing methods) . 

The carboxy groups to be converted into inner ester 
groups can be activated starting from polysaccharides 
containing free carboxy groups, or, preferably, from 
polysaccharides containing salified carboxy groups, for 
example metal salts, preferably alkaline or alkaline earth 
metals, and above all with quaternary ammonium salts, such 
as those descried hereafter. Salts with organic bases such 
as amines can however also be used as starting substances. 

The number of carboxy functions to be converted into 
inner esters is in proportion to the number of activated 
carboxy functions and this number depends on the quality of 
the activating agent used. In order to obtain total inner 
esters therefore, an excess of activating agents should be 
used, while in the case of partial esters, the quantity of 



WO 97/49412 



PCT/EP97/03238 



9 

this agent should be closed t according to the degree of 
esterif ication desired. 

The carboxy groups which are still free or salified 
after the cross -linking reaction can be exchanged in order 
to obtain opportune salts or can be esterified with 
monovalent or polyvalent alcohols thus obtaining mixed 
esters, partly cross- linked and partly externally 
esterified. Of course, partial esterif ication with alcohols 
can be effected before activation of part of the carboxy 
groups and subsequent conversion into inner esters . 

In the produced crosslinked products, the remaining 
free carboxy groups or those in the form of salts can be 
partially or totally esterified with mono- or polyvalent 
alcohols, thus obtaining esters mixed with bonds which are 
in part internal and in part external . The alcohols used 
for this esterif ication correspond to those described 
hereafter and from which mixed esters are derived. 

According to EP 0 216 453 Al, the external esters can 
be advantageously prepared by starting with quaternary 
ammonium salts with an etherifying agent in an aprotic 
solvent, such as dialkylsulf oxides, dialkylcarboxylamides, 
such as in particular lower alkyl dialkylsulf oxides with a 
maximum of 6 carbon atoms, particularly dimethylsulf oxide, 
and the lower alkyl dialkylamides of lower aliphatic acids, 
such as dimethyl or diethyl formamide or dimethyl or 
diethyl acetamide. Reaction should be effected preferably 
within a temperature range of between about. 25° and 75° , 
for example at about 30°. Esterif ication is effected 
preferably by gradually adding the etherifying agent to the 
above -said ammonium salt dissolved in one of the solvents 
mentioned, for example in dimethylsulf oxide . 

In the inner esters, the carboxy groups still left 
intact can be salified with organic or inorganic biases. The 
choice of bases for the formation of such salts is based on 
the intended use of the product. The inorganic salts are 
preferably those of alkaline metals, such as sodium or 
potassium salts or ammonium salts, cesium salts, salts of 



WO 97/49412 



PCT/EP97/03238 



10 



alkaline earth metals, such as calcium, magnesium or 
aluminum. 

The salts of organic bases are especially those of 
aliphatic, araliphatic, cycloaliphatic or heterocyclic 
amines. The ammonium salts of this type may derive from 
therapeutically acceptable, but inactive, amines, or from 
amines with a therapeutic action. Of the former, special 
consideration should be given to aliphatic amines, for 
example, mono, di and trialkylamines, with alkyl groups 
with a maximum of IB carbon atoms, or arylalkylamines with 
the same number of carbon atoms in the aliphatic part and 
where aryl means a benzene group possibly substituted by 
between 1 and 3 hydroxy groups. As therapeutically 
acceptable amines, but not active in themselves, cyclic 
amines are very suitable, such as alkylene amines with 
rings of between 4 and 6 carbon atoms, possibly interrupted 
in the ring by heteroatoms, such as oxygen, sulphur and 
nitrogen, such as piperidine, morpholine or piperazine, or 
may be substituted for example by amino or hydroxy 
functions, as in the case of aminoethanol , ethylene diamine 
or choline. 

In the ACP derivatives which also have carboxy 
functions esterified with monovalent or polyvalent 
alcohols, whether these functions be present in the 
starting materials of the above-mentioned procedure, or 
whether they be introduced at the end of the procedure, the 
alcohols may belong to the aliphatic, araliphatic, 
alicyclic or heterocyclic series. 

Alcohols of the aliphatic series for use as 
esterifying components are for example those with a maximum 
of 34 carbon atoms, which can be saturated or unsaturated 
and which can possibly also be substituted by other free 
functional or functionally modified groups, such as amino, 
hydroxyl, aldehydo, keto, mercapto, carboxy groups or by 
groups deriving from these, such as hydrocarbyl or 
dihydrocarbylamino groups (here and hereafter meaning by 
the term "hydrocarbyl" not only monovalent radicals of 



WO 97/49412 



PCT/EP97/03238 



11 

carbohydrates for example type C n H 2n+r , but also bivalent or 
trivalent radicals, such as "alkylenes" C n H 2n or 
"alkylidenes" C n H 2n ) , ether or ester groups, acetal or ketal 
groups, thioether or thioester groups, and esterified 
carboxy groups or carbamidic and substituted carbamidic 
groups by one or two hydrocarbyl groups, by nitrite groups 
or halogens . Of the above groups containing hydrocarbyl 
radicals, these should preferably be lower aliphatic 
radicals, such as alkylic, with a maximum of 6 carbon 
atoms. Such alcohols may then be interrupted in the carbon 
atom chain by heteroatoms, such as atoms of oxygen, 
nitrogen and sulfur. 

It is preferable to choose alcohols substituted with 
one or two of the above-said functional groups. Alcohols of 
the above group to be preferred for the purposes of the 
present invention are those with a maximum of 12 and 
especially 6 carbon atoms and in which the hydrocarbyl 
radicals in the above-said amino, ether, ester, thioether, 
thioester, acetal, ketal groups represent alkyl groups with 
a maximum of 4 carbon atoms, and also in the esterified 
carboxy groups or substituted carbamidic groups or 
hydrocarbyl groups are alkyls with the same number of 
carbon atoms, and in which the amino or carbamidic groups 
may be alkylene amine or alkyl^ne carbamidic groups with a 
maximum of 8 carbon atoms. Of these alcohols, of particular 
interest are those which are saturated and unsubstituted 
such as methyl, ethyl, propyl, isopropyl alcohols, n-butyl 
alcohol, isobutyl alcohol, tert-butyl alcohol, amyl 
alcohols, pentyl, hexyl, octyl, nonyl and dodecyl alcohols 
and above all those with a linear chain, such as n-octyl 
and n-dodecyl alcohols. Of the substituted alcohols of 
this group, preferred are: bivalent alcohols such as 
ethylene glycol, propylene glycol, butylene glycol, 
trivalent alcohols such as glycerin, aldehyde alcohols such 
as tartronic alcohol, carboxy alcohols such as lactic 
acids, for example glycolic acid, malic acid, tartaric 
acids, citric acid, aminoalcohols, such as aminoethanol , 



WO 97/49412 



PCT/EP97/03238 



12 



aminopropanol , n-aminopropanol , n-aminobutanol and their 
dimethyl and diethyl derivatives in the amine function, 
choline, pyrrolidinylethanol , piperidinylethanol , 
piperazinylethanol and the corresponding derivatives of 
n-propyl or n-butyl alcohols, monothioethyleneglycol and 
its alkyl derivatives, for example the ethyl derivative in 
the mercapto function. 

Of the higher aliphatic saturated alcohols, the 
following are examples: cetyl alcohol and myricyl alcohol, 
but of special importance for the purposes of the present 
invention are the higher unsaturated alcohols with one or 
two double bonds, such as especially those contained in 
many essential oils and having affinity with terpenes, such 
as citronellol, geraniol, nerol, nerolidol, linalool, 
farnesol, phytol. Of the lower unsaturated alcohols, the 
ones to be considered are allyl alcohol and propargyl 
alcohol . 

Of the araliphatic alcohols, preferred are those with 
only one benzene residue and in which the aliphatic chain 
has a maximum of 4 carbon atoms and in which the benzene 
residue may be substituted by between 1 and 3 methyl or 
hydroxy groups or by halogen atoms, especially by chlorine, 
bromine, iodine, and in which the aliphatic chain may be 
substituted by one or more functions chosen from the groups 
comprising free amino groups or mono or dimethyl groups or 
by pyrrolidine or piperidine groups. Of these alcohols, 
most preferred are benzyl alcohol and phenethyl alcohol. 

Alcohols of the cycloaliphatic or aliphatic 
cycloaliphatic series may derive from mono or polycyclic 
carbohydrates, may preferably have a maximum of 34 carbon 
atoms, may be unsubstituted and may contain one or more 
substituents, such as those mentioned above for the 
aliphatic alcohols. Of the alcohols derived from single- 
ringed cyclic carbohydrates, preferred are those with a 
maximum of 12 carbon atoms, the rings having preferably 
between 5 and 7 carbon atoms, which may be substituted for 
example by between one and three lower alkyl groups, such 



WO 97/49412 



PCI7EP97/03238 



13 

as methyl, ethyl, propyl, or isopropyl groups. As alcohols 
specific to this group, cyclohexanol , cyclohexanediol , 
1,2,3 cyclohexanetriol and 1,3,5 cyclohexanetriol 
(phloroglucitol) , inositol, should be mentioned, as well as 
the alcohols deriving from p-menthane, such as 
carvomenthol , menthol, a and 7-terpineol, 1-terpinenol, 
4-terpinenol and piperitol, or the mixture of these 
alcohols as " terpineol M , 1,4-and 1,8-terpin. Of the 
alcohols deriving from carbohydrates with condensed rings, 
for example those of the thujane, pinane or camphane group, 
useful also are thujanol, sabinol, pinol hydrate, D and 
L-borneol and D and L-isoborneol . 

Aliphatic-cycloaliphatic polycyclic alcohols to be 
used for the esters of the present invention are sterols, 
cholic acids and steroids, such as the sexual hormones and 
their synthetic analogues, and in particular corti- 
costeroids and their derivatives. Thus it is possible to 
use for example: cholesterol, dihydrocholesterol , 
epidihydrocholesterol , coprostanol , epicoprostanol , 
sitosterol, stigmasterol , ergosterol, cholic acid, 
deoxycholic acid, lithocholic acid, estriol, estradiol, 
equilenin, equilin and their alkyl derivatives, as well as 
the ethynyl or propynyl derivatives in position 17, for 
example 17-a-ethynyl-estradiol or 7-<y-methyl-17-cy-ethynyl- 
estradiol, pregnenolone, pregnanediol , testosterone and its 
derivatives, such as 17-<y-methyltestosterone, 1,2-dehydro- 
testosterone and 17-a-methyl-l , 2-dehydrqtestosterone , 
alkynyl derivatives in position 17 of testosterone and 
1, 2-dehydrotestosterone, such as 17 -a-ethynyl testosterone , 
17 -a -propynyl testosterone, norgestrel, hydroxyprogesterone, 
corticosterone, deoxycorticosterone, 19-nortestosterone, 
19-nor-17-a-methyltestosterone and 19 -nor- 17 -a-ethynyl - 
testosterone, cortisone, hydrocortisone, prednisone, 
prednisolone, fludrocortisone, dexamethasone, beta- 
methasone , paramethasone , f lumethasone , f luocinolone , 
f luprednyl idene , clobetasol , beclomethasone , aldosterone , 
desoxycorticosfcerone, alfaxalone, alfadolone, bolasterone. 



WO 97/49412 



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14 

Useful esterifying components for the esters of the 
present invention are genies, (aglycons) of cardioactive 
glycosides, such as digitoxigenin, gitoxigenin, digoxi- 
genin, strophanthidin, tigogenin, saponins. 
5 Other alcohols to be used according to the invention 

are vitamin alcohols such as axerophthol, vitamins D 2 and 
D 3 , aneurine, lactof lavine , ascorbic acid, riboflavine, 
thiamine, pantothenic acid. 

Heterocyclic alcohols may be considered to be 
10 derivatives of the above-said cycloaliphatic or aliphatic- 

cycloaliphatic alcohols, if their linear or cyclic chains 
are interrupted by one or more, for example between one and 
three ethero atoms chosen from the group formed by - O - , 
- S -, - N and -NH and in these there may be one or more 
15 unsaturated bonds for example double bonds, particularly 

between one and three, thus including also heterocyclic 
compounds with aromatic structures. The following are 
specific useful examples: furfuryl alcohol, alkaloids and 
derivatives such as atropine, scopolamine, cinchonine, 
20 cinchonidina, quinine, morphine, codeine, nalorphine, 

N-butylscopolammonium bromide, ajmaline; phenylethylamines 
such as ephedrine, isoproterenol, epinephrine; 
phenothiazine drugs such as perphenazine, pipothiazine, 
carphenazine , homof enazine , acetophenazine , f luphenazine , 
25 N-hydroxyethyl promethazine chloride; thioxanthene drugs 
such as flupenthizol and clopenthixol ; anticonvulsivants 
such as meprophendiol , antipsychotics such as opipramol; 
antiemetics such as oxypendil; analgesics such as 
carbetidine and phenoperidine and methadol ; hypnotics such 
30 as etodroxizine; anorexics such as benzhydrol and 

diphemethoxidine; mild tranquilizers such as hydroxyzine; 
muscle relaxants such as cinnamedrine , diphylline, 
mephenesin, methocarbamol, chlorphenesin, 2 , 2 -diethyl -1, 3- 
propanediol, guaifenesin, idrocilamide; coronary vaso- 
3 5 dilators such as dipyridamole and oxyfedrine; adrenergic 

blockers such as propanolol, timolol, pindolol, bupranolol, 
atenolol, metoprolol, practolol; antineoplastics such as 



WO 97/49412 



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15 

6-azauridine, cytarabine, floxuridine; antibiotics such as 
chloramphenicol, thiamphenicol , erythromycin, oleandomycin, 
lincomycin; antivirals such as idoxuridine; peripheral 
vasodilators such as isonicotinyl alcohol; carbonic 
anhydrase inhibitors such as sulocarbilate; antiasthmatics 
and antiinflammatories such as tiaramide; sulf amides such 
as 2-p-sulf anylanilinoethanol . 

2 . The Hyaluronic Acid 

In the present invention, hyaluronic acid (HA) serves 
as the starting material for making the ACP derivatives, or 
as a second component in combination with the ACP deriva- 
tives. The cross-linked HA may use, as starting substrate, 
any natural or synthetic HA. 

The substrate of hyaluronic acid can be of any origin, 
such as acids extracted from the above natural starting 
materials, for example from cocks' combs. The ACP/HA 
formulations of the present invention employ hyaluronic 
acid isolated from either bacterial (WO 95/04132) or animal 
sources (EP 0138572; WO 92/18543) or hyaluronic acid 
produced by in-vitro enzymatic synthesis (WO 95/24497) . 
According to the invention, it is preferable to use 
hyaluronic acids constituting molecular fractions of the 
integral acids obtained directly by extraction of organic 
materials with a wide range of molecular weights, for 
example between 90%-80% and 0.2% of the molecular weight of 
the integral acid, preferably between 5% and 0.2%. These 
fractions can be obtained by various procedures described 
in literature, and that is with hydrolyzing, oxidizing or 
enzymatic chemical agents or physical procedures, for 
example mechanical or irradiation procedures, and often 
during the same purification procedures, primordial 
extracts may be formed. Separation and purification of the 
molecular fractions obtained comes about by means of known 
techniques, such as by molecular filtration. One purified 
HY fraction suitable to be used according to the invention 
is for example the one known as "noninf lammatory-NIF-NaHA 



WO 97/49412 



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16 

sodium hyaluronate", described by Balazs in the pamphlet 
"Healon" - A guide to its use in Ophthalmic Surgery - D. 
Miller & R. Stegmann, eds . John Wiley & Sons N.Y 81983: 
p. 5. 

Also particularly important as starting materials for 
the ACP esters are two purified fractions which can be 
obtained from hyaluronic acid, for example the one 
extracted from cocks' combs, known by the names of 
"Hyalastine" and "Hyalectin". The fraction Hyalastine has 
an average molecular weight of about 50,000 to 100,000 
while the fraction Hyalectin has an average molecular 
weight of about 500,000 to 730,000. One combined fraction 
of these two fractions has also been isolated and 
characterized as having an average molecular weight of 
between about 250,000 and about 3 50,000. This combined 
fraction can be obtained with a yield of 80% of the total 
hyaluronic acid available in the particular starting 
material, while the fraction Hyalectin can be obtained with 
a yield of 30% and the fraction Hyalastine with a yield of 
50% of the starting HY . The preparation of these fractions 
is described in the above-mentioned European patent 
publication No. 0138572A3. 

The invention is illustrated by the following 
illustrative examples, without these in any way limiting 
its scope. 

3. Pr eparation of ACP Derivatives 

Example 1 : 

PREPARAT ION OF CROSS-LINKgD HYALURONIC ACID ffJV) 
Product description: 

1% of carboxy groups used in internal esterif ication . 

99% of carboxy groups salified with sodium. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.01 g (0.1 
mEq) of triethylamine are added and the resulting solution 
is agitated for 30 minutes. 



WO 97/49412 



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17 

A solution of 0.026 g (0.1 mEq) of 2 -chloro-1 -methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times with 100 ml of 
acetone/water 5:1 and three times with 100 ml of acetone 
and lastly vacuum-dried for 24 hours at 30 °C. 

3.97 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 2 : 

PREPARATION OF CROSS - LINKED HYALURONIC ACID (HY) 
Product description: 

5% of carboxy groups used in internal esterif ication. 

95% of carboxy groups salified with sodium. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 85,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C / 0.051 gr 
(0.5 mEq) of triethylamine are added and the resulting 
solution is agitated for 3 0 minutes. 

A solution of 0.128 gr (0.5 mEq) of 2 -chloro-1 -methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times in 100 ml of acetone/water 



WO 97/49412 



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18 

5:1 and three times with 100 ml of acetone and lastly 
vacuum-dried for 24 hours at 30 °C. 

3.95 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 3 : 

PREPARATION OF C ROSS -LINKED HYALURONIC ACTD fHV) 
Product description: 

10% of carboxy groups used in internal esterif ication . 
90% of carboxy groups salified with sodium. 
6.21 g of HY tetrabutyl ammonium salt with a molecular 
weight of 620,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.101 gr 
(1.0 mEq) of triethylamine is added and the resulting 
solution is agitated for 30 minutes. 

A solution of 0.255 gr (1.0 mEq) of 2 -chloro-1 -methyl - 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times in 100 ml of acetone/water 
5:1 and three times with 100 ml of acetone and lastly 
vacuum-dried for 24 hours at 30°C. 

3.93 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 



WO 97/49412 



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19 

Example 4 : 

PREPARATION OF CROSS-LINKED HYALURONIC ACID (HY) 
Product description: 

25% of carboxy groups used in internal esterif ication. 
75% of carboxy groups salified with sodium. 
6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.253 g 
(2.5 mEq) of triethylamine are added and the resulting 
solution is agitated for 30 minutes. 

A solution of 0.639 g (2.5 mEq) of 2 -chloro-1 -methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times in 100 ml of acetone/water 
5:1 and three times with 100 ml of acetone and lastly 
vacuum-dried for 24 hours at 3 0°C. 

3.85 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 5 : 

PREPARAT ION OF CROSS -LINKED HYALURONIC ACID (HY) 
Product description: 

50% of carboxy groups used in internal esterif icat ion . 

50% of carboxy groups salified with sodium. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 85,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.506 g 
(5.0 mEq) of triethylamine are added and the resulting 
solution is agitated for 30 minutes. 



WO 97/49412 



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20 

A solution of 1.28 gr (5 mEq) of 2 -chloro-1 -methyl - 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times in 100 ml of acetone/water 
5:1 and three times with 100 ml of acetone and lastly 
vacuum-dried for 24 hours at 30 °C. 

3.65 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 6 : 

PREPARATION OF CROSS-LINKED HYALURONIC: ACTD (HY) 
Product description: 

75% of carboxy groups used in internal esterif ication . 

25% of carboxy groups salified with sodium. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.759 gr 
{7.5 mEq) of triethylamine is added and the resulting 
solution is agitated for 30 minutes. 

A solution of 1.92 gr (7.5 mEq) of 2 -chloro-1 -methyl 
pyridinium iodide in 60 ml of DMSO is slowly added -drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times in 100 ml of acetone/water 



WO 97/49412 



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21 

5:1 and three times with 100 ml of acetone and lastly 
vacuum-dried for 24 hours at 30°C. 

3.54 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 7: 

PREPARATION OF CROSS -LINKED HYALURONIC ACID (HY) 
Product description: 

100% of carboxy groups used in internal 
esterif ication . 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 70,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 1.012 gr 
(10 mEq) of triethylamine are added and the resulting 
solution is agitated for 30 minutes. 

A solution of 2.55 gr (10 mEq) of 2 -chloro-1 -methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30 °C. 

The resulting mixture is slowly poured into 750 ml of 
acetone, maintaining continual agitation. A precipitate is 
formed which is filtered and washed six times with 100 ml 
of acetone and lastly vacuum-dried for 24 hours at 30°C. 

3.52 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 8 : 

PREPARATION OF THE PARTIAL E THYL ESTER OF CROSS -LTN TTPn 
HYALURONIC ACID (HY) 
Product description: 

25% of carboxy groups esterif ied with methanol; 



WO 97/49412 



PCT/EP97/03238 



22 

25% of carboxy groups used in internal esterif ication. 

50% of carboxy groups salified with sodium. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.390 gr 
(2.5 mEq) of ethyl iodide are added and the solution is 
kept for 12 hours at 30°C. 0.253 gr (2.5 mEq) of 
triethylamine are added and the solution is agitated for 30 
minutes . 

A solution of 0.639 g (2.5 mEq) of 2-chloro-l-methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
slowly poured into 750 ml of acetone, maintaining continual 
agitation. A precipitate is formed which is then filtered 
and washed three times in 100 ml of acetone/water 5:1 and 
three times with 100 ml of acetone and lastly vacuum-dried 
for 24 hours at 30°C. 

3.84 grs of the title compound are obtained. 
Quantitative determination of the ethoxy groups is carried 
out according to the method of R.H. Cundiff and P.C. 
Markunas (Anal. Chem. 13, 1028-1930 (1961)). Quantitative 
determination of the total ester groups is carried out 
according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 9 : 

PREPARATION OF THE PARTIAL ETHYL ESTER OF CROSS -LINKED 
HYALURONIC ACID (HY) 
Product description; 

50% of carboxy groups esterif ied with ethanol; 

25% of carboxy groups used in internal esterif ication . 

25% of carboxy groups salified with sodium. 



WO 97/49412 



PCT/EP97/03238 



23 

6,21 g of HY tetrabutylammonium salt with a molecular 
weight of 85,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.780 g 
{5.0 mEq) of ethyl iodide are added and the solution is 
kept for 12 hours at 30°C. 0.253 gr (2.5 mEq) of 
triethylamine are added and the solution is agitated for 3 0 
minutes. 

A solution of 0.639 g (2.5 mEq) of 2-chloro-l-methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times in 100 ml of acetone/water 
5:1 and three times with 100 ml of acetone and lastly 
vacuum-dried for 24 hours at 30°C. 

3.87 grs of the title compound are obtained. 
Quantitative determination of the ethoxy groups is carried 
out according to the method of R.H. Cundiff and P.C. 
Markunas (Anal. Chem. 23, 1028-1930 (1961)). Quantitative 
determination of the total ester groups is -carried out 
according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example 10: 

PREPARAT ION OF THE ETHYL ESTER OF CROSS - LINKED HYALURONIC 
ACID (HY) 

Product description: 

75% of carboxy groups esterified with ethanol; 

25% of carboxy groups used in internal esterif ication. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25 °C, 1.17 gr 
(7.5 mEq) of ethyl iodide are added and the solution is 



WO 97/49412 PCT/EP97/03238 

24 

kept for 12 hours at 30°C. 0.253 g (2.5 mEq) of 
triethylamine are added and the solution is agitated for 30 
minutes . 

A solution of 0.639 gr (2.5 mEq) of 2-chloro-l-methyl 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

The resulting mixture is slowly poured into 750 ml of 
acetone, maintaining continual agitation. A precipitate is 
formed which is filtered and washed five times with 100 ml 
of acetone and lastly vacuum-dried for 24 hours at 30°C. 

3.91 grs of the title compound are obtained. 
Determination of the ethoxy groups is carried out according 
to the method of R.H. Cundiff and P.C. Markunas (Anal. 
Chem. 33, 1028-1930 (1961)). Quantitative determination of 
the total ester groups is carried out according to the 
saponification method described on pp 169-172 of 
"Quantitative Organic Analysis Via Functional Groups" 4th 
Edition John Wiley and Sons Publication. 

Example 11: 

PREPARATION OF THE PARTIAL CORTISONE ESTER (C21) OF 
CROSS- LINKED HYALURONIC ACID (HY) 
Product description: 

20% of carboxy groups esterified with cortisone. 

25% of carboxy groups used in internal esterif ication. 

55% of. carboxy groups salified with sodium. 

6.21 gr of HY tetrabutylammdnium salt with a molecular 
weight of 70,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C. 0.85 gr (2 
mEq) 21-bromo-4-pregnene-17-<y-ol-3,ll,20-trion and the 
resulting solution is kept for 24 hours at 30°C. 0.253 gr 
(2.5 mEq) of triethylamine are added and the resulting 
solution is agitated for 30 minutes. 

A solution of 0.639 g (2.5 mEq) of 2 - chloro- 1 -methyl - 
pyridinium iodide in 6 0 ml of DMSO is slowly added drop by 



WO 97/49412 



PCI7EP97/03238 



25 

drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times with 100 ml of 
acetone/water 5:1 and three times with 100 ml of acetone 
and lastly vacuum-dried for 24 hours at 3 0°C. 

4.5 grs of the title compound are obtained. 
Quantitative determination of cortisone, mild alkaline 
hydrolysis with a hydroalcoholic solution of Na 2 C0 3 and 
extraction with chloroform, is carried out according to 
B.P. 

Quantitative determination of the total ester groups 
is carried out according to the saponification method 
described on pp 16 9-172 of "Quantitative Organic Analysis 
Via Functional Groups" 4th Edition John Wiley and Sons 
Publication. 



Example 12: 

PREPARAT ION OF THE MIXED ETHANOL AND CORTISONE PARTIAL 
ESTER ( C21) OF CROSS-LINKED HYALURONIC ACID IKY) 
Product description: 

20% of carboxy groups esterified with cortisone (C21) . 

25% of carboxy groups esterifi-ed with ethanol . 

25% of carboxy groups used in internal esterif ication. 

30% of carboxy groups salified with sodium. 

6.21 gr of HY tetrabutylammonium salt with a molecular 
weight of 85,000 corresponding to 10 mEq of a mbnomeric 
unit are solubilized in 248 ml of DMSO at 25°C. 0.39 gr 
(2.5 mEq) of ethyl iodide are added and the resulting 
solution is kept at 30°C for 12 hours. 0.85 gr (2 mEq) of 
21-bromo-4-pregnene-17-a-ol-3,ll,20-trion are added and the 
resulting solution is kept at 30°C for 24 hours. 0.253 gr 
(2.5 mEq) of triethylamine are added and the resulting 
solution is agitated for 30 minutes. 



WO 97/49412 PCT7EP97/03238 

26 

A solution of 0.639 g {2.5 mEq) of 2 -chloro-1 -methyl 
pyridinium iodide in 6 0 ml of DMSO is slowly added drop by 
drop over a period of 1 hour and the mixture is kept for 15 
hours at 3 0°C. 

A solution formed by 100 ml of water and 2.5 gr of 
sodium chloride is then added and the resulting mixture is 
then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times with 100 ml of 
acetone/water 5:1 and three times with 100 ml of acetone 
and lastly vacuum-dried for 24 hours at 30°C. 

4.41 grs of the title compound are obtained. 
Quantitative determination of cortisone, mild alkaline 
hydrolysis with a hydroalcoholic solution of Na 2 G0 3 and 
extraction with chloroform, is carried out according to 
B.P. 

Quantitative determination of the ethoxy groups is 
carried out according to the method of R.H. Cundiff and 
P.C. Markunas (Anal. Chem. 33, 1028-1930 (1961)). 
Quantitative determination of the total ester groups is 
carried out according to the saponification method 
described on pp 169-172 of "Quantitative Organic Analysis 
Via Functional Groups" 4th Edition John Wiley and Sons 
Publication . 

Example 13 : 

PREPARATION OF TH E MIXED ETHANOL AND CORTISONE ESTER ) 
OF CROSS -LINKED HYALURONIC ACID IKY) 
Product description: 

20% of carboxy groups esterified with cortisone (C21) . 

70% of carboxy groups esterified with ethanol. 

10% of carboxy groups used in internal esterif ication. 

6.21 g of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C. 1.09 g (7 
mEq) of ethyl iodide are added and the resulting solution 
is kept at 30°C for 12 hours. 0.85 gr (2 mEq) of 21-bromo- 



WO 97/49412 



PCT/EP97/03238 



27 

4-pregnerie-17-or-ol-3,ll,20-trion and the resulting solution 
is kept at 30°C for 24 hours. 0.101 gr (1.0 mEq) of 
triethylamine are added and the resulting solution is 
agitated for 30 minutes. 

A solution of 0.255 g (1.0 mEq) of 2-chloro-l-methyl- 
pyridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

The resulting mixture is slowly poured into 750 ml of 
acetone, maintaining continual agitation. A. precipitate is 
formed which is then filtered and washed five times with 
100 ml of acetone and lastly vacuum-dried for 24 hours at 
30°C. 

4.58 grs of the title compound are obtained. 
Quantitative determination of cortisone, mild alkaline 
hydrolysis with a hydroalcoholic solution of Na 2 C0 3 and 
extraction with chloroform, is carried out according to 
B.P. 

Quantitative determination of the ethoxy groups is 
carried out according to the method of R.H. Cundiff and 
P.C. Markunas (Anal. Chem. 33, 1028-1930 (1961)). 
Quantitative determination of the total ester groups is 
carried out according to the saponification method 
described on pp 169-172 of "Quantitative Organic Analysis 
Via Functional Groups" 4th Edition John Wiley and Sons 
Publication. 

Example: 14 

PREPARATION WITH K ANAMYCIN OF THE SALT OF A CROSS -LINKED 
HYALURONIC ACID 
Product description: 

25% of carboxy groups used in internal esterif ication . 

75% of carboxy groups with kanamycin. 

4.39 gr of partial tetrabutylammonium salt (25%) of 
hyaluronic acid corresponding to 10 mEq of a monomeric unit 
are solubilized in 248 ml of DMSO at 25°C, 0.253 gr (2.5 



WO 97/49412 



PCT/EP97/03238 



28 

mEq) of t r ie thy 1 amine are added and the resulting solution 
is agitated for 30 minutes. 

A solution of 0.639 g (2.5 mEq) of 2-chloro-l- 
methyl-pyridinium iodide in 60 ml of DMSO is slowly added 
drop by drop over a time interval of 1 hour and the mixture 
is kept for 15 hours at 30°C. 

The resulting mixture is slowly poured into 750 ml of 
acetone, maintaining continual agitation. A precipitate is 
formed which is then filtered and washed five times with 
100 ml di acetone and lastly vacuum-dried for 24 hours at 
30°C. 

The precipitate is suspended in 400 ml of distilled 
water and cooled to 5°C after which a solution obtained by 
solubilizing 1 . 1 gr of Kanamycin sulfate (7.5 mEq) in 25 ml 
of distilled H 2 0 and eluting in a column containing 15 ml of 
quaternary ammonium resin (Dowex 1x8) OH- form is added, 
while agitation is maintained for 30 minutes. The resulting 
mixture is f reeze-dried. 

4.6 grs of the title compound are obtained. Quanti- 
tative determination of the ester groups is carried out 
according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Microbiological quantitative determination of Kana- 
mycin is carried out on B. subtilis 6633 in comparison to 
standard Kanamycin. 

Example; 15 

PREPARATION WITH AMIKACIN OF A C ROSS -LTNlfSn HYALURONIC &rrn 
SALT 

Product description: 

25% of carboxy groups used in internal esterif ication. 
75% of carboxy groups with amikacin. 

4.39 gr of partial tetrabutylammonium salt (25%) of 
hyaluronic acid corresponding to 10 mEq of a monomeric unit 
are solubilized in 248 ml of DMSO at 25°C, 0.253 gr (2.5 



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mEq) of triethylamine are added and the resulting solution 
is agitated for 30 minutes. 

A solution of 0.639 gr (2.5 mEq) of 2 -chloro-1 -methyl - 
piridinium iodide in 60 ml of DMSO is slowly added drop by 
drop over a time interval of 1 hour and the mixture is kept 
for 15 hours at 30°C. 

The resulting mixture is slowly poured into 750 ml of 
acetone, maintaining continual agitation. A precipitate is 
formed which is then filtered and washed five times with 
100 ml of acetone and lastly vacuum-dried for 24 hours at 
30°C. 

The precipitate is suspended in 400 ml of distilled 
water and cooled to 5°C. 

1.1 gr {7.5 mEq) of basic amikacin are added while 
under constant agitation for 30 minutes. The resulting 
mixture is f reeze-dried. 

4.8 grs of the title compound are obtained. 
Quantitative determination of the ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Quantitative determination of amikacin is carried out 
microbiologically on S. aureus 29737, compared to standard 
Amikacin . 



Example: 16 

PREPARATION OF TH E PARTIAL ETHYL ESTER OF CROSS -LINKED 
HYALURONIC ACID (HY) 
Product description: 

50% of carboxy groups esterified with ethanol . 

10% of carboxy groups used in internal esterif ication. 

4 0% of carboxy groups salified with sodium. 

6.21 gr of HY tetrabutylammonium salt with a molecular 
weight of 85,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.780 gr 
(5.0 mEq) of ethyl iodide are added and the solution is 
kept for 12 hours at 30°C. 0.118 gr (1 mEq) of pyridine 



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chloride are added and the resulting solution is agitated 
for 30 minutes. 

A solution of 0.16 g (l mEq) of N-benzyl-N' -ethyl 
carbodiimmide in 20 ml of DMSO is slowly added drop by drop 
over a time interval of 1 hour and the mixture is kept for 
45 hours at 30°C. 

A solution is then added which is formed of 100 ml of 
water and 2.5 of sodium chloride and the resulting mixture 
is then poured slowly into 750 ml of acetone, maintaining 
continual agitation. A precipitate is formed which is then 
filtered and washed three times with 100 ml of acetone/H 2 0 
5:1 and three times with 100 ml of acetone and lastly 
vacuum- dried for 24 hours at 30°C. 

3.85 grs of the title compound are obtained. 
Quantitative determination of the ethoxy groups is carried 
out according to the method of R.H. Cundiff and P.C. 
Karkunas (Anal. Chem. 33, 1028-1930 (1961)). Quantitative 
determination of the total ester groups is carried out 
according to the saponification method described on pp 
16 9-172 of -Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

Example: 17 

PREPARATION OF C ROSS - LINKED HYALURONIC ACTD (HV) 

Product description: 

10% of carboxy groups used in internal esterif ication. 

90% of carboxy groups salified with sodium. 

6.21 gr of HY tetrabutylammonium salt with a molecular 
weight of 170,000 corresponding to 10 mEq of a monomeric 
unit are solubilized in 248 ml of DMSO at 25°C, 0.118 gr <1 
mEq) of pyridine chloride are added and the resulting 
solution is agitated for 30 minutes. 

A solution of 0.16 g (l mEq) of N-benzyl-N' -ethyl 
carbodiimmide in 20 ml of DMSO is slowly added drop by drop 
over a time interval of l hour and the mixture is kept at 
a temperature of 30 °C for 45 hours. 



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A solution made up of 100 ml of water and 2.5 of 
sodium chloride is added and the resulting mixture is 
slowly poured into 750 ml of acetone, maintaining continual 
agitation. A precipitate is formed which is then filtered 
and washed three times with 100 ml of acetone /H 2 0 5:1 and 
three times with 100 ml of acetone finally vacuum-dried for 
24 hours at a temperature of 30 °C. 

3.9 grs of the title compound are obtained. Quanti- 
tative determination of the total ester groups is carried 
out according to the saponification method described on pp 
169-172 of "Quantitative Organic Analysis Via Functional 
Groups" 4th Edition John Wiley and Sons Publication. 

4 . Formulations According to the Invention 

Example 18: 

PREPARATION OF A N INJECTABLE SUSPENSION CONTAINING AN 
ACTIVE PRINCIPLE BASED ON CROSS-LINKED HYALURONIC ACID 
(ACP) 

One 2 -ml vial contains: 



Example 19: 

PREPARAT ION OF AN INJECTABLE SUSPENSION CONTAINING AN 

ACTIVE PRINCIPLE WHICH IS A 75/25 MIXTURE BASED ON 

AUTOCROSS- LINKED HY ALURONIC ACID AND HYALURONIC ACID SODIUM 



autocross- linked hyaluronic acid (ACP) 
sodium chloride 

dihydrace monobasic sodium phosphate 
dodecahydrate bibasic sodium phosphate 
water for injection 



20 mg 
17 mg 
0 . 1 mg 
1 . 2 mg 
2 ml 



SALT 



One 2 -ml pre -filled syringe contains: 



autocross -linked hyaluronic acid (ACP) 15 mg 
hyaluronic acid sodium salt (hyalectin) 5 mg 
sodium chloride 17 mg 



dihydrate monobasic sodium phosphate 0 . 1 mg 
dodecahydrate bibasic sodium phosphate 1.2 mg 
water for injection 2 ml 



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32 

Example 20: 

PREPARATION OF AN TKLTprT ABLE SUSPENSION CONTAINING AN 
ACTIVE PRINCIPLE BASED ON A UTOCROSS- LINKED HYALURONIC ACID 
VEHICLING AN ANTIINFLA MMATORY AGENT SUCH AS METHYL - 
PREDNISOLONE 21 - SUCCINATE SODIUM SALT 

One 2-ml pre-filled syringe contains: 

autocross- linked hyaluronic acid (ACP) 20 mg 
methylprednisolone 21 -succinate sodium 

salt 10 mg 

sodium chloride 16 mg 

water for injection 2 ml 



Example 21: 

PREPARATION OF AN INJECTABLE SUSPENSION CONTAINING; AW 
ACTIVE PRINCIPLE WHICH IS A 75/25 MIXTURE RAfiRn nw 
AUTOCROSS -LINKED HYALURONIC A CID AND HYALURONIC ACID SODIUM 

SALT VEHICLIN G AN ANTIINFLAMMATORY AGENT SUCH AS 

TRIAMCINOLONE PHOSPHATE SODIUM fiAT.T 
One 2-ml vial contains: 

autocross-linked hyaluronic acid (ACP) 15 mg 
hyaluronic acid sodium salt (Hyalectin) 5 mg 
triamcinolone phosphate sodium salt 2 0 mg 

sodium chloride !8 mg 

water for injection 2 ml 

TeatB on Formul ations According to the Invention 

Example 22 : 

PREPARAT ION OF AN ACP/HA FORMULATION IN WHICH THE arp 
COMPONENT IS AUT OCROSS-LINKED TO A NOMINAL DEGREE OF 5* 

HA with a molecular weight range of 500-730 KDa was 
cross-linked to a nominal level of 5%. 

ACP/HA formulations were prepared at a final 
concentration of 1% w/w in phosphate buffer (NaCl 0.15M, 
phosphate salts 0.002M) at pH=6.5, by mixing various 
proportions of ACP/HA, ranging from 0/100 to 100/0 %. The 
suspensions were allowed to swell for 24 hours. 



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The rheological properties of the ACP/HA mixtures were 
measured on a Rheometrics Fluid Spectrometer (RFS-8S00) 
fitted with several geometries (50 mm diameter parallel 
plates: 1mm or 2mm gap and Couette : cup diameter 34 mm, bob 
diameter and length 32 respectively) at the fixed 
temperature of 25°C. From the oscillatory shear 
measurements (typically at 10% strain value) , the 
viscoelastic parameters G' (storage modulus) , G" (loss 
modulus) and y* {complex viscosity) were obtained over the 
frequency range of 0.01-100 rad/sec. 

The measurements indicated that ACP's, dispersed at 
sufficiently high polymer concentration and swollen in 
aqueous media, produce viscoelastic and transparent solid- 
like systems. The viscoelastic spectrum reported in Figure 
2A (ACP/HA 100/0) clearly shows a gel -like behaviour. In 
particular, G' (o>) > G" (a>) in the whole range of frequencies 
investigated and both G' and G" are slightly frequency- 
dependent. The ratio G'/G" (tan ) reaches a constant value 
(0.3) for frequencies lower than 2 rad/sec and it increases 
slightly (up to 0.4) with increasing frequency. The complex 
viscosity, , is strongly frequency- dependent following a 
power law in the whole range of frequencies investigated. 
The apparent power law exponent is - -0.82. 

By using the same applied strain (0.1 strain units), 
the measured absolute values of both moduli and complex 
viscosity, but not the viscoelastic behaviour, are 
appreciably affected by the geometry used.. This finding 
reflects the non- homogeneous character of the system. The 
gel -like response of ACP (100/0) is very different from the 
entangled network behaviour typical of the HA starting 
material (Kobayashi Y. et al . Biorheology, 1994, 31, 235- 
244) . 

As shown in Figure 2D (ACP/HA 0/100), the mechanical 
spectrum shows G" (u>) >G' (o>) and in the terminal region G'oe 
w2 and G"<x a>. Moreover, i?* (u>) is essentially independent 
from the frequency. By blending the two polymers in 
different ratios, while maintaining constant the total 



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polymer concentration (1% w/w) , it is possible to obtain a 
wide range of 2 -component systems. In particular, a HA-rich 
mixture (ACP/HA 30/70) may be regarded as a suspension in 
which the disperse component is constituted by discrete 
particles of swollen ACP and the continuous component is 
the HA aqueous solution. Conversely, the ACP-rich mixture 
(ACP/HA 75/25) may be considered as a "composite" in which 
the continuous component is much more rigid than the 
disperse component constituted by the HA aqueous solution. 

The mechanical response of such systems is expected to 
be dominated by the viscoelastic properties of the 
continuous component. In fact, the viscoelastic spectrum of 
the HA-rich mixture (Fig. 2C, 30/70) shows a liquid-like 
behaviour in the whole range of frequency investigated. 
However, in comparison with HA alone (Fig. 2D), G' and G" 
are enhanced (remarkable increase in the case of G # ) 
especially in the terminal region where the frequencies 
corresponding to joint movements at ordinary walking speed 
are included. On the other hand, the viscoelastic spectrum 
of the ACP-rich mixture (Fig. 2B, 75/25) shows a gel-like 
behaviour similar to that of ACP alone but with a decrease 
of both moduli especially at low frequency. In this case, 
G' and G" show a higher frequency-dependence. The changes 
at low frequency observed for both mixtures reflect the 
enormous differences between the moduli of the two 
components in the medium. 

As clearly shown in Fig. 3, at the frequency 
corresponding approximately to the movement of the joint in 
walking (0.72 rad/sec) (Kobayashi et al, Biorheology, 1994, 
supra) , and at T=25°C, G' and G" cross each other as 
function of the ACP content in the mixtures. In particular, 
a "transition" from liquid-like to solid-like behaviour 
occurring approximately in correspondence to 50% w/w of ACP 
content in the mixture is evident. 

Fig. 4 shows a comparison of the dynamic viscosity of 
ACP/HA formulations with ACP/HA ratios ranging from 100/0 
to 0/100. The improvement in the viscoelastic properties of 



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35 

the compositions with increasing content of ACP is clearly 
apparent . 

Example 23: 

PREPARATION AND T ESTING OF ACP /HA FORMULATIONS IN WHICH THE 
ACP COMPONENT IS AUTOCROSS- LINKED TO VARYING DEGREES . 

Autocross- linked carboxyl polysaccharides (ACP) 
synthesized from hyaluronic acid (HA) (640,000 Da) and used 
to prepare mixtures of ACP/HA included the following: 

ACP 20% 0.5% H 2 0 

ACP 10% 0.5% H 2 0 

ACP 5% 0.5% H 2 0 

The values 20, 10 and 5% refer to the nominal 
percentage of esterif ication, while 0.5% indicates the 
quantity of water added during synthesis. 

The formulations were prepared by mixing different 
quantities of ACP and HA (640,000 Da) in phosphate buffer 
(NaCl 0.15M and phosphate salts 0.002M) at pH=6.5. The 
mixtures all had final concentrations of 10 mg/ml and were 
prepared in a range of ACP/HA ratios of 100/0-0/100%. The 
suspensions were left to swell for 24 hours and then 
filtered on glass filters with a pore size of 100-40^m. 

Rheological measurements were made with a "Fluid 
Spectrometer RFS 8500" rheometer (Rheometrics) . Geometries 
were selected according to the viscosity of the solution: 
parallel plates (2 mm gap) for fairly viscous solutions and 
Couette (1 mm gap) for only slightly viscous . solutions . 

The studies were performed in dynamic frequency sweep 
(range = 100-0.05 rad/sec. strain = 10%, T = 25°C) . 

Formulations constituted by ACP/HA at 100/0 are 
generally characterized by G' being higher than G" for the 
whole range of frequencies considered (Fig. 5) . While 
maintaining the degree of cross- linking constant, the 
addition of greater amounts of HA in the mixture results in 
lower viscosity values, while G' and G " tend to get closer 
together (Fig. 6) . 



WO 97/49412 



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Particularly in the case of formulations of an ACP 
content of 50%, G' and G" may, according to the type of ACP 
being studied, overlap or cross over in correspondence to 
one or two different frequencies (Figs. 7, 8) . 

Figs. 9, 10, and 11 show the effect of the percentage 
of esterification on the viscosity of ACP/HA formulations 
with different ACP/HA ratios. 

It is possible to conclude, from the viscoelastic 
spectra, that for ACP/HA formulations with a high ACP 
content (eg 100/0) , the viscosity pattern is of the 
20%>10%>5% type (Fig. 9), while for 50/50 mixtures, the 
result is 20%>5%>10% (Fig. 10). Lastly, mixtures of ACP/HA 
4 0/60, starting from ACP 20% and ACP 5%, present only 
slight differences in viscosity (Fig. n) . 

If the objective is to reach high viscoelasticity 
values, ACP with a high degree of cross-linking (ACP 20%) 
alone (100/0) or mixed with small quantities of hyaluronic 
acid (e.g. 75/25) should be used. If, on the other hand, 
the desired viscoelasticity values are not high, (ACP/HA 
40/60, 30/70), then the percentage of cross-linking is a 
less decisive factor. 

The results described above indicate that the cross- 
linking of HA to form ACP yields a HA derivative with 
viscoelastic properties which are superior to those of 
unmodified HA. In addition, the Theological properties of 
ACP can be modulated by preparing compositions consisting 
of ACP/HA mixtures at varying weight/weight ratios. 

Even though ACP formulated in pharmaceutical 
excipients presents gel -like rheological profiles, 
interesting results have been obtained by mixing different 
quantities of ACP 10% 100/0 with synovial fluid from non- 
osteoarthritic horses. 

Figs. 12 and 13 show respectively the rheologioal 
profiles of equine synovial fluid and ACP 10% 100/0 
formulated in pharmaceutical excipients at a concentration 
of 10 mg/ml. 



WO 97/49412 



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Mixtures of ACP 10% with synovial fluid at final 
concentrations of 3.3 and 5 mg/ml of ACP gel (Figs. 14 and 
15) show not only a decisive increase in all viscoelastic 
parameters when compared to synovial fluid alone, but also 
a theoretically ideal rheological profile when compared to 
that of ACP alone. Indeed, G' and G" which run parallel in 
ACP-based formulations, cross over or tend to cross over in 
the presence of synovial fluid, according to the quantity 
of ACP added. 

These results indicate that, surprisingly, the 
addition of ACP to synovial fluid at a concentration which 
would be expected to exist following injection of ACP 
pharmaceutical compositions into articular joints, could 
modify the typical rheological profile of ACP's. 

Example 24 : 

COMPARISON OF T HE VISCOELASTIC PROPERTIES OF ACP ATVm 

EXISTING COMMERCI AL HA- BASED PRODUCTS FOR THE TREATMENT OF 

OSTEOAR THRITIS BY INTRA -ARTICULAR INJECTION 

The HA-based products currently on the market and used 

to treat arthropathies by intraarticular injection include: 
ART2 (Seikagaku, Japan) , an HA-based formulation 
with a molecular weight of between 600,000 and 
1,200,000 Da; 

SYNVISC (Biomatrix, U.S.A.), a two-component 
system composed of a mixture of two cross -linked 
HA derivatives, hylan fluid and hylan gel (US 
4, 713,448) . 

HYALGAN (Fidia) # an HA-based formulation with a 
molecular weight of between 500,000 and 730,000 
(EP 0138572 Bl) . 
The dynamic viscosity of ACP 20%, 0.5% water was 
compared to that of the above pharmaceutical products. The 
four formulations all had similar characteristics with 
respect to the final HA concentration and the 
pharmaceutical excipients present. The results of this 
comparison are shown in Fig. 16 and indicate that the ACP 



WO 97/49412 



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formulation has superior dynamic viscosity when compared to 
the three commercially available products. 

The development of ACP/HA pharmaceutical compositions 
was designed to provide compositions with improved 
viscoelastic properties, and consequently increased joint 
residence times, with respect to the presently available 
commercial HA-based products for the treatment of 
arthropathies. The variation of the ratio of ACP and HA 
contained in these compositions also permits the 
preparation of pharmaceutical compositions which have the 
optimal rheological properties for the treatment of 
arthropathies of various origins. 

The invention being thus described, it will be obvious 
that the same may be varied in many ways. Such variations 
are not to be regarded as a departure from the spirit and 
scope of the invention, and all such modifications as would 
be obvious to one skilled in the art are intended to be 
included within the scope of the following claims. 



WO 97/49412 



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39 
CLAIMS 

1 1. A pharmaceutical composition for the treatment of 

2 arthropathies comprising: 

3 (1) an autocross-linked hyaluronic acid, or a mixture 

4 of an autocross-linked hyaluronic acid and noncross- linked 

5 hyaluronic acid, and 

6 (2) a pharmaceutical^ acceptable excipient, diluent 

7 or carrier. 

1 2. The pharmaceutical composition of claim 1, 

2 wherein component (1) is an autocross-linked hyaluronic 

3 acid. 

1 3. The pharmaceutical composition of claim l, 

2 wherein component (l) is a mixture of an autocross-linked 

3 hyaluronic acid and a noncross-linked hyaluronic acid. 

1 4 . The pharmaceutical composition according to any 

2 one of claims 1-3 which further comprises at least one 

3 pharmacologically active substance. 

1 5. The pharmaceutical composition according to claim 

2 4, wherein the hyaluronic acid has a molecular weight of 

3 between 500,000 and 1,230,000 D. 

1 6. The pharmaceutical composition according to claim 

2 5, wherein said molecular weight is between 500,000 and 

3 730,000 D. 

1 7. The pharmaceutical composition according to claim 

2 4, wherein the pharmacologically active substance is an 

3 antibiotic. 

1 8. The pharmaceutical composition according to claim 

2 4, wherein the pharmacologically active substance is a 

3 steroidal antiinflammatory agent. 



WO 97/49412 



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40 

1 9. The pharmaceutical composition according to claim 

2 4, wherein the pharmacologically active substance is a 

3 non-steroidal antiinflammatory agent. 

1 10. The pharmaceutical composition according to claim 

2 4, wherein the pharmacologically active substance is an 

3 anaesthetic, an epitheliotrophic vitamin, a hormonal -type 

4 antiinflammatory/analgesic agent, a cytokine, a cytokine 

5 receptor, or a growth factor. 

1 11. The pharmaceutical composition according to claim 

2 4, wherein the pharmacologically active substance is 

3 salified hyaluronic acid. 

1 12. The pharmaceutical composition according to claim 

2 11, wherein the hyaluronic acid is salified with a silver, 

3 copper, zinc, calcium salt. 

1 13. The pharmaceutical composition according to claim 

2 3, wherein the relative proportions of the autocross-linked 

3 hyaluronic acid and the nonautocross- linked hyaluronic acid 

4 are about 95:05 to about 05:95. 

1 14. The pharmaceutical composition according to claim 

2 3, wherein the relative proportions of the autocross-linked 

3 hyaluronic acid and the nonautocross -linked hyaluronic acid 

4 are about 75:25 to about 25:75. 

1 15. A method of treatment which comprises administer - 

2 ing intraarticular^ to a patient affected by arthropathy 

3 an effective amount of a pharmaceutical composition of 

4 claim 1. 



1 

2 
3 



16. Use of a pharmaceutical composition comprised of 
an autocross-linked hyaluronic acid in the treatment of 
arthropathy. 



WO 97/49412 



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1 17. Use according to claim 16, wherein said compo- 

2 sition further comprises a noncross- linked hyaluronic acid. 



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INTERNATIONAL SEARCH REPORT 



Interm .tal Application No 

PCT/EP 97/03238 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 A61K31/715 



According to International Patent Classification (tPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched {classification system followed by classification symbols) 

IPC 6 A61K 



Documentation searched other than minimum documentation to the extent thai such documents are included m the Itelds searched 



Electronic data base consulted during the international search (name ol data base and. where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * Citation of document, with andicatton, where appropriate, of the relevant passages 



Relevant to datm No. 



WO 89 10941 A (FIDIA SPA) 16 November 1989 

cited in the application 

see page 38, line 10 - page 39, line 10 

see page 56 - page 70 

see examples 1-10 

see page 102 - page 116 

see examples 31-34,36-39 

EP 0 718 312 A (HERCULES INC) 26 June 1996 
see page 8, line 20 - line 36 



-/-- 



1-17 



1-17 



Further documents are listed in the continuation of box C. 



0 



Patent family members are listed in ennei. 



* Special categories ot cited documents : 

*A* document defining the general state of the art which is not 
considered to be of particular relevance 

"E* earlier document but published on or after the international 
fitng date 

"L* document which may throw doubts on priority claim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

*0* document referring to an oral disclosure, use. exhtoition or 
other means 

*P* document published prior to the international filing date but 
later than the priority date claimed 



*T later document published after the international filing date 
or priority dale and not in conflict with the application but 
chad to understand the pnnoipie or theory underlying the 
invention 

"X* document of particular relevance ; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y* document of particular relevance; the claimed invention 

cannot be considered to involve an mvefrtrve step when the 
dooument is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

*&* document member of the same patent family 



Date of the actual completion of the international search 



6 November 1997 



Date of mailing ot the international search report 



|9. tl 97 



Name and mailing address ot the ISA 

European Patent Office, P B 5818 Patentlaan 2 
NL - 2260 HV Rijawijk 
Tel. (+31*70) 340-2040. Tx 31 651 eponl. 
Foji:<+3i-70) 340-3016 



Authorized otttcer 



Seegert, K 



t-orrr PCT.W'tO 'second *h«*iv J July 199?) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Interhi. .al Application No 

. PCT/EP 97/03238 



C.( Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category ' 



Dilation of document, with indication, where appropriate, of the relevant pa&saqes 



Relevant to claim No 



ADAMS M.E. ET AL: "The role of 
viscosupplementation with hylan G-F 20 
(Synvisc) in the treatment of 
osteoarthritis of the knee: a Canadian 
multicenter trial comparing hylan G-F 20 
alone, hylan G-F 20 with non-steroidal 
anti-inf lanmatory drugs (NSAIDs) and 
NSAIDs alone" 

OSTEOARTHRITIS CARTILAGE (ENGLAND), 
vol. 3, no. 4, 1995, 
pages 213-225, XP002045971 
see abstract 



1-17 



1 



^orm PCT/1SA/210 (conttnuation o* tccond sti»«t) (July 1992} 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 

inlormation on patent family members 



Patent document 
cited in search report 



Publication 
date 



WO 8910941 A 



16-11-89 



Interna <al Application No 

PCT/EP 97/G3238 





Patent family 




Publication 




member(s) 




date 


AT 


115590 


T 


15-12-94 


AU 


631125 


B 


19-11-92 


AU 


3574789 


A 


29-11-89 


CA 


1339122 


A 


29-07-97 


0E 


68919900 


D 


26-01-95 


DE 


68919900 


T 


11-05-95 


DK 


10990 


A 


12-03-90 


EP 


G341745 


A 


15-11-89 


EP 


0614914 


A 


14-09-94 


ES 


2064378 


T 


01-02-95 


HU 


210926 


B 


28-09-95 


IL 


90274 


A 


12-09-96 


JP 


2504163 


T 


29-11-90 


NZ 


229100 


A 


28-08-95 


US 


5676964 


A 


14-10-97 



EP 0718312 A 


26-06-96 


AU 


4063495 A 


27-06-96 






CA 


2165890 A 


23-06-96 






CN 


1131675 A 


25-09-96 






JP 


8253504 A 


01-10-96 






PL 


312026 A 


24-06-96 



Form PCTflSA/210 (paienl lamtty anne«) {.Aity 1992) 



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