CA1291123C - Heavy metal salts of hyaluronic acid useful as antimicrobial agents - Google Patents

Abstract

HEAVY METAL SALTS OF HYALURONIC ACID
AND THEIR USE AS ANTIMICROBIAL AGENTS

ABSTRACT OF THE DISCLOSURE

Heavy metal salts of hyaluronic acid have been pre-pared. In particular, this invention is directed to silver, gold, cerium and tungsten salts of hyaluronic acid. These heavy metal salts of hyaluronic acid are useful as antimicrobial agents. Gold hyaluronate may also be used to treat arthritis.
This invention also concerns methods of making the silver salt of hyaluronic acid as well as compositions containing silver hyaluronate or gold hyaluronate.
The invention also concerns composition containing heavy metal salts having radioactively labelled hyalu-ronate moieties.

Description

HEAVY METAL SALTS OF HYALURONIC ACID
AND THEIR USE AS ANTIMICROBIAL AGENTS

BACKGROUN~ OF THE INVENTION

Throughout this application various publications are referenced by arabic numerals with parenthesesO Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are of interest, as they describe the state of art as known to those skilled therein as of the date of the invention described and claimed herein.

This invention concerns the heavy metal salts of hyaluronic acid.

Hyaluronic acid is present in various connective tissues of animals, such as skin and cartilage. Some organs are specifically rich in hyaluronic acid, such as the umbilical cord, synovial fluid, the vitreous humor and rooster combs. In addition, hyaluronic acid is produced by various microorganisms, such as streptococci Type A
and e . ~ ~

In skin and cartilage, the role of hyaluronic acid is to bind water and retain the tonicity and elasticity of ~ :

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the tissue. In joint fluids, the viscous hyaluronic acid solution serves as a lubricant to provide protec-tive environment to the cells. A aolution of ultrapure hyaluronic acid from rooster combs has been ln use for several years as a suppor~ive medium in ophthalmic 5 surgery, ~ee U.S~ Patent No. 4,141,973 of E.A. Balazs (1979). A similar preparation has been shown to be beneficial in the treatment of inflamed knee joints of race horses. Another use of hyaluronic acid results from it~ highly hydrophilic nature9 making it an ideal constituent of moisturization lotions ~or cosmetic use, U.S. Patent No. 4t303,676 of E. Balazs (1981~.

Hyaluronic acid has been isolated from the various b~ological sources, as described above, including mi-crobial broth. The isolation and characterization ofhyaluronic acid has been described by Meyer et al., J.
Biol. ChemO 107,629 (1934); J. Biol. Chem. 11~,689 (1936), and has recently been reviewed in Methods in Enzymol. 28, 73 (1972). The structure of hyaluronic acid was elucidated by Weissman et al., J. Am. Chem.
Soc. 76, 1753 (1954) and Meyer, Fed. Proc. 17, 1075 (1958). Other publications such as, U.S. Patent No.
4,141,973, February 27, 1979 by ~A. Balazs, concerned the production and purification of hyaluronic acid from the sources such as an~mal connective tissue.

Radioactively labelled hyaluronic acid and sodium salt thereof has been produced by growing streptococcus in fermentation broth containing radioact~vely labelled glucose. (3) Numerou~ silver co~p~unds having a wide range of uses are ~nown. These include silver acetate and silver chlorate which may be used as oxidizing ayents; silver . .

- 3 ~ 3 bromide and silver oxalate which are used in photogra-phy; silver difluoride for use in the flourination of hydrocarbons; silver chloride and silver cyanide ~or use in silver plating; and silver chromate (VI) and silver oxide which may be employed as catalysts, to name just a few.

The silver ion has also been shown to be an effective antimicrobial agent. It is not associated with signif~
icant side effects, is not an allergen, and is only rarely associated with the induction of resistant strains of bacteria. Many silver salts are useful as ~opical anti-infective~ or as antiseptics. These in-clude: silver flouride, silver iodide, silver lacta~e, mild silver protein and silver nitrate. Silver lacta~e and silver nitrate may also be employed as ~stringents and silver picrate and silver sulfadiazine may be used as antimicrobial or antibacterial agents.

It is believed that silver compounds produce their antimicrobial effects by ~he time-dependent release of silver ions and their effectiveness is directly related to the constant presence of the free ions in the tis-sues. The use of simple silver salts, such as silver nitrate, as an antibacterial agent has been limited by the requirement of frequent applications to achieve effective concen~rations of the silver ions ~10-20 ug/ml).

Although the use of silver sulfadiazine, a more com-plex silver compound, results in a more sustained re-lease of the silver metal ion~, the use of both silver nitrate and silver sulfadiazine i8 o~ten accompanied by adverse side-effects due to the anion part of the salts, namely nitrate and sulfadiazine. For example, : . .

_ ~ _ sulfadiazine hac such ide effects as leucopenia and immunosuppression activity. Other silver preparations, such as foils and silver nylon, have recently been offered for use, but their release of silver ion~ i8 limited and do not result in high enough concentra-tions. The use of salts is important because complexe~may not provide the steady release ions needed to ob-tain optimal concentrations.

Thus, the need for a silver compound providing 8U8-tained release of the silver metal ion, but having no adverse side effects is apparent.

Rheumatoid arthritis is characterized by severe inflam-mation of the joints which i8 followed by the appear-anoe of degraded hyaluronic acid in the joint fluid.In severe cases, anti-inflammatory agents such as cor-ticosteroids and gold salts, e.g. gold sodium thio-malate or gold sodium thiosulfate are administerea intra-articularly. However these agents are active for only a short duration, and there is a need to su~tain their action. Thus, the invention of an anti-inflamma-tory agent having sus~ained action would meet a long fel~ need and be a ~ignificant advance in the anti-inflammatory art.
The present invention i8 directed to heavy metal salt~
of hyaluronic acid, including silver hyaluronate, gold hyaluronate, cerium hyaluronate, and tungsten hyalu-ronate.
The invention is also directed to methodfi of inhibiting microbial growth utilizing these heavy met~ salts.

The present invention concerns methods o~ producing silver hyaluronate, compositions containing silver hyaluronate, and the treatment of wounds, burns and infections, especially soft-~issue ~nfections and gono-ccocal opth ~ malogical infections, wi h silver hyalu-ronate.

The invention further conoe rns a method for treating keratitis with silver hyaluronate and optionally in conjunction with antibiotics.
The invention further provides for incorporation of heavy meta} hyaluronate salts into deodorants and into cosmetic creams~ lotions and spray~.

The invention also concerns treating arthritis and joint inflammation with gold hyaluronate.

Finally, this invention is directed to compositions containing radioactively labelled heavy met~ sal~s e.g. 14C AgHA, which compositions may be used for diag-nostic purpo æ s.

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~QY~aEy~5~ INVE~ION

The invention concerns heavy metal alts of hyaluronic acid, in particular, silver hyaluronate, gold hyaluro-na~e cerium hyaluronate, and ~ungsten hyaluronate.

The invention also concern~ methods of making the 8il-ver hyaluronate by mixing aqueous sodium hyaluronate (NaHA) ~olution with a molar excess of aqueous silver nitrate (AgNO3) solution to form aqueous silver hyalu-ronate (Ag~A3 solution, precipitat~ng the silver hya-luronate from the solution and recovering the precipi-tated silver hya1uronate.

The~e heavy metal sal t8 of hyaluronic acid may be in-corporated into composi~ions, such as pharmaceuticalcompositions containing an effective amount o the heavy metal salt, e.g., silver hyaluronate or gold hyaluronate, and a ~harmaceutically acceptable carrier~

These heavy me~al ~alts of hyaluronic acid are useful as antimicrobial agent~. In particular, microbial grow~h may be inhibited by contacting the microbes with an effective amount of silver hyaluronate. Silver hyaluronate may also he used to inhibit microbial growth ~n infections, by topically applying an effec-tive amount of the silver hyaluronate to the infection.

The heavy metal sal~s are also useful when incorporated into deodorants and may also be used in co~metic creams, lotions and spray~

- 7 - ~ 3 Gold hyaluronate may be used to treat arthritis and joint inflammation by admini~tering an effective amount of the gold hyaluronate intra-articularly to the af-flicted subject.

This invention also concern~ compo~itions containing heavy met~ salts of radioactively labelled hyaluronic acid.

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~RI~F. ~ESC~I~N OF THE FIGURES

IG. 1: ~1Q~ Rel~se of Ag IQn~ fro~ A~a To test the Ag ion release properties of Ag~A, a Eample containing 1800 ug of Ag ion was dialyzed against dis-tilled water at room temperature. At points indicated in Figure 1, the dialysis bag containing Ag~A was transferred to 500 ml fresh distilled water. At each point a sample o~ the water wa~ removed and the concen-tration of Ag ions determined by atomic absorption.
The re~ults in Figure 1 show ~hat Ag ions are released in a slow manner, as 70% of the Ag ion concentration was retained in the dialysis bag after 48 h.

_ g _ DETAILED DESCRIPTION OF TIIE INVENTION

This invention concerns heavy metal salts of hyaluronic acid. More particularly, the salts encompaszed by this invention include the silver, gold, cerium, and tung-sten salts of hyaluronic acid.

The invention also concerns methods of making the sil-ver salt of hyaluronic acid by mixing aqueous sodium hyaluronate (NaHA) solution with a molar excess of aqueous silver nitrate (AgN03) solution to form aqueous silver hyaluronate (Ag~A) solution, precipitating the silver hyaluronate from the ~olution and recovering the precipitated silver hyaluronate. The silver hyaluro-nate precipitate may be recovered by separating the silver hyaluronate precipitate fram the aqueous silver hyaluronate solution, e.g., by centrifugation, wa~hing the separated precipitate with ethanol and drying the washed precipitate over nitrogen.

Preferably, the silver hyaluronate is prepared in the absence of light and the aqueous sodium hyaluronate and aqueous silver nitrate mixture is shaken for a suffi-cient period o~ time, ordinarily several hours, to form aqueou~ silver hyaluronate solution. The aqueous sil-ver hyaluronate solution is then treated to effectprecipitation of the silver hyaluronate which may be recovered by rinsing the silver hyaluronate precipitate with ethanol, drying the rinsed precipitate with nitro-gen and further drying the nitrogen-d~ied precipitate by high vacuum drying.

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The other heavy metal salt~ of hyaluronic acid may be prepared by methods analogous to the preparation of silver hyaluronate.

These heavy metal salts of hyaluronic acid may be com-bined with carriers to fonm composition~. ~he carrier or carriers may be any suitable carrier known to those of ordinary skill in the art. These compositions may be pharmaceutical compo~itions containing an effective amount of the heavy metal salt of hyaluronic acid, for example, silver hyaluronate, or gold hyaluronate and a pharmaceutically acceptable carrier.

The heavy metal salts of hyaluronic acid and composi-tions containing same are useful as antimicrobial agents. In particular, microbial growth may be inhib-ited by contactin~ the microbes with an effective amount of silver hyaluronate. Silver hyaluronate may also be used for treating burns, *ounds, soft tissue infections, for example, gonoccocal opthalmalogi Ql infec~ions or sepsis by topically applying an effec-tive amount of the silver hyaluronate to the site of the burn, wound, soft tissue infection or infection from which the sepsis stems. Silver hyaluronate compo-sitions are also used for treating keratitis infec-tions.
~' In another aspect of the invention, heavy metalbyaluronate salts are incorporated into d~odorant~, cosmetic creams, lotions and sprays.

Gold ~hyaluronate may also~be used for treating arthri-tis,~ rheumatoid ~arthriti~s, and joint inflammation in a ~ ~

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subject by administering an effective amount of the gold hyaluronate intra-articularly to the subject.

This invention also concerns composition~ containing the radioactively labelled heavy metal 8alt8 of hyalur-onic acid. These include ~alts wherein the hyaluronatemoiety is radioactively labelled, as well as salts wherein the heavy metal is in isotope form. Prefera~
bly, the radioactively labelled heavy metal salt is a heavy metal salt of radioac~ively labelled hyaluronic acid. More preferably, the hyaluronate mo~ety i8 ra-di~actively labelled with l4C. Preferably the heavy metal i5 silver. Most preferably ~he radioactively labelled heavy metal ~t is 14C radioactively la-belled silver hyaluronate. These radioactively la-belled heavy met~ salts and compositions containingthem may be used for diagnostic purposes.

As used throughout this application the term ~eavy metal" includes any metal in Period 5, 6, or 7 or the 4f (Lanthanide) or 5f (Actinide) series of the Periodic Table.

Hyaluronic acid (~A) is a major constitutent of connec-tive tissue, body flui~ds and skin, and hence is abso-lutely non-immunogenic. ; Due to their large size (M.W.
l.5xlO6 Daltonsj, HA salts form viscous solutions, and after injection into the tisfiue the polysaccharide diffuses extremely slowly from the injection site.
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Naturally occuring hyaluronic acid is a ~ ycosamino-glycan ~consisting of a ~linear polymer of molecular weight ~of 50,000-13,000,000 daltons. It is a polysac-. - 12 -charide made of a repeating units of glucuronic acid and N-acetyl-glucosamine, bound by alternating 1-3 and 1-4 bonds.

As employed throughout this application the term ~hya-luronic acidU includes substantially pure naturally occurring or synthetic hyaluronic acid, hyaluronic acid derivatives, hyaluronic acid cross-linked with itself, and hyaluronic acid cro~s linked with other substances such as collagen. See WO 86/00079 and WO
10 86/00912.

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Exp~RIMENTAL DETAIL~

~ E 1 PREP~RATIO~ OF SILVER HYA~RO~TE (~G~Al Silver hyaluronate has been prepared as follows:

Ao One volume of sodium hyaluronate solution in water (0.5% of purified bacterially-derived sodium hyalu-rona~e, M.W. 2.7x106 dalton~) was mixed with 1 volumeof 0.5 M AgN03 in water. Two vol umes of ethanol were then added, and ~he precipitated silver s~ t was oen-trifuged, washed with ethanol and dried over nitrogen.
The silver content of this hyaluronate salt wa~ found by atomic absorption to be 20~ w/w, i.e., corresponding to approximately 92% substitution of the carboxylic ions.

This preparation was found to effectively kill Staphy~
lococci, Pseudomona~, Candida Albicans and Candida Tropicans when applied as-a 0.1% solution (1:1, v/v) to cultures containing 106 microorganis~s per ml.

B. Preliminary studies have indicated that removal of traces of c~ oride ions is e~sential for obtaining a relatively stable, pure and clear A~HA product. This has~been achieved by~ethanolic precipitation of sodium HA ~rom a sodium nitrate soIution. The procedure for thè production of AgHA, starting with 1 gram of pure clinical grade sodium hyaluronate, i6 as follows:

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1. Pharmaceutical grade NaHA (1 gram) is dissolved in 200 ml of 0.5 M NaNO3.

2. 300 ml of ethanol is added, and the precipitated NaHA collected and washed thoroughly with 96~ ethanol.

The precipitate is redissolved in 200 ml of double distilled water.

4. 200 ml of 0.5 M AgNO3 is added in the dark and the mixture shaken for several hours. All subsequent oper-ations are performed in relative darkne~s.

5. Ethanol (600 ml) i8 added, and the precipitated AgHA is rinsed with 96% ethanol, dried with nitrogen, then followed by high vacuum drying.

Portions of the final material are taken and redis-solved in sterile, double-distilled water to give a working solution. A 1~ solution gives a viscous, clear liquid with a brownish color.

, E X~ 2 PROPERTIES OE' AGHA

Several batches of this material have been prepared.

:Analysis of the silver content, as determined by atomic absorption, was 82 grams per mole, which is 76~ of the theoretical stoichiometrical value, while the concen-tration of residual sodium ions was 0.5% of the theo-retical value.

Sta~ ty ~tudies:
Some preliminary observations of the stability of the 1% Ag~A solutions have been made. Storage in the dark under refrigeration had no effect on the appearance of the material: no changes in viscosity or darkening of the clear solution have developed during 3-4 monthsO
However, exposure of the solution to room temperature and light resulted in gradual browning, development of turbidity and a drop in viscosity within:a week to two weeks.

Molecul~ Weight:
The various~batches of AgHA were prepared from Na~A of M.W. of 3-3.5xl06. In order to measure the M.W. of AgHA, the material had to be converted to the Na form.
This was done by~ethanol precipitation from a concen-trated ~l M) solution of NaNO3. ~he M.W., a~ deduced from limiting viscosity measuremen~s with these precip-itates,~ was found to be:l.7-2xl06 daltons immediately after preparation of~ the AgHA solutions. On the shelf, at ro o t~emperature and l~ighting, the M.W. dropped to:

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- 16 ~ 3 1.27x106 dalton~ after 7 days, 0.96x106 daltons after 29 days.

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EXapPLE ~

SLQW RELEASE OF AG IONS FROM AG~A

Slow release of Ag ions is mandatory for a long-lasting 5 effect of the antimicrobial in the form of Ag complex.
To test the Ag ion release properties of Ag~A, a sample containing 1800 ug of Ag ion was dialyzed against dis-tilled water at room temperature. At points indicated in Figure 1, the dialy~is bag containing Ag~A was trans-10 ferred to 500 ml fresh distilled water. At each point asample of the water was removed and the concentration of Ag ions determined by atomic absorption. The res~ ts in Figure 1 sho~ that Ag ions are released in a ælow manner, as 70% of the Ag ion ooncentration was retained in the 15 dialysis bag after 48 h. The calculated dis~ociation constant of AgHA, assuming 100% dissocia~ion of free HA, is 6.7xlO 5M.
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EX~pL E 4 EFEI CACY pF AGH~

A. Mip~al Ir~hibitory ConceFItratio~. Minimal inhibitory 5 concentration was tested on eight species of bacteria isolated from hospitalized patients. The eight pecies are E. coli; Seratia; Pseudomonas; Proteus; R. pneu-moniae; Acinobacter; C. albicans; and M. morgani.

10 AgHA was dilu~ed in 0.25% Na~A to yield final concentra-tion in growth media of 100, 50, 40, 20, 10 ug/~l of silver ion equivalent~ The growth media wa~ L broth containing no NaCl and supplemented with NaHA to a final concentration of 0.25%. The latter NaHA concen~ration 15 was chosen as standard concentration for growth in liquid media because some bacteria did not grow as well at concentra~ion of 0.5% (Table 1). Concentrations of 0.5%
NaHA and above slowed the growth rate. This is attribut-ed to the high viscosity of the media which apparently 20 reduced oxygen solubility.

2 ml cultures were inoculated with approximately 105 cells and yrown on a rotary shaker at 37C for 72 h.
After 24, 48 and 72 hours, cultures were scored for 25 growth. A slight turbidity obser~ed in test tubes was scored as growth and marked in tables with the notation (plus) (Iakle 2).~

To compare the efficacy of AgHA to that of Ag-Sulfadia-30 zine, L ~broth media containing concentrations of silverions identical to tho~e u~ed ~or AgHA were prepared by serial dilutions. Growth and scoring methodology were .
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the same as described previou~ly (Table 3). For a con-trol experiment, L broth containing silver nitrate at identical concentrations of silver ions to those used for AgHA and Ag-Sulfadiazine were used (Table 4~. The re-sults of these experiments are presented in ~able 3 and 5 Table 4. ~he data obtained indicate that except for C, abicans and R. pneumoniae, all other clinical isolates were inhibited by a ~ncentration of 20 ug/ml silver ions. A concentration of about 40 ug/ml was needed to inhibit growth of C. Albicans and K. Pneumoniae. It is 10 clear that AgHA inhibits microbial growth.

B. Double Agar-Layer Test. Ag compounds are mo~t com-monly used as ointments. Ag ions have to be released and reach the target by diffusion. The methodology used to 15 test this aspect of AgHA was adopted from ~ettch et al.
(2) and modified by using petri plates. AgHA and Ag-Sulfadiazine were mixed with agar separately to yield a calculated concentration of 40 ug/ml silver ions. A
¦ layer of 2.2 mm silver containing agar was first poured 20 and after solidification a layer of L-agar of 2.2 or 2.0 mm was poured on top. ~he plates were aged for 24, 48, 72, and 96 h prior ~o use. Cultures of bacteria were grown overnight and diluted in L broth media to yield about 105-106 cells. 0.1 ml of diluted cultures were 25 applied to each plate to yield a final concentration of 104-105 cells per plate. No Ag ions were used in the control experiments when determining total viable cells per plate~ Table 5 and Table 6 show the results obtained for AgHA and Ay-Sulfadiazine. The results are shown in 30 fraction ~orm as the number of cells which grew on the silver ion plates (numerator) over the number of cells which grew on the control plates containing no silver .
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(denominator). The fraction is alzo expre~sed as a percentO In Table 5 the results correspond to the exper-iment where the upper layer oP L agar wa~ Z.0 mm. Except for C. albicans, more than 99% of the population of the different species were fully inhibited. The surviving 5 fraction was higher in the experiment shown in Table 6 than that ~hown in Table 5. This is attributed to the fac~ that the upper L-agar layer was 2.2 mm. Dettch found that each increase of 2 mm in height of the upper agar layer increa~ed ~urvival number tenfold. The in-10 crease of 0.2 mm in height of L-agar in our experiments is in agreement with the above finding, as the num~er of survivals did not exceed 2-3%. No ~ignificant dif~erence is observed between plates aged for 24-96 h, indicating that by 24 h the minimum concentration of silver ion~ has 15 reached the L-agar surface.

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TA~I,l~ 1 E~-ct of Hyaluronate on Bact~ l Groweh R4te~

Gen2ration Tim~ in Di~ferent Concontration~ of Hy~luronic Acid Sodiu~ S~lt ~lnut~) scsain 0 O.OS~ O.lO~ O.ZS~ 0.50 . . ~

15 E- coll 30 28 33 30 S0 P3eudo~ona~ 45 ~a:: ~3 ~ 60140, ~ ~ . -- -- : : :
Serat~a ~ 29~ ~ 30 ~ 31 3] ~ 30 :

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A~TIMICROBIAL ACT VITY OF SILVER HYALU~ONATE

Antimicrobial activity was tested against Staphylococcus 5 aureus, Escherichia coli, Pseudomona aeruginosa and Candida albicans.

The tests were performed using two different methods, a Minimum Inhibitory Concentration (MIC) test (1) where the 10 Ag-hyaluror.ate was added to a liquid growth medium and an Agar Cup Test (2) where the test substance was filled into small wells punched in agar plates.

The re ults indicates that a concentration o~ 1% of the 15 Ag-hyaluronate preparation inhibits the growth of the four test strains. In liquid medium P. aeruginosa was inhibited at a concentration of 0.1%.

The Ag-hyaluronate preparation which was used in the 20 tests contained 16 mg/ml of the silver salt.

~ imicrobiaI actlvity of Ag-hyaluronate:
Materials and Methods: Ag-hyaluronate.

25 Te~t strains: Escherichia coli ATCC 8739; Pseudomonas aeruginosa A~CC 9027; Staph~lococcus aureu~ ATCC 6538;
Candida albicans~ATCC 102 31.

m e microorganisms~ were grown on Trypticase Soy Broth 30 ~TSB) at 35-37C for 18-24 hours. Dilutions were made in physio10gical saline solution.
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~ 3 Te~t method 1. MIC-test:
~g-hyaluronate was added to TSB to a final concentration of 1.0%. Solution~ with Ag-hyaluronate concentration~ of 0.1% and 001% were made by further dilutions in TSB.
Test organism~ were added to the medium to a concentra-5 tion of approximately 103 org/ml. The te~t tubes werethen incubated at 35-37C and inspected for visible signs of growth during a period of one week. A turbid medium was recorded as growth. For Candida albicans the pres-ence of a white-colored bu~ton a~ the bottcm o~ the test 10 tube was recorded as growth.

Test method 2. Agar CuE~Test:
Overnight cultures of the test strain~ were diluted in ster$1e physiological saline~ me suspensions were 15 flushed over 14 cm diameter Trypticase Soy Agar plates and excess liquid was removed.

T~P plates were dried in a laminar air fl~w unit for 30 minutes~ 5 mm diameter wells were punched in the agar.
20 The test solutions were filled in the wells. The plates were incubated at 35-37C and were inspected after over-night incubat$on. Clear zones surrounding the wells indicated ant$microb$al activity. The d$ameter of the zones were measured.
The following solutions were tested:

Aqar Cup Test A:
Ag-hyaluronate (undiluted) Ag-hyaluronate d$1uted in distilled water 1:1 :

. :

Ag-hyaluronate diluted in horse serum (SBL) 1:1 0.01~ Thiomersal Reference Agar Cup Test B:
5 Ag hyal ur ona te 1 ~ in TSB
0.1 % ~u 0.01 %
0.001%
Thiomersal 0.01 %
10 " 0.00196 Resul t~ a re shown in Tabl es 7, ~ and 9:

.

:

30 ~
:. ~ : : :

`
:` ~ ~ : : :

1?~ 3 TABLE 7. MIC ~est Ag- hyal urona te con c .
Test organism 1% 0.196 0.01% 0.001% 0~6 .. .. . _ _ _ . .
5 Candi da - + + + +
Albicans - + + ~ +

E. Coli - + + + +
+ + +

P. Aerugino sa - - ~ + +
+ ~ +

~ . Aur e us - + + +
1 5 _ ~
... . . . .
+ Growth - No gr ow th Dupl icate æampleæ

:
:~
:
;

~:

TABLE 8. Agar Cup Test, A

I nh i bi ti on z one tmm) Test organism ~gHA AgHA/ AgHA/ 0.01%
undi- water 1:1 serum 1:1 T~iomer-1 ute d ~1 S. Aureus 15 13 12 29 C. Albicans 16 13 15 40 P. Aeruginosa 13 11 11 19 E. Coli 14 10 10 17 :: :
, ~ ~

~: :

~ : ; :: :

TA~LE 9. Agar Cup Test, B

Inhibition zone ~mm~

Test organism Ag~AAgE~A Ag8A Thiomer-1% 0.1%0.01% sal 0.01%

S. aureus 7 5 5 45 C.albican~9 5 5 22 P.aeruginosa 10 5 5 a2 E.coli 11 5 5 33 ~:
~ :

, ., . ~ ; : :
~ ~ :

~` ~

Dis~us~io~:
The results from the MIC-test in liquid medium indicates that a 1% solution of the test substance in Tryptic2fie Soy Broth inhibits the growth of S. aureus, C. albir~ ns and E~ coli. P. aeruginosa is inhibited at a concentra-5 tion of 0.1%, A 1~ solution contains 0.16 mg/ml of Ag-hyaluronate.

The tests with agar cup methodology confirms the results fram the tests in liquid medium~ though the lowest con~
10 centration for inhibition of P. aeruginosa wa~ 1% Ag-hyaluronate.

Nhen the Ag-hyaluronàte was mixed with serum before testing with the agar cup method no significant differ-ence could be measured as compared with Ag-hyaluronate mixed with dis$illed water.

A white-colored precipitate, probably AgCl, appears immediately when the Ag-hyaluronate comes in contact with 20 culture media or serum.

. . .

~P~E 6 The biosynthesis of HA by the bacteria involves the 5 utilization of exogeneous glucose. The sugar i~ used mainly as an energy source, while a small portion of it is converted into glucoronic acid and N-acetylglucos-amine. These are then incorporated into the HA molecule.
Thus, the incorporation of radioactivity can be achieved 10 by feeding carbon-14 labelled glucose into the fermenta-tion broth.

Various experiments were performed, firstly in shake flasks, then in a specially-made pH- and temperature-15 controlled vessel. It has been found that fermentationin flasks was not efficient resulting both in very low incorporation ratios and low molecular weights. A ~mini-fermentor" has been constructed, with a volume of 5 mL
of medium, and studies were performed in this system on 20 the rate of glucose incorporation and utilization. It has been found that at the optimal conditions for RA
production, about 5% of the exogeneous glucose i8 incor-porated into HA. The molecular weight of the ~A pro-duced in the mini-fermentor was about 2X106 daltons. The 25 æystem was then used for labelling the HA with 14C-glu-cose, and in one fermenta~ion about 20 mg of crude 14C_~A
wa~ obtained. This material was then purified. The yield of this procedure was ahout 50%, resulting in a final yield of 10 mg of HA of ~pecific activity of 1 uCi 30 of 14C per mg and a molecular weight of 2.2x106 daltonæ.

~ ~

The 14C radioactively labelled NaHA ~o purified may be converted into radioactively labelled AgHA following the procedures de~cribed in Example 1.

.

.

~ -E2~M PL IE 7 USE OF SILV~R HYALURONATE_A~AINST p~EUDOMON~S ~RUGI~OSA
INFECTION IN A RABBIT EYE MODEI,~

5 Introduction Reratitis produced by Pseu~s~nas aerugino&a i8 the most rapidly spreading and des~ructive bacterial disease with which the human oornea can be infected, as well as the 10 most disactrous (~aibson, 1972)~ The frequency of corne-al infections has greatly increased with the use of contact lens. Treatment of early-detected pseudomona~
in~ections with aminoglycosidic antibiotics such as gentamycin usually results in a good therapeutic re-15 sponse. Rowever, the increasing occurrence of antibiot-ic-resistant bacteria presents a problem, which becomes di astrous when such a situation is detected post-factum.
The use of an efficient wide range anti-bacterial agent, such as silver hyaluronate, might overcome this problem.
We used a rabbit model for the demonstration of the efficacy of silver hyaluronate for the treatment of an experimentally-induced pseudomonas keratitis (~yndiuk, 1981).
~QÇ~

Ract~ri~ -30 A pathogenic hospital i801ate of ~seudon~na~ ~erugi~
was used. It has been defined by the pseudomonas re~-erence laboratory at the Rambam ~edical Center, ~aifa, ~ ,.3 Israel a~ a polyagglu~inin serotype 6. Overnight cul-ture~ were grown in tryptic 80y broth without dex~rose at 37C with shaking. In the morning, an aliquot of this culture was diluted 20-fold in fresh broth which was further incubated unt~ a late logarithmic phase was 6 reached (2-3 h.; O.D.660 ~ 1.2). The concentration of bacteria was estimated fr~m the optical density at 660 nm (1 O.D. = 2 x 109 cells/ml). The cultures were diluted to the desired density with cold saline. The diluted suspensions were kept at 0 - 4C until use (1-2 h).
~nimal ~F~tme~:

New Zealand albino rabbits weighing 2-2.5 kg were used.
They were anesthetized by intramu cular injection of ketamine hydrochloride (35 mg/kg) and xylazine hydro-chloride (3 mg/kg). A 4 mm circular superficial cut wa~
applied on the cornea with a cork borer, ~hen 3 longitu-dinal parallel scratches were made inside the circle with a 21 gauge needle. The bacterial infection was infl~cted 20 by application of 2 drops (at a 10 min. interval) of the diluted bacterial suspension.

Preliminary studies were conducted in order to find the optimal concentration of bacteria needed for infection~
25 We found that suspensions of 8 x 107 bacteria per ml produc~d 90-100% infection within 30-40 h, whereas a two-fold reduction in bacterial number resul ted in lower and variable infection rates. Therefore, all subsequent experiments were performed with suspension~ of 8 x 107 bacteria per.ml.

Silver Hy~uronate Treatment:

Initial st~dies were performed with 0.5% w/v of silver hyaluronate in water prepared as de~cribed in Example l.
This solution is quite viscous, but its visco~ity does 5 not interfere with the drop-wise application into the eye. However, it was found that at this concentraton the treated eyes developed a marked irritation of the conjunctive, irrespective of the bacter~al infectionO
~ence, the 0.5~ ~gHA solution was diluted ~hree-fold with 10 0.53 Na~A aqueous solution (final Ag~A concentration 0.17%). A slight irritation respon~e was still apparent, but it wa~ transient and disappeared soon after cessation of treatment with the Ag~A preparation.

15 In all experiments, the control eyes were concurrently treated wi ~ a 0.5% ~odi~m hyaluronate solution.

Experimental d~sion:

20 In preliminary experiments, AgHA application was initi-ated 2, 4 and 8 hours after infection. An anti-barte-rial effect of the AgRA therapy was clearly evident with the use of the 4 and 2 h intervals. In the early experi-ments, hourly application o~ the Ag~A solution was con-25 tinued for 12 to 24 h only. This scheme resulted in asignificant protective effect for 24-48 h after infec-tion, but it did not prevent subsequent advancement of bacterial growth and de~erioration of the eye~ by 72 h.
Therefore, a treatment schedule was selected in which 30 hourly appli~ation of one drop of the 0.17% AgHA and 0.33% Na~A solution was used, starting 2 h after infec-tion and continuing for 40 h. Treatment was then contin-ued a~ 2 h intervals for additional 8-10 ho Two such experiments were performed using 12 or 16 rabbits. In each rabbit, the left eye was treated with the AgHA
preparation~ while the right eye received 0.5~ Na~A and served as a control.
~cori~ of the~es~ t~:

The severity of the infection was scored by: (1) mac-roscopic examination of the eye~; and (ii) coun~ing of 10 bacte~i~ in isolated cornea.

Visual evaluation was performed according to the fol-lowing scoring system:

- No reaction.
1 - Leucocyte infil~ration associated with the scratches only.
2 - Mo~rate infiltration at the scratches and around them, but not exceeding the 4 mm ring. 0 3 - Infiltration affecting about 30% of the corneal area.
4 - Inf il tration and turbidity of about 70% of th e corneal area and some cellular infiltration into the anterior chamber. 5 5 ~ Heavy infiltration of the tot~ area of the cornea and anterior chamber as well as develop-ment of a central corneal abscess.

The eyes were examined 24, 48 and 72 h after infection.
30 Concurrently, rabbits were sacrificed 48 and 72 h after infection and the cornea were removed and homogenized in saline. The homogenates were then serially diluted and ~ 3 - 4~

plated on McConkey agar plates for colony coun~ing.
Bacteria isolated from the cornea were identified as ~se.~dom,QD~s~aeru~i~Q~a by ~tandard biochemic~ tests and sensitivity to ant~biotics.

5 The scoring results were analyzed ~tatistically by the two tailed Wilcoxon rank ~um test.

Result~:

10 AgHA treatment was found to be highly effective in the prevention of P,s~do~ona~ keratiti~ in the rabbit eye.
Table 10 summarizes the results of two experiment~ per-formed with a 0.1796 AgHA preparation. As sh~wn, the intensive treatment with the 8il ver salt resulted in a 15 marked inhibition of bacterial grow~h in the treated, eyes. The scores of the control eyes were high as early as 42-45 h after infection (scoring index of 3.4 - 3.6) and increased during the nex~ days to ceach a ~ery ad-vanced stage (scoring index of 4.9) by 120 h. In con-20 trast, the mean scores of the treated eye~ were signifi-cantly lower than those of the control group throughout the experiment (Table 10), and they did not increase beyond 72 h (see Table 10, Experiment I). It should be emphasized that in both experiments, most of the scores 25 Of the AgH~ treated eyes did not exceed the value of 1.
the mean scores shown in Table 10 are somewhat higher than that because in each experiment there were one or two eyes that have escaped Ag~A protection.

30 The effectiveness of the Ag~A treatment was also appar-ent from the fact that no bacteria were found in i~o-lated cornea of treated eyes, whereas an abundant num-ber of pseudomonas microorganisms (105-106 bacteria per corneum; Table 10) were found in the control eyes 42-120 h after infection.

CQnU~1 usion:

AgRA was shown to have an effective therapeutic effect a~ainst the visco~ bacteria Pseudomonas aerugin~sa, in vivo. The use of such a preparation on its own or in conjunction wi~ the commonly-used antibiotics might 10 prove highly benefici~ in the topical treatment of bacteri~ keratitis.

~Q
Effect of AgHA T~eatment of Infected R~bblt ~ye~
on the Scoring Index and Bacterla Count TimeScoring Index No. of Bacteria/Corneum Exp. After (~e~n + SE~I~ (Ran~7e) No. Infection Control TreatedControl Treated Eye Eye Eye Eye 0 I 45 h3.62 + 1 11 1.44 + 0 97~*~ (a) (a) 72 h4.29 + 1.50 1.79 + 1.55~ ~b) (b) IN ~ 7)(N ~1 7) 120 h4.86 + 0.87 1.86 + 1.55~1.5x105 3 2.4x106 ~103 (N ~ 16) ~N ~ 16) (N ~ 4) ~N - 3 II 42h 3.41 + 1.700.84 i 0.65~ 5x105 - 4X106 ~103 ~N ~ 16) ~N - 16)(N - 4) (N = 5) 66 h4.22 + 1.64 1.50 1 1.20~r~ 3x104 - 5X106 <103 (N a 9)(N ~ 9)(N = 6)(N = 6) p 0.05: control vs. treated p 0.01 ~ P o.ool (a) samples lost ~b) note determined 9~

- ~3 -REFER~ S

1. Carr, H.S., T.J. Wlodkowski and ~.S. Rozen krantz, "Silver Sulfadiazine: In Vitro Anti-bacterial Activityn, Antimicrob. Agents and Chemoterhapy 4~ 585-587 (1973).

2. Dettch, E.A., A.A. Marino, ToE~ Gillespie and J.A. Albright, ~Silver-nylon: A New Antimicro-bial Agent ~, Antimicrob. Agents and Chemother-apy 23: 35~-359 (1982).

3. Ro~eman, S. et al, "8iosynthesis of Hyaluronic Acid ~y Group A Streptococcus~, Biol. Chem.
~Q~, 212-225 (1953).
~5 4. ~yndiuk, R.A., ~Experimental Pseudomonas Rera- :
titisn, Tr. Am. Ophth. Soc. 79: 541-624 (1981).

5. Laibson, P.R., ~Cornea and Scleran, Opthamol 88: 553-574 ~1 972) .

,~ ~

Claims (24)

1. A heavy metal salt of hyaluronic acid selected from the group consisting of silver, gold, cerium and tungsten.

2. A salt in accordance with claim 1, wherein the metal is silver.

3. A method of preparing the compound of claim 2 which comprises:

(a) admixing aqueous sodium hyaluronate solution with a molar excess of aqueous silver nitrate solution so as to form aqueous silver hyaluronate solution;

(b) treating the aqueous silver hyaluronate (AgHA) solution so formed to effect precipitation of the silver hyaluronate; and (c) recovering the precipitated silver hyaluronate.

4. The method in accordance with claim 3, wherein recovering the precipitated silver hyaluronate comprises separating the silver hyaluronate precipitate from the aqueous solution by contrifugation, washing the separated precipitate with ethanol and drying the washed precipitate over nitrogen.

5. A method in accordance with claim 3, wherein the silver hyaluronate is prepared in the absence of light.

6. A method in accordance with claim 5, wherein after admixing, the aqueous sodium hyaluronate and the aqueous silver nitrate mixture is shaken for a sufficient period of time to form aqueous silver hyaluronate.

7. A method in accordance with claim 1, wherein recovering the precipitated silver hyaluronate comprises rinsing the silver hyaluronate precipitate with ethanol, drying the rinsed precipitate with nitrogen and further drying the nitrogen dried precipitate by high vacuum drying.

8. A composition comprising the compound of claim 2 and a carrier.

9. A pharmaceutical composition comprising an effective amount of the compound of claim 2 and a pharmaceutically acceptable carrier.

10. A method of inhibiting microbial growth which comprises contacting microbes with an effective amount of the compound of claim 2.

11. The use of an effective topical amount of the compound of claim 2, for treating a burn victim.

12. The use of an effective topical amount of the compound of claim 2, for treating a wound victim.

13. The use of an effective topical amount of the compound of claim 2, for treating a subject having soft-tissue infection.

14. The use of an effective amount of the compound of claim 2, for treating a subject having gonococcal opthalmalogical infection.

15. The use of an effective amount of compound of claim 2, for treating a subject having sepsis.

16. A salt in accordance with claim 1, wherein the metal is gold.

17. A pharmaceutical composition comprising an effective amount of the compound of claim 16 and a pharmaceutically acceptable carrier.

18. A salt in accordance with claim 1, wherein the metal is cerium.

19. A salt in accordance with claim 1, wherein the metal is tungsten.

20. A composition comprising a heavy metal salt of claim 1 wherein is radioactively labeled.

21. A composition of claim 20, wherein the heavy metal salt of hyaluronic acid is silver hyaluronate.

22. A composition of claim 21, wherein the silver hyaluronate is radioactively labeled with 14C.

23. The use of an effective topical amount of a silver salt of hyaluronic acid for treating an infection in a subject having keratitis.

24. A use of claim 23, wherein an antibiotic is used in conjunction with the silver salt of hyaluronic acid.