HEAVY METAL SALTS OF HYALURONIC ACID USEFUL AS ANTIMICROBIAL AGENTS

Abstract: Heavy metal salts of hyaluronic acid have been prepared. 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 hyaluronate moieties.

Description: HEAVY METAL SALTS QF HYALURONIC ACID USEFUL AS ANTIMICROBIAL AGENTS This application is a continuation-in-part of Serial No. 840,419, filed March 14, 1986, the contents of which are hereby incorporated by reference into the present applicatilon. BACKGEOUND OF THE INVENTION Throughout this application various publications are referenced by arabic numerals with parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entire¬ ties are hereby incorporated by reference into this application in order to more fully 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 hyalu¬ ronic acid. Hyaluronic acid is present in various connective tis¬ sues 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 strepto¬ cocci Type A and C. In skin and cartilage, the role of hyaluronic acid is to bind water and retain the tonicity and elasticity of the tissue. In joint fluids, the viscous hyaluronic acid solution serves as a lubricant to provide protec¬ tive environment to the cells. 'A solution of ultrapure hyaluronic acid from rooster combs has been in use for several years as a supportive medium in ophthalmic surgery, see 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 its highly hydrophilic nature, making it an ideal constituent of moisturization lotions for cosmetic use, U.S. Patent No. 4,303,676 of E. Balazs (1981). Hyaluronic acid has been isolated from the various biological sources, as described above, including mi- crobial broth. The isolation and characterization of hyaluronic acid has been described by Meyer et al., J. Biol. Chem. H22,629 (1934); J. Biol. Chem. 111,689 (1936), and has recently been reviewed in Methods in Enzymol. 2ϋ, 73 (1972). The structure of hyaluronic acid was elucidated by Weissman et al., J. Am. Chem. Soc. 23., 1753 (1954) and Meyer, Fed. Proc. H, 1075 (1958). Other publications such as, U.S. Patent No. 4,141,973, February 27, 1979 by E.A. Balazs, concerned the production and purification of hyaluronic acid from the sources such as animal connective tissue. Radioactively labelled hyaluronic acid and sodium salt thereof has been produced by growing streptococcus in fermentation broth containing radioactively labelled glucose. (3) Numerous silver compounds having a wide range of uses are known. These include silver acetate and silver chlorate which may be used as oxidizing agents; silver bromide and silver oxalate which are used in photogra¬ phy; silver difluor ide for use in the flourination of hydrocarbons ; silver chloride and silver cyanide for 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 al so 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 sal ts are useful as topical an i-infectives or as antiseptics. These in¬ clude : silver flour ide , silver iodide , silver lactate, mild silver protein and silver nitrate. Silver lactate and silver nitrate may al so be employed as astringents and silver picrate and silver sulfadiazine may be used as antimicrobial or antibacterial agents. • It is believed that silver compounds produce thei r antimicrobial effect s by the time-dependent rel ease 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 concentrations of the silver ions (10-20 ug/ l) . Although the use of silver sul fadiaz ine, a more com- plex silver compound, results in a more sustained re¬ lease of the silver metal ions, the use of both silver nitrate and silver sulfadiazine is often accompanied by adverse side-effects due to the anion part of the salts, namely nitrate and sulfadiazine. For example, sulfadiazine has such side effects as leucopenia and immuno suppression activity. Other silver preparations, such as foils and silver nylon, have recently been offered for use, but their release of silver ions is l imited and do not resul t in high enough concentra- tions. The use of sal ts is important because complexes may not provide the steady release ions needed to ob¬ tain optimal concentrations. Thus, the need for a silver compound providing sus- 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 j oints which is followed by the appear- ance of degraded hyaluronic acid in the joint fluid. In severe case s, a nti- inflammatory agents such as cor- ticosteroids and gol d sal ts, e.g. gold sodium thio- malate or gold sodium thiogulfate are administered intra-articularly. However these agents are active for only a short duration, and there is a need to sustain their action. Thus , the invention of an anti- inflamma¬ tory agent having sustained action would meet a long felt need and be a significant advance in the anti- inflammatory art. The present invention is directed to heavy metal sal ts of hyaluronic acid, including silver hyaluronate, gold hyaluronate, cerium hyaluronate, and tungsten hyalu¬ ronate . The invention is al so directed to methods of inhibiting icrobial growth util izing these heavy metal salts. The present invention concerns methods of producing silver hyaluronate, compositions containing silver hyal uronate, and the treatment ' of wounds, burns and infections, especial ly sof t-tissue infections and gono- ccocal opthalmalogical infections, with silver hyalu- r ona te . The invention further concerns a method for treating kerati tis with silver hyaluronate and optionally in conjunction with antibiotics. The invention further provides for incorporation of heavy metal hyaluronate salts into deodorants and into cosmetic creams, lotions and sprays . The invention also, concerns treating arthritis and joint inflammation with gold hyaluronate. Finally, thi.s invention is directed to compositions containing radioactively labelled heavy metal salts e. g. "^C AgHA, which compositions may be used for diag¬ nostic purposes . SUMMARY OF THE INVENTION The invention concerns heavy metal sal ts of hyal uronic acid, in particular, silver hyaluronate, gold hyal uro¬ nate cerium hyaluronate, and tungsten hyaluronate. The invention also concerns methods of making the sil¬ ver hyaluronate by mixing aqueous sodium hyal uronate (NaHA) solution with a molar excess of aqueous silver nitrate (AgNO^) sol ution to form aqueous silver hyalu- ronate (AgHA) solution, precipitating the silver hya¬ luronate from the sol ution and recovering the precipi¬ tated silver hyaluronate. These heavy metal sal ts of hyaluronic acid may be in- corporated into compositions, such as pharmaceutical compositions containing an effective amount of the heavy metal sal t, e. g. , silver hyaluronate or gol d hyaluronate, and a pharmaceutically acceptable ca rrier. These heavy metal salts of hyal uronic acid are useful as antimicrobial agents. In particul ar, microbial growth may be inhibited by contacting the microbes with an effective amount of silver hyaluronate. Silver hyaluronate may also be used to inhibit microbial growth in infections, by topically applying an effec¬ tive amount of the silver hyaluronate to the infection. The heavy metal sal ts are also useful when incorporated into deodorants and may also be used in cosmetic creams, lotions and sprays. Gold hyaluronate may be used to treat arthritis and joint inflammation by administering an effective amount of the gold hyaluronate intra-articularly to the af¬ flicted subject. This invention also concerns compositions containing heavy metal salts of radioactively labelled hyaluronic acid. BRIEF DESCRI PTION OF THE FIGURES FIG. 1 : Sloy Release of Ag long from AgHA To test the Ag ion rel ease properties of AgHA, a sample containing 1800 ug of Ag ion was di alyzed against dis¬ tilled water at room temperature. At points indicated in Figure 1, the dialysis bag containing AgHA was transferred to 500 ml fresh distilled water. At each point a sample of the water was removed and the concen- tration of Ag ions determined by atomic absorption. The results in Figur e 1 show that Ag ions are released in a slow manner, as 70% of the Ag ion concentration was retained in the di alysis bag after 48 h. DETAILED DESCRI PTION OF THE INVENTION This invention concerns heavy metal salts of hyal uronic acid. More particul arly, the sal ts encompassed by this invention include the silver, gold, cerium, and tung- sten sal ts of hyaluronic acid. The invention al so 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 (AgN0 3 ) solution to form aqueous silver hyaluronate (AgHA) solution, precipitating the silver hyaluronate f rom the solution and recovering the precipitated silver hyaluronate. The silver hyaluro¬ nate precipitate may be recovered by separating the silver hyaluronate precipitate from the aqueous silver hyaluronate sol ution, e. g. , by centrifugation, washing the separated precipitate with ethanol and drying the washed precipitate over nitrogen. Preferably, the silver hyal uronate is prepared in the absence of l ight and the aqueous sodium hyaluronate and aqueous silver nitrate mixture is shaken for a suff i¬ cient period of time, ordinarily several hours, to form- aqueous silver hyal uronate solution. The aqueous sil- ver hyaluronate sol ution is then treated to effect precipitation of the silver hyaluronate which may be recovered by rinsing the silver hyaluronate precipitate with ethanol , drying the rinsed precipitate wi th nitro¬ gen and further drying the nitrogen-dried precipitate by high vacuum drying. The other heavy metal salts of hyaluronic acid may be prepared by methods analogous to the preparation of silver hyaluronate. These heavy metal sal ts of hyaluronic acid may be com- bined with carriers to form compositions. The carrier or carriers may be any suitable carrier known to those of ordinary skill in the ar t. These compositions may be pharmaceutical compositions 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 sal ts of hyaluronic acid and composi¬ tions containing same are useful as antimicrobial agents. In particular, microbial growth may be inhib¬ ited by contacting the microbes with an effective amount of silver hyaluronate. Silver hyaluronate may al so be used for treating burns, wounds, soft tissue infections, for example, gonoccocal opthalmalogical infections 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 f rom which the sepsis stems. Silver hyaluronate compo¬ sitions are also used for treating keratitis infec- tions. In another aspect of the invention, heavy metal hyaluronate salts are incorporated into deodorants, cosmetic creams, lotions and sprays. Gold hyaluronate may also be used for treating arthri¬ tis, rheumatoid arthritis, and joint inflammation in a subj ect by administering an effective amount of the gold hyaluronate intra-articul rly to the subj ect . This invention also concerns compositions containing the radioactively labelled heavy metal sal ts of hyalur- onic acid. These include salts wherein the hyaluronate moiety is radioactively labelled, as well as sal ts wherein the heavy metal is in isotope form. Prefera¬ bly, the radioactively labelled heavy metal sal t is a heavy metal salt of radioactively labelled hyal uronic acid. More preferably, the hyaluronate moiety is ra¬ dioactively labelled with 14 C. Preferably the heavy metal is silver. Most preferably the radioactively labelled heavy metal salt is 14 C radioactively la¬ belled silver hyaluronate. These radioactively la- belled heavy metal salts and compositions containing them may be used for diagnostic purposes. As used throughout this application the term "heavy metal " includes any metal in Period 5 , 6 , or 7 or the 4f (Lanthanide) or 5f (Actinide ) series of the Periodic Tabl e. Hyaluronic acid (HA) is a maj or constitutent of connec¬ tive tissue, body fluids and skin, and hence is abso- lutely non-immunogenic. Due to thei r large size (M . . 1.5xl0 6 Daltons) , HA salts form viscous solutions, and after inj ection into the tissues the polysaccharide diffuses extremely slowly from the injection site. Naturally occuring hyaluronic acid is a glycosamino- glycan consisting of a linear polymer of molecular weight of 50 ,000-13 ,000 ,000 dal tons. It is a polysac- charide made of a repeating units of glucuronic acid and N-acetyl-glucosamine , bound by al ternating 1-3 and 1- 4 bonds. As employed throughout this appl ication the term "hya- l uronic acid" includes substantially pure naturally occurring or synthetic hyaluronic acid, hyaluronic acid derivatives, hyal uronic acid cross-linked with itself, and hyaluronic acid cross linked with other substances such as collagen. See WO 86/00079 and WO 86/00912. EXPERIMENTAL DETAILS EXAMPLE 1 PREPARATION OF SILVER HYALURONATE (AGHA) Silver hyaluronate has been prepared as follows: A. One volume of sodium hyaluronate solution in water (0.5% of purified bacterial ly-de rived sodium hyalu- ronate, M.W. 2.7xl0 6 daltons) was mixed with 1 volume of 0.5 M AgN0 3 i n water. Two volumes of ethanol were then added, and the precipitated silver salt was cen- trifuged, washed with ethanol and dried over nitrogen. The silver content of this hyaluronate salt was 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, Pseudomonas, Candida Albicans and Candida Tropicans when applied as a 0.1% solution (1:1, v/v) to cultures containing 10 6 microorganisms per ml. B. Preliminary studies have indicated that removal of traces of chloride ions is essential for obtaining a relatively stable, pure and clear AgHA product;. This has been achieved by ethanolic precipitation of sodium HA from a sodium nitrate solution. The procedure for the production of AgHA, starting with 1 gram of pure clinical grade sodium hyaluronate, is as follows: 1. Pharmaceutical grade NaHA (1 gram) is dissolved in 200 ml of 0.5 M NaN0 3 . 2. 300 ml of ethanol is added, and the precipitated NaHA collected and washed thoroughly with 96% ethanol. 3. The precipitate is redissolved in 200 ml of double distilled wate r. 4. 200 ml of 0 .5 M AgN0 3 i s added in the dark and the mixture shaken for several hour s. All subsequent ope r¬ ations are performed in relative darkness. 5. Ethanol (600 ml) is 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 steril e, doubl e-distilled water to give a working solution. A 1 % sol ution gives a viscous, clear l iquid wi th a brownish color . EXAMINE 2 PROPERTIES OF AQ8A Several batches of this material have been prepared. Silver Content: 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. Stability S udies: Sane preliminary observations of the stability of the 1% AgHA 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 months. 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. Molecular Weight: The various batches of AgHA were prepared from NaHA of M.W. of 3-3.5x10 6* . In order to measure the M.W. of AgHA, the material had to be converted to the Na form. This was done by ethanol preci itation from a concen¬ trated (1 M) solution of NaN0 3 . The M.W. , as deduced from limiting viscosity measurements with these precip¬ itates, was found to be 1.7-2xl0 6 daltons immediately after preparation of the AgHA solutions. On the shelf, at room temperature and lighting, the M.W. dropped to: 1.27xl0 6 daltons after 7 days; 0.96xl0 6 daltons after 29 days, EXAMPLE 3 SLOW RELEASE OF AG IONS FROM AGHA Slow release of Ag ions is mandatory for a long-lasting effect of the antimicrobial in the form of Ag complex. To test the Ag ion release properties of AgHA, a sample 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 AgHA was trans- ferred to 500 ml fresh distilled water. At each point a sample of the water was removed and the concentration of Ag ions determined by atomic absorption. The results in Figure 1 show that Ag ions are released in a slow manner, as 70% of the Ag ion concentration was retained in the dialysis bag after 48 h. The calculated dissociation constant of AgHA, assuming 100% dissociation of free HA, is 6.7xlO~ 5 M. EXAMP E 4 EFFICACY OF AGHA A. Minimal Inhibitory Concentration. Minimal inhibitory concentration was tested on eight species of bacteria isolated from hospitalized patients. The eight species are E. coli; Seratia; Pseudomonas; Proteus; K. pneu- moniae; Acinobacter; C. albicans; and M. morgani. AgHA was diluted in 0.25% NaHA to yield final concentra¬ tion in growth media of 100, 50, 40, 20, 10 ug/ml of silver ion equivalent. The growth media was L broth containing no NaCl and supplemented with NaHA to a final concentration of 0.25%. The latter NaHA concentration was chosen as standard concentration for growth in liquid media because some bacteria did not grow as well at concentration 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 reduced oxygen solubility. 2 ml cultures were inoculated with approximately 10 5 cells and grown on a rotary shaker at 37°C for 72 In After 24, 48 and 72 hours, cultures were scored for growth. A slight turbidity observed in test tubes was scored as growth and marked in tables with the notation + (plus) (Table 2) . To compare the efficacy of AgHA to that of Ag-Sulfadia- zine, L broth media containing concentrations of silver ions identical to those used for AgHA were prepared by serial dilutions. Growth and scoring methodology were the same as described previously (Tabl e 3) . For a con¬ trol experiment, L broth containing silver nitrate at identical concentra tions of silver ions to those used for AgHA and Ag-Sul fadiaz ine were used (Table 4) . The re¬ sults of these experiments are presented in Tabl e 3 and Table 4 . The data obtained indicate that except for C. abicans and K. pneu oniae, all other clinical isolates were inhibi ted by a concentration 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 clear that AgHA inhibits microbial growth. B. Doubl e Aoar-Laγer Test. Ag compounds are most com¬ monly used as ointments. Ag ions have to be released and reach the target by diffusion. The methodology used to test this aspect of AgHA was adopted f rom Dettch et al. (2) and modified by using petri plates. AgHA and Ag- Sulfadiazine were mixed with agar separately to yield a calcul ated concentration of 40 ug/ml silver ions. A layer of 2 .2 mm silver containing agar was fi rst poured and after sol idif ication a layer of L-agar of 2 .2 or 2 .0 mm was poured on top. The plates were aged for 24 , 48 , 72, and 96 h prior to use . Cul tures of bacteria were grown overnight and dil uted in L broth media to yield about 10 5 -10 6 cells. 0 .1 ml of diluted cul tures were applied to each plate to yield a final concentration of 10 4 -lθ5 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 resul ts obtained for AgHA and Ag-Sulfadiazine. The results are shown in f raction form as the number of cells which grew on the silver ion plates (numerator) over the number of cell s which grew on the control plates containing no silver (denominator) . The f raction is also expre ssed as a percent. In Table 5 the resul ts correspond to the exper¬ iment where the upper layer of L -agar was 2.0 mm. Except for C. albicans, mor e than 99% of the popul ation of the different species were fully inhibited. The surviving f raction was higher in the experiment shown in Tabl e 6 than that shown in Tabl e 5. This is attributed to the fact 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 increased survival number tenfold. The in- crease of 0.2 mm in height of L-agar in our experiments is in agreement with the above finding, as the number of survivals did not exceed 2-3%. No significant difference is observed between plates aged for 24-96 h, indicating that by 24 h the minimum concentration of silver ions has reached the L-agar surface . - 21 - TAflLg 1 Effect of Hyaluronate on Bacterial Growth Rates Generation Time in Different Concentrations of Hyaluronic Acid Sodiuα Salt (minutes) Strain 0.051 0.10% 0.251 0.50% E. coli 30 28 33 30 50 Pseudomonas 45 48 48 60 140 Seratia 29 30 31 33 30 TABLE 2 Minimal Inhibitory Concentration in Liquid Medium Ag-HA Cone, of Ag* 100 ug/mlΛg+ 50 ug/ml 40 ug/ml Z0 ug/ml 10 ug/ml Hour Hour Hour Hour Hour Strain 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 E. Coli ♦ ♦ Seratia ♦ ♦ ♦ ♦ Paeudomonaa ♦ + Proteua ♦ ♦ + ♦ Acinobacter - ♦. * ♦ K. Pneumonia* — — — — — — 4 ♦ + ♦ ♦ ♦ ♦ M. Morgan! ♦ ♦ C. Tropicaila ■ ♦ + C. λlbicana TABLE 3 Minimal Inhibitory Concentration in Liquid Medium Ag-Sulfadiazine Cone, of Ag* 100 ug/mlAg* 50 ug/ml 40 ug/ml 20 ug/ml 10 ug/ml Hour Hour Hour Hour Hour Strain 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 E. Coli — — — — — — — .._ ... — — — — ♦ 4 ♦ Seratla — — — — — — — — — — — — 4 4 Proteua 4 4 4 Acinobacter — 4 4 K. Pneumoniae ♦ 4 . 4 4 M. Morgan! C. Tropicalla C. Albicanβ 4 4 4 4 TABLE 4 Minimal Inhibitory Concentration in Liquid Medium ΛgNO j - Control Cone.of Ag* 100 ug/mlAg4 50 ug/ml 40 ug/ml 20 ug/ml 10 ug/ml Hour Hour Hour Hour Hour Strain 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 E. Coli 4 4 Seratia 4 4 Pseudomonas 4 4 Proteus 4 4 Aci obacter 4 4 K. Pneumoniae 4 I. M. Morgan! 4 4 C. Tropicalia 4 4 C. Albicans 4 4 4 4 Cultures grown In liquid LB (no NaCl). Mlth concentrations of Ag ions indicated. TABLE 5 Efficacy of AgHA and Ag Sulfadiazine as Double Agar Layer (Thickness of upper layer - 2.0 mm) sFraTS Ag Hyaluronic Acid 140 μ,q/ml Λg4, A ~ ^" I TaJla ' zl Se ~ IWμcf/ X Λgt ) 21 hour II hour 73 hour 96 hour _T "" Rδu7 48 " libϋr 7? hour TRT Tiour " __ __ g __ ^ ..__ __ _._ _ .. R , coll • 0 • 0 ■ 0 0 ■ 0 * 0 "0 * 0 1.5-10* 1.5-105 l.flO 5 1.2«10* 1.5-105 I..-10* l.β-10* l.2-lθ5 S δ δ π ό ~~ "" o ό 6 " Seratla — - - 0 — - 0 — - 0 0.041 --- - 0 --- t p --- - 0 --- - 0 1.4-10* 1.9-10 5 2.0-10 5 l.0-10-> 1.4-10* 1.9-10» 2.0-I0 5 1.03-10* o s s o o o o o Pseudomoπas — - - 0 0 0 0 --- « 0 --- - 0 --- » 0 0 0.6-10* 1.8-10* . 7.J-10* 0.5-10* 0.6-10* I.8-10* 7.1-10* 0.5-10* 0 Proteus - 0 --—0.0-2 - 0 -- . o - 0 - 0 - 0 --- - 0 3.2 •10* 2.0'1-S 2.2 •lθ5 ,0-lθ5 3.2 '10 2.0 to * * 2.2 •10 s 2.0-IO* i 0 "" 600 0 0 0 . 0 0 0 K. Pneumoniae 0 -0. J ■ 0 0 0 0 * 0 * 0 1.1-10* 2.1-10 5 1.3-105 7.6-10* I.1-10* 2.1-105 I. J•10 7.6-10' ~ - ■ o ~""~ I J o δ ~ ~ "" o '"" o oo Λclrtobactcr 0 -0.004 -0.001 0 0 ■ 0 * 0 0. r » 2.7*10* 2.7-105 2.7-I0 5 1.0-lθ5 2.7-10* 2.7-I0 5 2.7-10^ 1.0-I0 r » ____ ΠΓQ δ JOU TJT T7β i iffβ C. Albicana 0.9 3 0 • 6 -12.2 7.4 -O.β 6 10* 0.5-10* 2.0-10* 6.5-10 3 10 3 0.5*10* 2.0-10* 6.5-10* 0 108 0 0 0 0 0 500 M. Morganl 0 - — "0.06 — - « 0 ■ 0 » 0 * 0 — — « 0 " 0.2 3.5-10* 1.7-10 5 2.2-10* 2.5-10* 3.5-10* l.7-lo5 2.2-10* 2.5-10* ' Reaultβ * are given In Percent TABLE 6 Eff icacy of AgHA and Ag Sulfadiazine as Double Agar Layer ( Thickness of upper layer - 2. 2 mm ) Strain Ag Hyaluronic Acid ( 40 μq/ml Ag+ ) Ag Sul fadiazine ( 0 ag/ml Ag ) 24 hour 48 hour 73 hour 96 hour 24 hour 48 hour 72 hour 96 houF __. 0 0 0 E. Coli — - 0 — * 0 = 0 = 0 — - = o = 0 — = o 1.5-10* 3.0-10* 2.6-10* 2.3-10* 1.5' 10 6 3.0 - 10* 2.6 -10* 2. 3 - 10* 0 26 160 2000 0 95 2000 5000 Seratia .__ « o =- 0.2 =0.07 --- =1.2 —- = 0 -— =0.03 --- = 1 --- = 3 1.4-10* 3.0-105 2.3-10* 1.6-10* 1.4-10* 3.0-10* 2.3-10* 1.6-10* ___ ^_ _ 60 253 576 260 600 Pseudomonas ■ at 11 — — — == 11.3 = 1.6 * 3.1 = 0 _ 46 0.6-10* 1.5-10* 3.0-10 3 1.0-10 3 0.6-10* 1.5-10* 3.0-10 3 1.3-10 3 0 24 2000 0 0 4000 2000 2000 K. Pneumoniae — - - 0 -— -0.02 — - 2.4 — - » 0 — «= o — » ___ »2.4 --- = 8.9 1 . 1 - 10* 10* 8. 2 -10* 4.0-10* 1 . 1 - 10* 10* 8.2-10* 2.3-10* ~ ~5 0 32 0 I " 12 2000 2000 " Acinobacter _.__ « o —- =0.01 --- * 0.1 — = 0 -—=0.004 -— = 0.2 --- = 6 --- = 89 2.7-10* 6.5-10* 3.3-10* 2.0-10* 2.7-10* 6.5-10 3 3.3-10* 2.3-10 3 152 600 300 560 130 144 900 840 C. Albicans -- =14.2 = 40 -— - 21 — =22.4 — = 13 30 = 64 -« = 33 10 3 1.5-10 3 1.4-10 3 2.5-10 3 10 3 1.5-10 3 1.4-10 3 2.5-10 3 0 0 0 0 3 600 960 2000 M. Morgani « 0 » 0 0 = 0 =0.01 0.2 =1.8 = 4 3.5-10* 2.4-105 5.2*104 4.6*10* 3.5-10* 2.4-lθ5 5.2-10* 4.6-10* Results are given in Percent EXAMPLE 5 ANTIMICROBIAL ACTIVITY OF SILVER HYALURONATE Antimicrobial activity was tested against Staphy loco ecus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The tests were performed using two different methods, a Minimum Inhibitory Concentration (MIC) test (1) where the Ag-hyaluronate 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 results indicates that a concentration of 1% of the 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 tests contained 16 mg/ml of the silver salt. Antimicrobial Activity of Aσ-hyaluronate; Materials and Methods: Ag-hyaluronate. Test strains: Escherichia coli ATCC 8739; Pseudomonas aeruginosa ATCC 9027; Staphylococcus aureus ATCC 6538; Candida albicans ATCC 102 31. The microorganisms were grown on Trypticase Soy Broth (TSB) at 35-37°C for 18-24 hours. Dilutions were made in physiological saline solution. Test method 1. MIC-test: Ag-hyaluronate was added to TSB to a final concentration of 1.0%. Solutions with Ag-hyaluronate concentrations of 0.1% and 001% were made by further dilutions in TSB. Test organisms were added to the medium to a concentra- tion of approximately 10^ org/ml. The test tubes were then incubated at 35-37°C 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 button at the bottom of the test tube was recorded as growth. Test method 2. Acar Cup Test: Overnight cultures of the test strains were diluted in sterile physiological saline. The suspensions were flushed over 14 cm diameter Trypticase Soy Agar plates and excess liquid was removed. Th * e plates were dried in a laminar air flow unit for 30 minutes. 5 mm diameter wells were punched in the agar. The test solutions were filled in the wells. The plates were incubated at 35-37°C and were inspected after over¬ night incubation. Clear zones surrounding the wells indicated antimicrobial activity. The diameter of the zones were measured. The following solutions were tested Aαar Cup Test A: Ag-hyaluronate (undiluted) Ag-hyaluronate diluted in distilled water 1:1 Ag-hyaluronate diluted in horse serum (SBL) 1:1 0.01% Thiomersal Reference Agar Cup T<?st B: Ag hyaluronate 1 % iin TSB _ 0.1 % _ 0.01 % _ 0.001% Thiomersal 0.01 % ■ 0.001% Results are shown in Tables 7, 8 and 9 - 30 - TABLE 7. MIC Test Ag-hyaluronate. cone. Test organism 1% 0.1% 0.01% 0.001% 0% Candida + + + + Albicans + + + + E. Coli + + + + + + P. Aeruginosa + + + + + + S. Aureus + + + + + + + + Growth No growth Duplicate samples TABLE 8. Agar Cup Test, A Inhibition zone (mm) Test organism AgHA AgHA/ AgHA/ 0.01% undi- water 1:1 serum 1:1 Thiomer- luted sal s. Aureus 15 13 12 29 C. Albicans 16 13 15 40 P. Aeruginosa 13 11 11 19 E. Coli 14 10 10 17 TABLE 9. Agar Cup Test, B Inhibition zone (mm) Test organism AgHA AgHA AgHA Thiomer- 1% 0.1% 0.01% sal 0.01% S. aureus 7 5 5 45 C. albicans 9 5 5 22 P. aeruginosa 10 5 5 22 E.coli 11 5 5 33 Discussion: The results from the MIC-test in liquid medium indicates that a 1% solution of the test -substance in Trypticase Soy Broth inhibits the growth of S. aureus, C. albicans and E. coli. P. aeruginosa is inhibited at a concentra- tion of 0.1%, A 1% solution contains 0.16 mg/ml of Ag- hyaluronate. The tests with agar cup methodology confirms the results from the tests in liquid medium, though the lowest con- centration for inhibition of P. aeruginosa was 1% Ag- hyaluronate. When the Ag-hyaluronate 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 distilled water. A white-colored precipitate, probably AgCl, appears immediately when the Ag-hyaluronate comes in contact with culture media or serum. EXAMPLE 6 PREPARATION OF 1 C LABELLED HYALURONIC ACID The biosynthesis of HA by the bacteria involves the util ization of exogeneoυs glucose. The sugar is 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 molecul e. Thus, the incorporation of radioactivity can be achi eved 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- controlled vessel . It has been found that fermentation in flasks was not effi cient resul ting both in very l ow incorporation ratios and l ow mol ecular weights. A "mini- fermentor " has been constructed, wi th a vol ume of 5 L of medium, and studies were performed in this system on the rate of glucose incorporation and util ization. It has been found that at the optimal condi tions for HA production, about 5% of the exogeneous glucose is incor¬ porated into HA. The molecular weight of the HA pro¬ duced in the mini-f ermentor was about 2xl0 6 daltons. The system was then used for labell ing the HA with ^-gl u¬ cose, and in one fermentation about 20 mg of crude 4 C-HA was obtained. This material was then pur if ied. The yield of this procedure was about 50% , resulting in a final yield of 10 mg of HA of specif ic activity of 1 uCi of 14 C per mg and a molecular weight of 2.2xl O δ daltons. The *C radioactively labelled NaHA so purified may be converted into radioactively labelled AgHA following the procedures described in Example L. EXAMPLE 7 USE OF S ILVER HYALURONATE AGAINST PS EODOMONAS AEROGINOSA INFECTION IN A RABBIT EYE MODEL Introduction Keratitis produced by Pseudomonas aeruginosa is the most rapidly spreading and destructive bacterial disease with which the human cornea can be inf ected, as well as the most disastrous (Laibson, 1972) . The frequency of corne- al inf ections has greatly increased with the use of contact lens. Treatment of ea rly-detected pseudomonas infections with aminoglycosidic antibiotics such as gentaπiycin usually results in a good therapeutic re- sponse. However, the increasing occur rence of antibiot¬ ic-resi stant bacteria presents a probl em, which become s disastrous when such a si tua tion is de tected po st-f actum . The use of an efficient wide range anti-bacterial agent , such as silver hyal uronate, might overcome th is probl em. We used a rabbit model for the demonstration of the eff icacy of silver hyal uronate for the treatment of an experimentally-induced pseudomonas kerati tis (Hyndiuk , 1981) . Experimental Bacteria : A pathogenic hospi tal isolate of Pseudomonas aeruginosa was used. It has been defined by the pseudomonas ref¬ erence laboratory at the Ram bam Medical Center, Haifa, Israel as a polyagglutinin serotype 6. Overnight cul¬ tures were grown in tryptic soy broth without dextrose at 37°C with shaking. In the morning, an aliquot of this culture was diluted 20-fold in fresh broth which was further incubated until a late logarithmic phase was reached (2-3 h. ; O.D. g6Q - 1.2). The concentration of bacteria was estimated from the optical density at 660 nm (1 O.D. * 2 x 10 9 cells/ml) . The cultures were diluted to the desired density with cold saline. The diluted suspensions were kept at 0 - 4°C until use (1-2 h) . Animal treatment: New Zealand albino rabbits weighing 2-2.5 kg were used. They were anesthetized by intramuscular injection of ketamine hydrochloride (35 mg/kg) and xylazine hydro¬ chloride (3 mgAg). A 4 mm circular superficial cut was applied on the cornea with a cork borer, then 3 longitu¬ dinal parallel scratches were made inside the circle with a 21 gauge needle. The bacterial infection was inflicted 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. We found that suspensions of 8 x 10 7 bacteria per ml produced 90-100% infection within 30-40 h, whereas a two¬ fold reduction in bacterial number resulted in lower and variable infection rates. Therefore, all subsequent experiments were performed with suspensions of 8 x 10' bacteria per. ml. Silver By?lug?n?te Treatment: Initial studies were performed with 0.5% w/v of silver hyaluronate in water prepared as described in Example 1. This solution is quite viscous, but its viscosity 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 bacterial infection. Hence, the 0.5% AgHA solution was diluted three-fold with 10 0.5% NaHA aqueous solution (final AgHA concentration 0.17%). A slight irritation response was still apparent, but it was transient and disappeared soon after cessation of treatment with the AgHA preparation. 15 In all experiments, the control eyes were concurrently treated with a 0.5% sodium hyaluronate solution. Experimental design: 20 In preliminary experiments, AgHA application was initi¬ ated 2, 4 and 8 hours after infection. An anti-bacte¬ rial effect of the AgHA therapy was clearly evident with the use of the 4 and 2 h intervals. In the early experi¬ ments, hourly application of the AgHA solution was con- 25. tinued for 12 to 24 h only. This scheme resulted in a significant protective effect for 24-48 h after infec¬ tion, but it did not prevent subsequent advancement of bacterial growth and deterioration of the eyes by 72 h. Therefore, a treatment schedule was selected in which 0 hourly application of one drop of the 0.17% AgHA and 0.33% NaHA solution was used, starting 2 h after infec¬ tion and continuing for 40 h. Treatment was then contin- 5 ued at 2 h intervals for additional 8-10 h. 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% NaHA and served as a control. Scoring of the results: The severity of the infection was scored by: (1) mac¬ roscopic examination of the eyes; and (ii) counting of bacteria in isolated cornea. Visual evaluation was performed according to the fol¬ lowing scoring system: 0 - No reaction. 1 - Leucocyte infiltration associated with the scratches only. 2 - Moderate infiltration at the scratches and around them, but not exceeding the 4 mm ring. 3 - Infiltration affecting about 30% of the corneal area. 4 - Infiltration and turbidity of about 70% of the corneal area and some cellular infiltration into the anterior chamber. 5 - Heavy infiltration of the total 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. 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 plated on McConkey agar plates for colony counting . Bacteria isol ated from the cornea were identif ied as Pseudomonas aerugino sa by standard biochemi cal tests and sensitivity to antibiotics. The scoring resul ts were analyzed statistically by the two tailed Wilcoxon rank sum test. Rgsul tg : AgHA treatment was found to be highly effective in the prevention of Pseudomonas kerati tis in the rabbit eye. Table 10 summarizes the resul ts of two experiments per¬ formed wi th a 0 .17% AgHA preparation. As shown, the intensive treatment with the silver sal t resulted in a marked inhibition of bacterial growth in the treated eyes. The scores of the control eyes were high as early a s 42-45 h after infection (scoring index of 3 .4 - 3 .6) and increased during the next days to reach a very ad¬ vanced stage (scoring index of 4 .9) by 120 h. In con- trast, the mean scores of the treated eyes were signif i¬ cantly lower than those of the control group throughout the experiment (Tabl e 10) , and they did not increase beyond . 72 h (see Tabl e 10 , Experiment I ) . It should be emphasized that in both experiments, most of the scores of the AgHA treated eyes did not exceed the val ue 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 AgHA protection. The effectiveness of the AgHA treatment was also appa r¬ ent from the fact that no bacteria were found in iso¬ lated cornea of treated eye s, whereas an abundant num- ber of pseudomonas microorganisms (10 5 -10 6 bacteria per corneum ; Tabl e 10) were found in the control eyes 42-120 h after infection. onclusion: AgHA was shown to have an effective therapeutic effect against the viscous bacteria Pseudomonas aeruginosa. in vivo. The use of such a preparation on i ts own or in conjunction with the commonly-used antibiotics might prove highly benef icial in the topical treatment of bacterial keratiti si. - 42 - _E_____L__S Effect of λgHλ Treatment of Infected Rabbit Eyes on the Scoring Index and Bacteria Count Time Scoring Index Ho. of Bacteria/Corneum Exp. λfter (jnean + SEM) (Ranαe) No. Infection Control Treated Control Treated Eye Eye Eye Eye I 45 h 3.62 ± 1.11 1.44 + 0.97*** (a) (a) 12) (N = 12) 72 h 4.29 ± 1.50 1.79 ± 1.55* (b) (b) (N = 7) (N - 7) 120 b 4.86 + 0.87 1.86 ± 1.55** 1.5xl0 5 - 2.4xl0 € <10 3 (V = 16) (N - 16) (N - 4) (N = 3) II 42 h 3.41 + 1.70 0.84 ± 0.65*** 5xl0 5 - 4x10 6 <10 3 (N - 16) (H * 16) (N - 4) (N « 5) 66 h 4.22 + 1.64 1.50 ± 1.20** 3x10* - 5xl0 6 <10 3 (N - 9) (N = 9) (N = 6) (N = 6) - • P o .05: control vs. treated * * P o .01 **• P o .001 (a) sampl' es lost (b) note ι determined REFERENCES 1. Carr, H.S., T.J. Wlodkowski and H.S. Rozen- krantz, "Silver Sulfadiazine: In Vitro Anti¬ bacterial Activity*, Antimicrob. Agents and 5 Chemoterhapy £: 585-587 (1973) . 2. Dettch, E.A., A. A. Marino, T.E. Gillespie and J.A. Albright, "Silver-nylon: A New Antimicro¬ bial Agent", Antimicrob. Agents and Chemother- 0 apy 21: 356-359 (1982). 3. Roseman, S. et al, "Biosynthesis of Hyaluronic Acid By Group A Streptococcus", Biol. Chem. 203. 212-225 (1953) . 5 4. Hyndiuk, R.A. , "Experimental Pseudomonas Kera¬ titis", Tr. Am. Ophth. Soc. 12: 541-624 (1981). _ 5. Laibson, P. R. , "Cornea and Sclera", Opthamol o 553-574 (1972). 5 0 5

Claims: WHAT IS CLAIMED IS: 1. A heavy metal salt of hyaluronic acid. 2. A salt in accordance with claim 1, wherein the metal is selected from the group consisting of silver, gold, cerium, and tungsten. 3. A salt in accordance with claim 2, wherein the metal is silver. 4. A salt in accordance with claim 2, wherein the metal is gold. 5. A salt in accordance with claim 2, wherein the metal is cerium. 6. A salt in accordance with claim 2, wherein the metal is tungsten. 7. A method of preparing the compound of claim 3 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 hyaluro¬ nate. 8. The method in accordance with claim 7, wherein recovering the precipitated silver hyaluronate com¬ pri ses separating the silver hyaluronate precipitate f rom the aqueous solution by centrifugation, washing the separated precipitate with ethanol and drying the washed precipitate over nitrogen. 9. A method in accordance with cl aim 7 , wherein the silver hyaluronate is prepared in the absence of light. 10. A method in accordance with claim 9 , wherein after admixing, the aqueous sodium hyaluronate and the aque¬ ous silver nitrate mixture is shaken for a sufficient period of time to form aqueous silver hyaluronate. 11. A method in accordance with claim 10, wherein recovering the precipitated silver hyaluronate compris¬ es rinsing the silver hyaluronate precipitate with ethanol , drying the rinsed precipitate with nitrogen and further drying the nitrogen dried precipitate by high vacuum drying. 12. A composition comprising the compound of claim 3 and a carrier. 13. A pharmaceutical composition comprising an effec¬ tive amount of the compound of claim 3 and a pharmaceu¬ tically acceptable carrier. 14. λ pharmaceutical composition comprising an effec¬ tive amount of the compound of cl aim 4 and a pharmaceu¬ tically acceptabl e carrier. 15. A method of inhibiting microbial growth which comprises contacting microbes with an effective amount of the compound of claim 3. 16. A method of treating a burn victim which comprises topically applying to the burn an effective amount of the compound of claim 3 . 17. A method of treating a wound victim which compris- es topically applying to the wound an effective amount of the compound of claim 3 . 18. A method of treating a subj ect having soft- tissue infection which comprises topically applying to the infection an effective amount of the compound of claim 3. 19. A method of treating a subj ect hav ing gonoccocal opthalmalogi cal infection which comprises topically applying to the inf ection an effective amount of the compound of cl aim 3. 20. A method of treating a subj ect having sepsi s which method comprises topically applying to the infection causing the sepsis an effective amount of compound of claim 3. 21. A method of treating a subj ect hav ing keratitis which method comprises typically applying to the infec- tion causing the keratitis an effective amount of com¬ pound of claim 3 . 22. A method of claim 21, wherein commonly used anti¬ biotics may optionally be used in conjunction with the compound. 23. A method of treating a subject afflicted with rheumatoid arthritis which comprises administering intra-articularly to the subject an effective amount of the compound of cl aim 4. 24. A method of treating a subject afflicted with inflammation of the joints which comprises administer¬ ing intra-articularly to the subject an effective amount of the compound of claim 4. 25. A composition comprising heavy metal salts of radioactively labelled hyaluronic acid. 26. A composition of claim 25, wherein the heavy metal salt of hyaluronic acid is silver hyaluronate. 27. A composition of claim 26, wherein the silver hyaluronate is radioactively labelled with 14 C.

Inventors: GREENMAN BENJAMIN, NIMROD ABRAHAM, GREENMAN, BENJAMIN, NIMROD, ABRAHAM
Applicants: BIO TECHNOLOGY GENERAL CORP, BIO-TECHNOLOGY GENERAL CORP.
Application year: 1987
Main classification: A61K43
IPC codes: A01N43, C08B37