NZ198416A - Anti-viral,anti-bacterial,and/or anti-fungal compositions containing metal oxycarboxylates - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number 1 98416 193416 </ Priority Dats^: 17".'&*39..

Complete 8poc ificQtion Filed: ?! .1 c'ass: Pubhcation Date: V. ).. 7 „ .

P.O. Journal Mo: . JT-fcP.

NEW ZEALAND PATENTS ACT, 1953 No.: Date: V -9FEBI984 COMPLETE SPECIFICATION ANTI VIRAL, ANTI BACTERIAL AND/OR ANTI FUNGAL COMPOSITION CONTAINING METAL" OXYCARBOXYLATE •*•17 We," BIO-SYSTEMS RESEARCH, INC. a corporation of Colorado, of 109 W. Rainbow Drive, Salida, Colorado 81201, United States of America, hereby declare the invention for which-f / we pray that a patent may be granted to pae/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1934'6 and the metal oxycarboxylate precipitate is recovered. Preferred metals include zinc, beryllium, magnesium, chromium, manganese, iron, cobalt, nickel, palladium, platinum, copper, silver, gold, cadmium and mercury. The structural formula for all of the compounds has not been confirmed. However, the following general formula is attributed: M2_8O(R-CO2)6 where M represents the metal cation, and R represents hydrogen or alkyl. The manganese and zinc compounds have been found to have the structural formula M^O(R-C02)g- Preferred acids include aliphatic carboxylic acids, preferably lower alkyl of up to 8 carbon atoms. Preferred acids include formic, acetic, propionic and butyric.

The Examples which follow illustrate the use of the compound as an anti virus, anti bacteria, or anti fungus agent.

Example 1 A stock (1%) solution is prepared by dissolving 0.1 gm of zinc oxyacetate in 10 ml of water. A 1/2 dilution (0.5% solution) is made by admixing 5 ml of the stock solution and 5 ml of water. A 1/4 dilution (0.25% solution) is made by admixing 5 ml of the 0.5% solution and 5 ml of water. A 1/10 dilution (0.1% solution) is made by admixing 1 ml of the stock (1%) solution and 9 ml of water.

Fine filter paper discs of 1 cm diameter are placed in each of four clean petri dishes. In dish #1, 0.1 ml of the 1% solution is dropped onto each filter disc and allowed to air dry. The same is done in dish #2 except that the 0.5% solution is used.

The same is done on dishes #3 and #4 except that the 0.25 and -0.1% solutions, respectively, are used. One ml of a previously -4 prepared 1 x 10 dilutionof a broth bacterial culture (Staphylococcus aureus) is then spread evenly over the entire surface of four agar plates. At the 12 o'clock position on each of the agar plates, a disc from dish.#1 (1% solution) is placed. 198416 The present invention relates to an anti viral, anti bacterial and/or anti fungal composition. The invention relates particularly to a composition suitable for treating Herpes virus, including Herpes simplex virus, types I and II (hereinafter referred to as "HSV-1" and "HSV-2").

In accordance with the invention, the reaction product of a metal, a carboxylic acid, and hydrogen peroxide, such as zinc, hexakis(acetato) oxotetra (hereinafter referred to as "zinc oxyacetate") is used to treat a virus, bacteria or fungus infection by administering an effective amount thereof to an infected patient. The compound can be administered topically or systemically, preferably in admixture with a pharmaceutical carrier. For topical use, the compound is preferably administered in an ointment or cream containing, preferably, from 0.1 to 20% by weight of the compound. For systemic use, daily dosage is generally about 15-50 mg of zinc for a person of average (70 kg) weight.

Zinc oxyacetate has the emperical formula C.-H.o0 Zn, and / ±Z X O J. J 4 the structural formula Zn .0 (CH-,CO_) , and the compound and its 4 J J. b preparation are described in the literature. See the Bulletin of the Chemical Society of Japan, March 1954, Volume 27, No. 2, pp. 112-114, in which the compound is prepared by slowly distilling powdered anhydrous zinc, acetate in a high vacuum. Zinc oxyacetate sublimes gradually and is collected as crystalline crust on a cool place in the distillation vessel. It is preferred, however, to make the compound in the manner described in New Zealand Patent Specification No. 198,418 and Entitled "Metal oxycarboxylates and Method of Making Same.

The disclosure of said application is herein incorporated by reference. . In that method, a metal, a carboxylic acid, and hydrogen peroxide are reacted in an aqueous reaction mixture *-9FEB1984^ Q\\ - 2 - 55385S! 1 984 Discs from dishes #2, #3 and #4 are similarly placed at the 3 o'clock, 6 o'clock, and 9 o'clock positions, respectively, of each of the four plates. Each disc is gently pressed to ensure good contact with the agar surface. For comparison purposes, a further stock solution of zinc sulphate (1%) is made up and an identical series of tests made. Each plate is incubated overnight at 37°C and the area of inhibition - the clear area around each disc - is measured.

Results are as follows: -Jc- I 984 f6 Compound P.lnLc ' No.

U • Zinc oxyacetate 1 1.85 Zinc oxyacetate 2 1.80 Zinc oxyacetate .3 1.78 Zinc oxyacetate 4 1.85 Zinc oxyacetate Average 1.82 Zinc sulphate 1 1.80 Zinc sulphate 2 1.72 Zinc sulphate 3 1.71 Zinc sulphate 4 1.42 Zinc sulphate • Average 1.66 . 51 1.70 1.52 1.61 1.72 1.64 1.50 1.45 .1.41 1.40 1.44 Clear ■ 251 1.32 1.35 1.40 1.42 1.37 1.40 1.40 1.39 1.31 1.37 Area (cm) .012 Control 1.25 0.31 1.22 1. 21 0. 61 N/A N/A N/A N/A N/A The results show that zinc oxyacetate has bacteriostatic qualities comparable to those of zinc sulphate. At 1% and 0.50% concentration of zinc sulphate, a visible precipitate formed under each sensitivity disc.

Example 2 Bread mold is innoculated on a nutrient plate. Discs of filter paper prepared as in Example 1 show good growth inhibition for the undiluted material and the 1% solution. The less concentrated solution does not noticeably inhibit growth.

Example 3 Evaluation of the compound is made using a simple neutralization of viral cytopathogenic effect (CPE) on Buffalo Green Monkey (BGM) cells using two tubes for each dilution of virus. The titer of — 4 + Herpes simplex virus type 1 (HSV-1) .is 10 based on 4 (100%) CPE, -2 * while that ofjHSV-2 is 10 . In order to evaluate zinc oxyacetate, the.compound (or the compound in equal-weight admixture with lauryl alcohol) is incubated for 1 hour .with the .virus prior to making " 1 98 4 t ten-fold serial dilutions and innoculatiny Llio UGM Lubes. 'iTic amount of virus used is 0.1 ml. The amount of test material used is 0.1 ml of a solution containing 0.14 of Twcen 80 and 0.1? of the test material. .

TEST f Titer based on 4+(100%) CPE HSV-1 HSV-2 1. Control 10 4 10 2 2. Zinc Oxyacetate 10 ^ (1 log'inhibition) 0 (2 log inhibition) 3. 50% Zinc Oxyacetate 10 * (3 log inhibition) 0 (2 log inhibition) 50% lauryl alcohol The. data shows that the compound is effective in inhibiting the virus and that the compound in admixture with, the lauryl alcohol carrier is. substantially 'more effective than the compound per sc in inhibiting the more virulent HSV-1 virus.

Example 4 .

In.a test similar to that of'Example 2, zinc oxyacetate is tested for inhibition of four bread molds. The amount of zinc oxyacetate is 0.1 ml of a 1% solution containing 0.1% Tween 80. The amount of mold is 0.1 ml. Test results are indicated in the table.

Mold Size of cleared area (mm) Candida albicans 9 Trichophyton mentagrophytes 9 Microsporum canis 1 8 Trichophyton rubrum 5'' 198416 The preferred carriers, for topical use, are petrolatum and alcohols of from 8-18 carbon atoms, preferably lauryl alcohol. An admixture of petrolatum and one or more of said alcohols may be used, preferably in a weight ratio of petrolatum to alcohol of 1:10 to 10:1. A suitable admixture is petrolatum 80% and lauryl alcohol 20%, by weight.

Examples 5-18 Results similar to those of Examples 104 are achieved by substituting the reaction products of acetic acid, hydrogen peroxide, and one or more of the followina metals accordina to the process as disclosed in said New Zealand Patent Specification No. 198,418; 5 - beryllium; 6 - magnesium 7 - chromium; 8 - manganese; 9 - iron; 10 - cobalt; 11 - nickel; 12 - palladium; 13 - platinum; 14 - copper; 15 - silver; 16 - gold; 17 - cadmium; 18 - mercury.

Examples 19-25 Manganese oxyacetate having the structural formula Mg^O(CH^CC^)g is prepared as disclosed in copending New Zealand Patent Specification No. 198,418. In this example, the manganese compound is compared, by plaque assays done on HSV-1 and HSV-2 with the zinc-lauryl acetate composition fo Example 3. All of the tests, including those of Examples 26-31 which follow, are with test substances dissolved in 2% FBS (fetal bovine serum)-MEM (Eagles Minimum Essential Medium). The solubility of the zinc containing compounds is poor and is reported as less than 1 mg/ml, The manganese compound is readily soluble and concentration is 1 my/ml. Results are as follows: HSV-2 Manganese Compound Zn composition Control Toxic ND"'" ND 4 0 39 0 0 , 5 Not Done # 1984 16 As indicated in the results, both substances inhibited HSV-2. However, the manganese compound was less effective than the zinc composition.

B. HSV-1 2%(1) 1.5% 0% 2% 1.5% 0% 2% 1.5% 0% 22 ~2 Irr(2) Irr 90 tnc(3) TNC TNC ND*4' ND TNC 23 ~3 46 45 11 24 105 31 115 TNC 34 24 *4 4 16 6 38 6 ~5 0 0 0 0 0 0 3 3 5 (1) % Methylcellulose in overlay medium (2%, 1.5% or 0%) (2) Irregular results (3) Too numerous to count (4) Not done The HSV-1 does not plaque as easily'as the HSV-2 strain. HSV-1 has a tendency to cause cell aggregate formation rather than distinct plaques. Consequently, two different concentrations of methylcellulose are used in the overlay medium, and finally, in the last trial a liquid overlay is used in an effort to get clear, distinct plaques. There are some variations in the results but, overall, with HSV-1, the Zn substance is less effective than the manganese compound in inhibiting HSV-1. This finding could be due to the lower solubility of the Zn material in 2% FBS-MEM.

Examples 26-28 Cytopathogenic effect (CPE) trials are carried out on the manganese compound and zinc composition of Examples 19-25. As shown in the following results, "these substances are not effective against the RNA viruses Echo 5 and Polio 1: - y- i

Claims (12)

1984 16 Example , Echo 5 Polio 1 -fi JS*1* -7 26 Control 10 °* 3 10 27 Manganese compound 10 10 i 2 8 Zn composition 10 10 5 (1) Approximate titer on BGM cells based on 4+ (100%) CPE. Examples 29-30 CPE trials for the Manganese compound of Examples 19-25 are conducted as in Examples 26-68 on HSV-1 and HSV-2. As is shown in the results which follow, the manganese compound is effective against HSV-2 but is probably not effective against HSV-1. The HSV-2 data are interesting because of the apparent viristatic action of the Mn compound rather than the viricidal activity seen with the Zn compound. Apparently at 10 ^ dilution of HSV-2, enough Mn compound is present to inhibit the virus completely and cause an increase in the size of the BGM -2 -3 cells. At 10 and 10 , one tube in each dilution showed early inhibition of virus but later the virus manifested itself in the typical syncytial formation. HSV-1 HSV-2 Control 10~3*5 ^ 10~2*5 Mn , -3.0 -r , ■ i - , • , 10 Inhibition compound - (1) Approximate titer on BGM cells based on 4+ (100%) CPE. Example 31 CPE trials for the manganese compound and zinc composition of Examples 19-25 are conducted as in Examples 26-30 on a DNA virus, Adenovirus type 4, using KB cells (human continuous cell line). The data clearly indicates that neither the test compound nor Zn carrier are effective in inhibiting the CPE of Adenovirus type 4 on KB cells. The titer in all cases in 10 3. * -V NAT^/vyfc i .A1?- 1 98416

1. A therapeutic composition comprising a metal oJtycarboxylate and a pharmaceutically acceptable carrier therefor.

2. A therapeutic composition according to claim 1 wherein said metal oxycarboxylate has the general formula K_ o0(R-C0_)c z — o z D wherein M is a metal and R is hydrogen or alkyl.

3., A composition according to claim 2 wherein said metal is selected from the group consisting of zinc, beryllium, magnesium, chromium, manganese, iron, cobalt, nickel, palladium, platinum, copper, silver, gold, cadmium, and mercury.

4. A composition according to claim 3 wherein the metal oxycarboxylate is present in an amount of from 0.1 to 20% by weight.

5. A composition according to claim 1 wherein said carrier comprises petrolatum.

6.. A composition according to claim 1 wherein said carrier comprises an alcohol having from 8 to 18 carbon atoms.

7- A composition according to claim 6 wherein said carrier comprises lauryl alcohol.

8- A composition according to claim 1 ' wherein said carrier comprises petrolatum and lauryl alcohol in a weight ratio of petrolatum-: lauryl alcohol of from 1:10 to 10:1. 9- A composition according to claim § comprising metal oxycarboxylate in an amount of 10% by weight, petrolatum in an amount of 80% by weight, and lauryl alcohol in an amount of 10% by weight.

9 19 3416

10- A composition according to claim xX wherein said metal ^4"' oxycarboxylate coraprises the reaction product of a metal, a carboxylic acid, and hydrogen peroxide.

11. a composition according to claim Vjt wherein said metal oxycarboxylate is selected from the group consisting of zinc oxyacetate and manganese oxyacetate.

12. A composition according to any one of claims 1 to 11 substantially as herein described. '!is/the!r s'_;nor.'sj • /.rents., A. J, PARK & SON. - 11 -