AP327A

AP327A - Method for inactivating enveloped viruses and sperm.

Info

Publication number: AP327A
Application number: APAP/P/1992/000370A
Authority: AP
Inventors: Edwin Bernard Michaels; Malamud Daniel Dr
Original assignee: Biosyn Inc
Priority date: 1991-03-22
Filing date: 1992-03-23
Publication date: 1994-03-21
Also published as: DE69130871T2; DE69130871D1; EP0733361A2; JP3940568B2; CA2106683C; EP0733361B1; DE69132621T2; EP0733361A3; JPH06505700A; KR100221486B1; RU2110256C1; ATE201593T1; AP9200370A0; ATE176399T1; JP3228928B2; OA09911A; EP0576425A1; DE69132621D1; AU8222791A; JP2002037733A

Abstract

There is provided methods for inhibiting the activity of enveloped viruses, treatment of viral infections and method inactivating sperm. There is provided articles for use in these methods.

Description

1· Field of the Invention

The present invention relates to a novel method and novel articles for inhibiting the activity of enveloped viruses. The invention also relates to treatment of viral infections. More particularly, the invention relates to a method for inhibiting the development of diseases and infections caused by enveloped viruses. In one aspect the invention particularly relates to a method of inhibiting the activity of viruses whose major mode of transmission is sexual. In other aspects the invention also relates to methods of inhibiting the activity of enveloped viruses whose mode of transmission is nonsexual. The compositions of use in the invention are also effective in the inhibition of bacteria and fungi which coexist with viruses or viral infections. Additionally, the invention relates to a treatment for virus related diseases, particularly sexually transmitted diseases related to AIDS, and to diseases related to this and other opportunistic infections of the immune-compromised host.

The method of the invention relates to using mixtures of betaines and amine oxides in the inhibition of viruses and treatment of viral infections. The betaine is selected from the group consisting of (a) an alkyl-N-betaine, an alkyl-N-sulfobetaine, an acyl-Nbetaine, an alkyl N-substituted aminopropionic acid and an alkylimidazolinium betaine and the amine oxide is

BAD ORIGINAL selected from the group consisting of (b) an alkyl-N, Ndimethylamine oxide, an alkyl-N, N-dihydroxyethylamine oxide and an acylamide t-amide oxide. The term betaine when used herein means an N-dimethyl glycine and their lower alkyl homologs. The term includes Ndinethyl amino propionic acids and sulfo betaines which are the sulfonic acid analogs of such compounds. Unless otherwise specified a N-dimethyl compound is intended. More particularly, the invention relates to a method for employing mixtures of alkyl N-di(lower alkyl) betaines and alkyl N-di(lower alkyl) amine oxides in the inhibition of enveloped viruses and use of these mixtures in the treatment of infections caused by these viruses. The mixtures may also be used as spermicides, either alone or in combination vith conventional spermicides. These mixtures may be used in combination with contraceptive devices or may be incorporated into the contraceptive device.

2. Description of the Prior Art

It is knovn that certain mixtures of amines are effective antimicrobial agents against gram negative and gram positive bacteria. For instance, it is knovn that certain mixtures of a) alkyl-N-betaine, alkyl-Nsulfobetaine, acyl-N-betaine, alkyl N-substituted aminopropionic acid or an alkylimidazolinium betaine and (b) alkyl-N,N-dimethylamine oxide, alkyl-N, Ndihydroxyethylamine oxide or acylamide t-amide oxide can be used for skin degerming, cleansing and deodorizing (see for example U.S. Patent 4,183,952, issued to E.B. Michaels; U.S. Patent No. 4,075,350 issued to E.B. Michaels, and U.S. Patent No. 4,107,328; issued to E.B. Michaels). These compositions are also useful for long term inhibition of body odor. Mixtures of the same active ingredients are also knovn to enhance oral hygiene by reducing oral microflora and inhibiting formation of dental plaque. (U.S. Patent 4,839,158 issued to E.B. Michaels). Compositions of this type are

AP 0 0 0 3 2 7

- 3 also known to be effective fungicides. Corner, A.M., et a 1. Antimicrobial Agents and Chemotherapy, 3 2 (3): 350 353 (March 1988) ; Corner, A.M. The Journal cf Clinical Dentistry, Vol. 2(2):34 - 38. (1990)). (See also U.S.

Patents 4,075,350, 4,107,328, 4,183,952 and 4,839,158 issued to E.B. Michaels.)

It is also known that nonionic surface-active agents possessing ether or amide linkages betveen the hydrophilic and hydrophobic portions of the molecule rapidly inactivate the infectivity of herpes simplex virus I and II. (Asculai, S.S., et al. Antimicrobial Agents Chemotherapy, Vol. 13:686 - 690 (1978)).

Asculai et al. also states that cytocidal activity has been used as an index of antiviral activity. The compositions utilized in this invention have an advantage over the compositions described in Asculai et al. in that these compositions inactivate viruses and bacteria while exhibiting low cytocidal activity.

The spermicide Nonoxynol-9 (N-9), has been found to have bactericidal and viricidal effects. For example, N-9 has been found to inactivate viruses related to AIDS (e.g. HIV, human immunodeficiency virus) and herpes simplex viruses (HSV I and II). In vitro, N9 has been shown to be effective against the organisms that cause gonorrhea, chlamydia, syphilis, trichomoniasis, and other sexually transmitted diseases. (U.S. Pharmacopeial Convention USPDI Update 1990 pps.

755 - 756).

SUMMARY OF THE INVENTION

It has now unexpectedly been found that certain hereinbelov defined mixtures of betaines such as (a) alkyl-N-betaines, alkyl-N-sulfobetaines, acyl-Nbetaines, alkyl-N-substituted aminopropionic acids and alkylimidazolinium betaines, and amine oxides such as (b) alkyl-N, N-dimethylamine oxides, alkyl-N, Ndihydroxyethylamine oxides or acylamide t-amide oxides in acceptable diluents, carriers and excipients are

BAD ORIGINAL 0 t

effective in inhibiting the activity of enveloped viruses. The invention provides a method of inhibiting the activity of enveloped viruses for vhich a major mode of transmission is sexual. The invention relates to a method for treating diseases and infections caused by enveloped viruses. These compositions are also effective in the inhibition of bacteria and fungi that co-exist vith viruses or viral infections and for treatment of bacterial and fungal infections which coexist vith viruses or viral infections. The invention also provides a method for disinfecting air and surfaces contaminated with enveloped viruses.

The invention also provides a method for preventing the transmission of enveloped viruses.

The compositions used in the method of the present invention can also be used as spermicides.

These compositions can be used alone, vith other knovn spermicides and vith or incorporated into contraceptive devices such as condoms, sponges, vaginal inserts, contraceptive films, diaphragms, suppositories, contraceptive patches or sustained release devices.

These compositions can also be incorporated into douches. The compositions can be incorporated into wipes. For use as viricides, these compositions can be applied alone; with other spermicides; and vith or incorporated into the contraceptive devices described above. The compositions can also be applied in the form of a foam, gel, cream, salve, jelly or lotion. The compositions can also be applied in the form of a liquid, aerosol, mist or spray.

DESCRIPTION OF THE DRAWINGS

Figure 1: is a graph showing the effect of C31G (a composition according to the present invention) and the effect of Nonoxynol-9 (N-9) on sperm motility.

Figure 2: is a graph showing the effect of C31G and the effect of Nonoxoynol-9 (N-9) on total sperm count.

AP 0 0 0 3 2 7

- 5 Figure 3: is a graph shoving the effect of C31G and the effect of Nonoxoynol(N-9) on the mean progressive velocity of sperm.

Figure 4: is a graph shoving in vitro toxicity of C31G vs. N-9 on sup-TI cells.

Figure 5: is a graph shoving a five day toxicity study of C31G vs. N-9 on OEM cells.

Figure 6: is a graph shoving comparing the effects on C31G vs. N-9 on HSV.

Figure 7: is a graph shoving the effect of C31G on HIV.

Figure 8: is a graph comparing inactivation of HIV by C31G and N-9.

Figure 9: is a chart comparing inactivation of HIV by C31G and N-9.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention relates to using mixtures of betaines and amine oxides in the inhibition of enveloped viruses and treatment of viral infections caused by enveloped viruses. The method of the invention also relates to using mixtures of betaines and amine oxides in the inactivation of enveloped viruses. The betaines that can be used in this invention are alkyl-N-betaines, alkyl-N-sulfobetaines, acyl-Nbetaines, alkyl N-substituted aminopropionic acids or alkylimidazolinium betaines or mixtures thereof. The amine oxides that can be used in this invention are alkyl-N, N-dimethylamir.e oxides_# alkyl-N, Ndihydroxyethylamine oxides or acylamide t-amide oxides or mixtures thereof. These compositions are also effective for the treatment of viral, fungal, and microbial infections and contaminations. These compositions are effective in inhibiting enveloped viruses that are transmitted sexually or nonsexually. These compositions are also effective in the inhibition of bacteria and fungi which coexist with viruses or viral infections. These compositions can also be used

BAD ORIGINAL ft topically.

Typically, these compositions are applied to areas where viruses are or can be transmitted. This includes application in the vagina, to mucous membranes, and to the skin.

These compositions can also be used as spermicides, either alone or in combination with other spermicides. These compositions can also be used with or incorporated into contraceptive devices such as, for example, condoms, diaphragms, sponges, contraceptive films, suppositories, sustained release devices, and contraceptive patches. For example, the composition may be incorporated into a lubricant applied to a condom or as part of a reservoir at the tip of a condom, or incorporated into a contraceptive sponge, contraceptive film, suppository, sustained release device or contraceptive patch. A contraceptive film can comprise the composition described above, together with, Type A gelatin, cellulose gum and a polyhydric alcohol. The compositions can also be used in contraceptive foams, gels, jellys, creams. The compositions can also be used in douches or use in preventing the transmission of viruses, these compositions can be used alone, with other spermicides and with or incorporated into a contraceptive device as described above. This invention also comprises a method for inhibiting the transmission of viruses that cause sexually transmitted diseases which comprises applying the compositions to those parts of the anatomy that are exposed to body fluids emanating from another during sexual activity.

These compositions have also unexpectedly been found to be less toxic to mammalian cells than

Nonoxoynol-9 (N-9) (See Figures 4 and 5) . Table 1 shows that these compositions have equivalent spermicidal activity to N-9, which is one of the few surfactants approved by the FDA for use as a spermicide.

For use in topical applications, the

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AP 0 0 0 3 2 7 coapositions can also be incorporated into liquids, creams, salves, lotions, foams and gels.

The invention also provides a method for disinfecting air and inanimate surfaces, for example, in an operating room or laboratory. More particularly, this invention provides a method for disinfecting areas which are contaminated with enveloped viruses. These compositions can also be incorporated into sprays, mists, wipes, aerosols or devices which produce sprays, mists or aerosols. These compositions can also be applied as liquids.

According to the method of this invention, these compositions may be used as viricides, fungicides and bactericides.

The compositions employed in the method of the irvention comprise an admixture of betaines and amine oxides. The betaines used in this invention are selected from the group consisting of (a) alkyl-Nbetaines, alkyl-N-sulfobetaines, acyl-N-betaines, alkyl N-substituted aminopropionic acid, alkylimidazolinium betaines and mixtures of two or more thereof. Typically the betaines have two lower alkyl groups bonded to the nitrogen atom. Most effectively they have two methyl substituents on the nitrogen atom. The amine oxides used in this invention are selected from the group consisting of b) a an alkyl-N,N-dinethylamine oxides, alkyl-N,N-dihydroxyethylamine oxide or acylamide t-amine oxides and mixtures of tvo or more thereof. Typically, the betaine and amine oxide components are present in a molar ratio of from 1:5 to 5:1, preferably in a molar ratio of 1:1. The alkyl-N-betaine, the alkyl-Nsulfobetaine, the acyl-N-betaine, the alkyl Nsubstituted 2-aminopropionic acid and alkylimidazolinium betaine (also referred to as cocoamphoacetates) employed as the components (a) of the composition of the invention have structures, respectively, as follows:

BAD ORIGINAL <fH₃ •N't-CH₂C00'

I ⁱ ch₃

CH3 itΪη•ch₂so₃'

Ο

II •C

CHCH₃

1+ Ii I.

N* CH₂COO or RC—*NH(CH₂)₃-N⁺έκ₃ itt₃

RNHCH₂CH₂COOH or RN(CH₂CH₂COOH)2 ch₂ch₂oh :h₂coo f?

RCNH οι

N

-CH-.COO’ ch₂

-ch₂ch₂or ch₂ch₂oh

I

N-CH₂COO' where R is a higher alkyl group having from 10 to 18 carbon atoms, preferably from 12 - 16 carbon atoms.

When used herein the term lower alkyl means an alkyl group of from 1 to 3 carbon atoms.

Illustrative of these aforementioned substances are: (1) coco-N-betaine, cetyl-N-betaine, stearyl-N-betaine, isostearyl-N-betaine, oleyl-N25 betaine; (2) coco-N-sulphobetaine, cetyl-Nsulphobetaine, stearyl-N-sulfobetaine, isostearyl-Nsulfobetaine, oleyl-N-sulfobetaine; (3) cocoamido-Nbetaine, cetylamido-N-betaine, stearylamido-N-betaine, isostearylamido-N-betaine, oleyl-awino-N-betaine; (4) N30 coco-2 aminopropionic acid, N-cetyl-2-aminopropionic acid, N-stearyl-2-aminopropionic acid, N-isostearyl-2aminopropionic acid, N-oleyl-2-aminopropionic acid,

N-stearyl-bis (2-aminopropionic acid), N-oleyl-bis (2-aminopropionic acid), N-coco-bis (2-aminopropionic acid), N-cetyl-bis(2-aminopropionic acic),

Λ

AP 0 0 0 3 2 7 (5) N-lauryl-bis (2-aminopropionic acid) 1-hydroxyethyl1-carboxymethyl-2-decylimidazolium betaine;

1- hydroxyethyl-1-carboxymethy1-2-dodecylimidazolium betaine; 1 -hydr oxy ethyl -1-carboxyme thy 1-2cocoimidazolium betaine; 1-hydroxyethyl-l-carboxymethy12- stearylimidazolium betaine; 1-hydroxyethyl-lcarboxymethyl-2-oleylimidazolium betaine; or mixtures of the same.

When used here the term coca is that used in the CTFA (designations of Cosmetic and Toiletry and Fragrance Association, Wash., D.C.) and is used to indicate alkyl groups present in coconut oil, i.e. a mixture of alkyl groups of from 10 to 18 carbon atoms. The designations of the compounds listed herein are those of the CTFA.

The (1) alkyl-N,N-diethylamine oxide, (2) alkyl-N, N-dihydroxylethylamine oxide, or (3) acylamide t-amine oxide employed as component (b) of the aforementioned mixture, respectively, have the structure:

O

R-i!-NH

CH-»

ch₃ where R is a higher alkyl group of from 10 to 18 carbon atoms, for instance, radicals such as decyl, undecyl, lauryl, tridecyl, myristyl, cetyl, stearyl, isostearyl or oleyl. Exemplary of the amine oxides are: decylΝ,Ν-dimethylamine oxide, lauryl-N,N-dimethylamine oxide, stearyl-N-N-dimethylamine oxide, oleyl-N,N-dimethylamine oxide, coco-Ν,Ν dihydroxyethylamine oxide, cetyl-N,NBAD OR1G

dihydroxyethylamine oxide, oleyl-N,N-dihydrcxyethylamine oxide, Ν,Ν-dihydroxyethylamine oxide, oleyl-N,-Ndihydroxyethyl-amine oxide and mixtures of the same.

The components (a) and (b) are usually admixed and acid is then added in an amount necessary to adjust the pH of a 0.5% solution to betveen 4-8, preferably to pH 4.5 - 5.5. The pH of an aqueous solution comprising the above enumerated components of the invention is determined by employing an aqueous solution of 0.5%, by weight, total of active components.typically at a glass electrode, to precisely define the acidity of the composition.

In general, the acid used to adjust the overall composition to the required pH is any organic or inorganic acid that is compatible with the intended use of the composition, for example, hydrochloric acid, phosphoric acid, sulfuric acid, citric acid, acetic acid or nicotinic acid. The balance of the composition, after allowing for the acid is usually an acceptable solvent, such as water or a lower (¢^-04) monohydric aliphatic alcohol, for a total of 100 parts or more. Where vater is employed, small amounts of a lower alkyl alcohol, such as ethanol or propanol, may also be added to provide ease in formulation. Acceptable diluents, carriers and excipients are, for example, ethyl or isopropyl alcohols, polyethylene glycol, povidone, polyhydric alcohols, glycerine, cellulose gums, gelatin, colorants and fragrances. If necessary, the pH of the total composition is then adjusted to the requisite pH by adding a suitable inorganic or organic acid thereto.

The result is a substantially uniform, homogeneous, relatively nontoxic composition having enhanced activity against enveloped viruses, bacteria and fungi.

It has been unexpectedly found that these compositions exhibit (1) low mammalian cell toxicity, a property which is known to correlate with low irritation

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- η and low toxicity when used in contact with mucous membranes (Journal Clinical Dentistry 2(2):34 - 38, (1990)), and (2) highly efficacious inactivation of enveloped viruses although cytocidal activity is low.

In the past, research has used cytocidal activity as an index of activity. (Asculai, S.S., Antimicrb. Agents Chemother. 13:678 - 690 (1978)).

In practice, the total amount of the components (a) and (b) of the overall composition can range from .01% - 40%, preferably from .03% - 30% depending on the intended means of use. For example, concentrations used are, approximately 20% - 30% in contraceptive films and 0.2% to 2% in gels.

Compositions for use in this invention comprise alkyl N-di(lover alkyl) glycines and alkyl Ndi (lower alkyl) a-aine oxides, wherein the lower alkyl is ^cl“3· ^{One class} betaines I have found to be partically useful in this invention are the alkyl Ndimethyl betaines, such as cocobetaines and lauryl betaines. Particularly useful amine oxides for use in this invention are the an alkyl N-dimethyl amine oxides, such as cocodimethyl amine oxides and lauryl dimethyl amine oxides.

One particular composition that can be used in this invention comprises cocobetaine, cocamine oxide and citric acid monohydrate.

Another composition that can be used in this invention comprises lauryl betaine, lauramine oxide and citric acid monohydrate.

In such compositions, the molar ratio of betaines to amine oxides is normally from 5:1 to 1:5, preferably in a molar ratio of 2:1 to 1:2, more preferably about 1:1.

Other compositions that can be used in this invention comprise mixtures of betaines, amine oxides, gelatin having a Bloom strength of 100 - 300 and a molecular weight from about 75,000 to about 300,000,

BAD ORIGINAL polyhydric alcohols and cellulose guns. Such compositions are described in a copending U.S.

application being filed simultaneously with the present application and having U.S. application Serial No. 07/673,631.

Compositions used in this invention can inhibit the activity of viruses that are related to AIDS. It is also expected that these compositions can inhibit the activity of HSV-1 and HSV-2. It is also expected that the compositions used in this invention can inhibit Hepatitis A, B and C. It is expected that the compositions can be used to inhibit viruses, bacteria and fungi which are associated with sexually transmitted diseases (STD's).

The compositions of this invention may be useful in relation to the following:

1) Transmission of HIV is often associated with the co-transmission of other viral and/or microbial pathogens. Indeed, some investigators have suggested that HIV may not be the sole agent responsible for AIDS (see Duesberg, P.H. (1991) Proc, Natl, Acad, Sci,

88:1575 - 1579; Lemaitre, M., Guetard, D., Henin, Y., Montagnier, L. and Zerial, A. (1990). Res, Virol, 141:5 - 16). For this reason, antimicrobial agents, such as those described in this application, with a broad spectrum of activities against viruses, bacteria, and yeasts such as Candida may be of particular value in the prevention and treatment of Acquired Immune Deficiency Syndrome (AIDS).

2) It is thought that certain bacteria known to cause STD's may aid in HIV transmission. In persons who have been exposed to HIV, certain bacteria which cause STD's often fail to respond to therapies that are otherwise highly effective. HIV infection may help the spread of a bacterial STD that in turn helps to spread HIV. STD's such as chlamydia, chancroid, syphilis, genital herpes and gonorrhea which cause ulcerations of

AP 0 0 0 3 2 7 the genital skin seem to increase the risk of acquiring or transmitting HIV infection sexually. Aral, S.O., et al. Scientific Arcricaru- 264 (2):62 - 69 (February 1991).

The compositions described above may also be of use to inactivate other enveloped viruses, including vaccinia, varicella, herpes zoster, cytomegalovirus, Epstein Barr virus, influenza, mumps, measles, rhinovirus, rabies and rubella. In order to facilitate a further understanding of the invention, the following examples are presented primarily for the purposes of illustrating more specific details thereof. The invention is not to be deemed as limited thereby except as defined in the claims.

Example. 1

The composition described below is a concentrate of C31G, an equimolar preparation of cocobetaine and cocodimethylamine oxides (designations of CTFA Cosmetic and Toiletry and Fragrance Association, Wash., D.c.) [CFTA] which can be used in a number of different configurations.

Cocobetaine; 31.5% active ingredient (Al); 183.9 kg (405.5 lb),

Cocamine oxide, 31.5% (Al): 147.4 kg (325 lb), citric acid monohydrate, USP; 11.8 kg (26 lb), Purified water, USP: 11.8 kg (26 lb),

To make about 354.9 kg (782.5 lb) C31G at

29.6% Al, at a dilution 1% Al; pH « 4.9.

Example 2

The spermicidal activity of C31G of example 1 was compared to the spermicidal activity of nonoxynol-9 (N-9).

The studies were conducted using the HamiltonThorn Sperm Motility Analyzer. Samples of pooled washed semen were incubated with dilutions of the two compounds for fifteen seconds and were analyzed (in triplicate) for determination of the minimum concentration for inactivation by the following criteria;

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1. Inhibition of sperm motility (Figure 1)

2. Decrease in total sperm count (Figure 2)

3. Inhibition of the mean progressive sperm velocity: (Figure 3)

As shown in Table 1, C31G and N-9 gave identical results:

Table 1

Minimum Concentration fcr Spermicidal Activity

C31G N-9

10 Total Inhibition of Motility	150	ppm	150	ppm
90% Decrease in Sperm Count	150	ppm	150	ppm
50% Decrease in Progressive
Velocity	125	ppm	125	ppm

£xarple_2

The cytotoxic effects of C31G and N-9 on mammalian cells were determined in several types of assays. Cell toxicity is an indication of the relative safety and comfort in use of surfactants in contraceptives or prophylactics.

As shown in Figure 4, a two week study monitoring drug effects on SUP-TI cells, (a human lymphocytic cell line), C31G demonstrated minimal toxicity at 0.001% [10 ppm] while N-9 at the same concentration was extremely toxic.

Example, 4

Figure 5 shows MMT reduction in CEM cells after five days of continuous exposure. Under these conditions C31G showed no toxicity to mammalian cells at 3 ppm [.0003%] while N-9 showing toxicity, reducing mammalian cell viability by 50% at the same concentration. It is unexpected that a compound having the same spermicidal activity as N-9 would be less toxic to mammalian cells than N-9.

Example 5

Antiviral activity of C31G was tested using HSV-1 and HIV-1.

To test the effect of C31G and N-9 on herpes

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- 15 simplex virus type-1 (KOS strain), the virus stock was prepared in Vero (African green monkey kidney) cells.

The virus was released from the cells by one freeze-thaw cycle followed by sonication. The virus titer was 4.5 X 10® plaque forming units (PFU)/ml, as determined on Vero cells. The drug formulations used were identical to the drugs in the spermicidal study.

The stock concentration of each drug was 51. Two-fold serial dilutions were made, down to 0.04$, in

PBS pH 7.4 at	room temperature.	24 ul of each dilution
was added to	220 ul of virus (10	⁸ PFU), giving final
concentrations of 0.5 - 0.004%.	These samples vere
incubated at	room temperature for 10 min, and then lOOul
of each was immediately diluted	to 1 ml with ice-cold
DMEM/5% FBS.	10-fold serial dilutions were then made,
down to 10-⁷	for titration of rerlining infectious virus
on Vero cells	in 24-well plates.	Plaques were counted
36 hours post	-infection.
	Table 2
Concentration	ill Residual virus fPFU/ml)
		C31S
0.004	3.5 X 10®	3.5 X 10®
0.008	2.7 X 10®	3.3 x 10®
0.015	1.1 X 10®	2.5 X 10⁵
0.03	3200	<167
0.06	<167	<167
0.13	<167	<167
0.25	<167	<167
0.5	<167	<167
Note that these are titers (ie.	PFU/ml) , rather than

absolute numbers of PFU. The titer determined for untreated virus was 4.7 x 10® PFU/ml in the experiment using N-9 and 4.5 x 10® PFU/ml in the experiment using C31G.

It is noted that complete inhibition of plague formation by C31G occurs at more than one dilution below that of N-9. See Figure 6.

θΑΟ ORIGINAL

ΊΛ

Example 6

The inactivation of HIV-l (AIDS) virus vas studied in two experiments. The first study compared the effects of C31G of HIV-l at two different exposure times, 2 minutes and 45 minutes. The next study compared the relative activity of C31G and N-9 on HIV using the same virus strain and measuring antiviral activity by reduction of virus titer as determined by reduction of P-24 HIV antigen.

The protocol and results of the first study follow: EFFECTS OF C31G ON HUMAN IMMUNODEFICIENCY VIRUS

Materials:

Virus: HIV-l strain 3B (5xl0⁵ ID50 units/ml)

Cells: SUP-ΤΙ cell line (CD4+ lymphoid cells which produce characteristic cell fusion with giant cells and syncytia when infected with HIV)

Protocol:

1. Serial fourfold dilutions of C31G in PBS (4.0% 0.004% and PBS alone as control) were prepared. pH was adjusted to 5.5.

2. Pooled aliquots of viral stock were prepared and C31G vas added at each concentration, at 1:9 detergent:virus ratio (1:10 detergent dilution). These vere incubated for either 2 or 45 minutes.

3. Serial fourfold dilutions of each virus/detergent mixture down to 1:4096 vere prepared.

4. 16.6 ul of each virus/detergent dilution from each series vere added to four replicate wells of sup-Tl cells (10⁴ cells in 150 ul volume; 1:10 detergent dilution followed by further fourfold dilutions).

5. The wells vere examined twice weekly for two weeks and each well was scored positive or negative for the presence of characteristic viral syncytia or non-syncytial ghost formation due to detergent lysis.

Note:

Virus vas exposed to C31G at a 1:10 dilution of

AP 0 0 0 3 2 7

- 17 initial concentration for either 2 or 45 minutes, followed by another 1:10 dilution and then serial four-fold dilutions for the entire period.

Cells were exposed to C31G at a 1:100 and then serial fourfold dilutions of initial concentration for entire period.

See Figure 7.

Swaplft-Z

The protocol and results of the second study follows:

Materials!

Cells: CEM cell line ATCC <119 T cell

Virus: ^HIVmb

Test compounds: Compounds C31G and N-9, supplied as 5% solutions, were filtered through a low binding 0.22 filter prior to diluting.

Protocol:

1. Serial 2 fold dilutions of compounds N-9 and C31G in PBS adjusted to pH 5.5 were prepared. The highest concentration vas 4% and the lowest was 0.03%. The initial concentrations were 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03%, as well as a control without drug. The control titration vas done twice.

2. 14 12 x 75mm sterile plastic tubes vith 0.9 ml each of concentrated virus (at least 1 x 10⁶ TCID⁵⁰/ml) were set-up. 0.1 ml of each compound dilution vere transferred to a tube (dilution of 1:10 of the initial drug concentrations).

3. Incubation was for 10 minutes at room temperature, and was terminated (or slowed) by diluting 1:10 in complete growth medium (on ice).

4. Additional serial 5 fold dilutions (7 dilutions, with the final dilution equal to 1:781250) in complete medium on ice were prepared.

5. 50 ul of the virus dilutions were added to quadruplicate wells of 96 well trays (U bottom) containing 3 X 10⁴ CEM cells in 50 ul.

BAD ORIGINAL

6. 60 minutes vere allowed for virus absorption. The cells vere washed twice by centrifuging the plates at 1500 rpm for 5 minutes and aspirating the supernatents. The final cell pellets were resuspended in 100 ul of medium and transferred to flat bottom 96 well trays. The results are shown in Figures 8 and 9.

Example 8

Formulations for Contraceptive/Antilnfective Preparations

Concentrate (CCon) comprises:

Lauryl Betaine (30% ai) 1000 pts

Lauramine Oxide (30% ai) 870 pts

Citric Acid monohydrate 69 pts

The above are stirred to a uniform solution. At a dilution of one part to 30, the composition should have a pH of 4.85 at the glass electrode. Putative concentration equal to 28.5% active ingredients (ai).

Example 9

Suppositories (inserts) for use with or without Condoms CCon (of Example 8) . 526 pts

PEG-32 4000 pts

PEG-20 1000 pts

Povidone 74 pts

The above are stirred to solution at 45*C. and injected into molds, cooled and ejected for packaging.

Example 10

Contraceptive Sponge

CCon (of Example 8) 3.5 gms

Added to each mold for curing urethane. Charge in mold provided vith polyester loop for post coital removal of the sponge.

Example_lA

Spermicidal Gels, Creams or Jellies for use with Cervical Caps Condoms, Diaphragms or alone prior to coitus.

Gel-1 CCon (of Example 8) 7.0 pts

Gelatin A200 1.5 pts

7.0 pts 1.5 pts

AP 0 0 0 3 2 7

- 19 Glycerine 10.0 pts

Hydroxyethyl Cellulose (high viscosity Grade) 0.4 pts

Water 81.1 pts

Gel-2 As above with the substitution of

1.0 pt of Nonoxynol-9 for 3.5 pts of CCon (of Example 8).

Procedure - The gelating and cellulose cum are triturated in the glycerine and added to the water at 45*C. and stirred to solution. The surfactants are added to the vessel and the warm solution removed for packaging. A uniform fluid high viscosity gel forms on cooling.

Jelly 1 and 2

Clear fluid contraceptive jellies are prepared by substitution of 1.5 pts of high viscosity grade hydroxypropyl or hydroxypropylmethyl cellulose for the gelatin and hydroxyethyl cellulose of the above examples.

Creme 1 and 2

The Gel formulations above are converted to Cream formulations by incorporation of 0.5 pts of cetyl alcohol in Gel 1 or 2 formulations by dissolving the alcohol in the glycerine at 49’C. before trituration of the gelating and cellulose gums.

Example 12

Contraceptive Film

6.2 lbs of gelatin Type A100 is triturated with 0.5 lbs of hydroxyethylcelluose. 31 lbs of glycerine are added. Solution is mixed thoroughly to form a slurry. Add 33 lbs of CCon of Example 8 and 15 lbs of water. Warm to 40*C. Mix until gums and gelatin are completely hydrated. Solution is poured on polyethylene sheet to cast a film of about 3 mm thick to be cut for films to be used as contraceptive films after cooling.

Claims

CLAIMS λ method of inhibiting activity of enveloped viruses comprising applying to a locus of the enveloped virus a composition comprising a mixture of a betaine and an amine oxide: said betaine being selected from an alkyl-Nbetaine, an alkyl-K-sulfobetaine, an acyl-K'-betaine, an alkyl N-substituted asinopropionic acid or an alkylimidazolinium betaine or a mixture of two or more thereof and said amine oxide being selected from an alkyl-N, N-dimethyl amine oxide, an alkyl-N, N-dihydrcxyethylamine oxide or an acylacide t-amine oxide or a mixture of two or core thereof, but excluding any medical method of treatment of the human or animal body.

1 - 11 , vherein the betaine and amine cxide composition is incorp-orated into or applied in the fora of a foam, gel, cream, salve, jelly, lotion, liquid, spray, mist, aerosol or wipe.

1 - 9, vherein the alkyl N-di(lover alkyl) betaine is cocctetair.e or lauryl-betaIne, and the alkyl N-di (lover alkyl) amine oxide is coccamine oxide or lauramine oxide.

1 - 8 , vherein the composition comprises:

a) an alkyl dimethyl glycine;

b) an alkyl dimethyl amine oxide and

c) acid in an amount sufficient to adjust the pH cf the composition from 4-8 vhen measured in an aqueous solution of components (a) and (b) in an acceptable carrier.

1-7, vherein the composition comprises alkyl N-di(lover alkyl) betaine, alkyl N-di(lover alkyl) amine oxide and acid in an amount sufficient to adjust the pH of the composition from 4-8 vhen measured in an aqueous solution in an acceptable carrier.

1-6, vherein the alkyl N-di(lover alkyl) betaines and alkyl N-di(lover alkyl) amine cxides are in a molar ratio cf from 1:5 to 5:1.

1-5, vherein the composition comprises an alkyl N-di (lever alkyl) betaine and an alkyl N-di(lover alkyl) oxide vhere the lover alkyl is C^-Cj.

2. A method of inhibiting activity of sperm comprising using as a spermicide a composition comprising a/ /'

SAX* AP Ο Ο Ο 3 2 7 mixture of a betaine and an amine oxide said betaine being selected iron an alkyl-N-betaine, an alkyl-N-sul fobetair.e, an acyl-N-betaine, an alkyl N-substituted aminopropionic acid or an alkyl imidazol inium betaine or a mixture of two or core thereof and said amine oxide being selected from an atkyl-N,N-dimethylamine oxide, an alkyl-N,Ndihydroxyethylamine oxide or an acylamide t-amine oxide or a mixture of two or more thereof, but excluding any medical method of treatment of the human or animal body.

3. A contraceptive device containing a composition comprising a mixture of a betaine and an amine oxide said betaine being selected from an alkyl-N-betaine, an alkyl-Nsulfobetaine, an acyl-N-betaine, an alkyl N-substituted aminopropionic acid or an alkylimidazolinium betaine or a mixture of two or more thereof and said amine oxide being selected from an alkyl-N, N-dimethylamine oxide, an alkyl-N, N-dihydroxycthylamine cxide or an acylaaide t-aaine oxide or a mixture of two or mere thereof.

4 r- 22

4. Use of a composition comprising a mixture of a betaine and an amine cxide; said betaine being selected frcm an alkyl-N-betaine, an alkyl-N-sulfobetaine, an acyl-Nbetaine, an alkyl N-substituted aminopropionic acid or an alkylimidazolinium betaine or a mixture of tvo or more thereof and said amine oxide being selected from an alkyl-N, N-dimethylamine oxide, an alkyl-N,N-dihydroxyethylamine oxide or an acylamide t-aaine oxide or a mixture of tvo or acre thereof for preparing a medicament for use in inhibiting the activity of enveloped viruses or for use as a spermicide.

5. A medicine for treating viral infections comprising a mixture of a betaine an,d an amine oxide; said betaine being selected froa an alkyl-N-betaine, an alkyl-Nsul fobeta ine , an acyl-N-betaine, an alkyl N-substituted aminopropionic acid or an alkylimidazolinium betaine or a mixture of tvo or aore thereof and said aaine oxide being selected froa an alkyl-N,N-dimethylanine oxide, an alkylN,N-dihydroxyethylamine oxide or an acylaaide t-aaine oxide or a mixture of two or more thereof.

BAD ORIGINAL

6. A method or device according to any one of claims

7. A method or device according to any one of claims

8. A method or device according to any one of claims

9. A method or device according to any one of claims

10. A method or device according to any one of claims

11. A method according to any one of claims 1, 2, or 4, vherein the composition is applied to a mucous membrane, to skin, to the vagina, to an inaminate surface or dispersed in air.

12. A method or device according to any one of claims

13. A method according to any one of claims 1, 4, or 5,vherein the virus is HIV, a virus associated vith

AP Ο Ο Ο 3 2 7 aids, HSVI, HSVII, Hepatitis A, Hepatitis B, Hepatitis C, vaccinia, varicella, herpes zester, cytcmega1ovirus, Epstein Barr Virus, Influenza, mumps, measles, rhinovirus, rabies or rubel1 a ,

14.

method according to any one of claims 1, 2, 410, wherein the composition is used vith or incorporated into a contraceptive device.

.

15.

A contraceptive device according to claim 3 wherein the composition is incorporated into a lubricant applied to a condom or the composition is part of a reservoir at the tip of a condom, the composition is incorporated into a contraceptive sponge, contraceptive film, sustained release device, suppository, foam, gel, cream or jelly.

16. A douche comprising a mixture of a betaine and an amine oxide said betaine selected from an alkyl-N-betaine, an alkyl-N-sulfcbetair.e, an acyl-K-beta ir.e, an alkyl Nsubstituted aminopropionic acid or an alkylimidazolinium betaine or a mixture of two or more thereof; and said omir.e oxide selected froa an alkyl-N,N-dieethylamine oxide, an alkyl-N, N-dihydrcxyethylaaine oxide or an acylamide t-amir.e oxide or a mixture of two or more thereof.

17. A wipe comprising a mixture of a betaine and an amine oxide said betaine selected from the an alkyl-Nbetaine, an alkyl-N-sulfobetaine, an acyl-N-betaine, an alkyl N -substituted aminopropionic acid or an a Iky1imidazolinium betaine and a mixture of two or more thereof; and said amine oxide selected from an alkyl-N, Ndimethylamine oxide, an alkyl-N,N-dihydrc xyethy1 amine oxide or an acylamide t-amine oxide cr a mixture of two or more thereof .

BAD OWGINAI

18.

- A cream comprising a mixture of a betaine and an amide oxide said betaine selected from an alkyl-N-betaine, an alkyl-N-sulfobetaine, an acyl-N-betaine, an alkyl N-substituted aminopropionic acid or an alky 1imidazoliniurr. betaine or a mixture of two or more thereof; and said amine oxide selected from an alkyl-N, Ndimethylamine oxide, an alkyl-N,Ν-dihydroxyethylamine oxide or an acylamide t-amine oxide or a mixture of two or more thereof.

19. A cream according to claim 20 further comprising gelatin having a Bloom strength of 100 - 300 and a molecular weight of about 75,000 to about 300000, a polyhydric alcohol and cellulose gums.