CA1177407A

CA1177407A - Antimicrobial agent

Info

Publication number: CA1177407A
Application number: CA000377816A
Authority: CA
Inventors: Peter Ladanyi
Original assignee: Vipont Laboratories Inc
Current assignee: Vipont Pharmaceutical Inc
Priority date: 1980-05-20
Filing date: 1981-05-19
Publication date: 1984-11-06
Also published as: SE8103152L; JPH0686380B2; CH645268A5; FR2482860A1; FR2482860B1; IT1136620B; AU548560B2; GB2078109A; DE3119746C2; AU7015681A; NL8102369A; DE3119746A1; JPS5756428A; IT8121849A0; GB2078109B

Abstract

ABSTRACT

An antimicrobial composition is disclosed which comprises a mineral acid salt of a benzophenanthridine alkaloid and a metal salt of an acid in a solvent.

Description

~l~774~7 BACKGROUND OF THE NVENTION

This invention relates to the composition and preparation of an antimicrobial agent which may be used in dental preparations, surgical and other soaps, various other topical preparations, injectable medicines, and other drug applications. In particular, the invention relates to com-positions containing mineral acid salts of benzophenanthridine alkaloids mixed with a metal salt, preferably a metal salt of a mineral acid, although salts of mono-or dicarboxylic acids, among others, may also be used.
One of the important sources of sanguinarine is a perennial herb native to North America called Sanguinaria canadensis Linne (Family: Papaveraceae~ commonly known as blood root, red root, puccoon, etc. The plant contains benzophenanthridine alkaloids including sanguinarine, chelerythrine, and several others. The major alkaloids present are sanguinarine and chelerythrine. The eighth edition of the Merck Index lists the alkaloids as sanguinarine, chelerythrine, protopine and homochelidonine. The pure chemicals sanguinarine, chelerythrine, and other benzophren-anthridine alkaloids can be isolated from other plants besides Sanguinar_a. Also, they are available, even though very rarely, from some chemical supply houses. Simi-purified forms of the alkaloids are commercially available, and these are generally referred to as sanguinarine nitrate and sanguinarine sulfate. These "salts" are the salts of the mixed alkaloids of the plant Sanguinaria: mainly sangui-narine, chelerythrine, and protopine. While few references can be found in the literature regarding the usage of any of the pure benzophenanthridine alkaloids, plants containing such compounds have been used for medical purposes for quite some time for a wide variety of ailments.
The principle use of sanguinarine up to rece~tly was a stimulant expectorant to cough syrups containing " sanguinarine nitrate" .

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~7~7~

The use of sanguinarine with thiophosphoric acid in various animal and human neoplasms is shown in French patents, number 70-22029 and 2,152,972.
The alkaloid sanguinarine in solution has been shown to have some antifungal and antiprotozoan properties. The sanguinarine is applied as an emulsion topically to fungal infections. The antibacterial activity of sanguinarine has been found to vary with the attached radicals, and various salts of sanguinarine have been shown to have some activity.
The hydrochloride and the sulfate salts have been found to have some activity against certain bacteria at various con-centrations. Sanguinarine nitrate is reported to have some weak bacteriostatic action on various types of bacteria.

SUMMAP.Y OF THE INVENTION
The present invention relates to the preparation and the use of antimicrobial agents, formed particularly from mineral acid salts of benzophenanthridine alkaloids and a metal salt and useful in dental preparations, mouthwashes, rinses, surgical soaps, shampoos, creams, lotionslpowders t injectables, etc., and other forms of drug preparation and disinfectants. The mineral acid salts of the benzophenan-thridine alkaloids may be used in various concentrations with a metal salt as an antimicrobial agent for use in treating both human and animal infections and diseases.
Metal salts particularly useful in the formulations of the present invention include metal salts of halogen acids.
Among these salts are zinc chloride, stannous fluoride, and sodium fluoride, although any metal salt can be used. This includes alkali, alkaline earth, and heavy metal fluorides, chlorides, bromides, and iodides.
Althouth non-toxic metal salts of halogen acids are preferred for use in the formulations according to the present invention, it has been found that non-toxic salts of other acids, such asmineral acids and mono-and dicar~
boxylic acids, are also effective. Examples of acids, 74~7 the non-toxic metal salts of which can be used, include sulfuric acid, nitric acid, and acetic acid.
Glycerol is the preferred vehicle of the formulations according to the present invention, although other vehicles that can be used include organic solvents such a propylene glycol, dimethyl sulfoxide ~DMSO), lower alcohols, and the like.
It is therefore, among the object and advantages of the present invention to provide an antimicrobial agent of mineral acid salts of a benzophenanthridine alkaloid and a metal salt, useful for topical administration, injectables and other forms of drug preparations.
Another object of the invention is to provide a benzophrenanthridine alkaloid salt-metal salt preparation for treatment of periodontal disease, prevention of dental caries and similar oral cavity impairmen's.
Still another object of the invention is to provide drug preparation of a benzophenanthridine alkaloid and a metal salt, uesful for treatment of ringworm infections, acne, cold sores and various parasitic infections.
Yet another object of the invention is to provide a drug preparation of a benzophenanthridine alkaloid and a metal salt, useful for treatment of scours in animals.
These and other objects and advantages of the invention may be readily ascertained by the following description and examples of the preparation of the invention.
The drug preparation of the present invention can be incorporated in dental preparations, toothpastes, mouth rinses, surgical and other soaps, vehicles for topical applications, vehicles for parenteral or intramuscular injection, and the like.

PREPARATION OF ANTIMICROBIAL AGENT
The pure chemical, either sanguinarine, chelerythrine, or other benzophenanthridine alkaloids, is dissolved in a chloroform/methanol mixture and acidified with a mineral ~1774(~7 acid such a hydrocholoric acid. The acidic mixture is evaporated to dryness and the residue is recrystallized from ethyl alcohol/chloroform, 50/50.
For use, the mineral acid salt of the benzophenan-thridine alkaloid is dissolved in either deionized water or Cl-C6 alcohols, glycerine, propylene glycol, petrolatum, or other organic solvents at 70 degrees C, and a metal salt or a salt-forming acid is added to the above solutions. The preparations generally contain 0.1% by weight and up to 20%
by weight of the benzophenanthridine alkaloid salt, and at least 1% and up to 60% by weight of a metal salt, with the remainder being the solvent. The material can be diluted to the desired concentration, depending on the type of use, with the solvents listed above~
The benzophenanthridine alkaloid salt is used in preparations containing 0.01%-10% benzophenanthridine by weight. The metal salt is present in amounts ranging from about 2% to about 60%. The lower concentrations are generally effective in the treatment of most diseases as explained below.
An example of a basic preparation:
sanguinarine chloride0.3%
glycerine U.S.P. 64.7%
zinc chloride AR. 35.0%
The basic preparation can be varied by using, in place of sanguinarine chloride, 0.3% of another mineral acid salt of a benzophenanthridine alkaloid, suchas chelerythrine chloride.
A second example of a preparation is:
sanguinarine chloride1.0%
glycerine U.S.P. 96.0%
zinc chloride AR. 3.0%
A third example of such a preparati~n is:
sanguinarine chloride 1.0%
glycerine U.S.P. 64~0%
zinc chloride AR. 35.0%

~77~

A fourth example of such a preparation for dental use is:
sanguinarine chloride1.0%
glycerine U.~.P. 95.6%
zinc chloride AR. 3.0%
stannous fluoride 0.4%
Additional formulations for a basic preparation are as follows:
sanguinarine chloride1.0%
stannous fluoride 0.4%
glycerine, U.S.P. 98.6%
and sanguinarine chloride1.0%
sodium fluoride 3.0%
glycerine, U.S.P. 96.0%
Numerous types of diseases were treated in humans and in animals with the composition of the present invention as follows:
Example 1 - Canine Ringworm A 5-10% solution of the first basic preparation was used, applièd directly to the infected area, one to three applications as indicated, 48 hours apart.
Example 2 - Feline Ringworm A 4-8% solution of the first basic preparations was applied directly to the infected area, 48 hours apart up to three applications.
Example 3 - Bovine Ringworm The etiological agent of this involvement is usually the mold known as Trychophyton album. The duration of the disease is 4-12 months.
A 30% dilution of the first basic preparation is used, applied directly to the involved areas, 48 hours apart. Three applications proved to be adequate in treating the condition successfully.

Example 4 - Bovine Neo-natal Diarrhea Twelve animals having diagnosed and confirmed neo-natal diarrhea were treated with 0.75 grams of the first basic preparation orally, once per animal, and showed clinical cure with one exception.
This constitutes an excellent result, considering that conventional antibiotic therapy currently in use has a much lower percentage of success.
Sanguinarine chloride and chelerythrine chloride have strong antimicrobial properties. Zinc chloride has anti-microbial properties only in high concentrations. It can be seen from Table I that sanguinarine chloride mixed with zinc chloride in a l:l ratio, as a rule, did not show a synergistic effect or even an additive action against most microorganisms tested n vitro. Further, it was found that in most cases .the antimicrobial action of the sanguinarine chloride and zinc chloride mixture depended mostly on the amount of sanguinarine chloride present in the mixture, and was relatively independent from the amount of zinc chloride.
TABLE I
Mean inactivating do~s in micro-grams per milliliter (ug/ml) of media.
Microorganism ZnCl2 Sanguinarine ZnCl~,& Sang-Chloride uin ine Chloride (l:l) Bacillus subtilis25,000 22 1,000 Escherichia coli6,250 270 500 Klebsiella pneumonia 3,125 540 l,000 Proteus vulgaris12,500 590 l,000 Staphylococcus aureus6,250 70 500 Streptococcus faecalis25,000 393 500 Streptococcus mutans 1,563 161 63 Candida albicans 150 Saccharomyces cerevisiae6,250 20 63 Pseudomonas aeruginosa3,500 7,000 400 ~774C~7 A separate test was conducted to determine the inhibitory concentration of sanguinarine chloride alone.
These concentrations, for microorganisms ln v tro, are as follows:
100 micrograms per milliliter for Escherichia coli 100 micrograms per milliliter for Candida albicans 50 micrograms per milliliter for Streptococcus mutans 10 micrograms per milliliter for Staphylococcus aureus It was further found that a concentration of sangui-narine chloride of 25 micrograms per milliliter caused a 100%
reduction of dental plaque by inactivating plaque forming microorganisms freshly collected from human dental plaque~
Sanguinarine Chloride compared favourably in vitro to chlorhexidine (Hibitane ~ , a material used as a standard in evaluating inhibition of human dental plaque forming micro-organisms.
However, under in vivo test conditions, sanguinarine chloride was found to be ineffective against plaque forming microorganisms.
When sanguinarine chloride was applied to the affected area of both dogs and humans, repeated treatment with sangui-narine chloride alone did not reduce dental plaque or alleviate the symptoms of gingivitis or periodontal disease.
Continuous treatment with sangunarine chloride did not prevent the accumulation of dental plaque on teeth or prevent the occurrence of periodontal disease. However, it has been found that a combination of sanguinarine chloride and zinc chloride in glycerine is effective in vivo in reducing dental plaque and the incidence of periodontal disease, and has shown definite promise in the treatment and prevention of human periodontal disease.
Results of tests on guinea pigs with induced ringworm infection, dogs with periodontal disease, and humans with periodontal disease have shown that glycerine preparations of sanguinarine chloride plus zinc chloride are far superior for ~.

~77~

the management of infections ln vivo than either zinc chloride or sanguinarine chloride alone.
When zinc chloride was replaced with a fluoride salt in the present preparations, the activity of the preparation against microorganisms believed to be associated with the causation of dental caries and periodontal disease ~Stre~-tococcus mutans), as well as other microorganisms, remained identical. Data supporting this phenomenon is presented in the following tables:
TABLE II
Minimum Inhibitory Concentrations (MIC) in ug/ml AEROBIC 1.0% sanguinarine 1.0% sanguinar-MICROORGANISM chloride ine chloride TESTED 3.0% zinc chlor- 0.4% stannous ide in glycerol fluoride in -- glycerol Escherichia coli 158 160 Klebsiella pneumoniae 158 160 Proteus Vulgaris Streptococcus mutans79 80 Streptococcus faecalis ~ 20 c20 Staphylococcus aureus ~20 ~20 Pseudomonas aeruginosa 630 1,280 Saccharomyces cerevisiae ~ 20 c20 Candida albicans 79 ~20 . . _ '~.

~77~(17 TABLE III
Minimum Inhibitory Concentrations (MIC) in ug/ml 1.0% sanguinarine 1.0% sangui-ANAEROBIC chlorîde narine chloride MICROORGANISMS 3.0% zinc chlor- 0.4% stannous TESTEDide in glycerol fluoride in glycerol Bacteroides melanino-genicus 8 8 Eikenella corrodens32 32 . . .
Actinomyces viscosus8 8 Actinobacillus actinomycetemcomitans 16 16 Capnocytophaga ~ingivalis 8 8 Capnocytophaga sputigena 8 8 TABLE IV
Minimum Bactericidal Concentrations (MBC) in ug/ml 1.0% sanguinarine 1.0% sanguinarine chloride chloride MICROORGANISMS 3.0% zinc chlor- 0.4% stannous TESTEDide in glycerol fluoride in glycerol Escherichia coli 158 160 Klebsiella pneumon1ae 1,261 640 Proteus vulgaris2,522 640 Streptococcus mutans 158 160 Streptococcus faecalis 315 160 Staphylococcus aureus 158 80 Pseudomonas aeruginosa 2,522 2,560 ~ ~ =. = . . . . =
Saccharamyces cervisiae 20 20 Candida albicans 79 160 _ g _ X .

11774(~7 The above phenomenon is not what would be expected in light of the in vitro test results. In most cases sanguin-arine chloride in vivo was only slightly effective or not at all. This was quite unexpected considering that in vitro all the antimicrobial activity of a sanguinarine chloride-metal salt mixture could be explained by the amount of sanguinarine present in the mixture.
Controlled clinical tests on 24 male beagle dogs showed that after four weeks of treatment the dogs treated with the zinc chloride-sanguinarine chloride-glycerine mixture had the lowest plaque and gingivitis scores, while the dogs treated with sanguinarine chloride alone had the highest. The dogs were treated topically once daily with the respective test formulation.
The results presented in Table ~ clearly indicate that the sanguinarine chloride-zinc chloride in glycerine treated group of dogs had lower gingivitis scores after four weeks of treatment than did the other groups. Zinc chloride alone was slightly active, but sanguinarine chloride alone showed no activity _ vivo at all. This result is unexpected, con-sidering that, ln vitro, sanguinarine chloride is quite effective against microorganisms.

~17~74~7 TABLE V
Oral Clinical Studies - Beagles (dogs) After Treatment 0 (start) 4 weeks Mean Ginyivitis Scores None 0.475 0.91 0.1% Sanguinarine chloride 0.495 0.942

2.7% Zinc chloride 0.44 0 75 0.1% Sanguinarine chloride with 2.7% zinc chloride in glycerine 0.418 0.548 Mean Plaque Scores None 7.478 12.408 0.1% Sanguinarine chloride 8.885 12.89 2.7% Zinc chloride 8.76 10.15 0.1% Sanguinarine chloride with 2.7% zinc chloride in glycerine 8.578 7.407 Mean Pocket Depths None 1.459 1.458 0.1% Sanguinarine chloride 1.415 1.48 2.7% 2inc chloride 1.46 1.37 0.1% Sanguinarine chloride with 2.7~ zinc chloride in glycerine 1.445 1.44 Similar results occurred, when the dogs were evaluated for the return of dental plaques (See Table V for plaque scores), sanguinarine chloride alone was not active in vivo.
Zinc chloride was slightly preventive but the preparation of sanguinarine chloride-zinc chloride in glycerine not only prevented the return and further proliferation of dental plaque but significantly further reduced it during the four weeks of treatment.

~7~4Q~

Data also indicate that even the pocket depth.s were reduced somewhat by the treatment materials containing zinc chloride in glycerine, or zinc chloride-sanguinarine chloride in glycerine.
In a clinical test involving twenty volunteer patients with periodontal disease, it was observed that after treatment with benzophenanthridine alkaloid chloride-zinc chloride in glycerine, there was rapid improvement. Inflammation, in-fection and pockets were eliminated, abcesses ceased, gingival tone greatly improved, ~issues healed, and in some cases normal tissue was restored and teeth mobility was reduced.
The clinical studies above showed that sanguinarine chloride in vlvo was only slightly antimicrobial, very slow acting, or had no effect on the course of the infection at all.
Zinc chloride in the concentrations required ln vivo to have antimicrobial action caused blanching of tissue and, in some cases, chemical burns and other tissue damage. Further, zinc chloride was found to be slow acting in vivo as an anti-microbial agent.
Glycerine preparations containing sanguinarine chloride or other benzophenanthridine alkaloids and zinc chloride or stannous fluoride or sodium fluoride were fast acting, re-quiring only one to three applications to clear out infections rapidly. Further, these preparations did not appear to have the undesirable side effects of the zinc chloride in the con-centration necessary to achieve antimicrobial effectiveness.
Example 5 - Human Periodontal Disease It has been reported that periodontal (gum) disease affects 2 out of 3 middle-aged ~mericans. Destruction of the tissue and structures that hold teeth fast in their sockets accounts for 75% of tooth loss after the age of 40. Most cases of periodontal disease are the result of neglect and can largely be prevented by a regular program of thorough hygiene. In the most common type of periodontal disease, the three chief culprits are bacteria, calculus (tartar), and food debris.

X

~'774(~7 The invention has been used by dentists in clinical management of over forty cases of various types of human periodontal disease.
In some cases, even a single treatment brought major improvements in the conditions of the diseased gums. Clinical cure resulting from the treatment was quite apparent and included: elimination of inflammation, normal tissue tone restored, pockets were eliminated, infections were cleared up, mobility was reduced, and gingival tone was greatly improved.
The materials and methods used were the following:
1. Undiluted Paste for Packing:
In cases of widespread tissue involvement, undiluted preparation was used in quantity sufficient (q.s.) to "cover"
or "pack" the infected or inflamed areas of gingival tissues~
The clinical procedure consisted of two treatments approximately two weeks apart, with the application of not less than l.0 mm thickness of the basic preparation to the diseased periodontium. In cases where undiluted material was used, it was either applied topically with a spatula or 0.25 ml was pressed through a 22 guage needle attached to a 1.0 ml pressure syringe. The material was introduced into the ging-ivae to the attachment (q.s.) to fill the pocket and the medication was left in place for 10-15 minutes.
2. String Technique:
Cotton String Saturated with the Drug Preparation.
In cases where individual teeth were to be treated, an ordinary soft cotton string, sterilized before use, or "gingipak" was used (gingipak contains racemic epinephrine hydrochloride 8-100 solution and 1% benzyl alcohol as preservative).
Cotton strings 1-1.5 cm in length were saturated with undiluted material by using a spatula and precut pieces of string which were then impregnated on a dentist's mixing pad.

~7~07 These strings, when properly impregnated with the preparation, weigh approximately 35 mg/cm.
One to three strings were used per tooth depending upon the circumference of the tooth. Up to three strings were some-times used for a total of approximately 10.5 mg of the pre-paration. Inpregnated strings were left in place 10-15 minutes.
Dental floss or similar substrate, such as synthetic hollow fiber string, can be impregnated with the basic pre-paration for use in treating the teeth and gums.

3. Dilution of Paste for Irrigation In addition to "packing" of the periodontium or employ-ing the "string technique" on individual teeth, irrigation was often used concurrently as part of regimen. Generally, the diseased periodontium was first packed with undiluted pre-paration or the teeth were individually treated by the string technique described above. These methods of treatment were followed by irrigation with a suspension of the preparation in either water or glycerine. Suspensions were prepared to contain 1 part of full strength material to 1 part of gly-cerine, or 1 part of water depending on whether a glycerine or water suspension was desired. The final suspension was V/V
mixture of 1 part material to 1 part diluent. Irrigation was accomplished by filling a 7.0 ml syringe to contain 420 mg of the suspension. For treatment of individual teeth, a total of 1.0 ml of material in suspension was used to irrigate the buccal, lingual, and interproximal areas about the tooth.
Where indicated, all teeth were irrigated with 840 mg of the suspension contained in two syringes.
Example 6 - Dental Caries Preparations containing about 0.3~ sanguinarine chloride and about 35% zinc chloride were used on seven patients with dental caries. Decay was removed from the teeth with a spoon excavator leaving a layer of carious ~3L7~9~0~

tissue about 1 mm to 1 1/2 mm in depth. The antimicrobial preparation was placed over the remaining decay, about 56.1 mg of preparation, with a ~ollenbeck carver and uniformly applied over the decayed area with a piece of cotton held in cotton tweezers. Intermediate restorative material (IRM) was used as a temporary restoration to seal the material in the cavity preparation.
After several weeks (6 weeks) specimens for bacteri-logical and histological studies, including electron micro scopy, were undertaken.
The conclusions of the investigators were that the preparation could be considered a cariostatic agent. Further, the material may enhance sclerotic dentine formation, thus forming a hard protective floor between the carious lesion and the pulp.
The benzophenanthrine chloride-zinc chloride-glycerine composition can be used in conjunction with a fluoride-providing compound. These compounds are characterized by the ability to release fluoride ions in water and by sub-stantial freedom from reaction with other compounds present in the oral preparation. Among these materials are inorganic fluoride salts such as suitable alkali metal, alkaline earth metal, and heavy metal salts. Alkali metal and tin fluorides, such as sodium and stannous fluorides, and mixtures thereof, are preferred.
The amount of the fluoride-providing compound is dependent to some extent upon the type of compounds, its solubility, and the type of oral preparation, but it must be a nontoxic amount. Any suitable minimum amount of such com-pound may be used, but it is preferable to employ sufficient compound to release from 0.005~ to 1~, most preferably about 0.1% by weight of fluoride ion. Typically, in the cases of alkali metal fluoride and stannous fluoride, this component 1~774~7 is present in an amount up to 3% by weight, based on the weight of the preparation, and preferably in the range of from 0.05% to 1%.
Example 7 - Treatment of Animal Skin Tumors Equine sarcoid (a locally malignant connective tissue tumor found on the skin of horses similar to fibrosarcomas which occur in other animals and man) are probably of viral origin transmissible between horses. These tumors are in-vasive and commonly result in sufficient debilitation of the horse that euthanasia is performed.
Surgical removal is the accepted means of therapy.
Recurrence of the tumor is the rule rather than the exception.
Sixty tumors were treated with the preparation employing the following treatment method:
~ Either spread the first basic preparation on the tumor with a wooden applicator, bandage and observe in 24 hour intervalsf~or place about 3 mm thickness of the preparation on a Telf~3pad covering an area slightly larger than the base area of the tumor, securing the Telfa pad on the tumor with a bandage. Observe in 24 hour intervals. Repeat appli-cation if needed after 48 hours.
Three to four or more applications may be necessary in cases of long duration and if the tumor is deep. Recurrence of the tumor occurred in only 25% of the cases.
In all cases, the tumors were removed by the excising action of the preparation and in no case thus treated did a post treatment infection (secondary infection) develop. This was true despite the fact that no covering was maintained on the lesions after the tumor came out, nor was any form of other antibacterial medication applied.
After the second or third application, the tumor sloughed out, a very firm scab formed on the tumor base, and healing proceeded under scab. The healing of the would was slow, but uneventful. Scars remaining were quite small.

~L1774~7 Example 8 - Equine Dermal Fibrosarcomas Seventy-five cases of equine dermal fibrosarcomas were treated topically as described in Example 7. Only 20~ of the tumors recurred within the one year observation period. While the cure rate is somewhat less than 80~, this compares favorably with the approximate 55% cure rate of this tumor following surgical removal and radiotherapy.
Example 9 - Bovine Squamous Cell Carcinoma Seven Hereford cattle diagnosed with the disease were treated with about 15 grams of the first basic preparation pasted right over the lesions. Improvement of clinical con-dition followed in all cases.
Example lO - Treatment of Squamous Cell and Basal Cell Carcinoma in Animals of Non-bovine Species Canine - Histologically confirmed squamous cell car-cinoma of both eyes, five applications with the first basic preparation in five days; in two weeks time, tumors reduced in size and were sloughing.
Equine - Histologically c~nfirmed basal cell carcinoma was treated by six direct topical applications of the pre-paration locally, once daily. In three weeks, most visible tumors were gone.
Equine - Histologically confirmed large hair follicle carcinoma on chest; tumor removed by surgery; ten topical applications of the preparation into the open wound. In three months lesion healed and no recurrence of the tumor was noted.
Example ll - Treatment of Human Skin Tumors ~ ascaI Cell Epithelioma:
A team of qualified dermatologists and surgeons treated sixty patients with diagnosed histologically confirmed basal ~'7~407 cell epithelioma (basal cell carcinoma).
The first basic preparation was directly applied topically to the involved area and held in place with a bandage. The lesions were observed in 48 hour intervals and the preparation was reapplied if it was indicated. In most cases, 2-3 applications were adequate. Clinical cure followed the treatment in all cases and no reappearance of the tumors was noted over a four year period.
Example 12 - Squamous Cell Carcinoma Sixteen cases of histologically confirmed squamous cell carcinoma were treated by the same method used in Example 11.
In all treated cases, clinical cure followed treatment and only in one case was tumor recurrence noted within four years.
Example 13 - Antimicrobial Activity - In order to illustrate the an~imicrobial effectiveness of the composition according to the present invention, a series of _ vitro and in vivo tests were performed. These tests were designed to demonstrate the lowest concentration of sanguin-arine chloride that will inactivate microorganisms.
Findings were as follows:
On the average a 22 ug/ml sanguinarine chloride solution will inactivate cultures of the bacteria Bacillus subtilis, a 270 ug/ml solution cultures of Escherichia coli, a 540 ug/ml solution cultures of Klebsiella pneumoniae, a 590 ug/ml solution cultures of Proteus vulgaris, a 70 ug/ml solution cultures of Staphylococcus aureus, a 393 ug/ml solution cultures of Streptococcus faecalis, and a 161 ug/ml solution cultures of Streptococcus mutans (see Table I).
Further on the average a 150 ug/ml and a 20 ug/ml solution also inactivates the cultures of yeasts Candida albicans and Saccharomyce_ cerevisiae. A considerably lower ~L~77~(~7 concentration than the inactivation concentration of the drug preparation is useful as a growth inhibitor of the same organism.
In addition, on the average a concentration of san-guinarine chloride presented in Table VI was sufficient per cubic centimeter of media, to inhibit growth of some fungi known to belong to the group of ringworm producing organisms.

TABLE VI
Mean Inhibiting Dose of Sangui-Microorganisms narine Chloride in ug/ml of Media Microsporum canis 867 Microsporum nanum 650 Trichophyton mentagrophytes 900 Trichophyton schoenleini 467 Trichophyton terrestre 467 . _ _ Trichophyton vanbreuseghemi 750 The basic preparations were, on numerous occasions, also tested on a variety of animals and also humans infected with ringworm and athletes foot fungus. It was found that preparations according to the present invention will consid-erably hasten recovery from ringworm infection and facilitate rapid healing of lesions.
Results of clinical tests run by numerous dentists and dental institutions confirm that regular application of the composition of the instant invention to teeth surfaces will reduce the incidence of dental caries and the use on gums either by packing or irrigations will prevent or rapidly cure periodontal disease or microbial infections of the gums and surrounding tissues.
Application of the compositions of the present in-vention to cold sores immensely hastens the healing process.
Cold sores dry up and heal in a few days.

~74~7 When animals suffering from scours are treated in-ternally with preparations according to the present invention, the recovery rate exceeds that of the antibiotics commonly used for the purposes of treating scours.

.,,,~ ~

Claims

WHAT IS CLAIMED IS:

1. A method for preparing an antimicrobial agent comprising a. Dissolving a benzophenanthridine alkaloid in a mixture of chloroform and methanol;
b. Acidifying the solution with a mineral acid to convert the alkaloid to an alkaloid salt;
c. Evaporating the acidic solution to dryness;
d. Recrystallizing the residue from a mixture of 50% ethanol and 50% chloroform;
e. Dissolving the crystals in a solvent to make a solution of at least 0.3% by weight of crystals;
f. Mixing the solution with at least 35% by weight of a metal salt of an acid.

2. The method of claim 1 wherein the solvent is selected from the group consisting of water, glycerine, propylene glycol, petrolatum, dimethyl sulfoxide, and C1-C6 alcohols.

3. The method of claim 1 wherein the benzophenanthridine alkaloid is selected from the group consisting of chelerythrine, sanguinarine, protopine, and homochelidonene.

4. The method of claim 1 wherein the alkaloid is chelerythrine.

5. The method of claim 1 wherein the alkaloid is sanguinarine.

6. An antimicrobial composition comprising a mineral acid salt of a benzophenanthridine alkaloid and a metal salt of an acid in a solvent selected from the group consisting of water, glycerine, propylene glycol, dimethyl sulfoxide, and C1-C6 alcohols.

7. The composition of claim 6 wherein the benzophenan-thridine alkaloid is selected from the group consisting of chelerythrine, sanguinarine, protopine, and homochelidonene.

8. The composition of claim 7 wherein the alkaloid is sanguinarine.

9. The composition of claim 6 wherein the solvent is glycexine.

10. The composition of claim 9 wherein the mineral acid salt is sanguinarine chloride.

11. The composition of claim 10 wherein the metal salt is a fluoride salt selected from the group consisting of stannous fluoride and sodium fluoride.

12. The composition of claim 11 wherein the fluoride salt is stannous fluoride.

13. The composition of claim 9 wherein the metal salt is zinc chloride.

14. The composition of claim 6 wherein the alkaloid is chelerythrine.

15. The composition of claim 6 wherein the mineral acid salt is chelerythrine chloride.

16. The composition of claim 13 including 0.1-3% of a fluoride salt selected from the group consisting of stannous fluoride and sodium fluoride.

17. An antimicrobial composition comprising a mineral acid salt of a benzophenanthridine alkaloid and a non-toxic metal salt of an acid in a suitable solvent vehicle.

18. The antimicrobial composition of claim 17 wherein the non-toxic metal salt is selected from the group consisting of fluorides, chlorides, bromides, iodides, sulfates, nitrates, and acetates.

19. The antimicrobial composition of claim 17 wherein the benzophenanthridine alkaloid is selected from the group consisting of chelerythrine, sanguinarine, protopine, and homo-chelidonene.