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Methods for Testing Effects of Fungistatic Compounds in Lacquer1
METHODS FOR TESTING EFFECTS OF FUNGISTATIC COMPOUNDS IN LACQTJER* LACQUER* RUDOLF H. KADEN, M.D.f
Most dermatomycoses and particularly onychomycosis are often very resistant to therapy. Moreover, successful treatment of the latter infection becomes more important because of its high incidence. White (1), for example, has considered 20 per cent of all nail disturbances to be of fungus origin. As a method of treatment for onychomycosis Rothman (2) and Langer and and Kaden Kaden (3) (3) have have used used nail nail
lacquer containing antimycotie antimycotic substances. Further investigation of certain aspects of the inhibitory effect of antimycotic antirnycotic lacquers is presented in this paper. MATERIALS AND METHODS METHOD5
An antifungal agent, halogenocyclohexane' was added to a nitrocellulose finger nail lacquer in a final concentration of 2 per cent. The anti-fungal effect of this was compared compared with a lithium bromide304) was compound in lacquer (Preparation (Preparation q 304) Asterol® base lacquer, (Ho), (Ro), proposed by Rothman Rothman (2) (2) and and with with aa common commonnail nail lacquer (B) to which no fungistatic agent had been added. The fungi used in the various tests included Penicillium Penicillium glaucurn, glaucurn, Aspergillus Aspergillus fumigatus, fumigatus,Trichophyton Trichophyton rubrum, Micros porumAudouini, Audouini,Epidermophyton Epidermophytonfloccosum, floccosum,and andCandida Candida Microsporum albicans.
antimycotic Three test methods were used to demonstrate the effect of the antimyeotio lacquers. (1) Slide film method: This method was described by Kaden (4). A strip of lacquer film is applied to the width of a sterile mieroslide microslide and allowed to dry for two hours. Molten Sabouraud's glucose agar was inoculated with the fungus to be tested and, with a sterile pipette, two strips of this this agar agar was was run run the the length length of of the theprepared preparedslides. slides.The Theslides slideswere were placed in Petri dishes for incubation at room temperature. Inhibition was determined by the extent of clear zones, in mm., from the lacquer strips. (2) Disc Method: This method method was was based based on on that that used usedby byRuggeri Ruggeri(1950) (1950)(5) (5)for fortesting testing the fungicidal activity of varnishes and lacquers. A Petri dish of Sabouraud's glucose agar was inoculated evenly over the entire surface with a suspension of the fungus to be tested. Lacquer samples were painted on one side of 3-" cover glasses which were used as discs. The lacquer film was allowed to dry for two hours after which the covers were placed lacquer
side up on the agar surface. The Petri dish cultures were incubated at room temperature and observed for a period of teu ten days. Inhibition of growth was determined by the size of the inhibitory zone, in mm., around the covers. Inhibition under the covers was noted also and termed an additional effect. (3) Evaporation test: Petri dishes were filled with enough Sabouraud's glucose agar (approx. 60 ml) to leave a 5 mm. space between the agar surface and the cover. The agar * The experimental investigation has been completed and the paper was written during a Fulbright Fellowship, spent with Dr. Norman F. Conant, Professor of Mycology, Department of Bacteriology, Duke University School of Medicine, Durham, North Carolina. t From the Hautklinik der Freien Universitaet, Berlin, Germany (Director: Dr. Erich Langer, Professor of Dermatology).
Received for publication August 8, 1955. 'Antimycotic-Lahopharma, Antirnycotic-Lahopharma, Lahopharma, Labopharma, Berlin, Germany. 221 221
222
THE JOURNAL JOURNALOF OFINVESTIGATIVE INVESTIGATIVEDERMATOLOGY DERMATOLOGY
surface was inoculated evenly with a suspension of the fungus to be tested. The entire inside of another Petri dish cover was painted with lacquer which was allowed to dry for two hours. This lacquered cover was placed on the test Petri dish. The results of inhibition, by continuing vaporization, was determined after an incubation period of four to ten days at room temperature. RESULTS RESULTS
Nail lacquer preparation 304 showed good inhibitory activity against a variety of fungi in the three tests described above. In the slide film test this lacquer suppressed growth of Penicillium glaucum, during a seven days incubation period for a distance of 6.5 mm. along the agar strip. Under the same conditions a similar effect was obtained with a nail lacquer (Ro) containing lithium bromide and Asterol®. Growth of the fungus in agar strips was not inhibited on slides "striped" with common nail lacquer (B) and on control (C) slides (figure 1).
The inhibitory effect of preparation 304 against Aspergillus fumigatus is demonstrated in figure 2. In this figure will be seen a wide zone of inhibition surrounding the cover glass previously painted with lacquer containing the antifungal agent. Fungus growth was also inhibited for some distance beneath this cover glass. No inhibition was noted around the cover glass painted with common lacquer or in the area covered by a clean unpainted cover glass.
The effect of preparation 304 on the growth of a variety of fungi using the "disc" method is shown in Table I. In this table it can be seen that the growth of T. rubrum, M. Audouini fioccosum was markedly inhibited in the presence Audoulni and and E. E.fioccosum of preparation *' 304. Complete inhibition of M. Andouini, T. rubrum, B. fioccosum, floccosum, C. albicans and ill. Avdouini,
FIG. 1. Slide film test method. method. Penic'iltium PenicilUum glaucum, glaucum, 77 days daysincubation. incubation.3O4 3O4}JalogenoIlalogenocyclohexane in iii lacquer; Rb Lithium bromide Asterol® in lacquer; B. Lacquer only; C. Control slide. slide.
223
FUNGISTATIC COMPOUNDS IN LACQUER
—C C
a
iv
304 3 Pt Aspergillus furnigalus,55days daysincubation incubationshowing showinginhibition inhibitionof of FJG. 2. Disc test method. Aspergillusfumigatos, FoG. 304 3O4Halogenocyclohexane Halogenocyclohexanein inlacquer; lacquer;B. B.Lacquer Lacqueronly; only;C. C.Control Controldisc. disc.Note Noteinhibition inhibition under "disc" in in 304. 304. under cover glass "disc"
TABLE I Zone of inhibition in millimeters in the areas adjoining the discs Samples applied applied to to the thediscs discsG8" (8" cover glasses)
Test fungus Preparation 304
Common nail lacquer
Trichophyton rubrum ]Iuicrosporu?n Audonini Audouini ]luicrospornm
15 14
Epidermophyton floccosum Epidermophylon flocco.vm...... Aspergillus Asper.qillus furnigatus fuinigatus
20
0 0 0 0 0
Candida albicans
8 3
Control
0 0 0 0 0
A. fumigatus was observed in Petri dishes when the inside of the cover had been painted with preparation 304. This effect is illustrated in figure 3. In figure 3 it can be seen that JVI. 111. Audonini failed to grow when the inoculated Petri dish
culture was covered with a cover painted on the inside with the antifungal lacquer. Growth was not inhibited in the control culture or in the culture covered with common nail lacquer. Growth of common contaminants such as Penicillium sp., (]andida sp. and bacteria were also inhibited in this test.
224
THE JOURNAL JOURNALOF OFINVESTIGATIVE INVESTIGATIVEDERMATOLOGY DERMATOLOGY
FIG. 3. Evaporation test. Microsporurn Audouini, 77 days days incubation. incubation. 3O4 304 Under Microporurn Audowini, surface of cover painted with Halogenocyclohexane lacquer; B. Painted with lacquer only; C. Control. DISCUSSION
The halogenocyclohexane derivative used in the experiments described above is chemically related to some of the known effective fungistatic agents. In the pentachlorphenol, in Germany, Rieth (6) United States, Ruggeri (5) mentioned pentachiorphenol, tested isomeres of hexachlorcyclohexane hexachiorcyclohexane and in France, Nedey (7) described tn-, tetra-, and pentachlorphenol as fungicidal agents. Attempts to incorporate these chemicals in nail lacquer, however, have not proved successful. Hexachiorcyclohexane Hexachlorcyclohexane and its derivatives was shown to act against insects, bacteria or fungi depending on a single change in their isomere structure (Czyzewski One of its derivatives, halogenocyclohexane, zewski (8) (8) and and Green Green (9). (9).One halogenocyclohexane, was was shown shown to have good fungistatic properties and it was also found found to to be be easily easily incorporated incorporated
into a nitrocellulose nail lacquer. Its fungistatic effect is dependent on the slow evaporation of the compound from the lacquer base. It can be used to inhibit immediately spore germination or, as Grimmer (10) has has shown, shown, itit can can be be used used to to inhibit further growth of a culture after varying periods of incubation. Cultures thus treated can be used for teaching and demonstration purposes. In addition to the laboratory tests with so-called fungistatic lacquers such materials have also been used in therapeutic tests. Rothman (2) reported good results in the treatment of onychomycosis with an antimycotic lacquer containing lithium bromide and Asterol®. However, poultices soaked in lithium bromide solution and special sprays were added to the therapeutic scheme. Kaden (3), using only halogenocyclohexane in lacquer (Preparation reportedsatisfactory satisfactoryresults results in in the the treatment treatment of skin and nail ation , 304)2 304)2 reported infections. Meyer (11) also reported successful clinical trials with the halogenocyclohexane derivative in mycotic infections of the skin. Preparation 3O4 is identical with "Mykotektan", Labopharma, Berlin, Germany and "Tektopal . .", Philadelphia Ampoule Laboratories, Philadelphia, Pennsylvania.
FUNGISTATIC COMPOUNDS IN LACQUER PUNGISTATIC
225
CONCLUSION
Halogenocyclohexarie derivative, incorporated in a nitrocellulose Halogenocyclohexane nitrocellulose nail nail lacquer lacquer T. rubrum, showed marked inhibitory effects effects against against M. M. Audouini, Audovini, T. fioccosum, rubrum, E. floccosum, C. albicans, Penicilhium Penicillium glaucum glaucum and and Aspergillus fumigatus. The fungistatic activity was due to a permanent evaporation of the effective agent as shown by three in vitro test methods. REFERENCES 1. WHITE, CLEVELAND, J.: Diseases of nails; 312 cases. Amer. Pract. Digest Treatm., 6: 226, 226, 1955. 1955. 2. 2.
disturbancesinin recalcitrant recalcitrant trichophyton trichophyton rubrum rubrum infections. infections. Systemic disturbances ROTHMAN, S.: ROTHMAN, S.: Systemic Arch. Dermat. & Syph.,
67: 239, 1953.
3. LANGER, UND KADEN,R.: R.:Lackfoermige Lackfoermigefungistatische fungistatische Wirkstoffe. Wirkstoffe. München KADEN, Münchenmed. med. 3. LANGER, E.E.UND
96: 674, 674, 1954. 1954. Wchnschr., 96: Wchnschr., 4. KADEN, R.:Antimykotiseher AntimykotischerLack LackininKlinik Klinik und und Experiment. Experiment. Zschr. Haut-Geschl. Haut.-Geschl. KADEN, R.: method. Paint Varnish Prod., krkh., 17:209, 209,1954. 1954.Testing Testingfungicides fungicidesby byslide slide film film method. Paint Varnish Prod., krkh., 17:
45: 32, 1955. 45: 5. 5. RUGGERI, RUGGERI,S.: S.:Fungicidal Fungicidalvarnishes Varnishes and andlacquers. lacquers. Paint. Paint. Varnish Varnish Prod., 40: 40: 8, 8, 1950. 1950.
6. KIMMIG, J. UND RIETH, H.: Antimykotica in Experiment und Klinik. ArzneirnittelArzneimittelForsch.,3:3:267, Forsch., 267, 1953. 1953. 7. 7. NEDEY, NxDEv, G.: Les agents fongicides. Peint. Pigin. Pigm. Vern., Vern., 31: 31: 791, 791, 1955. 1955. 8. CZYZEWSKI, CzYzEwSKI, A.: A.:Bestimmung BestimmungVon von Hexachiorcyclohexan Hexachiorcyclohexan im Hexa-Rauch. Hexa.Rauch. SchaedlingrbeSchaedlingrbekaernpfung, 43: 180, 1951. kaempfung,
9. GREEN, H. F.: Bacteriostatic and fungistatic properties of beuzene benzene hexachioride. Chem. & md., 8: 201, 1955. 10. GRIMMER, H.: Personal Communication, 1956.
11. MEYER, P. S.: 5.: TJeber die Pilzerkrankungen Pilzerkrankungen der der Genitaizonen. Genitalzonen. Zschr. Zschr.Haut-Geschl. Haut-Geschl. krkh., 18: 233, 1955. 12. CONANT, CONANT,N. N. F., F., SMITH, D. T., T., BAKER, BAKER, R. D., CALLAWAY, J. L. AND MARTIN, D. D. S.: S.: SMITH, D.
Manual of Clinical Mycology. Philadelphia and London, W. B. Saunders Co., 1954.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
Most dermatomycoses and particularly onychomycosis are often very resistant to therapy. Moreover, successful treatment of the latter infection becomes more important because of its high incidence. White (1), for example, has considered 20 per cent of all nail disturbances to be of fungus origin. As a method of treatment for onychomycosis Rothman (2) and Langer and and Kaden Kaden (3) (3) have have used used nail nail
lacquer containing antimycotie antimycotic substances. Further investigation of certain aspects of the inhibitory effect of antimycotic antirnycotic lacquers is presented in this paper. MATERIALS AND METHODS METHOD5
An antifungal agent, halogenocyclohexane' was added to a nitrocellulose finger nail lacquer in a final concentration of 2 per cent. The anti-fungal effect of this was compared compared with a lithium bromide304) was compound in lacquer (Preparation (Preparation q 304) Asterol® base lacquer, (Ho), (Ro), proposed by Rothman Rothman (2) (2) and and with with aa common commonnail nail lacquer (B) to which no fungistatic agent had been added. The fungi used in the various tests included Penicillium Penicillium glaucurn, glaucurn, Aspergillus Aspergillus fumigatus, fumigatus,Trichophyton Trichophyton rubrum, Micros porumAudouini, Audouini,Epidermophyton Epidermophytonfloccosum, floccosum,and andCandida Candida Microsporum albicans.
antimycotic Three test methods were used to demonstrate the effect of the antimyeotio lacquers. (1) Slide film method: This method was described by Kaden (4). A strip of lacquer film is applied to the width of a sterile mieroslide microslide and allowed to dry for two hours. Molten Sabouraud's glucose agar was inoculated with the fungus to be tested and, with a sterile pipette, two strips of this this agar agar was was run run the the length length of of the theprepared preparedslides. slides.The Theslides slideswere were placed in Petri dishes for incubation at room temperature. Inhibition was determined by the extent of clear zones, in mm., from the lacquer strips. (2) Disc Method: This method method was was based based on on that that used usedby byRuggeri Ruggeri(1950) (1950)(5) (5)for fortesting testing the fungicidal activity of varnishes and lacquers. A Petri dish of Sabouraud's glucose agar was inoculated evenly over the entire surface with a suspension of the fungus to be tested. Lacquer samples were painted on one side of 3-" cover glasses which were used as discs. The lacquer film was allowed to dry for two hours after which the covers were placed lacquer
side up on the agar surface. The Petri dish cultures were incubated at room temperature and observed for a period of teu ten days. Inhibition of growth was determined by the size of the inhibitory zone, in mm., around the covers. Inhibition under the covers was noted also and termed an additional effect. (3) Evaporation test: Petri dishes were filled with enough Sabouraud's glucose agar (approx. 60 ml) to leave a 5 mm. space between the agar surface and the cover. The agar * The experimental investigation has been completed and the paper was written during a Fulbright Fellowship, spent with Dr. Norman F. Conant, Professor of Mycology, Department of Bacteriology, Duke University School of Medicine, Durham, North Carolina. t From the Hautklinik der Freien Universitaet, Berlin, Germany (Director: Dr. Erich Langer, Professor of Dermatology).
Received for publication August 8, 1955. 'Antimycotic-Lahopharma, Antirnycotic-Lahopharma, Lahopharma, Labopharma, Berlin, Germany. 221 221
222
THE JOURNAL JOURNALOF OFINVESTIGATIVE INVESTIGATIVEDERMATOLOGY DERMATOLOGY
surface was inoculated evenly with a suspension of the fungus to be tested. The entire inside of another Petri dish cover was painted with lacquer which was allowed to dry for two hours. This lacquered cover was placed on the test Petri dish. The results of inhibition, by continuing vaporization, was determined after an incubation period of four to ten days at room temperature. RESULTS RESULTS
Nail lacquer preparation 304 showed good inhibitory activity against a variety of fungi in the three tests described above. In the slide film test this lacquer suppressed growth of Penicillium glaucum, during a seven days incubation period for a distance of 6.5 mm. along the agar strip. Under the same conditions a similar effect was obtained with a nail lacquer (Ro) containing lithium bromide and Asterol®. Growth of the fungus in agar strips was not inhibited on slides "striped" with common nail lacquer (B) and on control (C) slides (figure 1).
The inhibitory effect of preparation 304 against Aspergillus fumigatus is demonstrated in figure 2. In this figure will be seen a wide zone of inhibition surrounding the cover glass previously painted with lacquer containing the antifungal agent. Fungus growth was also inhibited for some distance beneath this cover glass. No inhibition was noted around the cover glass painted with common lacquer or in the area covered by a clean unpainted cover glass.
The effect of preparation 304 on the growth of a variety of fungi using the "disc" method is shown in Table I. In this table it can be seen that the growth of T. rubrum, M. Audouini fioccosum was markedly inhibited in the presence Audoulni and and E. E.fioccosum of preparation *' 304. Complete inhibition of M. Andouini, T. rubrum, B. fioccosum, floccosum, C. albicans and ill. Avdouini,
FIG. 1. Slide film test method. method. Penic'iltium PenicilUum glaucum, glaucum, 77 days daysincubation. incubation.3O4 3O4}JalogenoIlalogenocyclohexane in iii lacquer; Rb Lithium bromide Asterol® in lacquer; B. Lacquer only; C. Control slide. slide.
223
FUNGISTATIC COMPOUNDS IN LACQUER
—C C
a
iv
304 3 Pt Aspergillus furnigalus,55days daysincubation incubationshowing showinginhibition inhibitionof of FJG. 2. Disc test method. Aspergillusfumigatos, FoG. 304 3O4Halogenocyclohexane Halogenocyclohexanein inlacquer; lacquer;B. B.Lacquer Lacqueronly; only;C. C.Control Controldisc. disc.Note Noteinhibition inhibition under "disc" in in 304. 304. under cover glass "disc"
TABLE I Zone of inhibition in millimeters in the areas adjoining the discs Samples applied applied to to the thediscs discsG8" (8" cover glasses)
Test fungus Preparation 304
Common nail lacquer
Trichophyton rubrum ]Iuicrosporu?n Audonini Audouini ]luicrospornm
15 14
Epidermophyton floccosum Epidermophylon flocco.vm...... Aspergillus Asper.qillus furnigatus fuinigatus
20
0 0 0 0 0
Candida albicans
8 3
Control
0 0 0 0 0
A. fumigatus was observed in Petri dishes when the inside of the cover had been painted with preparation 304. This effect is illustrated in figure 3. In figure 3 it can be seen that JVI. 111. Audonini failed to grow when the inoculated Petri dish
culture was covered with a cover painted on the inside with the antifungal lacquer. Growth was not inhibited in the control culture or in the culture covered with common nail lacquer. Growth of common contaminants such as Penicillium sp., (]andida sp. and bacteria were also inhibited in this test.
224
THE JOURNAL JOURNALOF OFINVESTIGATIVE INVESTIGATIVEDERMATOLOGY DERMATOLOGY
FIG. 3. Evaporation test. Microsporurn Audouini, 77 days days incubation. incubation. 3O4 304 Under Microporurn Audowini, surface of cover painted with Halogenocyclohexane lacquer; B. Painted with lacquer only; C. Control. DISCUSSION
The halogenocyclohexane derivative used in the experiments described above is chemically related to some of the known effective fungistatic agents. In the pentachlorphenol, in Germany, Rieth (6) United States, Ruggeri (5) mentioned pentachiorphenol, tested isomeres of hexachlorcyclohexane hexachiorcyclohexane and in France, Nedey (7) described tn-, tetra-, and pentachlorphenol as fungicidal agents. Attempts to incorporate these chemicals in nail lacquer, however, have not proved successful. Hexachiorcyclohexane Hexachlorcyclohexane and its derivatives was shown to act against insects, bacteria or fungi depending on a single change in their isomere structure (Czyzewski One of its derivatives, halogenocyclohexane, zewski (8) (8) and and Green Green (9). (9).One halogenocyclohexane, was was shown shown to have good fungistatic properties and it was also found found to to be be easily easily incorporated incorporated
into a nitrocellulose nail lacquer. Its fungistatic effect is dependent on the slow evaporation of the compound from the lacquer base. It can be used to inhibit immediately spore germination or, as Grimmer (10) has has shown, shown, itit can can be be used used to to inhibit further growth of a culture after varying periods of incubation. Cultures thus treated can be used for teaching and demonstration purposes. In addition to the laboratory tests with so-called fungistatic lacquers such materials have also been used in therapeutic tests. Rothman (2) reported good results in the treatment of onychomycosis with an antimycotic lacquer containing lithium bromide and Asterol®. However, poultices soaked in lithium bromide solution and special sprays were added to the therapeutic scheme. Kaden (3), using only halogenocyclohexane in lacquer (Preparation reportedsatisfactory satisfactoryresults results in in the the treatment treatment of skin and nail ation , 304)2 304)2 reported infections. Meyer (11) also reported successful clinical trials with the halogenocyclohexane derivative in mycotic infections of the skin. Preparation 3O4 is identical with "Mykotektan", Labopharma, Berlin, Germany and "Tektopal . .", Philadelphia Ampoule Laboratories, Philadelphia, Pennsylvania.
FUNGISTATIC COMPOUNDS IN LACQUER PUNGISTATIC
225
CONCLUSION
Halogenocyclohexarie derivative, incorporated in a nitrocellulose Halogenocyclohexane nitrocellulose nail nail lacquer lacquer T. rubrum, showed marked inhibitory effects effects against against M. M. Audouini, Audovini, T. fioccosum, rubrum, E. floccosum, C. albicans, Penicilhium Penicillium glaucum glaucum and and Aspergillus fumigatus. The fungistatic activity was due to a permanent evaporation of the effective agent as shown by three in vitro test methods. REFERENCES 1. WHITE, CLEVELAND, J.: Diseases of nails; 312 cases. Amer. Pract. Digest Treatm., 6: 226, 226, 1955. 1955. 2. 2.
disturbancesinin recalcitrant recalcitrant trichophyton trichophyton rubrum rubrum infections. infections. Systemic disturbances ROTHMAN, S.: ROTHMAN, S.: Systemic Arch. Dermat. & Syph.,
67: 239, 1953.
3. LANGER, UND KADEN,R.: R.:Lackfoermige Lackfoermigefungistatische fungistatische Wirkstoffe. Wirkstoffe. München KADEN, Münchenmed. med. 3. LANGER, E.E.UND
96: 674, 674, 1954. 1954. Wchnschr., 96: Wchnschr., 4. KADEN, R.:Antimykotiseher AntimykotischerLack LackininKlinik Klinik und und Experiment. Experiment. Zschr. Haut-Geschl. Haut.-Geschl. KADEN, R.: method. Paint Varnish Prod., krkh., 17:209, 209,1954. 1954.Testing Testingfungicides fungicidesby byslide slide film film method. Paint Varnish Prod., krkh., 17:
45: 32, 1955. 45: 5. 5. RUGGERI, RUGGERI,S.: S.:Fungicidal Fungicidalvarnishes Varnishes and andlacquers. lacquers. Paint. Paint. Varnish Varnish Prod., 40: 40: 8, 8, 1950. 1950.
6. KIMMIG, J. UND RIETH, H.: Antimykotica in Experiment und Klinik. ArzneirnittelArzneimittelForsch.,3:3:267, Forsch., 267, 1953. 1953. 7. 7. NEDEY, NxDEv, G.: Les agents fongicides. Peint. Pigin. Pigm. Vern., Vern., 31: 31: 791, 791, 1955. 1955. 8. CZYZEWSKI, CzYzEwSKI, A.: A.:Bestimmung BestimmungVon von Hexachiorcyclohexan Hexachiorcyclohexan im Hexa-Rauch. Hexa.Rauch. SchaedlingrbeSchaedlingrbekaernpfung, 43: 180, 1951. kaempfung,
9. GREEN, H. F.: Bacteriostatic and fungistatic properties of beuzene benzene hexachioride. Chem. & md., 8: 201, 1955. 10. GRIMMER, H.: Personal Communication, 1956.
11. MEYER, P. S.: 5.: TJeber die Pilzerkrankungen Pilzerkrankungen der der Genitaizonen. Genitalzonen. Zschr. Zschr.Haut-Geschl. Haut-Geschl. krkh., 18: 233, 1955. 12. CONANT, CONANT,N. N. F., F., SMITH, D. T., T., BAKER, BAKER, R. D., CALLAWAY, J. L. AND MARTIN, D. D. S.: S.: SMITH, D.
Manual of Clinical Mycology. Philadelphia and London, W. B. Saunders Co., 1954.
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94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
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