mykosen [
`
`mykosen 25 (2) 74-80
`Eingegangen am 26. August 1981
`© Grosse Verlag Berlin 1982
`
`Bioavailability, Skin- and Nailpenetration of Topically
`Applied Antimycotics*
`Bioverfiigbarkeit, Haut- und Nagelpenetration lokal
`applizierter Antimykotica
`
`G. Sttittgen und Elke Bauer
`
`Hautklinik und Hautpoliklinik der Freien Universitat Berlin im Rudolf-Virchow-
`Krankenhaus, Berlin
`(Direktor: Prof. Dr. med. G. Sttittgen)
`
`Key words: Penetration - Econazole - Oxiconazole - Dimethylmorpholinohydrochlorid -
`Horny layer - Nails - DMSO
`SchliisselwGrter: Penetration - Econazol - Oxiconazol - Dimethylmorpholinohydrochlorid -
`Homschicht - N~igel - DMSO
`
`Summary: The penetration and permeation of econazol, oxiconazol and dimethylmorpholinohy-
`drochlofld in the human skin and nails have been performed using the measurement of radio la-
`belled substances (C14 ~-- 250 laCi/ml) in vitro (penetration chamber) and in vivo.
`The penetration of the antimycotics inside the horny layer of the skin and the layers of the nail
`(I0 lag/ml) ist sufficient enough to inhibit the growth of fungi by fungistatic mechanism.
`DMSO increases the penetration rate into the nail plate. Antimycotics solved in tinctures
`show a better penetration into the nails than the ointments which have advantages in penetrating
`the skin.
`The small fungicid effect and low dose in the deeper nails explains the long time topical treat-
`ment necessary at least for 6 months respectively the moulting process of the nail keratin.
`
`Zusammenfassung: Die Penetration und Permeation von Econazol, Oxiconazol und Dimethyl-
`morphoilnohydrocidofld in die menschliche Haut und Niigel wurde mit Hilfe marlderter Sub-
`stanzen (C14 ~ 250 pCi/ml) in vitro (Penetrationskammer) und in vivo untersucht.
`Die Penetration der untersuchten antimykotischen Substanzen in die Hornschicht der Haut
`und in die Keratinschichten der Niigel ist ansreichend, tun fungistatisch zn wirken.
`DMSO erhGht die Penetration in alas Nagelkeratin. Antimykotica in Tinkturen inkorporie~
`zeigen eine bessere Penetration in die Nligel als inkorporiert in Salben, die Vorzfige bei der
`Penetration in die Haut zeigen.
`Der geringe fungizide Effekt der meisten Antimykotica und die relativ geringe Dosis in den
`tiefen Nagelschichten erkl~’en die notwendig lange Behandlungszeit fiber 6 Monate, die den
`Mauserungseffekt des Nageikeratins berficksichtigt.
`
`*Cooperative team work of the study group "Permeability" Skin department of the Free University Ber-
`lin in Rudolf-Virchow-Hospital (Chairman: Prof. Dr. meal. G. StiJttgen) and Centre International de
`Recherches Dermatologiques, Valbonne (Directeur: Prof. Dr. rer. nat. H. Schaefer) and Bundesgesund-
`heitsamt Berlin (Direktor u. Professor Dr. med. A. Zesch).
`
`mykosen 25, Heft 2 (1982)
`
`ARGENTUM EX1016
`
`Page 1
`
`

`

`Topically Applied Antimycotics 75
`
`10"3
`
`~cZml
`
`!rpi lup.Corium [ lowerCorium I subcut Fat
`
`1 O* 2
`
`10"1
`
`10-o
`
`o
`
`10-1
`
`, i ,
`200 500
`
`I I I I i I i I
`*
`~ 1000 ’ 1400 mOO 22-- 2600
`
`um depth
`
`1000 min-Penetrotion in abdomen skin m vitro
`
`OxOconozol-ointment 1% (X-X)
`
`Oxiconozo[-hnc(ure 1% [¯ + +)
`
`Fig. 1
`
`Oimethyl - morphol~no - derivote t~ncture 0,5 %
`(o--o)
`
`Introduction
`
`It is obvious that an antimycotic agent, before it can exert its effect, should reach the infected
`tissue in sufficient concentrations. The way in which topically applied antimycotics migrate
`into the target organ skin and, after having acted, are removed from that area is a highly com-
`plex sequence of events (4).
`Liberation describes the diffusion process of a given substance out of the vehicle into
`which it has been incorporated. By penetration, the movement of a drug into a layer of the
`skin is described, while permeation means the migration through one or more layers.
`Resorption is the process by which the drug reaches the blood and lymph vessels, and by
`which it is removed from the local skin area. Absorption represent the sum of the above men-
`tioned processes (3).
`The best drug in preparation for topical application is useless, if the base in which the drug
`is incorporated does not allow the liberation process of the drug (Higuchi).
`Higuchl described the known correlation between liberation and the concentration, diffu-
`sion coefficient, saturation solubility, and dependency of time. Under constant conditions,
`the release of a drug from an vehicle is proportional to the square root of time observed.
`The problem of adsorption is correlated with a structural state of skin barrier namely the
`horny layer. The growth of fungi on the skin surface inside the horny layer induces toxic or
`
`mykosen 25, Heft 2 (1982)
`
`Page 2
`
`

`

`76
`
`G. Sttittgen and Elke Bauer
`
`....... .ON
`
`]1 ¯ .,0w0]~,~
`
`Fig. 2
`
`Table 1
`Permeation (lag/ml) of different antimycotics in the skin layer after variable exposition time
`
`Horny layer
`20 lam
`
`Epidermis Upper Corium Deeper Corium
`800 lam
`400 lam
`200 lam
`
`Subcutis
`1200 lam
`
`Time
`
`100’
`
`300’
`
`100’
`
`300’
`
`100’
`
`300’
`
`100’
`
`300’
`
`100’ 1000’
`
`Oxiconazole
`1000
`ointment 1% .....
`Tincture 1% ...... 3000
`Dimethyl-morpho-
`linoderivate
`ointment 1% .....
`Econazol-
`ointment 1%1) ....
`
`1200
`
`800
`
`Isoconazole-nitrate2)
`ointment 1% .....
`Clotrimazol-
`ointment 1% 3) .....
`
`1
`1
`
`2
`0.5
`
`2.5
`
`6
`
`1
`0.2
`
`2
`
`0.2
`
`0.7
`0.3
`
`5.5
`
`0.5
`
`0.3
`0.07
`
`1.5
`0.8
`
`0.1
`
`1.0
`
`0.1
`urin = 0.61%
`of the appli-
`cated dose
`
`12
`4
`
`10
`
`20
`
`10
`
`900
`2500
`
`2000
`
`1000
`
`15
`4
`
`14
`
`5
`
`9
`
`30
`
`~ Schaefer, H., Sttittgen, G., 1976 (in vivo experiments)
`2) Tiiuber, U., 1979
`3) Bayer-report, 1975
`
`allergenic substances irritating the skin especially the structure of the horny layer. The more
`horny layer is damaged, the more the process of permeation takes place.
`The quantitative measurement of absorption process of a given substance is a prerequisite
`for the therapeutical application later on. Such examinations have need ofstandarisation, be-
`muse only by respecting such standard methods it is possible to compare the different phar-
`macokinetic of antimycotics in relation to liberation and permeation inside the horny layer
`and into the deeper tissue. Measurement of the behaviour of radiolabelled substances is a
`method of choice. Tritium or C14 labelled substances with a specific activity of about 500
`laCi/ml were placed at our disposal (4).
`
`Processing of the skin respectively nails
`
`After 30, 100, 300 or 1000 min penetration time, the surplus cream on the surface was
`removed gently with dry cotton swabs. The specimen was then fastened on a rubber stopper
`and the horny layer removed by repeated stripping with adhesive tape (Tesafilm®). Each of
`approximately 20 strips was placed in a separate vial. A 28 mm sample was punched out and
`cut horizontally on a freeze microtome. The 16 slices ofl0 lam thickness first-sectioned re-
`presented the epidermis. The remaining part, representing the dermis, was cut into 40 lain
`slices.
`
`mykosen 25, Heft 2 (1982)
`
`Page 3
`
`

`

`Topically Applied Antimycotics 77
`
`104
`
`pg/ml
`
`Layer 1
`
`It
`Layer 2
`
`Layer 3
`
`\
`
`103
`
`102
`
`10
`
`Fig. 3
`
`1000 rain-Penetration in nail,= ,n vitro
`1% Oxlconazol- TINCTURE (÷ * .1
`
`I% Oxiconazol- OINTHENT(e ¯ e)
`
`The nail examination has been achieved b_y the same technic and same procedure putting
`the drug on the surface of nail area of 1 cm2 to obtain data about the absorption.
`The deep frozen nail was punched out and cut horizontally on a freeze microtom respect-
`ing the different layers of the nail (fig. 2). The samples were measured using a liquid scintil-
`lationcounter (Beckman LS 150 with an on-line teletype) whereby the maximum error was
`set to 10%. Subsequent calculations of decompositions per minute of percentage of the
`applied quantity and absolute quantities per layer were performed using a computer pro-
`gram.
`
`In viva examinations
`
`The surplus cream was removed and the horny layer sampled as indicated above. On the ex-
`posed skin an excision ofa 4 x 4 cm area was performed and a specimen of 28 mm2 was
`punched out from the excised area and siliced in the same manner.
`In viva studies of human skin are only possible in a limited number, because dermatologi-
`cal surgery allowing pretreatment and yielding suitable skin spacements is limited.
`In vitro results reflect in viva conditions predominantly down to the basal layer of the
`epidermis.
`Our studies were performed in vitro with a penetration chamber where the dainage or up-
`flow of the labelled substance through the blood and lymphatic vessels of the skin is simu-
`lated by a system with steady flowing at the temperature of 32° C which corresponds with the
`natural conditions.
`
`mykosen 25, Heft 2 (1982)
`
`Page 4
`
`

`

`78
`
`G. StiJttgen and Elke Bauer
`
`Table 2
`Penetration (pg/ml) of different antimycotics in the human nail at~er exposition time 1000 min
`in vitro
`
`dorsal nail plate
`(1)**
`- 200 pm
`
`ventral nail plate
`(2)**
`- 700 pm
`
`nail bed
`nail bed (3)**
`upper layers
`deeper layers
`till 1100 pm more than 1100 pm
`
`1000
`1500
`
`600
`
`1000
`
`1000
`
`800
`
`15
`50
`
`15
`
`10
`
`50
`
`70
`
`5
`80*
`
`8
`
`10
`
`70
`
`20
`
`7*
`80*
`
`15*
`
`12"
`
`80
`
`10
`
`Depth
`
`Oxiconazole
`ointment 1% .....
`Tincture 1% ......
`Dimethyl-morpho-
`linoderivate
`Tincture 0.5 % ....
`Econazol
`ointment 1% .....
`Econazollotion
`1% (99% DMSO)..
`Lotion 1%
`(49 % DMSO) .....
`
`**) see Fig. 2
`*) dam effect
`
`Our examinations were devided into two parts
`1. The normal and damaged human skin in vivo and vitro including stripping of the
`horny layers,
`2. the normal and damaged human nail in vitro including the effect of DMSO.
`To have exact data about the antimycotics it is necessary to compare the micrograms found in
`the different layers with the antimycotic as it was done by T~iuber with doing the microgram
`of isoconazole-nitrate found in the different layers of the skin in vitro.
`Using such technic it is possible to be informed about the skin layer in which the antimyco-
`tic has his antifungal activity.
`The antimycotic action of a substances is diminished by a binding capacity of proteins and
`membranes of the cell surface. By this facts the antimycotic action and the proved level of the
`antimycotic in the different skin layers needs further examinations which depends on the
`chemical characteristics of the free or bounded antimycotis. Skin (Tab. 1).
`
`Econazol
`
`After application time of 30 minutes in a depth of 200 micrometer the concentration of
`econazol was about 10 microgram, a concentration which is efficient to stop growth of fungi.
`By occlusion-dressing the level of econazol in the deeper layers of the skin is increased
`without over proportional value because the growth of keratophilic fungi takes place in the
`upper layer of the skin.
`
`Oxieonazol 1%
`
`Oxiconazol ointment shows in the epidermis a concentration which is higher than 10 micro-
`grams. In the deeper corium the concentration was about 1 microgram the level in ste subcu-
`tis ofl.5 microgram after exposition time 1000 min. shows surely the dam effect of the in vitro
`trials because the upflow of the oxiconazol simulated by the chamber technic is not aequi-
`valent to the natural conditions. The oxiconazol tincture shows lower concentrations in the
`epidermis and the corium (Fig. 1).
`
`mykosen 25, Heft 2 (1982)
`
`Page 5
`
`

`

`Topically Applied Antimycotics 79
`
`Dimethyhnorpholinohydrochlorid derivate 0.5 %
`
`Incorporated in an ointment this substance shows relatively high values in epidermis and
`deeper corium (Fig. 1).
`In comparison with isoconazolnitrate and clotrimazol it is obviously not necessary to
`increase the influx rate of antimycotic agent incorporated in a proved base to obtain better
`therapeutical results in skin affections with keratinophylic fungi. In the case of mycotic infec-
`tions which are situated in the deeper layers of the skin systemic treatment can be recom-
`mended. But also the use of salicylic acid and DMSO which are incorporated in the antimyco-
`tic ointments improve the penetration into the deeper horny layer, especially ff hy-
`perkeratosis have taken place.
`
`Nails
`
`An obvious unsolved therapeutical problem is the treatment of onychomycosis.
`In the last year we have given this problem the priority in our examinations.
`The fig. 2 shows a schematic representation of a normal nail. The different layers of the nail
`are made up of a dorsal solid hard nail plate, thickness, 200 micrometer, the ventral homo-
`genous and soft nail plate 700 micrometer and the inhomogeneous and soft nail bed about
`1100 micrometer and more.
`In tab. 2 the penetration of econa 7ol 1% in a lotion with 49% DMSO and a lotion with 99%
`DMSO is shown. Econazol with a higher percentage of DMSO show higher uptake in the
`deeper layers, but the permeation time trough the upper layers is markedly increased.
`Obviously a dam like effect of the deeper layers of the nails inhibit the upflow of the antimy-
`cotics; a saturation with the antifungal agent in this layer takes place.
`Concerning the comparison of 1% oxiconazol tincture with 1% oxiconazol ointment there
`is no doubt that the oxiconazol in the tincture shows a significant higher uptake into the dee-
`per layers of the nails (Fig. 3). Also the 3 rd layer, the nail bed, shows more than 10 micrograms
`oxiconazol, a fact which may be of therapeutical importance. Also the morpholino-derivate
`shows relatively high concentration in the deeper layers of the nailwhere as the absorption in
`the nail plate seems to be less developed. The unbroken but infected nail shows no signifi-
`cant higher penetration rate.
`The problem encountered in the therapy of nail mycoses is not the transport of the antimy-
`cotic agent from the exterior into the nail but rather, that the sporocidal concentration are
`maintained there (6). The growth of a toe nail from the root to the tip takes about more than 3
`months. An antimycotic agent which is unable to kill the spores must consequently be ap-
`plied regularly for 1 year until these spores are removed. In the horny layer of the epidermis
`there is a need of treatment about 1 month. By this reason a antimycotic therapy has to take
`into account this time. However, short-term therapy of the infection could be possible if the
`antimycotic agent is able to kill the spores. But anyhow the timefactor which include the
`moulting of the horny layers have to be respected.
`
`Conclusions
`
`What conclusions can be drawn from our own results and the review of the literature about
`permeation of antimycotic drugs.
`1. 90% of the preparations remains on the skin surface. We have defined this amount as
`the rejection rate.
`2. The reservoir function of the horny layer and also of the nail keratin is well demonstrated.
`DMSO can enhance the permeation inside the keratin layers.
`
`mykosen 25, Heft 2 (1982)
`
`Page 6
`
`

`

`80
`
`G. Stiittgen and Elke Bauer
`
`3. By occ!usion dressings a maximum penetration in the skin pertain, but in the most cases a
`penetration rate of only about 1% of applied doses of a antimycotic (in our experi-
`ments about 10 microgram/ml) is sufficient for a therapeutical purpose in the case ofder-
`matophytic infection.
`4. In the epidermis the highest quantity is found approximately 100 min. after application in
`vivo. The influx rate determines the different levels in dependance of the upflow of
`the substance. In the nails the highest levels are found 1000 rain. after application
`in vitro. The quicker the process of permeation the higher the removal rate from the
`skin surface. In this case a application of the drug has to be renewed in order to fill the
`reservoirs of the horny layers once again.
`5. The difference between the effect on the horny layer of the epidermis and the keratin
`of the nails has to be taken into account.
`
`References
`
`1. Higuchi, T. (1981): In vitro release from oint-
`ments and creams 1. Int. Symp. on Dermal
`and Transdermal Absorption, Munich, 12-14
`January 1981, Int. Ass. for pharmac. Tech-
`nology (proceedings in press).
`2. Schaefer, H. & G. Sti.ittgen (1976): Absolute
`Concentrations of an Antimycotic Agent,
`Econazole, in the Human Skin after Local
`Application Arzneim.-Forsch. (Drug Res.)
`26, 3,432-435.
`3. Schaefer, H. & G. StiJttgen (1978): Biophar-
`maceutical Problems in the Treatment of
`Superficial Mycoses, mykosen, Suppl. 1,164-
`170.
`4. Schaefer, H., A. Zesch & G. Sti.ittgen (1981):
`Skin Permeability. In: Handbuch der Haut-
`u. Geschlechtskrankheiten, Bd. 1, Teil 4 B,
`Sttittgen G., H. W. Spier & E. Schwarz (Hrsg.),
`Springer Verlag, Berlin - Heidelberg - New
`
`York, 541-827.
`5. T~iuber, U. (1980): Pharmakokinetische As-
`pekte bei der epikutanen Therapie von Haut-
`mykosen mit Travogen/Travocort. In: Der-
`matomykosen und ihre Therapie, Gartmann
`H., Z. S. ltani, M. L. Langen & E Schr6pl
`(Hrsg.), Exerpta Medica, Amsterdam - Ox-
`ford - Princeton, Teil I, 21-36.
`6. Voigt, W. H. (1975): Zur Wirkung von Anti-
`mykotika aufdie Ultrastruktur von Pilzzellen.
`In: Mykosen, Systematik, Klinik, Therapie,
`Hartung J. & D. Lubach (Hrsg.). Georg Thie-
`me Verlag Stuttgart, 115-120.
`
`Address: Prof. Dr. med. Gtinter StiJttgen, Direk-
`tot der Hautldinik und -Poliklinik der Freien
`Universit~t Berlin, im Rudolf-Virchow-
`Krankenhaus, Augustenburger Platz 1, D-IO00-
`Be flirt 65.
`
`mykosen
`
`Buchbesprechung
`
`Medical Mycology - Proceedings of Mycolo-
`gical Symposia of the XII International
`Congress of Microbiology - Munich, Sep-
`tember 3-8, 1978 Edited by Hans-Jfirgen
`Preusser. 157 figures and 99 tables. 394
`p., Gustav Fischer Verlag, Stuttgart - New
`York, 1980. Zentralblatt f’fir Bakteriologie
`Supplements Vol. 8, DM 178.-.
`
`Die mykologischen Beitdige des X/I.
`Internationalen Kogresses ftir Mikrobiolo-
`gie 1978 in Mfinchen sind 1980 als 8.
`Supplement des Zentralblattes [’dr Bakte-
`
`riologie erschienen. Die 7 Symposien be-
`fassen sich mit den Themen: Dimorphis-
`mus, Okologie von humanpathogenen Pil-
`zen, Kontamination und Elimination von
`Pilzen in Risikobereichen, Antigene Eigen-
`schaften von Pilzen und Anwendung in der
`Praxis, lokale und systemische Antimykoti-
`ca, Mykotoxine und Mykozoonosen.
`.. Die Herausgeber haben 46 Original- und
`Ubersichtsarbeiten, die yon eingeladenen
`Sprechern gehalten wu.rden, ausgew~ihlt.
`Sic geben einen guten Uberblick fiber die
`aktueUen wissenschaftlichen Fragestellun-
`gen auf dem Gebiet der Medizinischen
`Mykologie. Das Buch ist in erster Linie f’tir
`Spezialisten aufdem Gebiet d er Mykologie,
`Mikrobiologie, Hygiene, Immunologic und
`Serologic von Interesse, bedingt auch ffir
`mykologisch interessierte, klinisch ~tige
`
`mykosen 25, Heft 2 (1982)
`
`Page 7
`
`