Drug Development and Industrial Pharmacy, 24(7), 685---690 (1998)
`
`COMMUNICATION
`
`The Effect of Keratolytic Agents on the
`Permeability of Three Imidazole
`Antimycotic Drugs Through the Human
`Nail
`
`David Quintanar-Guerrero,1,2,3,* Adriana Ganem-
`Quintanar,1,2,3 Patricia Tapia-Olgufn,1 Yogeshvar N.
`Kalia,2,3 and Pierre Buri2,3
`
`1FES-Cuautitlan, Universidad Nacional Aut6noma de Mdxico, Mexico
`City
`2Faculte des Sciences, Section Pharmacie, Laboratoire de Pharmacie
`Galdnique, Universitd de Gen~ve, CH-1211 Gen~ve, Switzerland
`3Centre Interuniversitaire de Recherche et d’Enseignement,
`"Pharmapeptides," Campus Universitaire, Parc d’Affaires international,
`F-74166 Archarnps, France
`
`ABSTRACT
`
`The permeability of three imidazole antimycotics (miconazole nitrate, ketoconazole,
`and itraconazole) through the free edge of healthy human nail was evaluated in
`vitro using side-by-side diffusion cells. The influence of keratolytic substances (pa-
`pain, urea, and salicylic acid) on the permeability of the antimycotics was also
`studied. The results suggested that the nail constituted an impermeable barrier for
`these antimycotics; it could be considered that the nail behaved as a hydrophilic
`gel membrane, through which drugs of low solubility could not permeate. The use
`of ethanol did not promote the passage of any of the antimycotic drugs. Although
`scanning electron microscopy indicated that the keratolytic substances had a sig-
`nificant effect on the nail surface (papain > salicylic acid > urea), the passage of
`the three antimycotics was not improved by pretreatment with salicylic acid alone
`(20%for 10 days), or by the application of the drug in a 40% urea solution. It was
`found that only the combined effects of papain (15%for I day) and salicylic acid
`(20%for 10 days) were capable of enhancing the permeability of the antimycotic
`
`*To whom correspondence should be addressed. Centre Interuniversitaire de Recherche et d’Enseignement, "Pharmapeptides,"
`Campus Universitaire, Parc d’Affaires International, F-74166 Archamps, France.
`
`Copyright © 1998 by Marcel Dekker, Inc,
`
`685
`
`ACRUX DDS PTY LTD. et al.
`EXHIBIT 1020
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 6
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`

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`686
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`Quintanar-Guerrero et al.
`
`therapeutics. It is conceivable that the enhancement effect can be attributed to the
`formation of pores which create transport channels, through which the antimycotics
`are able to permeate.
`
`INTRODUCTION
`
`The nail! is the hard durable epidermal appendage
`that protects the end of the digit. It is composed of mul-
`tiple layers of horny cells joined in a tightly cemented
`continuous sheet. The nails cells are dead corneocytes
`without nuclei or organelles and filled with ~t-keratin,
`which constitutes almost the entire dry weight of the nail
`(the water content of the nail varies between 7 and 16%).
`The c~-keratin structure is underpinned by covalent
`crosslinkages (mainly disulfide bonds) between polypep-
`tide chains within the multihelical "ropes" and between
`adjacent partners (1-3). It is this biochemical construc-
`tion which defines the physical properties of the nail,
`e.g., the strength and its behavior as a permeability bar-
`rier for many substances including benefcial therapeu-
`tic agents.
`This impermeability of the nail constitutes a formi-
`dable obstacle for the treatment, principally, of fungal in-
`fections (onychomycoses) which are the most frequent
`cause of nail diseases and represent 30% of all mycotic
`infections of the skin (4,5). In terms of therapeutic prac-
`tice, it is generally accepted that substances applied lo-
`cally have little if any impact on arresting the spread of
`the infection. The pathogen will, as a rule, penetrate into
`the nail to such a depth within the keratin that no con-
`ventional drug will be able to achieve a corresponding
`in-depth fungicidal action capable of destroying the
`pathogens (6).
`A number of treatments such as a solution of
`miconazole (2% in alcohol) (6), tioconazole (28% with
`undecylenic acid), amonolfine (5% nail lacquer) (7), and
`"chemical removal" of the infected area with 40% urea
`paste (8) were reported to be effective in some cases.
`The efficiency of these therapeutic interventions depends
`on the degree of fungal localization, that is, the number
`of keratin layers that shelter the fungi from the antimy-
`cotic drug.
`Moreover, frequently the nail area lying under the
`dorsal groove of the nail bed is also infected, and it is in
`these cases, in particular, that treatment based exclu-
`sively on topical application of the therapeutic remains
`unsatisfactory (6). On the other hand, oral antifungicidal
`therapy should be avoided because of the possibility of
`
`~In this paper the term nail refers to the nail plate.
`
`recurrence and risk of provoking adverse side effects.
`Surgical avulsion of the nail is not curative and has re-
`sulted in disability, recurrence, or loss of the nail (9). It
`is therefore clear that the development of an effective
`topical treatment is needed. However, to achieve this, the
`keratin barrier has to be breached so that antifungal
`agents are able to reach the pathogens.
`In this work we have studied the in vitro permeabil-
`ity of the nail to three imidazole-based anfimycofic drugs
`(miconazole nitrate, ketoconazole, and itraconazole) and
`the effect of keratolytic agents (papain, urea, and sali-
`cylic acid) as permeability promoters, as well as the
`concomitant effects on nail structure.
`
`MATERIALS AND METHODS
`
`Materials
`
`Miconazole nitrate, ketoconazole, and itraconazole
`were provided by Janssen Farmacrutica (Puebla,
`Mexico). Salicylic acid, urea, papain, and bromocresol
`green were obtained from Sigma Chemical Co. (St.
`Louis, MO). All other chemicals used were reagent
`grade and were obtained from J. Baker S.A. de C.V.
`(D.F., Mexico). Sections of healthy human fingernails
`(the free edge), with an average thickness of 0.027 cm
`± 10%, as measured using a gauge scale (Metromex,
`Mexico), were washed with distilled water and used
`immediately after being obtained from the donor. The
`volunteers were told to avoid contact with solvents or
`detergent solutions and to refrain from using nail varnish
`or varnish remover for a period of I week before the
`study.
`
`Analysis of Antimyeotie Concentration
`
`An assay using bromocresol green was used to quan-
`tify the concentration of the antimycotic drugs. Repeated
`extraction (n = 3) with chloroform at pH 3.0 (using a
`eitrate-HCl bufl’er system) resulted in a reaction between
`the imidazole moiety and bromocresol green causing a
`yellow complex to be formed. The concentration of the
`complex product was followed by spectrophotometric
`analyses of the chloroformic extracts, at L = 420 nm for
`
`2 of 6
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`Permeability of Imidazole Antimycotic Drugs
`
`687
`
`miconazole nitrate and ketoconazole and at k = 422 nm
`for itraconazole. This method has been validated for the
`three antimycotic drugs used in this study (10).
`
`Experimental Protocol
`
`Effect of Keratolytic Agents on Permeation
`
`These studies were carried out using a modified ver-
`sion of the horizontal cell design (t I). Circular sections
`of the nail plate were filed slightly on their external sur-
`face (until the shiny appearance had been removed) be-
`fore being securely attached to the cell by two silicon O-
`rings. The hermeticity of the system was continuously
`monitored. The donor and receptor solutions (30 ml
`ethanol:water, 60:40) were continuously stirred at 50
`rpm using a glass paddle propeller and maintained at 37
`+ 0.5°C by using a sand bath. The experiments were
`conducted with attention being paid at all times to the
`solubility and stability of the antimycotic agents in the
`presence of the keratolytic substances. In some cases, the
`nail was pretreated with 3 ml of papain (15% w/v) in
`phosphate buffer (pH 6.7) for 1 day or as the first step
`in a pretreatment procedure where the second phase
`consisted of treatment with salicylic acid (20% w/v) for
`10 days. These experiments were conducted in order to
`verify previously obtained results (12). Samples (1 ml)
`of the solution were periodically withdrawn from the
`receptor compartment (care was taken to restore the
`volume to 30 ml) and analyzed to detect the presence of
`any antimycotic using the assay described above. All
`experiments were carried out at least in duplicate.
`
`Effect of Keratolytic Agents on Nail Plate Surface
`Morphology
`
`Scanning electron microscopy was used to examine
`the changes in nail plate ultrastructure following expo-
`sure to the keratolyte. The external surfaces of nail plate
`sections (filed and unfiled) were subjected to keratoly-
`sis by urea (40%) and salicylic acid (20%) in
`ethanol:water (60:40), and by papain (15%) in phosphate
`buffer at pH 6.2 for 4 days at 37°C. Following this treat-
`ment period, the nails were rinsed with distilled water,
`dried under vacuum at ambient temperature, and subse-
`quently analyzed with a scanning electron microscope
`(JSM 2S SII, Jeol Instruments, Japan).
`
`RESULTS AND DISCUSSION
`
`riod of 60 days, in the experiments performed in the
`absence of a keratolytic agent. Waiters et al. (13) and
`recently Mertin (14) suggested that the human nail be-
`haves like a hydrogel of high ionic strength, the perme-
`ability of which depends on the solubility of the drug in
`water or in the swollen keratin matrix. Considering that
`the imidazole derivatives used in this study are only
`slightly or practically insoluble in water, it is easy to
`understand their low capacity to pass through a barrier
`with essentially hydrophilic characteristics. These results
`agree with the clinical ineffectiveness reported for topi-
`cal formulatiofis containing imidazole derivatives (9). It
`should also be noted that the presence of ethanol (used
`as cosolvent) did not promote the passage of the anti-
`mycotics. Although ethanol has been reported as a skin
`permeation enhancer (15), it does not appear to have the
`same effect on the nail. Walters et al. (16) observed that
`solvents which tend to promote the diffusion through the
`stratum eornetnn have little promise as accelerants of the
`nail permeability. Most of these enhancers, such as etha-
`nol, have an effect on the intercellular stratum corneum
`lipids; however, the lipid content of the nail is only
`0.15--0.76% of the total weight of the nail (17). Further-
`more, the corueocytes are joined in a tightly cemented
`continuous sheet, with overlap of their borders (1),
`which constitutes a barrier that is insensitive to the effect
`of ethanol.
`The addition of urea (40%) to the donor solution and
`the pretreatment with salicylic acid (20%) for 10 days,
`did not induce the transport of any of the antimycotic
`drugs. Thus, although both substances have been re-
`ported as good keratolytic agents for the detachment of
`scales, softening of cornified material (18), and chemi-
`
`16.
`
`14’
`
`12’
`
`4.
`
`2,
`
`0
`
`0
`
`I
`
`3
`
`’ I
`
`6
`
`I
`
`9
`
`I
`
`12
`
`I
`
`15
`
`time ((Jays)
`
`The results showed that no permeation through the
`nail was detected for the three antimycotics, over a pe-
`
`Figure 1. Permeation profile for miconazole nitrate through
`nail pretreated with papain t5%/1 day and salicylic acid 20%/
`10 days (n = 3).
`
`3 of 6
`
`

`
`688
`
`4
`
`g
`! 2-
`
`Quintanar-Guerrero et al,
`
`1,5,
`
`1,2¸
`
`,~0,9¸
`
`0,3,
`
`t
`
`4
`
`5
`
`1 I I
`10
`S
`time (~)
`
`’ I
`
`12
`
`I
`
`14
`
`0 "
`25
`
`I
`30
`
`I
`35
`
`I I I
`40 4,5 5O
`nm (m,ys)
`
`I
`55
`
`1
`5O
`
`I
`
`Figure 2. Permeation profile for ketoconazole nitrate through
`nail pretreated with papain 15%/1 day and salicylic acid 20%/
`10 days (n = 3).
`
`Figure 3. Permeation profile for itraconazole nitrate through
`nail pretreated with papain 15%/1 day and salicylic acid 20%/
`10 days (n = 2).
`
`cal dissolution of the diseased nail [e.g., 40% urea paste
`(8)], they are not capable of promoting the passage of
`the antimycotics through the nail structure.
`Figures 1-3 and Tables 1 and 2 give the permeation
`profiles and the permeability data for the three
`antimycotics when the nail was pretreated with papain
`(15%) for 1 day, followed by salicylic acid (20%) for 10
`days. As is shown by the data, only this two-step pre-
`treatment allowed the permeation of the antimycotics.
`After short lag times, between 0.53 and 1.12 hr, zero-
`
`order kinetics were observed for the three antimycotics.
`This behavior is explained by the formation of pores in
`the nai! matrix, as a consequence of the keratolytic ac-
`tion. It is possible that the antimycotic molecules can
`pass through these pores than via interconnected chan-
`nels permeate into the nail. This hypothesis is supported
`by the SEM images shown in Fig. 4. The normal scaly
`surface of the nail was modified by the keratolytic action
`of the permeation enhancers, as evidenced by the obser-
`vation of a more fractured scaly surface. This effect was
`
`Experimental Conditions Employed for the Nail Permeation Studies
`
`Table I
`
`Donor (mg/ml)
`
`Pretreatment
`
`Miconazole nitrate (60)
`
`Ketoconazole (37.5)
`
`Itraconazole (0.8)
`
`None
`None
`None
`Type Ia
`Type IIb
`None
`None
`None
`Type I
`Type II
`None
`None
`None
`Type I
`Type II
`
`Keratolytic in the
`Donor
`(mg/ml)
`
`None
`Urea (40)
`Salicylic acid (20)
`Urea (40)
`None
`None
`Urea (40)
`Salicylic acid (20)
`Urea (40)
`None
`None
`None
`Urea (40)
`Urea (40)
`None
`
`Sampling
`(Days)
`
`60
`60
`60
`60
`30
`60
`60
`60
`60
`30
`60
`60
`60
`60
`30
`
`"Pretreatment type I with papain (15% w/v) for I day.
`bPretreatment type 11 with papain (15% w/v) for I day and salicylic acid (20% w/v) for 10 days,
`
`4 of 6
`
`

`
`Permeability of Imidazole Antimycotic Drugs
`
`Figure 4. Scanning electron microphotographs showing the nail surface after exposure to keratolytic substances for 4 days at 37°C.
`(a) Control; (b) urea; (c) salicylic acid, and (d) papain (× 1500).
`
`most pronounced in the case of papain, followed by sali-
`cylic acid, and was less significant with urea. Note that
`these observations only showed the effect of the kera-
`tolytic substances on the nail surface and not in the
`
`deeper regions. Thus, we can only say that the treatment
`with papain (15%) for 1 day and salicylic acid (20%) for
`10 days allowed the formation of pathways into the nail.
`This long and aggressive pretreatment was chosen, after
`
`Table 2
`
`Nail Permeability Data for the Antimycotics Assayed After Nail Pretreatment with (15% w/v)/1 Day and Salicylic
`Acid (20%)/10 Days
`
`Antimycotic
`
`Miconazole nitrate
`Ketoconazole
`Itraeonazole
`
`Fluxa
`(rag cm-2sec-l) x 105
`
`Lag Time (TL)
`(see)
`
`Effective Diffusion Constant (Deft)b
`(em2 sec-1) × l0s
`
`6.66 + 0.42
`1.15 + 0.21
`0.13 ± 0.08
`
`1929 ± 208
`3373 + 425
`4050 + 632
`
`6.29
`3.60
`3.00
`
`n = 3 (miconazole nitrate and ketoconazole); n = 2 (itraconazole).
`~From the steady-state values,
`bFrom Dar = h2/6TL (mean value for h = 0.027 cm).
`
`5 of 6
`
`

`
`69(1
`
`Quintanar-Guerrero et al.
`
`previous reports describing in vitro results were consid-
`ered (12), because although the nail lost elasticity, no
`visual degradation was observed. The enhanced perme-
`ability for the three antimycotics, by the papainisalieylic
`acid pretreatment proposed in this paper, may have use-
`ful implications in onyehomycosis treatment, in particu-
`lar for proximal nail disease or severe nail bed involve-
`ment, which is extremely unlikely to respond to topical
`and/or oral treatment. However, it is clear that in vivo
`studies with infected nails are necessary.
`
`CONCLUSIONS
`
`The nail constitutes an impermeable membrane for
`the administration of the imidazole antimycotics--
`miconazole nitrate, ketoconazole, and itraconazole. The
`nail appears to behave as a hydrophilic gel membrane,
`through which drugs of low aqueous solubility cannot
`permeate. Pretreatment with salicylic acid (20%) and the
`use of urea (40%) did not induce any antimycotic trans-
`port. Only a combined pretreatment consisting ofpapain
`(15%) for 1 day and salicylic acid (20%) for 10 days
`enhanced the permeability of the three antimycotics. The
`proposed permeation route consists of the formation of
`pores, as a consequence of the keratolytic action, which
`are interconnected by transport channels. The enhanced
`permeability of these antimycotics could have important
`implications in onychomycosis treatment. Studies inves-
`tigating the efficiency and aggressiveness of the papain/
`salicylic acid pretreatment in vivo are now under way.
`
`ACKNOWLEDGMENTS
`
`D. Quintanar-Guerrero and A. Ganem-Quintanar
`would like to thank CONACYT and the Facultad de
`Estudios Superiores Cuautitlan, UNAM, Mexico, for fi-
`nancial support. The authors are grateful to Mr. Rodolfo
`Robles-G6mez for assistance with the scanning electron
`microscope.
`
`REFERENCES
`
`1.
`
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