Expert Opinion on Investigational Drugs
`
`ISSN: 1354-3784 (Print) 1744-7658 (Online) Journal homepage: http://www.tandfonline.com/loi/ieid20
`
`Recent progress on the topical therapy of
`onychomycosis
`
`Michael RK Alley, Stephen J Baker, Karl R Beutner & Jacob Plattner
`
`To cite this article: Michael RK Alley, Stephen J Baker, Karl R Beutner & Jacob Plattner (2007)
`Recent progress on the topical therapy of onychomycosis, Expert Opinion on Investigational Drugs,
`16:2, 157-167, DOI: 10.1517/13543784.16.2.157
`
`To link to this article: http://dx.doi.org/10.1517/13543784.16.2.157
`
`Published online: 24 Jan 2007.
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`Date: 17 April 2017, At: 15:36
`
`FlatWing Ex. 1036, p. 1
`
`

`

`1. Introduction
`
`2. Description of onychomycosis
`
`3. Challenges to the therapy of
`onychomycosis
`
`4. Drugs currently approved for
`onychomycosis in the main
`markets
`
`5. New antifungal compounds in
`development for onychomycosis
`
`6. Discovery and characterization
`of AN-2690
`
`7. Clinical summary
`
`8. Expert opinion
`
`Review
`
`Anti-Infectives
`
`Recent progress on the topical
`therapy of onychomycosis
`Michael RK Alley, Stephen J Baker, Karl R Beutner & Jacob Plattner†
`†Anacor Pharmaceuticals, 1060 East Meadow Circle, Palo Alto, CA 94303, USA
`
`Onychomycosis is a fungal infection of the fingernails and toenails that
`results in thickening, discoloration, splitting of the nails and lifting of the nail
`from the nail bed. The disease is caused by dermatophytes and has a high
`incidence within the general population, especially among older individuals.
`Present treatment options include both oral and topical drugs, with oral
`therapies giving better outcomes; however, neither of these treatment
`options provides high cure rates that are durable. The difficulty in treating
`onychomycosis results from the deep-seated nature of the infection within
`the nail unit (nail plate, nail bed and surrounding tissue) and the inability of
`drugs to effectively reach all sites. Ongoing drug development activities have
`focused on novel delivery technologies to facilitate penetration of existing
`antifungal drugs through the nail plate and on the discovery of inherently
`penetrable antifungals. AN-2690 represents an oxaborole antifungal that is
`designed to penetrate the nail plate and is showing promising results in
`clinical trials.
`
`Keywords: antifungal agent, dermatophyte, fungal infection, leucyl-tRNA synthetase,
`nail penetration, onychomycosis, oxaborole, tinea unguium
`
`Expert Opin. Investig. Drugs (2007) 16(2):157-167
`
`1. Introduction
`
`Onychomycosisis is a progressive fungal infection of the nail unit that leads to the
`destruction and deformity of toenails and (less frequently) fingernails. This con-
`dition is common and represents ∼ 50% of all nail disorders. Onychomycosis has a
`high occurrence throughout the world, with recent epidemiological data indicat-
`ing a prevalence of 6.5 – 13.8% in North America [1]. The infection shows an
`increasing incidence in older individuals and 1 study reported that 48% of people
`[2]. The susceptibility to
`aged 70 years are infected with onychomycosis
`onychomycosis is higher in men than in women, although women seek medical
`treatment more frequently.
`The infection is caused by fungi that infect the nail unit (the nail bed, the nail
`plate and surrounding tissue) and these include yeasts, dermatophytes and other
`molds. By far the most common fungi that cause onychomycosis are the dermato-
`phytes, which account for ∼ 90% of all cases. Dermatophytes are also the cause of
`skin fungal infections [3] and many patients with a nail infection also have a co-exist-
`ing skin infection. The Trichophyton spp., Microsporum spp. and Epidermophyton
`spp. are the main causative dermatophytes, with Trichophyton rubrum and
`T. mentagraphytes representing the two most common isolates [3-5].
`
`2. Description of onychomycosis
`
`Tinea unguium is the medical term that is used to describe a nail infection caused by
`a dermatophyte, whereas onychomycosis is used more broadly to characterize all
`fungal nail infections. Distal subungual onychomycosis represents the most
`
`10.1517/13543784.16.2.157 © 2007 Informa UK Ltd ISSN 1354-3784
`
`157
`
`FlatWing Ex. 1036, p. 2
`
`

`

`Recent progress on the topical therapy of onychomycosis
`
`common presentation of tinea unguium [6]. Distal subungual
`onychomycosis starts by the microorganism (usually a
`dermatophyte) invading the stratum corneum of the hypo-
`nychium and distal nail bed. Subsequently, the infection
`moves proximally in the nail bed and invades the ventral nail
`surface of the nail plate. The infection is characterized by dis-
`coloration, separation of the nail plate from the nail bed (ony-
`cholysis), accumulation of subungual debris and nail plate
`dystrophy. Proximal subungual onychomycosis is the least
`common variant of onychomycosis. This condition starts by
`fungal invasion of the stratum corneum of the proximal nail
`fold and subsequently of the nail plate. White superficial ony-
`chomycosis occurs when the nail plate is invaded directly by
`the causative organism and is characterized by the presence of
`white, chalky patches on the nail plate. The patches may coa-
`lesce to cover the whole nail plate. The potential end point of
`all forms of onychomycosis is total dystrophic onychomycosis
`and occurs when the entire nail plate and nail bed are invaded
`by the fungus.
`Although onychomycosis is generally not life threatening,
`the disease adversely affects the quality of life of its victims. In
`those cases in which the nail unit is seriously compromised,
`patients can experience pain and discomfort at the site of
`infection. Because of the high incidence of onychomycosis
`and the inadequacy of present treatment modalities, con-
`siderable research efforts have been directed to finding
`improved therapeutic options. This review summarizes the
`recent developments for new treatments of onychomycosis.
`
`3. Challenges to the therapy of onychomycosis
`
`Onychomycosis has proven to be a challenging infection to
`treat, with treatment failures and relapses being common
`occurrences [7-9]. In a study reported in 1998, it was found
`that 22.2% of patients whose toenail onychomycosis had been
`cured by oral therapy experienced a relapse during a 3-year
`follow-up period [10]. For an antifungal drug to be effective, it
`must disseminate throughout the nail unit and kill the patho-
`gen. When a sample taken from the nail bed of an infected
`patient shows a negative culture and negative microscopy, this
`is termed a mycological cure; however, a clinical cure includes
`not only elimination of the fungi from the nail unit but also
`the formation of clear, new nail growth that is absent of dys-
`trophic characteristics. Because of the slow rate of growth of
`toenails (∼ 1 mm/month), evidence of a clinical cure can take
`9 – 12 months. For this reason, drug treatment periods for
`onychomycosis are lengthy and require 3 – 12 months.
`During this treatment period, the infected nails can be
`monitored for growth of new clear nail and for the presence of
`viable dermatophytes.
`The current treatment modalities for onychomycosis
`include mechanical procedures, systemically administered
`antifungals and topical drug therapy. Although infected nails
`can be surgically removed, recurrence of the onychomycosis
`can occur as the new nail grows back and there is no evidence
`
`that the surgical approach is effective in producing a sustained
`disease-free period. In addition, because surgical avulsion is
`quite traumatic, this procedure is rarely used today. An alter-
`native procedure of chemically removing the nail using a urea
`ointment is also not widely used by current practitioners. In
`preference, podiatrists perform periodic trimming and debri-
`dement of affected nails as a means of reducing symptoms,
`with the added hope of minimizing progression by stabilizing
`the disease.
`Whereas mechanical procedures to treat onychomycosis
`only offer marginal benefit to the patient, drug therapies have
`provided some advancement in the attempt to effectively treat
`this disease; however, the overall success in treating ony-
`chomycosis is still far from optimal (as mentioned in
`Section 3) and this almost certainly results from the unique
`anatomical features of the nail unit and its pervasive coloniza-
`tion by dermatophytes during infection. The varied anatomy
`of the nail unit provides an opportunity for the fungal patho-
`gen to establish a deep-seated infection by invading and pro-
`liferating into the nail plate, the nail bed and the surrounding
`tissue. Systemically administered drugs must not only reach
`the nail bed but must also achieve sufficient concentration in
`the nail plate to eliminate dermatophytes at this location. It is
`more likely that systemically administered drugs reach the nail
`plate via the nail matrix, which is a continually proliferating
`epidermal tissue that serves as the origin of new nail growth.
`Access to the nail plate from the newly formed cells in the
`matrix is a slow process due to the very slow growth rate of
`toenails. As a consequence of this slow growth rate, oral ther-
`apy must be continued for some time and this may have the
`inherent disadvantage of causing systemic adverse effects.
`On the other hand, topically administered drugs face even
`more challenges to reach all of the required sites of these
`deep-seated infections. This difficulty in achieving relevant
`fungicidal concentrations throughout the nail unit directly
`relates to the nail plate’s unique properties, its thickness and
`relatively compact structure. The nail plate is a hard yet
`slightly elastic convex structure that consists of ∼ 25 layers of
`dead, keratinized, flattened cells that are tightly bound
`together. The nail plate itself consists of three layers: the dor-
`sal and intermediate layers derived from the matrix, and the
`ventral layer derived from the nail bed [11]. The upper (dorsal)
`layer is a few cell layers thick and consists of hard keratin. It
`constitutes the main barrier to drug diffusion into and
`through the nail plate. The intermediate layer constitutes
`75% of the whole nail thickness and consists of soft keratin.
`Below the intermediate layer is the ventral layer of soft kera-
`tin, a layer that is a few cells thick, that connects to the under-
`lying nail bed in which many pathological changes can occur.
`Thus achieving an effective drug concentration in the ventral
`nail plate and the nail bed is of great importance in the
`treatment of nail diseases.
`Chemically, the nail plate mainly consists of fibrous
`proteins – keratins – which are highly cross-linked with
`disulfide bonds. Coupled with the highly compact structure
`
`158
`
`Expert Opin. Investig. Drugs (2007) 16(2)
`
`FlatWing Ex. 1036, p. 3
`
`

`

`of the keratinized cells in the nail plate, these highly
`cross-linked proteins within the cells present a formidable bar-
`rier to the entry of topically applied agents [12]. In 1 study, the
`concentration of an applied drug across the nail dropped
`∼ 1000 times from the outer surface to the inner surface [13].
`As a result, the drug concentration had presumably not
`reached a therapeutically effective
`level
`in the
`inner
`ventral layer.
`Another factor contributing to the difficulty of topically
`applied antifungal drugs to penetrate the nail plate is the
`apparent mismatch of drug physicochemical properties with
`the biophysical properties of the nail plate [14]. Most existing
`antifungal drugs were originally designed for oral and/or skin
`applications and are consequently fairly lipophilic molecules,
`only sparingly soluble in water and have molecular weights of
`≥ 300 Da. On the other hand, with its dense keratin fabric
`network and a high capacity for flux of water, the nail plate
`has been described as a hydrophilic gel [15-17]. Thus most of
`the known antifungal agents will not find a compatible
`environment in traversing the nail plate.
`
`4. Drugs currently approved for
`onychomycosis in the main markets
`
`4.1 Oral agents
`Systemic drug treatment is currently the most effective
`method of treating onychomycosis. Even so, 20 – 25% of the
`patients fail to respond [18] and recurrence of disease after suc-
`cessful treatment is common. Terbinafine and itraconazole
`(Figure 1) are the two systemic treatments of choice, with ter-
`binafine showing greater efficacy and lower rates of recurrence
`than itraconazole [19].
`Terbinafine, a member of the allylamine family of
`antifungals, has a broad spectrum of activity and exerts fungi-
`cidal activity against most fungal pathogens. This compound
`also inhibits squalene epoxidase, an important enzyme in the
`biosynthesis of
`the
`essential membrane
`component
`ergosterol [20]. Terbinafine is active against dermatophytes,
`Malassezia furfur, Aspergillus spp. and some Candida spp.
`(including Candida parapsilosis). A single dose of terbinafine
`250 mg p.o. administered to humans produces peak plasma
`concentrations of 1 µg/ml within 2 h. It is > 99% protein
`bound and has a half-life of ∼ 36 h. It is administered at a
`dose of 250 mg q.d. with treatment duration of 6 and
`12 weeks for fungal infection of the fingernail and toenail,
`respectively. Using this regimen, 38.2% of toenails showed a
`clinical cure when examined at 48 weeks [21]. Therapeutic lev-
`els of drug persist in the nail for 3 – 6 months after therapy is
`discontinued. Liver toxicity has been reported for terbinafine
`and hepatic function tests are recommended for patients who
`use terbinafine continuously for > 6 weeks. Terbinafine is
`metabolized by CYP enzymes and has been noted to have a
`number of drug interactions [22].
`Itraconazole, which is from the azole class of antifungal
`agents, inhibits lanosterol 14α-demethylase and thus stops
`
`Alley, Baker, Beutner & Plattner
`
`the biosynthesis of ergosterol. It has a broad spectrum of
`activity against species including dermatophytes, Candida
`spp., Aspergillus spp. and M. furfur [23]. Blood levels of
`itraconazole after a single dose of 200 mg administered to
`humans reached a peak level of 0.2 – 0.3 µg/ml after 4 – 5 h.
`It is 99.8% protein bound and has a half-life of 21 h. It is
`administered as either 200 mg q.d. for 12 weeks or 200 mg
`b.i.d. for 7 days, followed by 3 weeks with no treatment and
`repeated for 3 months [22]. Therapeutic levels of itraconazole
`persist in the nail for 3 – 6 weeks after therapy is discontin-
`ued. Itraconazole has also been associated with liver damage
`and liver function tests are required if continuous treatment
`exceeds 1 month. This agent specifically inhibits the CYP3A4
`isoenzyme system and may consequently increase plasma
`concentrations of drugs metabolized by this pathway [22].
`Griseofulvin (Figure 1) is a natural product that was isolated
`from Penicillium griseofulvin in 1939 [24] and has been used
`for treating dermatophytosis since its introduction in 1958.
`Griseofulvin acts by binding to microtubular proteins, which
`results in the inhibition of cell mitosis and the formation of
`multinucleated fungal cells [25]. The drug is administered
`orally and is effective against a wide variety of dermatophytes.
`Griseofulvin has been used in complicated, difficult to treat
`tinea capitis and onychomycosis.
`Fluconazole is an orally active, synthetic, bis-triazole anti-
`fungal agent with activity against a wide range of fungi,
`including most Candida spp. and (as with other azoles) inhib-
`its lanosterol 14α-demethylase. Although fluconazole is not
`approved in the US or Europe for the treatment of ony-
`chomycosis, it is used off-label for this indication. A number
`of clinical trials studying the kinetics and efficacy of
`fluconazole have been reported [22].
`
`4.2 Topical agents
`Treatment of onychomycosis by topical methods has been met
`with limited success for the reasons described in Section 3.
`The two main topical treatments that are used today are
`ciclopirox and amorolfine (Figure 2), both of which are formu-
`lated in lacquers that are painted onto the infected nails. The
`lacquer dries to leave a water-insoluble film on top of the
`infected nail, which subsequently acts like a drug depot releas-
`ing the drug into the nail plate. Ciclopirox is a synthetic com-
`pound and is a member of the hydroxypyridone family of
`antifungal agents. The hydroxypyridone antifungals are active
`against many pathogenic fungi such as dermatophytes,
`M. furfur and Candida spp. Ciclopirox is believed to work by
`inhibiting metal-dependant enzymes by chelating the poly-
`valent cations (Fe3+ or Al3+) [26,27]. Ciclopirox has antifungal,
`antibacterial and anti-inflammatory activities. It is admin-
`istered to the infected nails daily and this treatment regimen
`continues for ≥ 6 months. Ciclopirox persists in the nail for
`14 days after therapy is completed. Clinical response rates for
`the treatment of onychomycosis are in the range of 7 – 10%.
`Amorolfine belongs to the morpholine group of synthetic
`antifungal agents. Its mechanism of action involves the
`
`Expert Opin. Investig. Drugs (2007) 16(2)
`
`159
`
`FlatWing Ex. 1036, p. 4
`
`

`

`Recent progress on the topical therapy of onychomycosis
`
`Cl
`
`Cl
`
`O
`
`O
`
`N
`
`N
`
`O
`
`N
`
`N
`N
`
`O
`
`N
`
`N
`
`N
`
`O
`
`Cl
`
`O
`
`Itraconazole
`
`N
`
`O
`
`O
`
`O
`
`O
`
`Griseofulvin
`
`Terbinafine
`
`Figure 1. Chemical structures of itraconazole, griseofulvin and terbinafine.
`
`N
`
`O
`
`O
`
`N O
`
`H
`
`Ciclopirox
`
`Amorolfine
`
`Figure 2. Chemical structures of ciclopirox and amorolfine.
`
`the ergosterol
`in
`important steps
`two
`inhibition of
`pathway [28]. Amorolfine is fungicidal against C. albicans and
`T. mentagrophytes. It is administered once or twice weekly to
`the infected nails for 6 – 12 months and (as with ciclopirox)
`persists in the nail for 14 days after therapy is completed.
`
`5. New antifungal compounds in development
`for onychomycosis
`
`Due to the treatment limitations described in Section 3 for
`onychomycosis, it is not surprising that significant R&D
`efforts have focused on the discovery of improved therapies.
`In general, these efforts have emphasized eliminating the defi-
`ciencies of currently approved drugs, which include long
`treatment times, concurrent side effects of oral drugs and poor
`efficacy of topical agents. The new treatment modalities that
`are presently in clinical development for onychomycosis are
`listed in Table 1.
`As noted from the entries in Table 1, most of the
`approaches represent re-formulations of known antifungal
`compounds with penetration enhancers in an effort to
`
`increase penetration of the active agent into and through the
`nail plate. The only systemic drug in development is itracon-
`azole (Hyphanox™), which is being developed by Barrier
`Therapeutics as a once-daily oral formulation. With respect
`to topical treatments for onychomycosis, the use of novel
`delivery vehicles and permeation enhancers applied to exist-
`ing classes of antifungals (e.g., azoles or allyl amines) repre-
`sents the main R&D approach that has been pursued over
`the past 10 years. Very little work has been reported on
`approaches focusing on discovering antifungal compounds
`that have intrinsic nail-penetrating properties. S-291-ND is
`one such compound and involves the use of a topical
`NO-generating formulation that can be applied directly to
`the toenail. NO is a highly reactive molecule that has anti-
`microbial activity [29] and may also have intrinsic nail-pene-
`trating capabilities due to is low molecular weight. The
`other novel approach is represented by the boron-containing
`antifungal compound, AN-2690. Now in clinical trials, this
`agent was specifically designed to overcome the nail barrier
`when applied topically so that
`it can achieve high
`concentrations in the nail bed [30].
`
`160
`
`Expert Opin. Investig. Drugs (2007) 16(2)
`
`FlatWing Ex. 1036, p. 5
`
`

`

`Alley, Baker, Beutner & Plattner
`
`Table 1. Onychomycosis treatments in clinical development.
`
`Compound
`
`Abafungin (Abasol™,
`York Pharma)
`
`Company
`
`York Pharma
`
`Topical butenafine gel
`
`Mylan
`
`AN-2690
`
`Anacor
`Pharmaceuticals
`
`Stage
`
`Description
`
`Pre-registration
`(in UK)
`
`Phase III
`
`Phase II
`
`A topical 2-aminothiazole membrane intergity antagonist
`
`Topical formulation of ergosterol synthesis inhibitor butenafine
`
`Topical boron-containing antifungal with inherent nail-penetrating
`characteristics
`
`NM-100060
`
`NexMed/Novartis
`
`Phase II
`
`Topical formulation of terbinafine with penetration enhancer
`
`Itraconazole (Hyphanox™,
`Barrier Therapeutics)
`
`Barrier
`Therapeutics
`
`Organo-gel delivery of
`allylamine antifungals
`
`MediQuest
`Therapeutics
`
`Phase II
`
`Once-daily oral formulation of itraconazole in 200-mg tablets
`
`Phase II
`
`Proprietary organo-gel delivery of naftifine or terbinafine
`
`Topical NO (S-291-ND)
`
`ProStrakan
`
`Phase II (in UK)
`
`Cream formulation of NO precursors that liberate NO on the nail
`
`Onycofitex
`
`Bentley Pharma
`
`Phase I/II
`
`Antifungal nail lacquer formulation of clotrimazole
`
`Transdermal SEPA®
`
`MacroChem
`
`Phase I
`
`Econazole in a nail lacquer with SEPA, a transdermal absorption
`enhancer
`
`6. Discovery and characterization of AN-2690
`
`6.1 Designing a compound to penetrate the nail plate
`Given the many failures and poor efficacy of topical treat-
`ments using known antifungal agents [31], a completely new
`approach was contemplated. Topical antifungal agents are
`applied to the outer surface (dorsal layer) of the nail plate and
`treatment is then dependent on the passive diffusion of the
`drug through this thick, keratin-rich, lipid-containing barrier.
`Several reviews on onychomycosis therapy suggest that topical
`antifungal drugs fail in clinical trials because of the lack of
`penetration of the drug through the nail plate [11,32,33]. To the
`authors’ knowledge, there has been no reported chemical opti-
`mization effort to identify an antifungal agent for delivery
`through a nail plate to treat onychomycosis topically.
`Studies have shown that nail penetration is dependent on
`molecular weight, octanol:water partition coefficient and
`water solubility, with smaller, more polar, water-soluble mol-
`ecules capable of penetrating to a greater extent than larger
`lipophilic molecules [15,34]. In addition to keratin, the nail
`plate contains lipids but (unlike the skin) these are not at such
`high concentrations; furthermore, they seem to be localized in
`the thin upper dorsal and lower ventral layers with a much
`lower concentration throughout the remaining nail plate [17].
`Another consideration was the suggestion in the literature
`that the antifungal potency of certain drugs may be reduced
`in the presence of keratin [35]. This is believed to be due to the
`drug binding to keratin, thus requiring a higher drug
`concentration to compensate for this loss.
`With this knowledge, a medicinal chemistry-based
`project was initiated to develop a small-molecule antifungal
`agent with appropriate physicochemical properties to treat
`onychomycosis topically. The criteria that was used for such
`a molecule included low molecular weight (< 250 Da), polar
`(cLogP < 2.5), good water solubility (> 0.1 mg/ml) and
`
`retention of antifungal activity in the presence of 5%
`keratin powder.
`
`6.2 Structure–activity relationship and chemical
`properties of AN-2690
`Initially, 1-phenyl-substituted benzoxaboroles (1; Figure 3)
`were identified as having broad-spectrum antifungal activity
`with reasonable minimum inhibitory concentration (MIC)
`values [30]. These had a molecular weight of ∼ 200 Da; how-
`ever, these 1-phenylbenzoxaboroles had a high cLogP (> 3.5),
`and were not very water soluble. Initially, 1-phenylbenzoxabo-
`roles were synthesized with hydrophilic substituents appended
`to the 1-phenyl group. Although these modifications
`improved water solubility, they did not reduce the cLogP by
`any significant margin. Furthermore, all of these substitutions
`led to an undesirable gain in molecular weight.
`To lower molecular weight further, the 1-phenyl group
`was
`replaced with
`a 1-hydroxyl
`group
`to
`give
`compound 2. This immediately lowered molecular weight to
`∼ 130 – 180 Da and retained equivalent broad-spectrum
`antifungal activity to the 1-phenyl derivatives. In addition to
`the large reduction in molecular weight, these compounds
`also had lower cLogP values as an aromatic ring had been
`replaced by a hydroxyl group. Furthermore, this modifica-
`tion increased water solubility. Several of these derivatives
`were synthesized and the most potent compounds were
`found to contain a halogen para to the boron. Activity was
`lost by replacing the halogen or by moving the fluoro group
`from the 5-position to another position on the aromatic
`ring. This study resulted in the identification of AN-2690 as
`having the best combination of potency and optimum
`physicochemical properties.
`AN-2690 has a low molecular weight (152 Da), good
`solubility in water (∼ 1 mg/ml) and a cLogP of 1.24 (cLogP
`calculated using CS ChemDraw Ultra 10).
`It has
`
`Expert Opin. Investig. Drugs (2007) 16(2)
`
`161
`
`FlatWing Ex. 1036, p. 6
`
`

`

`Recent progress on the topical therapy of onychomycosis
`
`R2
`
`1
`
`R1
`
`B
`
`O
`
`R1
`
`OH
`B
`
`O
`
`2
`
`OH
`B
`
`O
`
`F
`
`AN-2690
`
`Figure 3. Chemical structures of 1-phenyl substituted benzoxaboroles.
`
`Br
`
`(A)
`
`OH
`
`F
`
`F
`
`3
`
`(B, C)
`
`Br
`
`4
`
`O
`
`O
`
`F
`
`AN-2690
`
`OH
`B
`
`O
`
`Figure 4. The typical synthesis of AN-2690. Conditions: (A) CH3OCH2Cl, i-Pr2NEt and CH2Cl2 at room temperature; (B) n-BuLi,
`(i-PrO)3B and THF at -78ºC to room temperature; and (C) 6N HCl and THF at room temperature [30].
`
`broad-spectrum antifungal activity against dermatophytes,
`yeast, molds and other filamentous fungi with MIC values in
`the
`low microgram per milliliter range. AN-2690
`is
`fungicidal against T. rubrum at 4 to 8 times its MIC.
`AN-2690 is synthesized using the method shown in
`Figure 4. The scale-up development of this compound has
`been relatively simple and it has been synthesized at the 30-kg
`scale using a modification of this scheme.
`The stability of these boron-containing oxaboroles is
`good and comparable with other small non-boron-contain-
`ing organic molecules. They are easy to handle and require
`no specialized equipment. They are purified and analyzed
`using standard methods including recrystallization, column
`chromatography, HPLC and LCMS. They are stable when
`stored at room temperature for prolonged periods and are
`also stable under acidic or basic conditions as well as at
`elevated temperatures.
`To determine whether the activity of AN-2690 was affected
`by the presence of keratin, a MIC study was performed
`against T. rubrum in the presence and absence of 5% pow-
`dered keratin. This showed that the MIC of AN-2690 was
`essentially the same, leading to the conclusion that the activity
`of this compound is not affected by keratin binding.
`
`6.3 Nail-penetration studies
`Nail penetration of AN-2690 and ciclopirox was performed
`by
`the group of Maibach using
`their published
`methodology [36]. In this study, human cadaver finger nails
`were mounted in a one-cell diffusion chamber on top of a cot-
`ton ball wetted with saline to act as the nail bed. AN-2690
`was formulated at 10% w/v in ethanol:propylene glycol (4:1)
`
`as a vehicle and compared with ciclopirox (8% w/w in
`commercial lacquer) [37]. These test articles were applied at a
`dose of 10 µl to the top of the nail plates once daily for
`14 days. At the study end, the top and bottom sections of the
`dosed area of the nail plate were micro-dissected and analyzed
`for test compound. The result of the cotton ball analysis (i.e.,
`the amount of radiolabeled material to penetrate through the
`nail plate and deposit into the cotton ball as determined by
`scintillation counting) is shown in Figure 5. AN-2690 pene-
`trated through the nail plate in remarkably high con-
`centrations and the degree of penetration through the nail
`plate was far superior to ciclopirox. A total of 3 other
`AN-2690 derivative compounds were also studied for nail
`penetration, 1 analog whereby the fluoro group was replaced
`by chloro and 2 1-arylbenzoxaborole derivatives. The chloro
`analog showed almost equal penetration to AN-2690, whereas
`the 1-arylbenzoxaboroles did not show such good penetration
`as could be expected as these congeners had a higher mol-
`ecular weight and were more lipophilic. This result showed
`that the small, polar, water-soluble nature of AN-2690
`matched the requirements to penetrate the nail plate pro-
`viding an antifungal agent with an unprecedented ability to
`penetrate human nails.
`In a final in vitro demonstration of activity, AN-2690,
`ciclopirox and amorolfine were studied in a model of
`infection using the TurChub® cells (MedPharm Ltd;
`Figure 6) [101,102]. In this study, the antifungal agent must
`first pass through full thickness human nail plates to show a
`zone-of-inhibition against the dermatophyte cultured in the
`flask below the nail plate. AN-2690 (10% w/v in ethyl
`acetate:propylene glycol 1:1) was compared with ciclopirox
`
`162
`
`Expert Opin. Investig. Drugs (2007) 16(2)
`
`FlatWing Ex. 1036, p. 7
`
`

`

`Alley, Baker, Beutner & Plattner
`
`human heptocytes (HepG2) is > 1000 µg/ml as measured in
`an MTS cell viability assay. As expected, the difference
`between the bacterial and human AARS enzymes is much
`larger so it is not surprising that the archetypal AARS inhibi-
`tor, mupirocin, is an antibacterial [41]. However, the relatively
`close homology between the fungal and human AARS
`enzymes has not prevented antifungal AARS inhibitors being
`found; for example, even mupirocin, which is used solely as
`an antibacterial topical drug, does have some antifungal activ-
`ity as demonstrated by its efficacy in a guinea-pig T. mentagro-
`[42]. Other examples of more
`phytes ringworm model
`well-known
`specific
`antifungal AARS
`inhibitors
`are
`icofungipen and cispentacin [43-46].
`The AARS enzymes perform the first step in protein syn-
`thesis by attaching their cognate amino acid to either the 2′-
`or 3′-hydroxyl group of the terminal adenosine of tRNA. This
`is performed in a 2-step process: first, the amino acid is acti-
`vated by the attachment of the α-carboxyl group to the
`5′-phosphate of AMP; and second, this activated amino acid
`is transferred to either the 2′- or 3′-hydroxyl group of the
`adenosine on the 3′-terminus of the tRNA with the corre-
`sponding anticodon. The accuracy of this reaction in protein
`synthesis is essential as it is the linchpin in the translation of
`the genetic code into protein, linking the triplet codon to the
`amino acid. Leucyl-tRNA synthetase, as for the AARS for iso-
`leucine, valine, methionine, alanine, lysine, proline and
`phenylalanine, possess additional proofreading mechanisms,
`which can edit either the incorrect amino acid–AMP inter-
`mediate or the incorrect aminoacylated tRNA product, to
`improve the fidelity of tRNA aminoacylation [47]. The homo-
`logous AARS enzymes for leucine, isoleucine and valine pos-
`sess two structurally distinct domains: one domain contains
`the site of aminoacylation and the other domain bears an edit-
`ing site that proofreads the aminoacylated tRNA [48-50]. The
`archetypal AARS
`inhibitor mupirocin
`inhibits
`iso-
`leucyl-tRNA synthetase [51] by binding to the aminoacylation
`active site as an isoleucine adenylate analog [52]. The AARS
`inhibitors cispentacin and icofungipen are thought to act as
`amino acid analogs and (as with mupirocin) probably bind to
`the aminoacylation site. Although AN-2690 inhibits an
`AARS, its mechanism of action is very different to these
`known AARS inhibitors. This was made apparent early in
`genetic studies in Saccharomyces cerevisiae and C. albicans
`when all of the AN-2690 resistance mutations were mapped
`to the editing domain of leucyl-tRNA synthetase. As would
`be expected for a drug that bound to the editing site,
`AN-2690 is a non-competitive inhibitor with respect to leu-
`cine, ATP or tRNA. The oxaborole AN-2690 represents a
`new class of inhibitors that exerts their action on AARS via
`the editing domain. As the editing active site has evolved to
`accommodate a larger repertoire of compounds than the
`aminoacylation active site, the editing site may prove to be a
`better target for designing compounds that inhibit this
`validated drug target.
`
`*
`
`‡
`15
`
`AN2690
`Ciclopirox
`
`3
`
`6
`
`9
`Time (days)
`
`12
`
`2.5
`
`2
`
`1.5
`
`1
`
`0.5
`
`0
`
`cotton ball (mg)
`Amount of compound found in
`
`
`
`Figure 5. Cumulative total amount of AN-2690 and
`ciclopirox found under the nail plate in cotton ball supports
`at each time point following a 14-day, multiple-dose
`nail-penetration study.
`*2.24 mg. ‡0.009 mg.
`
`(8% w/w in commercial lacquer) and amorolfine (5% w/w
`in commercial lacquer). Using 5 – 6 replicates, test articles
`were dosed daily for 5 days at a concentration of 40 µl/cm2.
`The vehicle showed no zone-of-inhibition nor did ciclopirox
`or amorolfine in their com