JPP 2009, 61: 431–437
`ß 2009 The Authors
`Received August 16, 2008
`Accepted January 7, 2009
`DOI 10.1211/jpp/61.04.0003
`ISSN 0022-3573
`
`Correspondence: Dr S. Narasimha
`Murthy, 113, Faser Hall,
`Department of Pharmaceutics,
`The University of Mississippi,
`University, MS 38677, USA.
`E-mail: murthy@olemiss.edu
`
`Research Paper
`
`A study on the effect of inorganic salts in transungual
`drug delivery of terbinafine
`
`Anroop B. Nair, Srinivasa M. Sammeta, Siva Ram K. Vaka and
`S. Narasimha Murthy
`
`Department of Pharmaceutics, The University of Mississippi, University, Mississippi, USA
`
`Abstract
`
`Objectives The poor success rate of topical therapy in nail disorders is mainly because of
`the low permeability of keratinized nail plates. This can be overcome by utilizing potent
`perungual drug penetration enhancers that facilitate the drug permeation across the nail
`plate. This study evaluated the efficacy of inorganic salts in enhancing the trans-nail
`permeation using a model potent antifungal agent, terbinafine hydrochloride.
`Methods Permeation studies were carried out across human cadaver nail in a Franz
`diffusion cell using terbinafine solution (1 mg/ml; pH 3). Preliminary studies were carried
`out to assess the effect of salts (0.5 M) on the terbinafine permeation into and through the
`nail. Further, the influence of salt concentration (0.25–3 M) on permeation, the mechanism
`for the enhancement and the suitability of developing a formulation were also studied.
`Key findings Terbinafine permeation (3–5 fold) through the nail and drug load (4–7 fold)
`in the nail were enhanced significantly when salts were used at 0.5 M concentration.
`Increase in salt concentration up to 1 M increased the permeation, which decreased with
`the
`further increase in salt concentration (>1 M). Mechanistic studies revealed that
`enhanced permeation by salts was mainly due to their ability to increase the nail hydration
`and also to increase the thermodynamic activity of the drug. The cumulative amount of
`(9.70 ± 0.93 mg/cm2) was
`terbinafine permeated at 24 h from the formulated gel
`comparable with that of a solution (11.45 ± 1.62 mg/cm2).
`Conclusions Given the promising results from the permeation and drug load studies, it was
`concluded that inorganic salts could be used as potent transungual permeation enhancers.
`Keywords hydration; nail; permeation enhancer; salts; terbinafine
`
`Introduction
`
`infections and psoriasis.
`The most common causes of nail diseases are fungal
`Onychomycosis, fungal infection of the nail, is the most prevalent (approximately 50%) of
`all nail diseases. Dermatophytes are the main causative organism for onychomycosis, while
`non-dermatophytic molds and yeasts are also involved.[1] Existing treatment modalities for
`onychomycosis include oral delivery and topical application. Topical drug delivery is the
`most appropriate therapy as it offers higher patient compliance and better proximity to the
`infected site and avoids potential systemic adverse effects.[2,3] Currently, topical formulations
`are available as nail lacquers, creams, ointments, gels, solutions and lotions.[4] However, the
`efficiency of these formulations is limited due to their inability to deliver a therapeutically
`effective amount of drug into and across the impermeable nail plate. Therefore, this therapy
`is limited for the treatment of superficial and minor subungual onychomycosis.[5,6]
`Topical delivery of drugs by the usage of permeation enhancers is well known. A wide
`variety of proven permeation enhancers are available and are used in enhancing drug delivery
`across the skin. The skin contains about 15% of lipids arranged as lamellae in the
`intercellular space. Most skin penetration enhancers act by modulation of lipid structures.
`The nail plate has less lipid content (<1%) than the skin.[7] The major component of the nail
`plate is keratin. Enhancers of skin drug penetration that act predominantly on the
`keratinocyte pathway are relatively few, thus it is obvious that most skin permeation
`enhancers are unsuccessful in transungual drug delivery. Therefore, research is being carried
`out to screen novel entities for their potential transungual permeation enhancement ability.
`Many chemical agents, such as acetylcysteine, urea, salicylic acid, 2-n-nonyl-1,3-dioxalone,
`
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`Journal of Pharmacy and Pharmacology 2009; 61: 431–437
`
`mercaptoethanol, thioglycolic acid and glycolic acid, have
`been screened for their ungual permeation enhancement
`capacity.[8–12] Reports on the capability of these agents to
`enhance transungual delivery are inconsistent.[2] Recently
`Malhotra and Zatz[10] reported the ineffectiveness of kerato-
`lytic agents, even at high concentration, when delivered from
`gel formulation. The authors also observed the failure of
`sodium metabisulfite to enhance penetration. Also,
`the
`efficiency of acetylcysteine was limited only on the dorsal
`nail layer and it was inactive in the intermediate and lower
`nail layers.[8]
`It is well known that the cure rates with existing topical
`therapy remain much lower than those achieved by systemic
`antifungal therapy. Successful treatment of nail diseases by the
`topical route can be achieved by identifying potent perungual
`penetration enhancers to deliver drugs across the nail plate in
`effective amounts. It is also known that the efficiency of
`penetration enhancers vary with different formulation condi-
`tions and with different drugs. Therefore, one needs to identify
`the most appropriate enhancers for the given drug and
`formulation. In this direction, we developed a high-throughput
`technique for screening penetration enhancers, called Trans-
`Screen-N. It is a rapid microwell plate-based method that
`involves two different treatment procedures – simultaneous
`exposure treatment and sequential exposure treatment. The
`technique was developed to screen permeation enhancers for
`the delivery of terbinafine hydrochloride, a potent antifungal
`agent, which is the current treatment of choice in onychomy-
`cosis. During this process the efficiency of inorganic salts to act
`as perungual permeation enhancers was discovered. The drug
`load in nail pieces soaked in formulations containing salts as
`enhancers (sodium sulfite, sodium phosphate, potassium
`phosphate, calcium phosphate and ammonium carbonate)
`were found to be significantly higher than the control.
`TransScreen-N is a screening method only and the results are
`suggestive. The method does not reveal the drug permeation
`profile, flux, lag time or the possible mechanisms. Encouraged
`by results that suggested the potential permeation enhancement
`efficiency of salts in TransScreen-N, further research on this
`phenomenon was undertaken. The objective of this study was to
`evaluate the efficacy of inorganic salts in enhancing the
`transungual delivery of terbinafine hydrochloride.
`
`Materials and Methods
`
`Materials
`Terbinafine hydrochloride was procured from Uquifa
`(Jiutepec, Mexico). Sodium sulfite, sodium phosphate
`monobasic, potassium phosphate monobasic, calcium phos-
`phate monobasic and ammonium carbonate were purchased
`from Sigma (MO, US). Human cadaver nails (second, third
`and fourth finger nails), both male and female with varying
`thickness (0.4–0.7 mm), were procured from Science Care
`(AZ, US) and were stored at 4∞C and used within a week. All
`solutions were prepared in de-ionized water.
`
`Analytical method
`The amount of terbinafine in the samples was quantified by
`high-performance liquid chromatography (HPLC) system
`
`(Waters 1525) with an autosampler (Waters, 717 plus)
`consisting of a Phenomenex C18 (2) 100 R analytical column
`(4.6 ¥ 150 mm; Luna 5.0 mm) and a variable wavelength dual l
`absorbance detector (Waters 2487). The mobile phase consisted
`of aqueous solution (0.096 M triethyl amine, 0.183 M orthopho-
`sphoric acid) and acetonitrile (60 : 40) adjusted to pH 2 with
`orthophosphoric acid. Elution was performed isocratically at
`32∞C at a flow rate of 1.0 ml/min. The injection volume
`was 20 ml and the column effluent was monitored at 224 nm.
`The method was validated by determination of linearity,
`precision and accuracy. The range for the calibration curve was
`2–1000 ng/ml (R2 = 0.99). The coefficient of variation and the
`accuracy (relative mean error) was in the range of 1.03–6.08%
`and -0.54 to -6.96%, respectively.
`
`Preliminary in-vitro permeation studies
`Nails were cleaned and adherent tissue was removed with a
`pair of scissors and scalpel and the nail was rinsed in water.
`Each of the nail plates was soaked in water for 1 h and
`mounted on a nail adapter (PermeGear, PA, US). The whole
`assembly was sandwiched between the two chambers of a
`Franz diffusion cell
`(Logan Instruments Ltd, NJ, US).
`Terbinafine hydrochloride solution (500 ml, 1 mg/ml with
`0.5 M enhancer) was placed in the donor compartment and
`the receiver chamber was filled with 5 ml of acidified water
`(adjusted to pH 3 using 0.1 M hydrochloric acid or sodium
`hydroxide as the initial study data revealed that an ionic
`strength within 0.15 M does not affect the drug load and
`permeation). The enhancers used were ammonium carbonate,
`calcium phosphate, potassium phosphate, sodium phosphate
`and sodium sulfite. The active permeation area was 0.2 cm2.
`The solution in the receiver compartment was stirred at
`600 rev/min with a 3-mm magnetic stir bar. Samples were
`withdrawn at regular intervals for a period of 24 h from
`the receiver compartment and analysed. Similarly, control
`experiments (formulation with drug and no enhancer) were
`run in parallel for comparison.
`
`Amount of drug in nail
`The amount of terbinafine loaded in the nail was determined
`after the permeation study (24 h). Briefly, after in-vitro
`diffusion studies the nail plates were washed by a
`standardized procedure using water and 95% ethanol, until
`the surface was free from drug. The active diffusion area was
`cut into small pieces, weighed and placed in screw-cap pyrex
`vials. Sodium hydroxide (1.5 ml of 1 M) was added and the
`vials were incubated for 24 h in a shaker water bath at room
`temperature to allow the nail to dissolve. Extraction of drug
`was carried out by a slight modification of the method
`described by Dykes et al.[13] Briefly, after dissolving the
`nails in vials, 200 ml of 5 M hydrochloric acid was added to
`neutralize the mixture. Hexane (3 ml) was added to the vial
`to extract the drug and the vials were shaken manually for
`30 min. The mixtures were transferred into centrifuge tubes
`and centrifuged at 4000 rev/min for 10 min. The hexane
`layer was collected, 1 ml of 0.5 M sulfuric acid–isopropyl
`alcohol (85 : 15) was added and the mixture was shaken
`vigorously for 30 min. The lower acidic aqueous layer, which
`holds the majority of terbinafine, was collected separately
`and the amount of drug in the nail was determined. This
`
`

`

`Enhanced nail drug delivery by inorganic salts
`
`Anroop B. Nair et al.
`
`433
`
`extraction procedure was validated by spiking different drug
`concentrations (2–20 mg/ml) into sodium hydroxide solution
`in which the nail was previously dissolved. The recovery was
`found to be 84 ± 7%.
`The amount of terbinafine diffused into the peripheral
`region (which was not in contact with the formulation or
`enhancer) was determined by dissecting the peripheral nail
`area (4–5 mm surrounding), which was washed, dried and
`weighed. The amount of drug was determined as described
`above.
`
`Solubility measurements
`A normal equilibrium solubility determination was carried
`out by the method of Okumara et al.[14] An excess amount of
`terbinafine hydrochloride was added and dissolved in a
`measured amount of solution containing different concentra-
`tions of sodium phosphate (0, 0.25, 0.5, 1.0, 2.0 and 3.0 M)
`in a glass vial to obtain a saturated solution. The pH of the
`solution was adjusted to pH 3 or 5 (using 0.1 M HCl or 0.1 M
`sodium hydroxide) and was monitored occasionally through-
`out the study period. The system was stirred for 24 h at room
`temperature and kept at rest for 1 h to assist the attainment
`of equilibrium. The solution was then filtered through a
`membrane filter (pore size 0.22 mm) and, after dilution, the
`solubility was determined.
`
`Effect of salt concentration on permeation
`Permeation of terbinafine in the presence of different
`concentrations of sodium phosphate (0.25, 0.5, 1, 2 and 3 M)
`was carried out as described previously in preliminary in-vitro
`permeation studies. The donor drug concentration was 1 mg/ml
`(pH 3). The amount of drug permeated after 24 h was measured
`by HPLC.
`
`Effect of pretreatment on permeation
`Pretreatment of nails was carried out by soaking the nail
`plates in 0.5 M sodium phosphate solution (pH 3) for 24 h.
`The nail plates were then washed with 10 ml of water (pH 3)
`five times and mounted on a nail adapter. Drug solution
`(500 ml; 1 mg/ml; pH 3) was placed in the donor compart-
`ment. For control trials, the nail was soaked in water (pH 3).
`Permeation studies were carried out as described in
`preliminary in-vitro permeation studies.
`
`Effect of pH
`The influence of pH on the permeation of drug was assessed
`using 1 mg/ml of terbinafine hydrochloride solution (pH 5) in
`the absence of salt. Permeation was carried out as described
`previously in preliminary in-vitro permeation studies and the
`amount of terbinafine permeated after 24 h was determined.
`
`Permeation studies using dialysis membrane
`Permeation studies were carried out across the dialysis
`membrane (MWCO 1000, 7 Spectra/Por; Spectrum Labora-
`tories, Inc., CA, US) with varying concentration of sodium
`phosphate (0.25, 0.5, 1, 2 and 3 M). The membrane was
`mounted between the donor and receiver chamber of a Franz
`diffusion cell (Logan Instruments Ltd., NJ, US). Drug–
`enhancer solution (500 ml; 1 mg/ml; pH 3) was placed in the
`donor compartment while the receiver chamber was filled
`
`with 5 ml of the respective sodium phosphate solutions
`without drug (pH 3), to maintain uniform osmotic pressure.
`The permeation area was 0.64 cm2. The solution in the
`receiver compartment was stirred at 600 rev/min with a
`3-mm magnetic stir bar. Samples were withdrawn at regular
`intervals from the receiver compartment and analysed.
`Control experiments were run in parallel for comparison
`using terbinafine solution (1 mg/ml; pH 3).
`
`Water uptake
`Nail pieces (4 ¥ 4 mm) were incubated at 37 ± 0.5∞C for
`24 h, weighed (W1) and soaked for 24 h in water (pH 3) or
`0.5 M sodium phosphate solution (pH 3). Then the nail plates
`were removed and wiped with Kimwipe to remove the
`surface water. Nails were placed in a screw-cap Pyrex vial
`containing pre-weighed anhydrous sodium sulfate (W2) and
`kept at room temperature until the weight of sodium sulfate
`(W3) remained constant (48 h). The increase in the weight of
`sodium sulfate was noted and the percentage water uptake of
`the nail was calculated by the following equation.
`
`% Water uptake = [(W3 - W2)/W1] ¥ 100
`
`(1)
`
`Permeation using poloxamer gel
`The poloxamer gel was prepared by a ‘cold process’. Drug
`solution (1 mg/ml; pH 3) was slowly added to poloxamer
`(21% w/v) with constant stirring at low temperature (4–5∞C)
`until the polymers were uniformly dispersed. The required
`amount of permeation enhancer (0.5 M sodium phosphate)
`was incorporated into the drug dispersion while mixing, and
`the pH was readjusted to 3 using 0.1 M HCl. The dispersion
`was mixed and then kept in a refrigerator (4–5∞C) overnight
`(12 h).
`The drug content was determined by accurately weighing
`1 g of each gel, which was diluted with ethanol (95%),
`filtered and assayed by HPLC. The drug content in the gel
`was found to be 0.84–0.89 mg/g.
`Nail plates were soaked in water for 1 h and mounted on
`a nail adaptor. Five-hundred milligrams of the viscous gel
`(with or without enhancer) was placed in the donor
`compartment and the permeation studies were carried out
`for a period of 24 h, as previously described in preliminary
`in-vitro permeation studies.
`
`Statistical analysis
`Statistical analysis on the effect of different salts on the
`permeation of terbinafine through the nail, drug load in the
`nail and the influence of concentration of sodium phosphate
`on the drug permeation across the nail and dialysis membrane
`was performed using two tailed Kruskal–Wallis test (Graph-
`pad Prism 5; Graphpad software, Inc., CA, US). In all cases,
`post-hoc comparisons of the means of individual groups were
`performed using Dunn’s test. The effect on enhancement of
`terbinafine permeation across nail and dialysis membrane with
`different concentrations of sodium phosphate was analysed
`using Mann–Whitney U-test. The data points provided in the
`graphs are an average of four trials. The error bars represents
`the standard deviation. P < 0.05 denoted significance in
`all cases.
`
`

`

`434
`
`Journal of Pharmacy and Pharmacology 2009; 61: 431–437
`
`when compared with the control. The cumulative amount of
`drug permeated after 24 h with different salts (ammonium
`(8.09 ± 1.85 mg/cm2),
`sodium phosphate
`carbonate
`(11.44 ± 1.62 mg/cm2), calcium phosphate (7.75 ± 0.59 mg/
`cm2), potassium phosphate (10.47 ± 1.92 mg/cm2), sodium
`sulfite (6.93 ± 0.72 mg/cm2)) was found to be higher than the
`control (2.24 ± 0.72 mg/cm2). The profiles also indicated
`that the drug permeation was higher in the initial period
`(up to 12 h) and followed slow release afterwards, with all
`the enhancers studied.
`In contrast, control experiments
`exhibited much less permeation and the lag time to attain
`steady-state diffusion was ˜8 h.
`The drug load in the active diffusion area and peripheral
`area of the nail plate after 24 h of drug permeation studies
`with different enhancers and the control are represented in
`Figure 2. The inorganic salts enhanced the drug load in both
`the active diffusion area (4–7 fold, Kruskal–Wallis test,
`Dunn’s test, P = 0.011) and peripheral area (5–10 fold,
`Kruskal–Wallis test, P = 0.0275).
`Table 1 summarizes the solubility of terbinafine with
`different concentration of sodium phosphate at pH 3 and pH 5.
`The solubility decreased with increasing salt concentration at
`both pH 3 and pH 5.
`In the next step, permeation studies were carried out to
`evaluate the effect of the concentration of sodium phosphate
`(0, 0.25, 0.5, 1.0, 2.0 and 3.0 M) on drug permeation across
`the nail plate using terbinafine solution (1 mg/ml pH 3), and
`the data obtained are presented in Figure 3. The drug
`permeation increased with increase in salt concentration
`increase in salt
`up to 1 M and decreased with further
`concentration (2–3 M) (Kruskal–Wallis test, P = 0.001). A
`similar effect was also observed when the permeation was
`carried out at different salt concentrations across the dialysis
`membrane (Figure 4).
`
`Results
`
`Figure 1 represents the effect of different inorganic salts at
`terbinafine
`0.5 M concentration on the permeation of
`hydrochloride across the nail plate. It is evident that the
`permeation was significantly enhanced (3–5 fold, Kruskal–
`Wallis test, Dunn’s test, P = 0.008) in the presence of salts
`
`0
`
`5
`
`10
`
`15
`Time (h)
`
`20
`
`25
`
`30
`
`14
`
`12
`
`10
`
`8 6 4 2 0
`
`Cumulative amount permeated (␮g/cm2)
`
`Figure 1 Permeation profile of
`terbinafine hydrochloride in the
`presence of different salts. Permeation was carried out using terbinafine
`hydrochloride solution (1 mg/ml; pH 3) and the salt concentration was
`0.5 M. #, control; ♦, ammonium carbonate; ♢, potassium phosphate;
`■, sodium phosphate; !, calcium phosphate; , sodium sulfite. Data are
`expressed as mean ± SD, n = 4.
`
`Control
`
`m oniu m
`carbonate
`
`A m
`
`Calciu m
`phosphate
`
`Potassiu m
`phosphate
`
`Sodiu m
`phosphate
`
`Sodiu m
`sulfite
`
`(b)
`
`0.02
`
`0.015
`
`0.01
`
`0.005
`
`Amount of terbinafine (␮g/mg)
`
`0
`
`Control
`
`m oniu m
`carbonate
`
`A m
`
`Calciu m
`phosphate
`
`Potassiu m
`phosphate
`
`Sodiu m
`phosphate
`
`Sodiu m
`sulfite
`
`0.7
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0
`
`(a)
`
`Amount of terbinafine (␮g/mg)
`
`Figure 2 The amount of terbinafine hydrochloride loaded (mg/mg) in the nail diffusion area (a) and peripheral nail (b) during permeation studies in
`the presence of different salts (0.5 M) in 24 h. The diffusion area was 0.2 cm2. Data are expressed as mean ± SD, n = 4.
`
`

`

`Enhanced nail drug delivery by inorganic salts
`
`Anroop B. Nair et al.
`
`435
`
`0.25 M
`
`1 M
`0.5 M
`2 M
`Concn of sodium phosphate
`
`3 M
`
`9 8 7 6 5
`
`4 3 2 1 0
`
`Enhancement factor
`
`Table 1 Solubility of terbinafine hydrochloride in water (pH 3 and 5)
`with different concentration of sodium phosphate
`
`Concentration
`of sodium phosphate (M)
`
`Terbinafine hydrochloride
`solubility (mg/ml)
`
`pH 5
`pH 3
`1.12 ± 0.16
`1.57 ± 0.23
`0
`0.86 ± 0.10
`1.41 ± 0.18
`0.25
`0.74 ± 0.14
`1.19 ± 0.25
`0.5
`0.63 ± 0.08
`0.98 ± 0.20
`1.0
`0.54 ± 0.12
`0.79 ± 0.16
`2.0
`0.42 ± 0.11
`0.69 ± 0.11
`3.0
`Solubility studies were carried out at room temperature (25 ± 1∞C). Each
`value represents the mean ± SD, n = 5.
`
`Figure 4 Comparison of enhancement
`in terbinafine hydrochloride
`permeation across nail and dialysis membrane in the presence of different
`concentration of sodium phosphate. Enhancement was calculated with
`respect to control (1 mg/ml, pH 3 water). The donor constituted 1 mg/ml of
`terbinafine hydrochloride. Open columns, dialysis membrane; shaded
`columns, nail. Data are expressed as means ± SD, n = 4.
`
`0
`
`5
`
`10
`
`15
`Time (h)
`
`20
`
`25
`
`30
`
`12
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`Cumulative amount permeated (␮g/cm2)
`
`Figure 5 Effect of sodium phosphate on the permeation of terbinafine
`hydrochloride from poloxamer gel across the nail plate. The concen-
`tration of sodium phosphate used was 0.5 M. ♦, sodium phosphate;
`#, control. Data are expressed as mean ± SD, n = 4.
`
`Amount permeated in nail (␮g/cm2)
`
`18
`16
`14
`12
`10
`
`8 6 4 2 0
`
`0 M
`
`2 M
`1 M
`0.5 M
`0.25 M
`Concn of sodium phosphate
`
`3 M
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Amount permeated in dialysis
`
`membrane (␮g/cm2)
`
`Figure 3 Comparison of
`terbinafine
`the cumulative amount of
`permeated across the dialysis membrane and nail at the end of 24 h
`with different concentrations (0, 0.25, 0.5, 1.0, 2.0 and 3 M) of sodium
`phosphate. The diffusion area of the dialysis membrane and nail were
`0.64 cm2 and 0.2 cm2, respectively. Open columns, dialysis membrane;
`shaded columns, nail. Data are expressed as mean ± SD, n = 4.
`
`Mechanistic aspects on the enhancement of terbinafine
`permeation by inorganic salts were studied by pretreatment
`of nails with sodium phosphate solution (0.5 M for 24 h) and
`then followed by the permeation studies. The amount of
`terbinafine permeated after 24 h across the treated and
`untreated nail plate was 3.03 ± 0.52 and 2.39 ± 0.44 mg/cm2,
`respectively. Water uptake study indicated a higher percen-
`tage water uptake by the nail in the presence of inorganic
`(36.34 ± 4.68%) when compared with control
`salt
`(26.27 ± 4.55%). The cumulative amount of
`terbinafine
`permeated after 24 h from the formulated gel containing
`0.5 M sodium phosphate (9.70 ± 0.93 mg/cm2) was found to
`be comparable with the amount of drug permeated from
`solution (11.44 ± 1.62 mg/cm2) (Figure 5).
`
`Discussion
`
`Preliminary in-vitro permeation studies were carried out
`across the nail plate with different inorganic salts using
`terbinafine hydrochloride solution (1 mg/ml; pH 3). The
`concentration of all salts was kept at 0.5 M for the purpose of
`comparison. The donor vehicle was adjusted to pH 3 as the
`drug possesses high solubility at this pH as compared with
`
`higher pH. From Figure 1 the ability of salts to enhance the
`permeation of terbinafine across the nail plate and reduce
`the lag time to attain steady-state diffusion is evident. All the
`salts were comparable in their enhancement efficiency (3–5
`fold), although with sodium phosphate the difference to the
`control was significant (˜5-fold enhancement). This is the
`first report to our knowledge on the enhancement effect of
`inorganic salts in transungual drug delivery.
`
`

`

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`Journal of Pharmacy and Pharmacology 2009; 61: 431–437
`
`During permeation across biological membranes, a certain
`amount of drug is retained in the barrier. The drug retention is
`a factor of its solubility in the membrane, pH–pKa conditions
`of the experiment and concentration of the drug. Retention due
`to irreversible binding generally hampers the permeation of a
`drug into deeper tissues in topical drug delivery. However, in
`the case of nail, we observed that the terbinafine loaded in
`the nail during the permeation process formed a depot and
`released subsequently to provide prolonged therapeutic effect
`in the affected target tissue underneath the nail plate.[15] It is
`evident from Figure 2a in our study that the presence of salts
`significantly enhanced the drug load (4–7 fold) compared
`with the control. Like permeation, the highest drug load was
`observed with sodium phosphate (0.47 ± 0.16 mg/mg).
`Further, the amount of drug permeated across the nail plate
`was found to be proportional to the drug load in the nail.
`The amount of drug loaded in the active diffusion area
`represents the area of nail that is in direct contact with the
`formulation. In vivo, this would be the exposed part of the nail
`available for application of drug formulation. Onychomycosis
`affects the whole nail apparatus, which includes the nail that is
`not accessible due to overlap by the nail fold. Therefore, the
`drug loaded in the active diffusion area is required to diffuse
`peripherally into the area that is unexposed to formulation.
`This peripheral diffusion, in turn, plays a crucial role in the
`success of the onychomycosis therapy. In our permeation
`experiments, the amount of drug loaded into the surroundings
`of the active diffusion nail area was also assessed. The amount
`of drug reaching the peripheral nail area depends on the
`concentration gradient between the active diffusion area and
`peripheral nail area. It is obvious that the salts enhanced the
`drug load in the peripheral nail as they could enhance the drug
`load in the active diffusion area (Figure 2b).
`Given the promising results in the preliminary permeation
`studies, further studies on the effect of salt on transungual drug
`delivery was investigated using sodium phosphate as a
`representative salt enhancer. An increase in salt concentration
`did lead to increased drug permeation. However, the drug
`permeation decreased at higher salt concentrations (2–3 M)
`(Figure 3). To obtain further insight into the mechanisms
`responsible for the enhancement of drug permeation by
`inorganic salts, further studies were carried out. Generally,
`drug penetration enhancers act by modifying the structure of
`the barrier or by driving the drug into the barrier. If the
`permeation enhancers are capable of bringing about irrever-
`sible changes in the barrier property of the tissue, then the
`permeability of the nail plate would be enhanced upon
`pretreatment of the nail plate with the penetration enhancer.
`Hence to assess the effect of salts on the barrier property of
`nail, permeation studies were carried out across nails that had
`been pretreated with sodium phosphate solution (0.5 M for
`24 h). The permeation of terbinafine in 24 h across the treated
`(3.03 ± 0.52 mg/cm2) and control (2.39 ± 0.44 mg/cm2) nail
`plate suggests that the terbinafine permeation enhancement
`observed (in preliminary studies and the effect of salt
`concentration in the subsequent studies) was not due to any
`irreversible structural alteration of the nail plate.
`The other possible mechanism responsible for the penetra-
`tion enhancement
`is thermodynamic driving due to the
`concentration gradient. To explain the enhancement of
`
`permeation based on thermodynamic activity, one should
`know the solubility of drug in different concentrations of sodium
`phosphate solution. From Table 1, it can be noted that the
`solubility of terbinafine hydrochloride decreased with an
`increase in salt concentration. It is well known that permeation
`flux is maximal from saturated solutions of drug due to the
`maximum thermodynamic activity. In this study the donor
`concentration used was 1 mg/ml and from Table 1 it is apparent
`that
`the thermodynamic activity of terbinafine increased
`in the salt solution in the order of 0.25 < 0.5 < 1 M. At 1 M
`concentration, the solubility was 0.98 ± 0.20 mg/ml, which has
`the maximum thermodynamic activity of unity. Thus, it appears
`that increase in thermodynamic activity is one of the mechan-
`isms for the enhanced terbinafine permeation (cumulative
`amount permeated in 24 h) across the nail plate. At concentra-
`tions >1 M the thermodynamic activity still remained one, but
`the absolute solubility of drug decreased with increasing salt
`concentration. This is the likely reason for the decrease in drug
`permeation from salt solution concentrations above 1 M. Although
`there was a meaningful relationship interpreted between the per-
`meation data and thermodynamic activity, this experiment does
`not rule out the possible real time effect of salts on the nail plate.
`To understand the relative contribution of thermodynamic
`activity and the potential real time effect of salts on the nail
`plate, two sets of permeation studies were carried out. In the first
`set, permeation studies were carried out across the nail using
`terbinafine hydrochloride solution (1 mg/ml) at pH 5 (in the
`absence of salt) in which the thermodynamic activity of the
`solution would be higher than at pH 3 due to low drug solubility.
`The drug solubility at pH 3 and 5 was 1.57 ± 0.23 and
`1.12 ± 0.16 mg/ml, respectively. The cumulative amount of
`drug permeated across the nail at pH 5 was 1.5-fold
`(3.38 ± 0.39 mg/cm2) that observed at pH 3. This study further
`confirms that the thermodynamic activity has a significant
`role to play in the transungual drug permeation enhancement
`by salts. Here the important point to note is that the solubility
`of terbinafine at pH 5 in the absence of any salt (1.12 ±
`0.16 mg/ml) was comparable with its solubility at pH 3 in the
`presence of 0.5 M sodium phosphate (1.19 ± 0.25 mg/ml).
`However, the enhancement in drug permeation observed in the
`latter case was significantly higher (5 fold) than in the former.
`In the second set of studies, the permeation experiments were
`carried out at different salt concentrations across a dialysis
`membrane. The salt concentration on both sides of the
`membrane was the same so as to avoid a concentration gradient.
`In this case, the cumulative amount permeated showed a similar
`salt-concentration-dependent trend as observed with the nail
`permeation studies (Figure 3). However, it is noteworthy that the
`permeation enhancement factor (in dialysis membrane) over the
`control was lower (Mann–Whitney U-test, P = 0.0286) than that
`observed across the nail plate (Figure 4). The above two sets of
`studies suggests that there could be additional mechanisms
`contributing to the permeation enhancement by salts, besides
`thermodynamic activity.
`Water is known to be a plasticizer for nail as it hydrates
`up to ~25% of the nail weight.[16] It is well known that most
`of the permeation enhancers generally increase the hydration
`level of nail keratin molecules. Increased hydration of keratin
`molecules is believed to provide a continuous pathway to
`the diffusion of molecules across the nail plate and thereby
`
`

`

`Enhanced nail drug delivery by inorganic salts
`
`Anroop B. Nair et al.
`
`437
`
`enhances the permeation and drug load.[17] Moreover, it is
`also reported that the pH of the solution does not influence
`the nail hydration level.[18] Hence it was thought rational to
`determine the water uptake of nail
`in the presence and
`absence of salt at pH 3. The calculated percentage of water
`uptake by nail
`in the presence of salt enhancer was
`36.34 ± 4.68%, and for the control it was 26.27 ± 4.55%.
`The apparent difference in hydration level due to the
`presence of sodium phosphate is one of the key mechanisms
`responsible for the transungual drug permeation enhance-
`ment effect of salts. However, quantifying the water content
`in the nail plate at different salt concentrations would be a
`challenging task due to the difference between the tissues and
`due to the lack of any simple and sensitive method of
`measuring water in a way applicable to the present case.
`Neverthless, from this study we can speculate that
`the
`increase in salt concentration increases the hydration of
`keratin in the nail plate. When the drug concentration in the
`salt solution is constant (as in the case of salt concentra-
`tions <1 M), the increased hydration would in turn increase
`the drug loading in the nail. At salt concentrations above 1 M,
`the absolute drug concentration is less, which is likely the
`reason for the relative decrease in the drug load, despite the
`increased hydration of nail plate at higher salt concentration.
`The last phase of study was intended to develop a suitable
`formulation and assess the permeation across the nail plate.
`As mentioned earlier there are different formulations available
`for topical application to the nails. Most of these are nail
`lacquers in which a film-forming polymer is dissolved in a
`rapidly evapo