Comparison of the Antifungal Efficacy of Terbinafine Hydrochloride

Feb 3, 2014 - Onychomycosis is a fungal infection mostly induced by dermatophytes such as Trichophyton rubrum. Due to slow nail growth, the treatment ...
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Comparison of the Antifungal Efficacy of Terbinafine Hydrochloride and Ciclopirox Olamine Containing Formulations against the Dermatophyte Trichophyton rubrum in an Infected Nail Plate Model Anja Taü ber and Christel C. Müller-Goymann* Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany ABSTRACT: Onychomycosis is a fungal infection mostly induced by dermatophytes such as Trichophyton rubrum. Due to slow nail growth, the treatment takes 3−9 months depending on the nail size and infected area. Hence, high efficacy of the active ingredient without systemic side effects is of major interest. To test the efficacy of an antifungal formulation, an appropriate in vitro model reflecting the in vivo situation as close as possible is required. In this study, a variety of antifungal formulations, i.e., commercial ones (Ciclopoli and Lamisil cream), those used in compounding pharmacies (Pentravan) as well as poloxamer 407-based systems, have been evaluated in an infected nail plate model. The active pharmaceutical ingredients (APIs) were ciclopirox olamine and terbinafine hydrochloride. The poloxamer 407-based formulations consisted of poloxamer 407, double distilled water, propylene glycol, isopropyl alcohol, medium chain triglycerides and either 1% ciclopirox olamine or 1% terbinafine hydrochloride as API, respectively. Former studies have shown high permeation rates of terbinafine hydrochloride from similar poloxamer 407-based formulations with dimethyl isosorbide instead of propylene glycol. The present contribution shows superior inhibition of T. rubrum growth from poloxamer 407-based formulations in comparison to the commercial Lamisil cream. Moreover, poloxamer 407-based formulations were equally effective as the nail lacquer Ciclopoli even though the poloxamer formulations contained only 1% of the drug instead of 8% in the marketed lacquer. Poloxamer 407-based systems containing ciclopirox olamine proved to be about as effective as similar terbinafine hydrochloride systems. KEYWORDS: ciclopirox olamine, terbinafine hydrochloride, Trichophyton rubrum, onychomycosis, poloxamer 407, Ciclopoli, Lamisil, Pentravan

1. INTRODUCTION Onychomycosis is defined as a nail infection caused by fungi and is accountable for more than 50% of all nail disorders.1 Infections can be caused by yeasts, dermatophytes and nondermatophyte molds. Toenails are 4−10 times more prone to infection in comparison to fingernails. More than 90% of all toenail disorders are induced by dermatophytes such as Trichophyton rubrum and Trichophyton mentagrophytes.2 Nail infections display a thickened and discoloured nail plate and affect the quality of life due to aches and pain. In addition, untreated nails are more vulnerable to secondary infections.3 The onychomycosis therapy may be systemic, topical, chemical, mechanical (surgical avulsion) or a combination thereof.4 The longer the duration of therapy, the higher the risk of side effects and the lower the patient compliance. A combination of oral and local drug application proved superior5 in reducing the time until a full removal of symptoms. The advantage of a topical onychomycosis treatment over a systemic therapy includes reduced drug interaction and less hepatotoxicity and gastrointestinal and systemic side effects.6 However, the keratinized structure of the nail shows a high resistance to drug permeation. As a result, it is challenging to © 2014 American Chemical Society

ensure sufficient penetration into and permeation through the nail. In vitro tests are important to gain information regarding the efficacy of antimycotic agents. In this study, the infected nail plate model established by Lusiana et al. was used.7 During the entire therapy, the concentration of the drug has to be kept above the minimum inhibitory concentration (MIC). Active pharmaceutical ingredients (APIs) being available for topical onychomycosis treatment are ciclopirox, amorolfine, econazole, bifonazole2 and octopirox.8 All are lipophilic and of low molecular weight.9,10 In addition to the API itself, an appropriate carrier is required to support the release and permeation of the API. Nail lacquer or gel-like formulations are preferred dosage forms9 due to their high water content. The water evokes nail swelling and aids Special Issue: Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation Received: Revised: Accepted: Published: 1991

November 26, 2013 January 24, 2014 January 29, 2014 February 3, 2014 dx.doi.org/10.1021/mp400711q | Mol. Pharmaceutics 2014, 11, 1991−1996

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percentage and either 1% ciclopirox olamine or 1% terbinafine hydrochloride, respectively (Table 1).

drug permeation. In our group, poloxamer 407-based hydrogels with thermoreversible gelation have been studied extensively with a variety of APIs such as 5-aminolevulinic acid and terbinafine hydrochloride.7,11 The composition of such systems included poloxamer 407 (POX), dimethyl isosorbide (DMIS), isopropyl alcohol (IPA), medium chain triglycerides (MKT), double distilled water and 1% terbinafine hydrochloride. 5Component systems have been represented within a pseudo ternary phase diagram with double distilled water, a fixed 1:1 ratio of IPA and DMIS and a fixed ratio of 4:1 for POX and MKT as the vertices of a triangle.11 In this contribution, liquid and gel-like poloxamer 407-based formulations are of particular interest. Due to an incompatibility between terbinafine hydrochloride and DMIS recognized as yellow discolouration,12 DMIS should be replaced by propylene glycol (PG). PG is favorable, since it is monographed in the Ph. Eur. and was available in pharmaceutical grade.

Table 1. Composition of Poloxamer 407-Based Formulations

1P/T2525 1P/T3040 1P/T4030

water [%]

IPA [%]

PG [%]

MKT [%]

POX 407 [%]

API [%]

49.50 29.70 29.70

12.38 19.80 14.85

12.38 19.80 14.85

4.95 5.94 7.92

19.80 23.76 31.68

1.00 1.00 1.00

1P2525 represents a formulation loaded with 1% CPX, while the vehicle (P2525) itself contained 25% POX/MKT (4:1), 25% IPA/PG (1:1) and 50% double distilled water; 1T2525 contained 1% TBF instead of CPX. All ingredients were weighed in an Unguator jar and automatically stirred at 1440 rpm for 1.5 min with an Unguator e/s GAKO Konietzko GmbH (Bamberg, Germany). Subsequent storage of the poloxamer 407-based formulations was done for 24 h in a temperature controlled room at 20 °C to ensure sufficient equilibration prior to the study. The vehicles P2525, P3040 and P4030 were chosen because of their different macroscopic appearances. P2525 had a creamlike appearance. Incorporation of API led to liquid and translucent emulsions with saturation solubility of 4% for CPX and 2.5% for TBF, respectively. Saturation solubility was defined as the concentration with first appearance of API crystals under a polarizing microscope Leica DM LM (Leica Microsystems GmbH, Wetzlar, Germany). The vehicle P3040 and the P/T3040 formulations were liquid and translucent emulsions. The saturation concentration was determined at 10% for CPX and 3% for TBF. The vehicle P4030 as well as the P/T4030 poloxamer 407-based formulations showed a gel-like appearance of moderate consistency with a ringing effect when the jar was knocked onto a hard surface. A maximum of either 5% CPX or 3.5% TBF was dissolved in the formulation, respectively. With increasing drug concentrations, the formulations became softer. Furthermore, it should be mentioned that 1P/T2525 had a ratio of POX/MKT:IPA/PG (1:1), while 1P/T3040 and 1P/ T4030 displayed a 1:1 ratio of POX/MKT:H2O and IPA/ PG:H2O, respectively. 2.2.2. Pentravan Fagron. Pentravan (= penetration enhancing vanishing) cream manufactured by Fagron (Barsbüttel, Germany) is an oil-in-water emulsion with a liposomal matrix.13 It is claimed to enhance the penetration of an API from the formulation into the skin. Consequently, API should become available in a shorter time and to a higher extent. The cream is slightly yellow to off-white with a pH of 4.0− 5.5. The water content is 62% (w/w), and the oil content accounts for 28% (w/w).14 In this study, either 1% terbinafine hydrochloride or 1% ciclopirox olamine was incorporated using the Unguator e/s with 1440 rpm for 1.5 min, respectively. After 24 h of equilibration, the formulations were tested for antifungal activity in the in vitro infected nail plate model. 2.2.3. Commercial Pharmaceutical Products. Ciclopoli from Taurus Pharma GmbH (Bad Homburg, Germany) is a nail lacquer containing 8% ciclopirox. It contains hydroxypropyl chitosan (HPCH), i.e., a water-soluble ingredient that is able to build a film on the nail plate.15 Ciclopirox acts against a broad

2. MATERIALS AND METHODS 2.1. Materials. Ciclopirox olamine (CPX), propylene glycol (PG), medium chain triglycerides (MKT) and poloxamer 407 (POX) were purchased from Fagron (Barsbüttel, Germany); 2propanol (IPA) was received from VWR International (Leuven, Belgium); terbinafine hydrochloride (TBF), Ph. Eur. 6.0 grade, was purchased from Suzhou Leader (Chemical Co., Ltd. China) and water was used in double distilled quality. Phosphate buffered saline (PBS) was prepared with PBS tablets from MP (Illkirch, France) according to the package instructions. For PB pH 5.8 (Ph. Eur. 7.0, 4002100), disodium hydrogen phosphate dihydrate and potassium dihydrogen phosphate were purchased from Merck KGaA (Darmstadt, Germany). Blond hair was received from a local hairdresser and washed with water containing 0.5% sodium dodecyl sulfate (SDS) before being used. SDS was purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Bovine hooves were ordered via an online pet shop (Edingershops, Germany). For the keratin film manufacture, urea, thiourea and glycerol were received from Carl Roth GmbH + Co. KG (Karlsruhe, Germany); 2-mercaptoethanol and Trizma base were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Silicone paste (Baysilone-Paste, high viscous from GE Bayer Silicones) was delivered by Carl Roth GmbH (Karlsruhe, Germany). For dialysis tubing, a Spectra/Por Dialysis Membrane (MWCO: 6−8,000 Da, Spectrum Laboratories, Inc., Rancho Dominguez, California) was used. The Trichophytum rubrum strain DSM 19959 was obtained from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and rejuvenated on a malt extract charcoal medium. For the manufacture of the different agars, medical charcoal was received from Caesar & Loretz GmbH (Hilden, Germany), agar−agar, Kobe I from Carl Roth GmbH + Co. KG (Karlsruhe, Germany) and malt extract, potato glucose agar and Sabouraud dextrose broth were purchased from Fluca Analytical (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). 2.2. Formulations Involved in This Assay. 2.2.1. Preparation of the Poloxamer 407-Based Formulations. The tested formulations 1P/T2525, 1P/T3040 and 1P/T4030 consisted of double distilled water, 2-propanol, propylene glycol, medium chain triglycerides, poloxamer 407 in a specific 1992

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Table 2. Excipients of the Commercial Products product Ciclopoli Lamisil cream Pentravan cream

excipient ethyl acetate, ethanol 96%, cetyl stearyl alcohol, hydroxypropyl chitosan, water (1 g of Ciclopoli contains 80 mg of ciclopirox) isopropyl myristate, polysorbate 60, stearyl alcohol, cetyl alcohol, cetyl palmitate, sorbitan stearate, benzyl alcohol, water, sodium hydroxide (1 g of Lamisil cream contains 10 mg of terbinafine hydrochloride) water, isopropyl myristate, stearic acid 50, glycerol monostearate, polyoxyl 40 stearate, isopropyl palmitate, lecithin, simethicone, urea, cetyl alcohol, stearyl alcohol, potassium sorbate, sorbic acid, benzoic acid, butyl hydroxy toluene, disodium edetate dihydrate, carbomer 980, hydrochloric acid 35%

A volume of 2 mL (1 mL was added first, the next 1 mL after 4 h) was sufficient to create approximately 100 μm thick films. The films were allowed to dry for 24 h at 40 °C before being baked at 110 °C for 3 h to oxidize the disulfide bridges between keratin molecules. The finished films were water-insoluble and refractory. Afterward, they were hydrated in PBS for 1 h. Then circles of 17 mm diameter were cut out and the thickness of the films was determined with a micrometer screw gauge. The dried films had diameters of 15 mm. In this study, keratin films with a thickness of 120 μm were used exclusively. 2.5. Preparation of the Bovine Hooves. The bovine hoof plates were produced from bovine hoof ordered via an online pet shop. The sole part of the hooves was cut into 2 × 2 cm squares in our workshop. After being submerged in double distilled water for 1−3 days, the hooves were soft enough to be sliced into 100 μm thick plates with a rotational microtome MICROM HM 355S (Walldorf, Germany). Afterward, circles (diameter: 15 mm) were cut out and inserted in PBS for one hour to check the thickness of the plates. 2.6. Experimental Setup of Infected Nail Plate Cultivation. The setup was done as described by Lusiana et al.7 In brief, PGA was inoculated with 7 day old T. rubrum by streaking a cotton swab in three directions over the agar. The plate was allowed to dry for 15 min before 5 hoof plates or keratin films were placed in it. The incubation was done for 7 days at 30 °C to enable full confluence of T. rubrum. The membranes were then transferred to SDA (one membrane per agar), and a small polyamide (PA) ring (height 5 mm, outer diameter 12.5 mm, thickness 3 mm; created in our workshop) was glued onto the membrane with silicone paste to avoid leakage of liquid formulations. The formulation was injected with a sterile syringe (volume: 2 mL) from B. Braun Melsungen AG (Melsungen, Germany) into the PA ring. To prevent evaporation of elusive compounds, a PET foil was glued with silicone paste onto the ring. This composition was further incubated for 6 days at 30 °C. Each batch of 5 membranes from 1 PGA included 1 positive control (without treatment), which showed complete fungal growth. All utilized materials were autoclaved before usage at 121 °C, 2 bar for 15 min. 2.7. Evaluation of the Result. Results were evaluated with a score of 0 to 10. 10 represented full fungal growth at and on the border of the membranes. Assemblies without fungi were scored with 0. Partial fungal growth was scored with values on a percentage basis, e.g., 1 indicated that 10% of the membrane border was populated with T. rubrum. 2.8. Statistical Evaluation. Statistical analysis was performed using the software IBM SPSS Statistics 21. The scores obtained for each formulation on KF and on hooves were compared. Due to not normally distributed scores,

spectrum of fungi in a sporicidal manner, even in the passive state of fungi when the ergosterol synthesis is not active.16 Lamisil cream (Novartis GmbH (Nürnberg, Germany)) is a marketed cream against tinea pedis and contains 1% terbinafine hydrochloride among other ingredients (Table 2). Both commercial pharmaceutical products were purchased from a local pharmacy and were stored according to the package information leaflet. 2.3. Agar. Malt extract charcoal medium (MCM) was manufactured with 3% (w/w) malt extract, 0.30% (w/w) medical charcoal, 1.65% (w/w) agar−agar and double distilled water. MCM was used for rejuvenation of T. rubrum. The rejuvenated culture was stored for 7 days at 30 °C, before being placed in a refrigerator. Medical charcoal served as carbon source for T. rubrum. Potato glucose agar (PGA) was prepared according to the package instructions (15.60 g of powder and 400 mL of double distilled water). This agar induced the production of conidia.17 Sabouraud dextrose agar (SDA) contained 2.86% (w/w) Sabouraud dextrose broth, 1.90% (w/w) agar−agar and double distilled water. SDA supported the growth of T. rubrum18 and served as nail bed model.7 Media were autoclaved at 121 °C, 2 bar for 15 min. Following the procedure, 12 mL of PGA and 20 mL of SDA were pipetted on disposable Petri dishes (92 × 16) with cams (Sarstedt AG, Nümbrecht, Germany) under a laminar air flow. After cooling, the Petri dishes were stored at 6−8 °C in a sterile bag. For the MCM, 7 mL was pipetted into test glasses and cooled aslant to enlarge the agar surface. 2.4. Preparation of Keratin Films. The keratin films (KF) were produced according to Lusiana et al.19 Briefly, 25 g of washed hair was cut into 1−3 cm long parts and given 500 mL of Shindai solution (containing 25 mM Tris pH 8.5, 2.6 M thiourea, 5 M urea and 5% (v/v) 2mercaptoethanol). This mixture was extracted for 72 h at 50 °C. Afterward, the insoluble hair was filtered off with three layers of medical gauze, and then centrifuged at 5,000 rpm for 15 min. The supernatant was filtered with filter paper (2.5 μm pore size) using a waterjet pump. The filtrate was filled into a dialysis tube (Spectra/Por Dialysis Membrane (MWCO: 6−8,000 Da, Spectrum Laboratories, Inc., Rancho Dominguez, California)). The dialysis against water was done for 72 h at ambient temperature to remove remaining components of the extraction process. Afterward, the dialysate was centrifuged 5 times at 10000g for 30 min to eliminate aggregates. The supernatant was mixed with 1% (w/w) glycerol to gain soft films. The mixture was degassed under vacuum to remove possible air bubbles, which could cause holes in the dried films. To create the keratin films, a Teflon ring (constructed in our workshop) was glued onto a PET foil with silicone paste, which was stabilized with a sheet metal. 1993

dx.doi.org/10.1021/mp400711q | Mol. Pharmaceutics 2014, 11, 1991−1996

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Table 3. Score after the Treatment with Antifungal Formulation at Day 6 of Incubation KF treatment

score

n

mean ± SD

hoof median

score

mean rank

n

mean ± SD

median

KFb

hoofb

p valuec

positive control (untreated) Ciclopoli Lamisil 1T2525 1P2525 Pentravan + 1% CPX

10

10

10

10

10

16

10 ± 0

10

ndd

nd

nd

0; 0; 0; 0 7; 9; 8; 10 0; 0; 0.5; 0 0.5; 0; 0; 0 10; 9; 10; 10

4 4 4 4 4

0 8.50 ± 1.29 0.125 ± 0.25 0.125 ± 0.25 9.75 ± 0.5

0 8.5 0.25 0.25 9.5

8 8 8 8 8

0.125 ± 0.35 5.25 ± 2.25 0.125 ± 0.35 0.188 ± 0.37 8.25 ± 3.49

0.5 5 0 0 10

6.00 9.75 6.88 6.38 7.25

6.75 4.88 6.31 6.56 6.13

0.480 0.022 0.695 0.911 0.543

Pentravan + 1% TBF 1P3040 1P4030 1T3040 1T4030

9; 0; 0; 0; 0;

4 4 4 4 4

7.75 ± 1.5 0 0 0 0

8 0 0 0 0

0; 0; 1; 0; 0; 0; 0; 0 9; 7; 1; 5; 5; 5; 5; 5 1; 0; 0; 0; 0; 0; 0; 0 0; 1; 0.5; 0; 0; 0; 0; 0 9; 10; 0;a 10;a 10; 10; 7; 10 8; 8; 9; 10; 10; 10; 10; 7 0; 0; 0; 0; 0.1; 0 0; 0; 0; 0 0; 0; 0; 0 0; 0; 0; 0

8 6 4 4 4

9.00 ± 1.20 0.017 ± 0.04 0 0 0

9.5 0 0 0 0

4.38 5.00 4.50 4.50 4.50

7.56 5.83 4.50 4.50 4.50

0.137 0.414 1.000 1.000 1.000

a

7; 0; 0; 0; 0;

9; 0; 0; 0; 0;

6 0 0 0 0

Incubation for 14 days, at day 6: scores 0 and 0.5, respectively. bAccording to Mann−Whitney U test. cTwo tails. dNot determined.

Figure 1. Hoof (B−D) and KF (E, F) after treatment with antifungal formulations; A, positive control (hoof) without treatment; B, treated with Ciclopoli (score: 0); C, treated with Pentravan cream containing 1% terbinafine hydrochloride (score: 10); D, treated with 1T2525 (score: 0); E, treated with Lamisil cream (score: 10); F, treated with poloxamer 407-based formulation containing 1% ciclopirox olamine (1P3040) (score: 0).

According to former studies by Lusiana,12 the 120 μm thick keratin films showed the same permeability coefficients and accumulated amounts in the membranes as 100 μm thick hoof plates for two different molecules (sodium fluorescein and fluorescein isothiocyanate-dextran MW 4000). The results of the present study are shown in Table 3. Except for Lamisil cream (p < 0.05), the scores of the tested formulations on KF and on hoof plates did not show any statistically significant differences between the two models.

the nonparametrical Mann−Whitney U test was used. A p value of 0.05 (Table 3), although keratin films and hoof plates differ highly in their water absorption capacity.19 Regarding the median score of 1P2525, we received a score of 0.25 (n = 8) for KF and a score of 0 (n = 8) for hooves with p = 0.911.

The poloxamer 407-based formulations 1P4030, 1T4030 and 1T3040 inhibited fungal growth completely. 1P3040 showed total inhibition by using keratin films and minimal fungal growth on hoof membranes (score: 0.017, n = 6). Minimal growth of T. rubrum was detected for 1P2525 and 1T2525 on hooves (Figure 1D) as well as on KF. All poloxamer 407-based formulations had a high water content (30−50%). In accordance with the literature, high water contents support nail hydration and increase drug permeation as well as nail’s mean porosity.20 TBF and CPX are lipophilic molecules, which can be released more quickly from a hydrophilic formulation than from a multiphase cream. In addition, CPX has a low molecular weight (207.27 g/mol), which may facilitate drug penetration into the nail plate.9 The Pentravan formulations with either 1% TBF or 1% CPX, respectively, did not inhibit growth of T. rubrum at all and showed mean fungal growth scores between 7.75 and 9.75 on KF and hooves (Figure 1C). Regarding the test series with Pentravan containing 1% CPX, there were two agar plates with scores of 0 and 0.5 after 6 days of incubation whereas six plates showed high fungal growth scores. To analyze whether fungal growth would still occur, the incubation time was enlarged to 14 days. Finally, scores of 0 and 10 were observed. The latter result suggests that the agar plate did not sufficiently nourish the culture to allow fungal growth. Although the manufacturer Fagron advertises its product Pentravan cream as having both a high penetration rate and bioavailability,13 our findings did not reflect the company’s claim. The high scores showed that the drugs TBF and CPX were not released from the multiphase formulation. Therefore, the growth of the fungal cultures was not inhibited. Regarding the commercial products, Lamisil cream reached a score of 8.50 on KF (Figure 1E) and 5.25 on hooves, respectively. Consequently, it did not inhibit fungal growth. Lamisil cream is a creamy formulation containing 1% terbinafine hydrochloride. Terbinafine hydrochloride is an antifungal drug belonging to the allylamine class, which inhibits the fungal squalene epoxidase. Consequently, the fungus does not produce ergosterol any longer, which finally kills the cells. TBF is a widely accepted API and evaluated to be a highly potent drug against onychomycosis.21 However, its highly lipophilic nature reduced release from the vehicle and availability within tissue. Skin consists of three different layers (epidermis, dermis and subcutaneous fat tissue) with different properties. The main permeation barrier is the epidermis, especially the stratum corneum. It comprises dead cells (corneocytes) being embedded in a lipid matrix and can be considered as lipophilic barrier due to its low water content.22 Nail plates also consist of three layers: the dorsal, the intermediate and the ventral nail plate. The ventral and subungual keratin is the most likely to be infected. The nail plate behaves as a hydrophilic gel membrane with a suggested additional lipophilic route.23 Lamisil cream is only approved for fungal skin infections and nail beds after, e.g., a keratolytic antifungal treatment.24 Skin has a different structure in comparison to nail. This could be the reason why Lamisil cream was not effective against T. rubrum on the artificial nail model. Nevertheless, the scores for Lamisil cream concerning KF and hooves showed differences, yet with high standard deviations. The p exact value is 0.022, which means that the

4. CONCLUSION The infected nail plate model has successfully been adopted to analyze the efficacy of various antifungal formulations. Excluding Lamisil cream, KF and hooves yielded similar scores. The poloxamer 407-based formulations 1P4030, 1T4030 and 1T3040 have been slightly more effective than Ciclopoli in inhibiting the growth of T. rubrum and considerably more effective than Lamisil cream. Terbinafine hydrochloride and ciclopirox olamine showed approximately the same efficacy, if these APIs were incorporated into the same compounded poloxamer 407based formulations. In conclusion, we have developed highly effective antifungal formulations despite low drug content.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +49 531 3915650. Fax: +49 531 3918180. E-mail: c. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

A.T. would like to thank Lusiana for advice throughout this study as well as Carolin Rattunde (Institut für Pharmakologie, Toxikologie und Klinische Pharmazie, Technische Universität Braunschweig, Germany) for technical assistance. 1995

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dx.doi.org/10.1021/mp400711q | Mol. Pharmaceutics 2014, 11, 1991−1996