Antimicrobial Activity against Propionibacterium acnes and

Apr 1, 2013 - ... of Isotretinoin: Antimicrobial Activity against. Propionibacterium acnes and Dermatokinetic Modeling. Kaisar Raza,. †. Bhupinder S...
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Nanocolloidal Carriers of Isotretinoin: Antimicrobial Activity against Propionibacterium acnes and Dermatokinetic Modeling Kaisar Raza,† Bhupinder Singh,†,‡ Saloni Singla,§ Sheetu Wadhwa,‡ Babita Garg,‡ Sanjay Chhibber,§ and Om Prakash Katare*,‡ †

UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and Nanocomposites, Panjab University, Chandigarh, India 160014 ‡ Division of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India 160014 § Department of Microbiology, Panjab University, Chandigarh, India 160014 ABSTRACT: Acne, a common skin disease in teenagers, is caused by Propionibacterium acnes (P. acnes). Isotretinoin (ITR) is though reported to have immense antiacne potential, yet there are hardly any reports vouching its antimicrobial activity. The present study, therefore, was undertaken to study the antimicrobial activity of ITR and evaluate the effect of its encasement in nanocarriers on its minimum inhibitory concentration (MIC). The nanocarriers were also evaluated for the skin transport characteristics. MICs of pure drug and entrapped drug in nanolipid carriers (ITR-NLCs) and in solid lipid nanoparticles (ITRSLNs) were determined by broth dilution method against clindamycin phosphate as the reference antibiotic. It was observed that ITR possessed marked antimicrobial activity against anaerobic pathogen, P. acnes. Nanocarriers loaded with ITR, that is, SLNs and NLCs, enhanced the antimicrobial activity even at lower concentrations vis-à-vis the drug alone and improved drug transport potential vis-à-vis the commercial gel. The unique findings could be the result of effective adhesion of ITR-loaded nanocarriers to the bacterial membranes and release of drug directly to the target. Besides establishing ITR as an antimicrobial agent against acne-causing bacteria, the current work ratifies immense potential of nanocolloidal carriers like SLNs and NLCs to treat acne in a more efficient manner. KEYWORDS: minimum inhibitory concentration (MIC), solid lipid nanoparticles (SLNs), nanolipid carriers (NLCs), Propionibacterium acnes, anaerobic pathogen, acne vulgaris, comedolytic

1. INTRODUCTION

Though acne is seldom associated with mortality, it substantially affects the psychological and social status of the affected population.6,7 Other concerns associated with acne are the long tenure of treatment along with economic issues.8 Topical retinoids alone, or in combination with antibiotics, are widely prescribed for the treatment of mild to moderate acne.9,10 For severe acne, however, oral isotretinoin (ITR) is frequently prescribed.11 Use of antibiotics alone for the treatment of acne has yielded promising results.12−14 Nevertheless, issues of microbial resistance against antibiotics are of great concern.15,16 It is also advised that clinicians should limit the prescription of antibiotics, wherever and whenever possible.15 Hence, there are adequate reasons to search for alternative therapy or to improve the efficacy of the present treatment approaches.

Acne vulgaris, commonly known as acne, affects around 85% of teenagers, reaching its acme during adulthood.1,2 According to WHO, acne is an inflammatory disease of the pilosebaceous units in the skin of the face, neck, chest, and upper back. Typically, it first appears during early puberty when androgenic stimulation triggers excessive production of sebum and abnormal follicular keratinization, colonization by a Grampositive bacterium (i.e., Propionibacterium acnes), and local inflammation. P. acnes is one of the principal factors of acne pathogenesis. P. acnes produces inflammation through the production of extracellular products such as lipases, proteases, hyaluronidases, and chemotactic factors.1 It is the predominant microorganism of the infected pilosebaceous glands of acne skin, as nearly as 107 viable organisms are reported to be isolated from a single sebaceous unit.3,4 P. acnes typically grows in the infrainfundibulum of the pilosebaceous units and digests the oily substance produced by the sebaceous glands.3 Apart from acne, P. acnes overgrowth is also reported to be associated with diseases like toxic shock syndrome and endocarditis.2,5 © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1958

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dispersed in a portion of water along with Tween 80 (7.0 g) and SMBS (0.5 g). Compritol (1.2 g) and IPM (0.2 g) were melted at 70 °C. The lipid phase, aqueous phase, and drug solution were mixed isothermally to obtain a clear microemulsion. The remaining portion of water (about 80 mL) was cooled at 4 °C. The hot microemulsion formed was poured into cold water previously maintained at 4 °C, and the dispersion formed (100 mL) was stirred continuously for 20 min at 3000 rpm.23 2.3. Preparation of Bacteria. P. acnes (MTCC 1951) (Microbial Type Culture Collection, IMTECH, Chandigarh, India) was cultured on brain heart infusion media under anaerobic conditions using Anaerogas Pack at 37 °C. Single colonies were inoculated and incubated at 37 °C until reaching around OD600 = 0.3 (logarithmic growth phase) under anaerobic conditions. The bacteria were harvested by centrifugation at 5000g for 10 min, washed with PBS, and suspended in an appropriate amount of PBS.2 2.4. Antimicrobial Activity of ITR and Its Nanocarriers against P. acnes. P. acnes was incubated in BHI broth with 1% glucose for 48 h under anaerobic conditions and adjusted to yield approximately 1 × 108 CFU/mL. 2-fold serial dilutions were made in the broth over a range to give concentrations of 8−500 μg/mL of isotretinoin. In sterile 96-well microtiter plates, 100 μL of the drug solution (in 40% dimethyl sulfoxide, DMSO)/SLN-dispersion/NLC-dispersion/blank SLN-dispersion/blank NLC-dispersion/40% DMSO solution was diluted with broth and added to wells containing 100 μL of bacterial suspension in broth. To adjust the interference by nanoparticle components, a parallel series of mixtures with uninoculated broth was prepared. Triplicate samples were performed for each test concentration. After incubation for 48 h at 37 °C under anaerobic conditions, the quantum of microbial growth was determined by absorbance at 600 nm using a microplate reader (Biotek Instruments, Winooski, VT, USA). Aqueous solution of clindamycin phosphate was also studied analogously to serve as the control owing to its marked antimicrobial activity against P. acnes.24 MIC was taken as the lowest concentration of a test compound (i.e., ITR) which inhibited the growth of P. acnes.25 2.5. Drug Permeation Studies. Hairless Laca mice were employed for the skin permeation studies on Franz diffusion cells (M/s Permegear, Inc., PA, USA). After sacrificing the animals, the hairs on the dorsal side of animals were removed. The skin was harvested, freed of adhering fat layers, and mounted on Franz diffusion cells having a cross-sectional area of 3.142 cm2 and receptor volume of 30.0 mL. The diffusion medium in the receptor compartment was composed of Isotonic Palitzsch Buffer26 containing Tween 80 (2.7% w/w) and ethanol (20.0% v/v). The assembly was maintained at 37 ± 1 °C with the help of thermo-regulated outer water jacket, while the diffusion medium was stirred continuously using a magnetic stirrer. Various formulations (ITR-SLN gel, ITR-NLC gel, and commercial gel), each containing ITR equivalent to 0.6 mg, were applied onto the mice skin in the donor compartment. Aliquots of 1 mL each were periodically withdrawn at suitable time intervals from the sampling port and replaced with an equal volume of fresh diffusion medium to maintain the constant receptor volume. The samples were analyzed by a validated reverse phase high-performance liquid chromatographic (RP-HPLC) method.27 ITR was quantified at 355 nm using a liquid chromatograph LC-2010CHT (Schimadzu, Japan), equipped with a UV detector and a software LC

Novel drug delivery systems are known to enhance the overall performance of the drug at the target site vis-à-vis the conventional dosage forms.17 Solid lipid nanoparticles (SLNs) and nanolipid carriers (NLCs) are nanocolloidal systems composed of biocompatible lipids.18,19 The biocompatible lipid-based carriers can transport the drugs to the target site and maintain drug concentrations higher than that with the conventional formulations.2,20 These agents do not employ solvents like dimethyl sulfoxide (DMSO), which are commonly employed in other topical products as penetration enhancer(s) or solubilizer(s). Such penetration enhancers are reported to cause dermal irritation and at times, serious side effects.2 ITR is now-a-days also prescribed in the topical forms and is believed to affect all of the causative mechanisms of acne, including infestation by P. acnes.10 The hypothesis of ITR antimicrobial activity against P. acnes has not yet been proved in vitro or in vivo. Apart from this, it is a newly reported fact that use of novel carrier systems can result in substantial antimicrobial activity of the drug even at lower concentrations.2,21 Hence, the present study endeavors to establish the antimicrobial activity of ITR against P. acnes and to study the influence of encapsulation in nanocarriers SLNs and NLCs on the skin transport characteristics, as well as on the minimum inhibitory concentration (MIC). The developmental studies have been published elsewhere along with antiacne activity, biocompatibility, and biochemical evaluation.22,23

2. MATERIALS AND METHODS 2.1. Materials. Isotretinoin (ITR; M/s Ipca Laboratories Ltd., Ratlam, India), phosphatidylcholine (PC; Phospholipon 90 G; M/s Phospholipid GmbH, Nattermannallee, Germany), clindamycin phosphate (M/s Psycoremedies, Ludhiana, India), and Compritol 888 ATO GF3123 (M/s Gattefosse, Hauptstrasse, Germany; M/s Colorcorn, Mumbai, India) were provided ex-gratis by the respective organizations. Isopropyl myristate (IPM; M/s LobaChemie, Mumbai, India), butylated hydroxy toluene (BHT; M/s SD. Fine Chemicals Ltd., Mumbai, India), Anaerogas Pack 3.5 L (M/s Hi Media Lab. Ltd., Mumbai, India), brain heart infusion media (BHI; M/s Hi Media Lab. Ltd., Mumbai, India), and tocopherol acetate (M/s E-Merck (India) Ltd., Mumbai, India were procured from the corresponding sources. Microbial culture of P. acnes (MTCC 1951) was procured from Institute of Microbial Technology (IMTECH), Chandigarh, India. All other chemicals employed in various studies were of analytical grade and were used as such. Ultrapure water (Milli-Q Integral system; M/s Merck Millipore, Billerica, USA) was used throughout the study. 2.2. Preparation of ITR-Loaded Nanocarriers. 2.2.1. ITR-Loaded SLNs. The microemulsification method was employed for the preparation of ITR-loaded SLNs.22 In brief, ITR (50.0 mg) and the lipophilic antioxidant (BHT; 138.7 mg) were dissolved in ethanol (6.0 g). The PC (208.5 mg) was dispersed in a portion of water along with Tween 80 (6.5 g) and sodium metabisulfite (SMBS; 0.5 g). Compritol (674.0 mg) was melted at 70 °C. The lipid phase, aqueous phase, and drug solution were mixed at same temperature to give a clear microemulsion. The hot microemulsion formed was poured into the remaining portion of water (i.e., about 80% of the total), previously cooled and maintained at 4 °C. The resulting mixture (100 g) was stirred continuously at 3000 rpm for 20 min.23 2.2.2. ITR-Loaded NLCs. Drug (50.0 mg) and BHT (BHT; 138.7 mg) were dissolved in ethanol (5.0 g). PC (0.6 g) was 1959

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Figure 1. TEM photomicrographs (a) ITR-loaded SLNs (2 000 000×); (b) ITR-loaded NLCs (1 000 000×).

solutions through a reverse phase C-18 column of length 150 × 4.6 mm, 3 μ ODS1 Spherisorb (M/s Waters Corporation, Milford, USA). The mobile phase consisted of glacial acetic acid 1.96% v/v: methanol (27:73 v/v), delivered at a flow rate of 1.0 mL/min. The permeation flux values were calculated for all of the formulations by plotting the amount of drug released per unit surface area vs time. The slope of the regressed line of the straight portion of the graph was reported as the permeation flux value.28 2.6. Skin Retention Studies. Skin mounted on the diffusion cell was removed carefully after completion of permeation studies. The formulation remaining adhered on the skin was scrapped off carefully with the help of a spatula and subsequently analyzed for drug content. The cleaned skin tissue was washed thrice with ultrapure water and dried using lint-free cotton swab. The skin was chopped into small pieces and macerated in methanol (5 mL) for 24 h for complete drug extraction to take place. After filtering the solution through a membrane (0.45 μm), the filtrate was analyzed using the validated RP-HPLC technique. 2.7. Dermatokinetic Modeling. The excised skins of Wistar rats were used for the studies. The skin tissue was prepared as discussed in section 2.5. Franz cell diffusion assembly was used for the studies as discussed under permeation studies with a dose of application equivalent to 1 mg of ITR. In this case, the whole skin was removed from the Franz cell at the respective sampling times. The skin was washed thrice to remove any adhering formulation and subsequently soaked in hot water (60 °C) to detach the epidermis from dermis.29,30 Both of the sections were chopped into small pieces, separately, and macerated in methanol (5 mL) for 24 h for complete drug extraction to take place. After filtering the solution through a membrane (0.45 μm), the filtrate was analyzed using the validated RP-HPLC technique. The obtained data were fitted into one-compartment open model, as per the following equation (eq 1) Cskin =

skin K p·Cmax

(K p − Ke)

concentration achieved in skin, and Ke is the skin elimination constant. Win-Nonlin Ver 5.0 software was employed to compute various dermatokinetic parameters , namely, Kp, Cskin max, skin (time required to achieve C ) and area under the Ke, and Tskin max max curve (AUC0−6h) using the Wagner−Nelson method. Data analysis was accomplished using nonlinear function minimization employing Gauss-theorem algorithms built into the software. Statistical validity of the results was discerned on the basis of minimization of various model fitness parameters like Akaike Information Criterion, Schwartz Criterion, sum of squares due to residuals, and maximization of Pearsonian correlation coefficient. 2.8. Estimation of Drug in Blood Portal. Six animals (Healthy male Wistar rats, 9−10 weeks old, 280−320 g) each were divided into three groups. Animals were caged and fed as per the norms of Institutional Animal Ethics Committee. Group A received marketed product while Group B and Group C received ITR-SLN gel and ITR-NLC gel, respectively. Each group received ITR equivalent to 0.6 g by topical application on the shaven skin. After dose application, serial blood samples (0.5 mL aliquots) were withdrawn from the tail vein at 0, 2, 4, and 6 h postdosing. Plasma was harvested by centrifugation and stored at −20 °C until analysis.31 2.9. Ethical Compliance. All animal protocols were duly approved by Institutional Animal Ethics Committee, Panjab University, Chandigarh, India (ref. letter no. CAH/09/70; IAEC/170-175). 2.10. Statistical Analysis. Multiple comparisons were made using one-way ANOVA followed by post hoc analysis using Student’s t test. Statistical significance was considered at P < 0.05.

3. RESULTS AND DISCUSSION 3.1. Formulation Details. Average particle size of both the colloidal carriers (i.e., SLNs and NLCs) was found to be below 100 nm, i.e., 75.3 nm (SLNs) and 80.0 nm (NLCs), respectively. Entrapment efficiency of these carriers was found to be above 75%, i.e., 89.49% for SLNs and 78.60% for NLCs. Both the carriers possessed significantly high magnitude of zeta-potential, i.e., −22.4 mV for SLNs and −15.0 mV for NLCs, the negative sign corroborating the anionic surface

(e−K pt − e−Ket ) (1)

where Cskin is the concentration of drug in skin at time t, Kp is skin the dermal permeation constant, Cmax is the maximum 1960

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components of the nanocarriers and better interaction of the carriers with the skin components.17 The significant difference (p < 0.001) in the drug transport characteristics of SLNs and NLCs both may be assigned to the difference in the composition and physicochemical properties of these lipidic colloidal carriers. 3.4. Skin Retention Studies. Skin retention values (μg cm−2 ± SD) from the studied formulations were observed as follows:

charge on the colloidal carriers. ITR-loaded nanoconstructs were observed to be spherical in nature, as confirmed by transmission electron microscopic (TEM) photographs (Figure 1). 3.2. Antimicrobial Activity. The order of MIC values was observed to be as follows: ITR(125.0 μg/mL) > ITR−SLNs ≈ ITR−NLCs(62.5 μg/mL)

ITR−NLCs(22.98 ± 1.98) > ITR−SLNs(15.79 ± 1.32)

> clindamycin phosphate(31.75 μg/mL)

> commercial gel(9.78 ± 0.59)

It is clearly vivid from the results that ITR also possesses its own antimicrobial activity with MIC value of 125.0 μg/mL visà-vis 31.75 μg/mL for the control antibiotic, i.e., clindamycin phosphate. Interestingly, incorporation of ITR in the lipidbased nanocarriers resulted in marked 50% reduction of MIC to 62.50 μg/mL for both ITR-SLNs and ITR-NLCs. This twofold enhancement in the antimicrobial activity of the nanoentrapped ITR can be ascribed to the better interaction of the lipid-based nanocarriers with the bacterial cell wall, resulting in increased contact time and sustained delivery of the drug to the bacteria.2 3.3. Drug Permeation Studies. Figure 2 shows the drug permeation profile of ITR from various studied systems.

Nanocolloidal carriers, namely, SLNs and NLCs, were able to significantly (p < 0.001) enhance the skin deposition of ITR in comparison to that of the commercial product. This can be attributed to the fusion of the biocompatible drug loaded carriers with the skin components and formation of micro drug reservoirs.20 These micro drug-depots in the skin ensure the sustained drug delivery to the target site in a controlled manner. 3.5. Dermatokinetic Modeling. Figures 3 and 4 show the distribution of drug in epidermis and dermis of the Wistar rat

Figure 3. Graph showing the amount of drug present in the epidermis of Wistar rats at various time points.

Figure 2. Drug permeation profile of ITR from various studied formulations across Laca mice skin.

Percent drug permeation (±SD) after 24 h was found to observe the following order: ITR−SLN gel(84.91 ± 4.25) > ITR−NLC gel(75.21 ± 3.76) > commercial gel(43.88 ± 2.19)

The pattern of skin permeation flux values (μg cm−2 h−1 ± SD) was found to observe the following: ITR−SLN gel(19.75 ± 1.21) > ITR−NLC gel(14.39 ± 1.37) Figure 4. Graph showing the amount of drug present in the dermis of Wistar rats at various time points.

> commercial gel(9.66 ± 0.89)

The extent of permeation, as reflected from the corresponding permeation flux values, were found to be significantly (p < 0.001) greater in case of nanocarriers vis-à-vis the marketed product. This can be attributed to the biocompatible

skin, respectively. Delivery of ITR in the skin layers by SLNs and NLCs was found to be significantly greater (p < 0.05) than that from the commercial gel. However, the drug delivery 1961

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Table 1. Various Dermatokinetic Parameters (Mean ± SD) of ITR Topical Formulations in Epidermis and Dermis of Wistar Rats (n = 6) isotretinoin-SLNs

isotretinoin-NLCs

commercial gel

dermatokinetic parameters

epidermis

dermis

epidermis

dermis

epidermis

dermis

AUC0−6h (μg cm−2 h) −2 Cskin max (μg cm ) skin Tmax (h) Kp (h−1) Ke (h−1)

176.99 ± 12.31 156.84 ± 7.23 0.35 ± 0.02 34.36 ± 2.97 1.03 ± 0.45

496.84 ± 31.09 109.62 ± 9.98 2.25 ± 0.02 0.89 ± 0.07 0.18 ± 0.01

192.89 ± 14.14 114.86 ± 9.65 0.55 ± 0.03 7.48 ± 0.79 0.98 ± 0.08

381.72 ± 20.12 88.97 ± 3.98 2.11 ± 0.02 0.67 ± 0.05 0.32 ± 0.02

142.31 ± 7.98 26.21 ± 0.04 0.75 ± 0.03 0.38 ± 0.02 0.33 ± 0.02

97.33 ± 7.81 25.93 ± 1.45 1.42 ± 0.08 1.46 ± 0.07 0.27 ± 0.01

edged. Generous help provided by Mr. Aman Singla in dermatokinetic data treatment is highly appreciated.

potential in the skin layers by NLCs was observed to be superior to that of SLNs. Table 1 gives the numeric values of skin AUC0−6h, Cskin max, Tmax, skin penetration rate constant (Kp), and skin elimination rate constant (Ke). The nanocolloidal systems (SLNs and NLCs) significantly enhanced the ITR delivery in the skin layers vis-à-vis the commercial product (p < 0.001 each). AUC of ITR was also found to be significantly greater (p < 0.001 each) in the dermis than in the epidermis for both of the colloidal carriers. The results, therefore, unequivocally vouch that the carriers delivered ITR to the dermis layer quite efficiently ascertaining adequate drug supply to the sebaceous glands, i.e., the afflicted site of acne. 3.6. Estimation of Drug in Blood Portal. Plasma samples of all of the studied groups were found to be devoid of retinoids after single dose administration at the studied sampling intervals. Therefore, the developed nanocarriers and the marketed product possibly will not cause the side effects related to the systemic distribution of ITR like psychological disorders.31

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4. CONCLUSIONS The current studies successfully embarked upon the demonstration of the antimicrobial activity of ITR against the acne causing pathogen, i.e., P. acnes. The findings indicate highly encouraging results of the lipidic nanocarriers, i.e., SLNs and NLCs, on the antimicrobial activity of ITR. These lipid-based nanocarriers not only decreased the MIC of ITR but also helped to retain the drug at the desired sites like various skin layers. Therefore, lipid-based naocarriers hold immense promise to enhance the efficacy and dermal delivery of antimicrobials in a quite strategic manner. Significant findings of the work can be rationally extrapolated to other similar drugs and analogous lipidic carriers.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 91-172-2541142. Notes

The authors declare no competing financial interest.

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REFERENCES

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ACKNOWLEDGMENTS

Authors are thankful to M/s Ipca Laboratories, Ratlam, MP, India for generously providing the gift samples of ITR where to M/s Gattefosse, Germany along with M/s Colorcon, India and M/s Phospholipid GmbH, Nattermannallee, Germany, for the ex-gratis supply of Compritol 888 ATO GF3123 and phospholipids, respectively. Financial grants obtained from University Grants Commission (UGC), New Delhi, India and M/s Ipca Laboratories, Mumbai, India are gratefully acknowl1962

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