http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.950586
RESEARCH ARTICLE
Nanostructured lipid carriers based nanogel for meloxicam delivery: mechanistic, in-vivo and stability evaluation S. Khurana1, N. K. Jain2, and P. M. S. Bedi1 Drug Dev Ind Pharm Downloaded from informahealthcare.com by Michigan University on 11/02/14 For personal use only.
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Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab, India and 2Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, Madhya Pradesh, India Abstract
Keywords
Aim: Our investigation was aimed to investigate the potential suitability of meloxicam-loaded nanostructured lipid carriers (MLX-NLC) gel for topical application. Main methods: MLX-NLC gel was prepared and in vivo skin penetration ability of the NLC gel was evaluated using confocal laser scanning microscopy. We studied the effect of MLX-NLC gel on the changes in lipid profile of skin to get an insight into its skin penetration enhancement mechanism. Acetic acid induced writhing test was performed to evaluate the analgesic effect. Drug concentration-time profile of MLX in rat plasma and skin after topical and oral treatment with MLX-NLC gel and oral MLX-solution, respectively, was observed. MLX-NLC gel was subjected to primary skin irritation test, sub-acute dermal toxicity study. Storage stability of MLX-NLC gel was also assessed for 90 days. Key findings: NLC gel was effective in permeating Rhodamine 123 to deeper layers of rat skin. Changes in skin lipid prolife were observed in the rat skin on treatment with MLX-NLC gel and the results supported skin lipid extraction as a possible penetration enhancement mechanism. MLX-NLC gel demonstrated sustained pain inhibitory effect. Pharmacokinetics study established that topical application of MLX-NLC gel had the potential to avoid systemic uptake and hence the risk of systemic adverse effects. MLX-NLC gel demonstrated good skin tolerability and biosafety. Excellent physical stability of nanogel was observed at 4 ± 2 C. Significance: The study revealed that NLC gel is a promising carrier system for the topical application of MLX without side effects.
Analgesic, meloxicam, nanostructured lipid carriers, skin tolerance, storage stability
Introduction Topical drug delivery is usually the most common route of administration since systemic load of drug and the drug-related side effects are reduced as compared to oral and parenteral route. However, one major drawback of topical drug delivery is the low percutaneous absorption of drugs because of the barrier functions of stratum corneum (SC), the topmost skin layer. Considerable research has now been centered on improving percutaneous absorption of drugs by utilizing advantages of lipidbased nanocarriers like nanoemulsion (NE), soild-lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)1. Different nanocarrier possesses their own way of facilitating percutaneous absorption. These nanocarriers can change the physicochemical properties of the entrapped drug and therefore can modify skin permeation profile. These carriers have the potential improvement of drug bioavailability either by facilitating skin penetration or by prolonging the skin residence time. Permeation of drug from lipid-based nanocarrier can be attributed to the high specific
Address for correspondence: Dr P. M. S. Bedi, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar – 143104, Punjab, India. Tel/Fax: + 91-183-2258819. E-mail: [email protected]
History Received 26 April 2014 Revised 26 July 2014 Accepted 28 July 2014 Published online 25 August 2014
surface area of nanoparticles that facilitates the strong adhesion of nanoparticles to the skin surface and offer an occlusive effect to the skin. The occlusive effect can eventually lead to an increase in skin hydration and can promote the deposition of drugs into the viable skin by reducing corneocytes packing and widening intercorneocyte gaps2,3. Recently published literature suggests NLC with solid lipid matrix composed of a mixture of spatially different lipid molecules, generally blend of solid and liquid lipid, and possess a number of features like small size, biocompatibility, controlled release properties, occlusive properties and improved skin targeting potential, which are advantageous for the topical route of application. Meloxicam (MLX) is a highly potent, non-steroidal antiinflammatory drug (NSAID) used for treatment of arthritis4–6. Its main limitations include poor aqueous solubility, poor incorporation into formulations and poor skin permeation7,8. To address this concern, several studies7–13 have been carried out in order to improve the percutaneous absorption and bioavailability of MLX, avoiding the systemic side effect associated with oral route. The aim of our study was to study the potential suitability of MLX-NLC gel for topical application. We evaluated the analgesic effect, in vivo skin penetration ability and the preclinical safety of MLX-NLC gel through various animal studies, including toxicity study and skin irritation study. Additionally, we assessed the storage stability of MLX-NLC nanogel for 90 days with repeated
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particle size, polydispersity index (PI), zeta potential, drug entrapment measurements at regular time intervals.
Materials and methods Drugs and chemicals Meloxicam was received as a gift sample from M/s Lupin Pharmaceuticals Ltd., Goa, India. Acetic acid, cetyl palmitate, caprylic acid, propylene glycol (PG), Tween 80, polyethylene glycol 400 (PEG 400) and triethanolamine were purchased from S. D. Fine Chemicals, Mumbai, India. Carbopol 940, were purchased from Sigma-Aldrich, Mumbai, India. Dialysis membrane (MWCO 12-14000), triton X-100 was procured from HiMedia, Mumbai, India. All other chemicals were of the analytical grade and used as received.
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Animals All animal care and experimental procedures were conducted as per guidelines of the committee for the purpose of control and supervision of experimental animals (CPCSEA) and were approved by the Institutional Animal Ethical Committee of Guru Nanak Dev University, Amritsar, India. For the experiments, the animals were housed in standard cages and maintained at an ambient temperature under conditions of optimum light (12 h light and 12 h dark) with food (standard laboratory rodent’s chow) and water provided ad libitum. For the experiments, they were acclimatized to the laboratory conditions for 5 days. Preparation of meloxicam-loaded nanostructured lipid carriers based gel Nanostructured lipid carrier-based gel was prepared using cetyl palmitate and caprylic acid as the matrix material as previously described14. An appropriate amount of distilled water was added to the heated (65 C) mixture of cetyl palmitate (65 mg) and caprylic acid (35 mg) under magnetic stirring to obtain homogeneous milky slurry to which a calculated volume of Tween 80 (0.2 ml) and the propylene glycol (1 ml) were added. The mixture turned transparent within seconds. For the preparation of NLC gel (plain-NLC gel), Carbopol 940 (5 mg) was completely dispersed in the NLC dispersion with constant stirring and triethanolamine was added drop wise to adjust the pH (6.0). Meloxicam and Rhodamine 123 containing NLC gel (MLX-NLC gel) were prepared after replacing a definite amount of the lipid phase by MLX (5% with regard to lipid content) and Rhodamine 123, respectively, and processed in the same way as mentioned above. Meloxicam gel (MLX-gel) was prepared by dispersing Carbopol 940 to the solution of meloxicam in water containing Tween 80 and PG under stirring followed by neutralization with triethanolamine. Storage stability study MLX-NLC gel was stored in screw capped glass vials and stored under different conditions (Table 1) as per ICH Harmonised Tripartite Guideline for the stability testing of new drug substances and products Q1A (R2) (ICH 2003). The formulation
Table 1. Storage conditions as per ICH Harmonised Tripartite Guideline for the stability testing. Study Refrigeration Long term Accelerated
Storage condition
4±2 C 25 ± 2 C/60 ± 5% RH 40 ± 2 C/75 ± 5% RH
Time points 0, 30 and 60 days 0, 30 and 60 days 0, 30 and 60 days
was analyzed with respect to particle size, PI, zeta potential and drug entrapment efficiency at regular time intervals (Table 1) and compared with fresh MLX-NLC gel formulation. Photon correlation spectroscopy (PCS) with a Malvern Nanosizer ZS (Malvern Instruments, Malvern, UK) was used to determine the average particle size and PI. The analyses were carried out by diluting the MLX-NLC gel with distilled water to weak opalescence to produce a suitable scattering intensity. The value of the zeta potential was measured using a Malvern Nanosizer ZS (Malvern Instruments, UK). The conductivity of the diluted formulation was adjusted to 50 mS/cm with 0.9% w/v sodium chloride solution for zeta potential measurement. The efficiency of MLX entrapment into NLC gel was determined15–17 after removing the free drug from samples of NLC gel containing MLX by ultra dialysis against acetate buffer (pH 6.0) containing 30% PEG 400 (v/v) at 4 C for 4 h using dialysis bag (Sigma, St Louis, MO; MWCO 12 000–14 000). The dialyzed formulation was lysed using TritonX 100 (0.1% v/v) and subsequently analyzed for drug content (A2) by UV spectrophotometry (Hitachi U-2800 spectrophotometer, Tokyo, Japan) at 362 nm. Drug entrapment efficiency (EE) of NLC gel was calculated by Equation (1): EEð%Þ ¼
A2 100 A1
ð1Þ
where A1 is the total amount of drug added in the formulation. Visualization of skin penetration in vivo Three Albino rats of either sex, weighing 150–200 g were taken. The skin of the dorsal region was trimmed free of hair by shaving. NLC gel formulation loaded with rhodamine 123 (5 mg/ml) was applied in a marked area of 1 cm2 at the dorsal site of animals for 24 h. Thereafter, animals were sacrificed by cervical dislocation. The excised skin was blotted in inert paper, washed thrice with ethanol. The application area was then sectioned into the pieces of 1 mm2 size and evaluated for depth of probe penetration by confocal microscopy using confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany)18. Effect of MLX-NLC gel on the skin lipid profile The excised abdominal rat skin was washed with isotonic NaCl. The epidermis was separated and treated with 250 mg of the formulation for 24 h (s) in a Franz diffusion cell at 32 ± 0.5 C. After incubation, the remnants of formulation sticking on the surface of treated epidermis were cleaned off and the skin lipids were extracted19. One hundred milligrams of epidermis was hydrated by incubation with 500 ml NaCl solution (300 mM) for 15 m. Chloroform/methanol (2:1) was added at a volume of 5 ml. After shaking for 2 h (s) at room temperature, 100 ml NaCl solution (300 mM) was added and incubated at room temperature for 10 m. Centrifugation at 2500 rpm for 10 m led to the appearance of two phases: an upper hydrophilic layer and an underlying layer containing lipid in an organic solution. 3.5 ml of the later was transferred through cotton wool into a lipid-free glass vial and evaporated to dryness at room temperature19. Cholesterol, triacylglycerols, free fatty acid, total lipid content in each sample was determined using their respective kits (Erba Diagnostic, Mannheim, Germany) method and autoanalyzer (Erba Chem 5 Plus V2, Mannheim, Germany). Percentage of lipid extracted was calculated by using following formula : ¼
1 Content in treated skin 100 Content in normal skin
Nanostructured lipid carriers
DOI: 10.3109/03639045.2014.950586
Analgesic effect 20
Acetic acid induced writhing was used to evaluate the analgesic effect of the formulations. Prior to dosing, the abdomen hair of Swiss albino mice (3–4 months old, weighing 20 to 25 g) was removed carefully. Mice were divided into four groups of six animals each. MLX-NLC gel, plain-NLC gel and MLX-gel (250 mg each) were applied on the hair free skin of the abdominal site (approximately 2.26 cm2 areas) of the animals of group I, group II and group III, respectively. Fourth group was untreated and served as control. After 24 h, the writhing was induced by intraperitoneal injection of 0.6%, (v/v) acetic acid at a dose of 10 mg/kg body weight. The analgesic response was assessed by counting the number of writhing (W) for a period of 20 min and pain inhibition ratio (PIR) was calculated using Equation (2):
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PIR ð%Þ ¼
Wc Wt 100 Wc
ð2Þ
where Wc is the writhing numbers of control group and Wt is the writhing numbers of test group. Pharmacokinetic study Albino rats (either sex) (150–200 g) were divided into two groups of 18 animals each. The first group was treated with MLX-NLC gel. MLX-NLC gel at a dose level of 10 mg/kg was applied on the shaved hind paw region of the rat, covering the knee and proximal part of the fumer muscles in the group I. MLX was dissolved in phosphate buffer (pH 7.4) and was administered orally (10 mg/kg) using oral feeding cannula to the animals of group II. Blood sample was withdrawn from tail vein of the rat into a centrifuge tube containing heparin sodium as anticoagulant according to a programed schedule at 0, 1, 3, 6, 12 and 24 h after dosing. Each blood sample was centrifuged at 4000 rpm for 10 min to separate plasma, which was stored at 20 C until analysis. The plasma sample was homogenized with a mixture of acetonitrile and water (9:1 v/v). The drug from the de-proteinized homogenate was extracted with methanol and the samples were centrifuged (3200 rpm, 10 min). The supernatants collected were analyzed for drug content by HPLC. The shaved hind paw skin of rats (n ¼ 3) in different treated groups was collected at 0, 1, 3, 6, 12 and 24 h time intervals after sacrifice and skin samples were stored at 20 C until further analysis. The collected skin sample was placed in methanol and sonicated for 20 min to extract the drug. All the samples were then centrifuged; the supernatant was transferred to vials. An aliquots (20 ml) of the supernatant was directly injected into HPLC for analysis. Pharmacokinetic parameters were analyzed by Thermo Kinetica 5.0 software (Thermo Fisher Scientific, Philadelphia, PA). Preparation of calibration curve of meloxicam in plasma Blood sample (2 ml) was collected from retro-orbital plexus of rat using heparinized capillary tube and centrifuged at 4000 rpm for 10 min. Equal volume of acetonitrile was added to the obtained plasma for deproteinization. After 15 min, the plasma was centrifuged at 4000 rpm for 10 min to remove the precipitated proteins. Supernatant was passed through a 0.2 mm filter. Accurately weighed MLX (2 mg) was transferred to a 100 volumetric flask and the volume was made up with mobile phase (methanol:water:phosphoric acid::69.9:30:0.1) resulting in a stock solution of 20 mg/ml. From the stock solution, accurately measured aliquots of 0.2, 0.4, 0.6 up to 2.0 ml were withdrawn in a series of 10.0 ml volumetric flasks and 100 ml of plasma was added in each sample. The samples were diluted to 10.0 ml with mobile phase to prepare solutions in the concentration range of 0.2–2.0 mg/ml. These samples were subjected to HPLC analysis
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by injecting 20 ml of sample into injection port using a mobile phase delivered at a flow rate of 1 ml/m. UV detection wavelength was 362 nm. All operations were carried out at an ambient temperature. Peak area was then plotted against concentration to make the calibration curve. All experiments were performed in triplicate. Toxicity study Repeated dose subacute dermal toxicity study was conducted in Swiss albino mice at dose levels of 8, 16 and 32 mg/kg for 21 days according to the Organization for Economic Cooperation and Development guidelines 41021. Swiss albino mice (3–4 months old) weighing 20 to 25 g were randomly divided into one control (untreated) and one treatment groups, with five males and five females per group. Twenty-four hours before the experiment, the mice dorsal skin was trimmed free of hair by shaving. MLX-NLC gel was smeared onto the hair free skin areas (7 cm2) of mice of treated group once a day on a 5-day per week basis, for a period of 21 days. Body weight and food consumption were measured and recorded weekly. The treated areas were covered with porous gauze, which was held in place with non-irritating tape (Transpore 3M surgical tape, 3M India Ltd, Bangalore, India). The general physical condition of the mice was monitored daily. Before and after 21 days of the formulation application, blood samples were collected for hematology (Sysmex KX-21 automated hematology analyzer, Sysmex America, Inc., Lincolnshire, IL), biochemical analyses (auto analyzer, Erba Chem 5 Plus V2, Germany), and sodium and potassium measurements (EasyLyte Na/K Analyzer, Minnesota). Histological examinations were performed on the organs and tissues of the high-dose group and the untreated group. The animals were sacrificed by cervical dislocation. Skin, heart, liver, lungs, kidney and spleen were taken out. Then, all the tissues were fixed and preserved in 10% formalin for histopathological examination. Sections were fixed and blocks were made using the procedure as reported22. The sections were stained with eosin– hematoxylin to determine gross histopathology. Histological sections were examined using phase contrast microscope with photographic arrangement Zeiss Primostar, Germany). Skin irritation study Skin irritation potential of MLX-NLC gel was assessed by carrying out skin irritation test on male albino rabbits (weighing 2.0–2.5 kg) as per the OECD guidelines 40423 for testing of acute dermal irritation/corrosion. Rabbits were randomly divided into two groups, with three rabbits per group. Each rabbit was caged individually. The fur on both the sides of the dorsal surface of the trunk of each test animal was removed carefully with electronic hair remover, 24 h prior to the experiment. Three areas (A, B and C) each with approximately 6 cm2 area were marked on the hair free area of every test animal. 20% sodium lauryl sulfate (SLS) solution was used as positive control (Manosroi et al., 2013). The animals were treated as follows: Group I – 20% w/v SLS solution (area A, B), untreated (area C); Group II – plain NLC gel (area A), MLX-NLC gel (area B) and untreated (area C). Five hundred milligrams of the test formulation was applied to the marked area A and area B in each group. The treated area was covered with a gauze patch which is held in place with nonirritating tape (Transpore 3M surgical tape, 3M India Ltd, India). C area in each group was used as the control. At the end of 24 h exposure period, the residual formulation on the skin was removed carefully with purified water without altering the existing response or the integrity of the epidermis. Observations were
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made at 1, 24, 48, and 72 h, and the dermal reactions (erythema and edema) were scored between 0 and 4 on the basis of degree of severity and grading of skin reactions was ascertained to determine the total dermatitis score as per OECD guideline 404 (OECD 2002). The scores for erythema and edema were summed up for all the rabbits at 1, 24, 48 and 72 h. From the grades, primary irritation index (PII) was calculated using Equation (3): Sum of erythema grade at 1=24=48=72h þSum of edema grade at 1=24=48=72h 100 ð3Þ PII ¼ Number of animals The irritation degree was categorized based on the PII values as negligible (PII ¼ 0–0.4), slight (PII ¼ 0.5–1.9), moderate (PII ¼ 2–4.9) or severe (PII ¼ 5–8) irritation24.
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Statistical analysis All the data are expressed as mean ± standard deviation (SD). All statistical analysis was performed using Sigma Stat Software, 3.5 (Systat Software Inc, San Jose, CA). Data were analyzed using the one-way analysis of variance (ANOVA) with the Holm–Sidak correction. p Values 50.05 were considered as statistically significant.
Results and discussion The aim of our study was to develop a topical MLX formulation to control the delivery of MLX across the skin. A preliminary assessment of NLC gel as drug carriers for MLX was undertaken and motivating results were obtained14. In this study, we further investigated the drug delivery potential and safety of MLX-NLC gel on topical application. Stability study Stability is mainly evaluated to ensure that the quality of the product will be maintained throughout its shelf life25. An important limitation of nanodispersion is their poor storage stability. To improve the storage stability, NLC dispersion is commonly transformed into semisolid gel. MLX-NLC gel stored at different temperature and humidity conditions (Table 1) was evaluated for any changes in particle size, PI, zeta potential and drug content at 30, 60, 90 days time points to assess the effect of storage conditions on the stability as a function of time. The particle size was monitored at regular time intervals during storage to assess particle aggregation. The nanoparticles are thermodynamically unstable system and for their stability, a zeta potential value between 30 mV and 60 mV is desirable to avoid aggregation of particles26. Thus, zeta potential measurement gives us the estimation of the storage stability of nanoparticles. Stability of MLX-NLC gel was evaluated in terms of their MLX entrapment efficiency. Log(E.E %) was plotted against time and the slopes (m) were calculated by linear regression. The slopes (m) were then substituted into the following equation for the determination of k values: k ¼ m 2:303 Shelf-life values (the time for 10% loss, i.e. t90) were then calculated by the following equation: t90 ¼ 0:105=k In case of MLX-NLC gel, no significant (p40.05) change of particle size, PI, zeta potential, drug entrapment efficiency was observed at 4 ± 2 C over the period of 90 days (Table 2). An increasing trend of particle size and PI, decreasing trend of zeta
Table 2. Effect of storage conditions on the particle size, polydispersity index, zeta potential and drug entrapment efficiency. Storage condition Parameter Particle size (nm)
Day
0 30 60 90 Polydispersity 0 index 30 60 90 Zeta potential 0 (mV) 30 60 90 Drug entrapment 0 efficiency (%) 30 60 90
4 C
25 ± 2 C and 60 ± 5% RH
40 ± 2 C and 75 ± 5% RH
210.66 ± 1.79 210.66 ± 1.79 210.66 ± 1.79 211.99 ± 2.80 229.45 ± 3.04 240.12 ± 3.18 213.33 ± 2.82 335.12 ± 4.43 513.54 ± 6.79 214.21 ± 2.83 414.65 ± 5.49 762.42 ± 10.09 0.213 ± 0.00600 0.213 ± 0.00600 0.213 ± 0.00600 0.215 ± 0.00284 0.291 ± 0.00385 0.318 ± 0.00420 0.218 ± 0.00288 0.309 ± 0.00409 0.365 ± 0.00482 0.220 ± 0.00291 0.333 ± 0.00441 0.410 ± 0.00542 27.33 ± 0.620 27.33 ± 0.620 27.33 ± 0.620 27.1 ± 0.358 25.23 ± 0.333 21.43 ± 0.283 26.95 ± 0.356 21.53 ± 0.284 17.89 ± 0.236 26.92 ± 0.355 19.43 ± 0.257 14.87 ± 0.196 85.61 ± 1.35 85.61 ± 1.35 85.61 ± 1.35 84.56 ± 1.12 81.01 ± 1.07 78.33 ± 1.04 83.98 ± 1.11 77.05 ± 1.02 72.54 ± 0.96 82.67 ± 1.09 71.32 ± 0.94 60.23 ± 0.80
The data reported are mean ± SD (n ¼ 3).
potential and drug entrapment efficiency were observed with storage time at 25 ± 2 C/60% ± 5% RH and 40 ± 2 C/75 ± 5% RH. The increase of particle size and polydispersity index, and diminution of zeta potential and drug entrapment efficiency were comparatively higher at 40 ± 2 C/75%±5% RH. This demonstrated the physical stability of the MLX-NLC gel at 4 ± 2 C over the period of 90 days. The results are in agreement with previous results27 suggesting the good stability of MLX-NLC gel. Upon storage at high temperature, the change in the particle size in MLX-NLC gel can be attributed to the increase in the kinetic energy of system at high temperature that may accelerate the collision between particles, and consequently can increase the chances of aggregation of particles28. This decrease in entrapment efficiency at high temperature could also be attributed to lipid transformation of the solid lipid used in NLC over time, leading to the formation of a highly ordered lipid structure resulting in drug expulsion. Shelf-life values, i.e. the time at which the drug concentration is lost by 10% were calculated. A good chemical stability of MLX-NLC gel (t90, 281.90 days) was observed when stored at 4 ± 2 C It is evident from the data that MLX-NLC gel had good long-term physical and chemical stability at 4 ± 2 C and the recommended storage temperature is 4 ± 2 C. Visualization of skin penetration in vivo Confocal laser scanning microscopy (CLSM) provides information about the localization and the permeation pathway of fluorescent model compound in the tissue. The major advantage of CLSM is that the distribution of the fluorescent model compound in the sample can be visualized without cryo-fixing or embedding the tissue29. The depth of Rhodamine 123 (lipophilic fluorescence marker) penetration into skin from NLC gel (Figure 1) was measured by CLSM. NLC gel was found to be effective in permeating Rhodamine 123 to a skin depth of 160 mm (Figure 1). Our results indicate that NLC gel is effective in improving the skin penetration of MLX and the findings are consistent with the in vitro skin permeation study14 supporting the high permeation of MLX to deeper layers of the skin from NLC gel. These characteristics are significant for its use as topical dosage form.
Nanostructured lipid carriers
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DOI: 10.3109/03639045.2014.950586
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Figure 1. Photomicrograhs (a) and fluorescence intensity (b) measured by confocal laser scanning microscopy of rat skin after 24 h treatment with Rhodamine 123 containing nanostructured lipid carriers gel.
Similar results were reported by Teeranachaideekul et al.30. They suggested that the high amount and deep penetration of dye into skin from NLC could be attributed to its high occlusion factor. In addition to the occlusion effect, the observed effect may be a consequence of the NLC components, such as CA (short chain fatty acid), Tween 80 and PG that could significantly reduce the barrier properties of SC by modifying the thermodynamic activity of the drug in the vehicle. Tween 80 and PG can change the diffusional driving force for solvent partition into the tissue and thereby can facilitate the drug permeation into skin31,32. Further, the permeation of nanoparticles also rely on the characteristic features like surface properties, zeta potential, flexibility and particle size of the system. Shah et al.33 investigated the effect of surface modification of fluorescent dye encapsulated NLC on topical delivery by in vivo confocal microscopy across the skin and suggested that the surface modification of NLC with a peptide containing 11 arginines (R11) improved transport of spantide II (SP) and ketoprofen (KP) across the deeper skin layers. Biochemical investigation It is recognized that the lipid composition of the skin plays a key role in determining the barrier functions34. The effect of the skin lipids content on the rate of skin permeation has been recognized by a strong relationship between the quantity of extracted skin lipids with acetone/methanol and the quantity of drug
permeation19. Skin lipids may affect drug permeation, as they can interact with the formulation. Thus to elucidate penetration enhancement mechanism of nanogel, biochemical studies were carried out to examine the changes in the lipid profile of skin as a result of the interaction with the nanogel formulation. Skin is the outermost largest organ and mainly provides barrier attributes to the skin and makes the topical/transdermal delivery challenging. It is composed of hypodermis, hydrophilic dermis and epidermis. Epidermis is divided into living hydrophilic layer and a hydrophobic horny layer composed of dead cells (stratum corneum, SC)35,36. The application of MLX-NLC gel caused changes in the lipid content of skin and as a result, the barrier property of skin. The percentage of cholesterol, triacylglycerol, free fatty acids and total lipids extracted from excised rat skin treated with MLX-NLC gel and delipidized (negative control) is shown in Table 3. These findings imply that the formulation induced an alteration in lipid content and suggest that acted via lipid interactions with skin that lead to the disruption of the barrier properties and enhanced the permeation of the drug across the skin. Our results support the reports that a decrease in skin lipid level may result in decrease of skin barrier property37,38. Analgesic effect Both MLX-NLC gel and MLX-gel produced appreciable analgesic activity with PIE of 42.86 ± 2.79 and 8.82 ± 0.57%,
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Table 3. Percentage of lipids extracted from excised rat skin after 24 h (s) of treatment. Skin stratum corneum sample
Cholesterol (%)
Triacylglycerol (%)
Free fatty acids (%)
Total lipid content
39.13 ± 2.54 98.98 ± 0.86
25.37 ± 1.02 99.40 ± 0.86
11.31 ± 1.49 98.97 ± 0.86
23.11 ± 2.47 99.45 ± 0.86
MLX-NLC gel treated Delipidized The data reported are mean ± SD (n ¼ 3).
35 Plasma concentration (µg/ml)
Figure 2. Plasma concentration–time profiles of meloxicam in rat plasma after topical and oral treatment with nanogel and oral solution respectively each at 10 mg/kg dose level. The data reported are mean ± SD (n ¼ 3).
MLX oral solution
30 25 20 15 10 5 0
0
5
10
15
20
Time (h)
Figure 3. Meloxicam concentration–time profiles in rat skin after topical and oral treatment with nanogel and oral solution respectively each at 10 mg/kg dose level. The data reported are mean ± SD (n ¼ 3).
30
MLX-NLC gel
MLX oral solution
25 Concentration (µg/g)
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MLX-NLC gel
20 15 10 5 0 1
3
6
12
24
Time (h)
respectively, after 24 h and also that the analgesic effect was more pronounced in MLX-NLC gel compared to MLX gel. Eventually; MLX-NLC gel could be appropriate for topical pain reduction. Pharmacokinetic study Figure 2 shows the plasma concentration–time profile of MLX in rat plasma after topical and oral treatment with nanogel and oral solution, respectively, each at a dose level of 10 mg/kg. The related pharmacokinetic parameters were determined and it was observed that the maximum concentration (Cmax) in plasma appeared at 3 h and 24 h after oral administration and topical treatment, respectively. Cmax value in blood plasma was found to be much lower after topical treatment with MLX-NLC gel compared to that obtained after oral administration of MLXsolution indicating the potential of MLX-NLC-gel to reduce high levels in plasma that might be associated with side effects. The plasma AUC0 t after topical treatment with MLX-NLC gel was 14.46 times lesser than that observed after oral administration of MLX-solution indicating that lower fraction of MLX were absorbed in the circulation after MLX-NLC gel application compared with MLX oral solution. This can probably be ascribed to the drug reservoir forming potential of skin. Further, the drug deposition in skin following treatment with MLX-NLC gel was higher than that following MLX-oral solution administration (Figure 3). Cmax and AUC0 t values were higher in skin after topical application of MLX-NLC gel as compared to
that obtained after oral administration of MLX-solution. The AUC0 t in skin after topical administration of MLX-NLC gel was 79.03-fold, higher than that obtained after oral administration of MLX-solution. Our results indicated the potential of MLXNLC gel to avoid systemic uptake and to form drug reservoir in the skin that could act as the force for delivering drug into local deeper tissues. The lower systemic exposure would avoid GIT irritation and reduce the risk of adverse systemic effects. In the final outcome, the site-specific drug delivery of MLX from the proposed drug delivery system and consequently lowered side effects will facilitate the design of an optimized drug delivery system, overwhelmingly superior to the existing oral administration. Our results are in agreement with literature39,40. The authors found the lower systemic exposure of the drug after topical administration of flurbiprofen (FB)-loaded NLC gel than after oral administration of FB solution. Toxicity study As reported earlier, MLX is associated with risk of arterial thrombotic events, a functional renal failure, liver dysfunction and skin reactions, particularly at high doses and on long-term treatment41–43. In view of this, it became necessary to establish the safety of developed NLC-based nanogel formulation against the local and systemic toxic effects. Repeated application of MLX-NLC gel did not show any unusual behavioral changes in
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DOI: 10.3109/03639045.2014.950586
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Figure 4. Photomicrogrphs (400) of hematoxylin–eosin stained skin (A), heart (B), liver (C), lungs (D), kidney (E) and spleen (F) sections of untreated mice in repeated dose toxicity study.
Figure 5. Photomicrogrphs (400) of hematoxylin–eosin stained skin (A), heart (B), liver (C), lungs (D), kidney (E) and spleen (F) sections of mice treated with MLX-NLC gel in repeated dose toxicity study at 32 mg/kg dose.
the treated animals. No mortality was observed in any of the animal group. On the site of application of the MLX-NLC gel, no reaction was seen. The animals belonging to the test substancetreated group did not show significant change in body weight as compared with control. All the animals showed normal weight gains and revealed no toxic effects during the observation period. Following a 21-day repeated administration, the values of all the hematological and biochemistry parameters were within the normal limits and demonstrated normal kidney, liver, pancreas function and absence of bone marrow toxicity. In order to check out the potential toxicity on internal organs frequently coupled with the oral COX-2 selective anti-inflammatory drugs, histopathological examination of skin, heart, liver, lungs, kidney and spleen tissues, of high-dose treated group was performed. Compared to control (Figure 4A–F), microscopic examinations of skin, heart, liver, lungs, kidney and spleen in treated animals (Figure 5A–F) did not show any abnormality in their natural architecture. In addition, no evidence of inflammation, cell lysis or lesions were seen in the histological evaluation of organ tissues of any of the treated animals when compared to control. These results clearly established that the developed gel was non-irritant to the skin as well as non-toxic to the internal organs and illustrated the absence of the local and systemic toxicities of MLX-NLC gel at high doses in mice. Thus, the biocompatibility and safety of the developed MLX-NLC gel was confirmed. The safety of MLX-NLC gel for skin application can be attributed to the fact that the primary fraction of MLX remained in the skin and extremely low amount of MLX was absorbed in the circulation subsequent to application of nanogel. This eventually could reduce the chances of drug-associated undesirable systemic effects. Furthermore, the lipids selected for NLC gel formulation were reported to be biocompatible, safe and effective in topical/ transdermal formulations. Skin irritation study In order to further demonstrate the skin tolerability of the MLX-NLC gel, skin irritation study was conducted. According to
the PII scores, the degree of irritation was classified as negligible (PII ¼ 0–0.4), slight (PII ¼ 0.5–1.9), moderate (PII ¼ 2–4.9) or severe (PII ¼ 5–8) irritation24. The study indicated that MLXNLC gel had no significant effect on the animal weights at all time points (p40.05). MLX-NLC gel showed no erythema or edema on the rabbit skin with PII 0.0. However, 20% SLS treatment resulted in moderate skin irritation with PII value 2.33 and 2.0 after 1 h and 24 h, respectively. The skin tolerability of MLX-NLC gel could be due to the entrapment of drug in NLC leading to avoidance of direct contact of drug with the skin. Thus, MLX-NLC gel formulation would prove to be highly advantageous for topical use with improved safety profile.
Conclusions It can be concluded that MLX-NLC gel has the potential to form drug reservoir in the skin that can act as the force for delivering drug into local deeper tissues, and hence offers MLX-NLC gel promising platform as topical drug delivery system. The results of the toxicity study further substantiate the role of NLC gel as a promising topical carrier for MLX in order to achieve better therapeutic efficacy without systemic side effects. Storage stability study confirmed good physical stability of MLX-NLC gel at 4 ± 2 C. These preclinical observations necessitate to be extrapolated to humans for establishing their efficacy in clinical use. Scale up and pilot plant studies will have to be conducted in order to establish the industrial feasibility of the proposed delivery system. We anticipate that there is a bright perspective on the future development of the NLC gel in controlled and targeted drug delivery.
Declaration of interest The authors report no declarations of interest.
References 1. Joshi M, Patravale V. Formulation and evaluation of nanostructured lipid carrier (NLC)-based gel of valdecoxib. Drug Dev Ind Pharm 2006;32:911–18.
Drug Dev Ind Pharm Downloaded from informahealthcare.com by Michigan University on 11/02/14 For personal use only.
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2. Muller RH, Radtke M, Wissing SA. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Adv Drug Deliv Rev 2002;54:S131–55. 3. Schafer-Korting M, Mehnert W, Korting HC. Lipid nanoparticles for improved topical application of drugs for skin diseases. Adv Drug Deliv Rev 2007;59:427–43. 4. Burke A, Smyth E, FitzGerald GA. Analgesic-anti pyretic and anti inflammatory agents; pharmacotherapy of gout. In: Bruntom LL, Lazo JS, Parker KL, eds. Goodman’s and Gilman’s the pharmacological basis of therapeutics. New York: McGraw Hill; 2006: 676–700. 5. Abramson SB, Yazici Y. Biologics in development for rheumatoid arthritis: relevance to osteoarthritis. Adv Drug Deliv Rev 2006;58: 212–25. 6. Quan LD, Thiele GM, Tian J, Wang D. The development of novel therapies for rheumatoid arthritis. Expert Opin Ther Pat 2008;18: 723–38. 7. Yuan Y, Li S, Mo F, Zhong D. Investigation of microemulsion system for transdermal delivery of meloxicam. Int J Pharm 2006;32: 1117–23. 8. Jantharaprapap R, Stagni G. Effects of penetration enhancers on invitro permeability of meloxicam gels. Int J Pharm 2007;343:26–33. 9. Ruiz Martinez MA, Lopez-Viota Gallardo J, de Benavides MM, et al. Rheological behavior of gels and meloxicam release. Int J Pharm 2007;333:17–23. 10. Yuan Y, Chen X, Zhong D. Determination of meloxicam in human plasma by liquid chromatography–tandem mass spectrometry following transdermal administration. J Chromatogr 2007;B852: 650–4. 11. Yuan Y, Li S, Yu L, et al. Physicochemical properties and evaluation of microemulsion systems for transdermal delivery of meloxicam. Chem Res Chin Univ 2007;23:81–6. 12. Kasliwal N, Derle D, Negi J, Gohil K. Effect of permeation enhancers on the release and permeation kinetics of meloxicam gel formulations through rat skin. Asian J Pharm Sci 2008;3:3193–9. 13. Ah YC, Choi JK, Choi YK, et al. A novel transdermal patch incorporating meloxicam: in vitro and in vivo characterization. Int J Pharm 2010;385:12–19. 14. Khurana S, Bedi PMS, Jain NK. Development and characterization of a novel controlled release drug delivery system based on nanostructured lipid carriers gel for meloxicam. Life Sci 2013;93: 763–72. 15. Utreja S, Khopade AJ, Jain NK. Lipoprotein-mimicking biovectorized systems for methotrexate delivery. Pharm Acta Helv 1999;73: 275–9. 16. Khurana S, Bedi PMS, Jain NK. Development of nanostructured lipid carriers (NLC) for controlled delivery of meloxicam. Int J Biomed Nanosci Nanotechnol 2010;1:247–66. 17. Khurana S, Bedi PMS, Jain NK. Development of nanostructured lipid carriers (NLC) for controlled delivery of mefenamic acid. Int J Biomed Nanosci Nanotechnol 2012;2:232–50. 18. Dubey V, Mishra D, Dutta T, et al. Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J Control Release 2007;123:148–54. 19. Stahl J, Niedorf F, Kietzmann M. Characterisation of epidermal lipid composition and skin morphology of animal skin ex vivo. Eur J Pharm Biopharm 2009;72:310–16. 20. Xi H, Cun D, Xiang R, et al. Intra-articular drug delivery from an optimized topical patch containing teriflunomide and lornoxicam for rheumatoid arthritis treatment: does the topical patch really enhance a local treatment? J Control Release 2013;169:73–81. 21. Organization for Economic Cooperation and Development (OECD). Guidelines for testing chemicals 410 repeated dose dermal toxicity: 21/28-day study. Paris: OECD; 1981.
Drug Dev Ind Pharm, Early Online: 1–8
22. Lee BJ, Lee TS, Cha BJ, et al. Percutaneous absorption and histopathology of a poloxamer-based formulation of capsaicin analog. Int J Pharm 1997;159:105–14. 23. Organization for Economic Cooperation and Development (OECD) Guidelines. Test guideline 404 acute dermal irritation/corrosion. Paris: OECD; 2002. 24. Manosroi A, Chankhampan C, Manosroi W, Manosroi J. Transdermal absorption enhancement of papain loaded in elastic niosomes incorporated in gel for scar treatment. Eur J Pharm Sci 2013;48:474–83. 25. ICH Stability testing of new drug substances and products. International Conference on Harmonization (ICH) Guidelines Q1A (R2). European Medicines Agency, CPMP/ICH/2736/99; 2003. 26. Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art. Eur J Pharm Biopharm 2000;50:161–77. 27. Junyaprasert VB, Teeranachaideekul V, Souto EB, et al. Q10-loaded NLC versus nanoemulsions: stability rheology and in vitro skin permeation. Int J Pharm 2009;377:207–14. 28. Freitas C, Muller RH. Stability determination of solid lipid nanoparticles (SLN) in aqueous dispersion after addition of electrolyte. J Microencapsul 1999;16:59–71. 29. Chen H, Chang, X, Du D, et al. Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. J Control Release 2006;110: 296–306. 30. Teeranachaideekul V, Boonme P, Souto EB, et al. Influence of oil content on physicochemical properties and skin distribution of Nile red-loaded NLC. J Control Release 2008;128:134–41. 31. Williams AC, Barry BW. Penetration enhancers. Adv Drug Del Rev 2004;56:603–18. 32. Fox LT, Gerber M, Plessis JD, Hamman JH. Transdermal drug delivery enhancement by compounds of natural origin. Mol 2011;16: 10507–40. 33. Shah PP, Desai PR, Patel AR, et al. Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs. Biomaterials 2012;33:1607–17. 34. Elias PM, Menon GK. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv Lipid Res 1991;24:1–26. 35. Babita K, Kumar V, Rana V, et al. Thermotropic and spectroscopic behavior of skin: relationship with percutaneous permeation enhancement. Curr Drug Deliv 2006;3:95–113. 36. Karadzovska D, Brooks JD, Monteiro-Rivierem NA, Riviere JE. Predicting skin permeability from complex vehicles. Adv Drug Deliv Rev 2013;65:265–77. 37. Elias PM, Cooper ER, Korc A, Brown BE. Percutaneous transport in relation to stratum corneum structure and lipid composition. J Invest Dermatol 1981;76:297–301. 38. Kostiuk NV, Zhigulina VV, Belyakova MB, et al. Effect of melatonin on lipid barrier in rats’ skin. Am J Biochem 2012;2: 67–73. 39. Han F, Yin R, Che X, et al. Nanostructured lipid carriers (NLC) based topical gel of flurbiprofen: design characterization and in vivo evaluation. Int J Pharm 2012;439:349–57. 40. Chen-yu G, Chun-fen Y, Qi-lu L, et al. Development of a quercetinloaded nanostructured lipid carrier formulation for topical delivery. Int J Pharm 2012;430:292–8. 41. Distel M, Mueller C, Bluhmkij E, Fries J. Safety of meloxicam: a global analysis of clinical trials. Br J Rheumatol 1996;35:68–77. 42. Lanes SF, Rodriguez LA, Hwang GE. Baseline risk of gastrointestinal disorders among new users of meloxicam ibuprofen diclofenac naproxen and indomethacin. Pharmacoepidemiol Drug Saf 2000;9: 113–17. 43. Villegas I, Alarcon de la, Lastra C, et al. Effects of food intake and oxidative stress on intestinal lesions caused by meloxicam and piroxicam in rats. Eur J Pharmacol 2001;414:79–86.
RESEARCH ARTICLE
Nanostructured lipid carriers based nanogel for meloxicam delivery: mechanistic, in-vivo and stability evaluation S. Khurana1, N. K. Jain2, and P. M. S. Bedi1 Drug Dev Ind Pharm Downloaded from informahealthcare.com by Michigan University on 11/02/14 For personal use only.
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Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab, India and 2Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, Madhya Pradesh, India Abstract
Keywords
Aim: Our investigation was aimed to investigate the potential suitability of meloxicam-loaded nanostructured lipid carriers (MLX-NLC) gel for topical application. Main methods: MLX-NLC gel was prepared and in vivo skin penetration ability of the NLC gel was evaluated using confocal laser scanning microscopy. We studied the effect of MLX-NLC gel on the changes in lipid profile of skin to get an insight into its skin penetration enhancement mechanism. Acetic acid induced writhing test was performed to evaluate the analgesic effect. Drug concentration-time profile of MLX in rat plasma and skin after topical and oral treatment with MLX-NLC gel and oral MLX-solution, respectively, was observed. MLX-NLC gel was subjected to primary skin irritation test, sub-acute dermal toxicity study. Storage stability of MLX-NLC gel was also assessed for 90 days. Key findings: NLC gel was effective in permeating Rhodamine 123 to deeper layers of rat skin. Changes in skin lipid prolife were observed in the rat skin on treatment with MLX-NLC gel and the results supported skin lipid extraction as a possible penetration enhancement mechanism. MLX-NLC gel demonstrated sustained pain inhibitory effect. Pharmacokinetics study established that topical application of MLX-NLC gel had the potential to avoid systemic uptake and hence the risk of systemic adverse effects. MLX-NLC gel demonstrated good skin tolerability and biosafety. Excellent physical stability of nanogel was observed at 4 ± 2 C. Significance: The study revealed that NLC gel is a promising carrier system for the topical application of MLX without side effects.
Analgesic, meloxicam, nanostructured lipid carriers, skin tolerance, storage stability
Introduction Topical drug delivery is usually the most common route of administration since systemic load of drug and the drug-related side effects are reduced as compared to oral and parenteral route. However, one major drawback of topical drug delivery is the low percutaneous absorption of drugs because of the barrier functions of stratum corneum (SC), the topmost skin layer. Considerable research has now been centered on improving percutaneous absorption of drugs by utilizing advantages of lipidbased nanocarriers like nanoemulsion (NE), soild-lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)1. Different nanocarrier possesses their own way of facilitating percutaneous absorption. These nanocarriers can change the physicochemical properties of the entrapped drug and therefore can modify skin permeation profile. These carriers have the potential improvement of drug bioavailability either by facilitating skin penetration or by prolonging the skin residence time. Permeation of drug from lipid-based nanocarrier can be attributed to the high specific
Address for correspondence: Dr P. M. S. Bedi, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar – 143104, Punjab, India. Tel/Fax: + 91-183-2258819. E-mail: [email protected]
History Received 26 April 2014 Revised 26 July 2014 Accepted 28 July 2014 Published online 25 August 2014
surface area of nanoparticles that facilitates the strong adhesion of nanoparticles to the skin surface and offer an occlusive effect to the skin. The occlusive effect can eventually lead to an increase in skin hydration and can promote the deposition of drugs into the viable skin by reducing corneocytes packing and widening intercorneocyte gaps2,3. Recently published literature suggests NLC with solid lipid matrix composed of a mixture of spatially different lipid molecules, generally blend of solid and liquid lipid, and possess a number of features like small size, biocompatibility, controlled release properties, occlusive properties and improved skin targeting potential, which are advantageous for the topical route of application. Meloxicam (MLX) is a highly potent, non-steroidal antiinflammatory drug (NSAID) used for treatment of arthritis4–6. Its main limitations include poor aqueous solubility, poor incorporation into formulations and poor skin permeation7,8. To address this concern, several studies7–13 have been carried out in order to improve the percutaneous absorption and bioavailability of MLX, avoiding the systemic side effect associated with oral route. The aim of our study was to study the potential suitability of MLX-NLC gel for topical application. We evaluated the analgesic effect, in vivo skin penetration ability and the preclinical safety of MLX-NLC gel through various animal studies, including toxicity study and skin irritation study. Additionally, we assessed the storage stability of MLX-NLC nanogel for 90 days with repeated
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particle size, polydispersity index (PI), zeta potential, drug entrapment measurements at regular time intervals.
Materials and methods Drugs and chemicals Meloxicam was received as a gift sample from M/s Lupin Pharmaceuticals Ltd., Goa, India. Acetic acid, cetyl palmitate, caprylic acid, propylene glycol (PG), Tween 80, polyethylene glycol 400 (PEG 400) and triethanolamine were purchased from S. D. Fine Chemicals, Mumbai, India. Carbopol 940, were purchased from Sigma-Aldrich, Mumbai, India. Dialysis membrane (MWCO 12-14000), triton X-100 was procured from HiMedia, Mumbai, India. All other chemicals were of the analytical grade and used as received.
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Animals All animal care and experimental procedures were conducted as per guidelines of the committee for the purpose of control and supervision of experimental animals (CPCSEA) and were approved by the Institutional Animal Ethical Committee of Guru Nanak Dev University, Amritsar, India. For the experiments, the animals were housed in standard cages and maintained at an ambient temperature under conditions of optimum light (12 h light and 12 h dark) with food (standard laboratory rodent’s chow) and water provided ad libitum. For the experiments, they were acclimatized to the laboratory conditions for 5 days. Preparation of meloxicam-loaded nanostructured lipid carriers based gel Nanostructured lipid carrier-based gel was prepared using cetyl palmitate and caprylic acid as the matrix material as previously described14. An appropriate amount of distilled water was added to the heated (65 C) mixture of cetyl palmitate (65 mg) and caprylic acid (35 mg) under magnetic stirring to obtain homogeneous milky slurry to which a calculated volume of Tween 80 (0.2 ml) and the propylene glycol (1 ml) were added. The mixture turned transparent within seconds. For the preparation of NLC gel (plain-NLC gel), Carbopol 940 (5 mg) was completely dispersed in the NLC dispersion with constant stirring and triethanolamine was added drop wise to adjust the pH (6.0). Meloxicam and Rhodamine 123 containing NLC gel (MLX-NLC gel) were prepared after replacing a definite amount of the lipid phase by MLX (5% with regard to lipid content) and Rhodamine 123, respectively, and processed in the same way as mentioned above. Meloxicam gel (MLX-gel) was prepared by dispersing Carbopol 940 to the solution of meloxicam in water containing Tween 80 and PG under stirring followed by neutralization with triethanolamine. Storage stability study MLX-NLC gel was stored in screw capped glass vials and stored under different conditions (Table 1) as per ICH Harmonised Tripartite Guideline for the stability testing of new drug substances and products Q1A (R2) (ICH 2003). The formulation
Table 1. Storage conditions as per ICH Harmonised Tripartite Guideline for the stability testing. Study Refrigeration Long term Accelerated
Storage condition
4±2 C 25 ± 2 C/60 ± 5% RH 40 ± 2 C/75 ± 5% RH
Time points 0, 30 and 60 days 0, 30 and 60 days 0, 30 and 60 days
was analyzed with respect to particle size, PI, zeta potential and drug entrapment efficiency at regular time intervals (Table 1) and compared with fresh MLX-NLC gel formulation. Photon correlation spectroscopy (PCS) with a Malvern Nanosizer ZS (Malvern Instruments, Malvern, UK) was used to determine the average particle size and PI. The analyses were carried out by diluting the MLX-NLC gel with distilled water to weak opalescence to produce a suitable scattering intensity. The value of the zeta potential was measured using a Malvern Nanosizer ZS (Malvern Instruments, UK). The conductivity of the diluted formulation was adjusted to 50 mS/cm with 0.9% w/v sodium chloride solution for zeta potential measurement. The efficiency of MLX entrapment into NLC gel was determined15–17 after removing the free drug from samples of NLC gel containing MLX by ultra dialysis against acetate buffer (pH 6.0) containing 30% PEG 400 (v/v) at 4 C for 4 h using dialysis bag (Sigma, St Louis, MO; MWCO 12 000–14 000). The dialyzed formulation was lysed using TritonX 100 (0.1% v/v) and subsequently analyzed for drug content (A2) by UV spectrophotometry (Hitachi U-2800 spectrophotometer, Tokyo, Japan) at 362 nm. Drug entrapment efficiency (EE) of NLC gel was calculated by Equation (1): EEð%Þ ¼
A2 100 A1
ð1Þ
where A1 is the total amount of drug added in the formulation. Visualization of skin penetration in vivo Three Albino rats of either sex, weighing 150–200 g were taken. The skin of the dorsal region was trimmed free of hair by shaving. NLC gel formulation loaded with rhodamine 123 (5 mg/ml) was applied in a marked area of 1 cm2 at the dorsal site of animals for 24 h. Thereafter, animals were sacrificed by cervical dislocation. The excised skin was blotted in inert paper, washed thrice with ethanol. The application area was then sectioned into the pieces of 1 mm2 size and evaluated for depth of probe penetration by confocal microscopy using confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany)18. Effect of MLX-NLC gel on the skin lipid profile The excised abdominal rat skin was washed with isotonic NaCl. The epidermis was separated and treated with 250 mg of the formulation for 24 h (s) in a Franz diffusion cell at 32 ± 0.5 C. After incubation, the remnants of formulation sticking on the surface of treated epidermis were cleaned off and the skin lipids were extracted19. One hundred milligrams of epidermis was hydrated by incubation with 500 ml NaCl solution (300 mM) for 15 m. Chloroform/methanol (2:1) was added at a volume of 5 ml. After shaking for 2 h (s) at room temperature, 100 ml NaCl solution (300 mM) was added and incubated at room temperature for 10 m. Centrifugation at 2500 rpm for 10 m led to the appearance of two phases: an upper hydrophilic layer and an underlying layer containing lipid in an organic solution. 3.5 ml of the later was transferred through cotton wool into a lipid-free glass vial and evaporated to dryness at room temperature19. Cholesterol, triacylglycerols, free fatty acid, total lipid content in each sample was determined using their respective kits (Erba Diagnostic, Mannheim, Germany) method and autoanalyzer (Erba Chem 5 Plus V2, Mannheim, Germany). Percentage of lipid extracted was calculated by using following formula : ¼
1 Content in treated skin 100 Content in normal skin
Nanostructured lipid carriers
DOI: 10.3109/03639045.2014.950586
Analgesic effect 20
Acetic acid induced writhing was used to evaluate the analgesic effect of the formulations. Prior to dosing, the abdomen hair of Swiss albino mice (3–4 months old, weighing 20 to 25 g) was removed carefully. Mice were divided into four groups of six animals each. MLX-NLC gel, plain-NLC gel and MLX-gel (250 mg each) were applied on the hair free skin of the abdominal site (approximately 2.26 cm2 areas) of the animals of group I, group II and group III, respectively. Fourth group was untreated and served as control. After 24 h, the writhing was induced by intraperitoneal injection of 0.6%, (v/v) acetic acid at a dose of 10 mg/kg body weight. The analgesic response was assessed by counting the number of writhing (W) for a period of 20 min and pain inhibition ratio (PIR) was calculated using Equation (2):
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PIR ð%Þ ¼
Wc Wt 100 Wc
ð2Þ
where Wc is the writhing numbers of control group and Wt is the writhing numbers of test group. Pharmacokinetic study Albino rats (either sex) (150–200 g) were divided into two groups of 18 animals each. The first group was treated with MLX-NLC gel. MLX-NLC gel at a dose level of 10 mg/kg was applied on the shaved hind paw region of the rat, covering the knee and proximal part of the fumer muscles in the group I. MLX was dissolved in phosphate buffer (pH 7.4) and was administered orally (10 mg/kg) using oral feeding cannula to the animals of group II. Blood sample was withdrawn from tail vein of the rat into a centrifuge tube containing heparin sodium as anticoagulant according to a programed schedule at 0, 1, 3, 6, 12 and 24 h after dosing. Each blood sample was centrifuged at 4000 rpm for 10 min to separate plasma, which was stored at 20 C until analysis. The plasma sample was homogenized with a mixture of acetonitrile and water (9:1 v/v). The drug from the de-proteinized homogenate was extracted with methanol and the samples were centrifuged (3200 rpm, 10 min). The supernatants collected were analyzed for drug content by HPLC. The shaved hind paw skin of rats (n ¼ 3) in different treated groups was collected at 0, 1, 3, 6, 12 and 24 h time intervals after sacrifice and skin samples were stored at 20 C until further analysis. The collected skin sample was placed in methanol and sonicated for 20 min to extract the drug. All the samples were then centrifuged; the supernatant was transferred to vials. An aliquots (20 ml) of the supernatant was directly injected into HPLC for analysis. Pharmacokinetic parameters were analyzed by Thermo Kinetica 5.0 software (Thermo Fisher Scientific, Philadelphia, PA). Preparation of calibration curve of meloxicam in plasma Blood sample (2 ml) was collected from retro-orbital plexus of rat using heparinized capillary tube and centrifuged at 4000 rpm for 10 min. Equal volume of acetonitrile was added to the obtained plasma for deproteinization. After 15 min, the plasma was centrifuged at 4000 rpm for 10 min to remove the precipitated proteins. Supernatant was passed through a 0.2 mm filter. Accurately weighed MLX (2 mg) was transferred to a 100 volumetric flask and the volume was made up with mobile phase (methanol:water:phosphoric acid::69.9:30:0.1) resulting in a stock solution of 20 mg/ml. From the stock solution, accurately measured aliquots of 0.2, 0.4, 0.6 up to 2.0 ml were withdrawn in a series of 10.0 ml volumetric flasks and 100 ml of plasma was added in each sample. The samples were diluted to 10.0 ml with mobile phase to prepare solutions in the concentration range of 0.2–2.0 mg/ml. These samples were subjected to HPLC analysis
3
by injecting 20 ml of sample into injection port using a mobile phase delivered at a flow rate of 1 ml/m. UV detection wavelength was 362 nm. All operations were carried out at an ambient temperature. Peak area was then plotted against concentration to make the calibration curve. All experiments were performed in triplicate. Toxicity study Repeated dose subacute dermal toxicity study was conducted in Swiss albino mice at dose levels of 8, 16 and 32 mg/kg for 21 days according to the Organization for Economic Cooperation and Development guidelines 41021. Swiss albino mice (3–4 months old) weighing 20 to 25 g were randomly divided into one control (untreated) and one treatment groups, with five males and five females per group. Twenty-four hours before the experiment, the mice dorsal skin was trimmed free of hair by shaving. MLX-NLC gel was smeared onto the hair free skin areas (7 cm2) of mice of treated group once a day on a 5-day per week basis, for a period of 21 days. Body weight and food consumption were measured and recorded weekly. The treated areas were covered with porous gauze, which was held in place with non-irritating tape (Transpore 3M surgical tape, 3M India Ltd, Bangalore, India). The general physical condition of the mice was monitored daily. Before and after 21 days of the formulation application, blood samples were collected for hematology (Sysmex KX-21 automated hematology analyzer, Sysmex America, Inc., Lincolnshire, IL), biochemical analyses (auto analyzer, Erba Chem 5 Plus V2, Germany), and sodium and potassium measurements (EasyLyte Na/K Analyzer, Minnesota). Histological examinations were performed on the organs and tissues of the high-dose group and the untreated group. The animals were sacrificed by cervical dislocation. Skin, heart, liver, lungs, kidney and spleen were taken out. Then, all the tissues were fixed and preserved in 10% formalin for histopathological examination. Sections were fixed and blocks were made using the procedure as reported22. The sections were stained with eosin– hematoxylin to determine gross histopathology. Histological sections were examined using phase contrast microscope with photographic arrangement Zeiss Primostar, Germany). Skin irritation study Skin irritation potential of MLX-NLC gel was assessed by carrying out skin irritation test on male albino rabbits (weighing 2.0–2.5 kg) as per the OECD guidelines 40423 for testing of acute dermal irritation/corrosion. Rabbits were randomly divided into two groups, with three rabbits per group. Each rabbit was caged individually. The fur on both the sides of the dorsal surface of the trunk of each test animal was removed carefully with electronic hair remover, 24 h prior to the experiment. Three areas (A, B and C) each with approximately 6 cm2 area were marked on the hair free area of every test animal. 20% sodium lauryl sulfate (SLS) solution was used as positive control (Manosroi et al., 2013). The animals were treated as follows: Group I – 20% w/v SLS solution (area A, B), untreated (area C); Group II – plain NLC gel (area A), MLX-NLC gel (area B) and untreated (area C). Five hundred milligrams of the test formulation was applied to the marked area A and area B in each group. The treated area was covered with a gauze patch which is held in place with nonirritating tape (Transpore 3M surgical tape, 3M India Ltd, India). C area in each group was used as the control. At the end of 24 h exposure period, the residual formulation on the skin was removed carefully with purified water without altering the existing response or the integrity of the epidermis. Observations were
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made at 1, 24, 48, and 72 h, and the dermal reactions (erythema and edema) were scored between 0 and 4 on the basis of degree of severity and grading of skin reactions was ascertained to determine the total dermatitis score as per OECD guideline 404 (OECD 2002). The scores for erythema and edema were summed up for all the rabbits at 1, 24, 48 and 72 h. From the grades, primary irritation index (PII) was calculated using Equation (3): Sum of erythema grade at 1=24=48=72h þSum of edema grade at 1=24=48=72h 100 ð3Þ PII ¼ Number of animals The irritation degree was categorized based on the PII values as negligible (PII ¼ 0–0.4), slight (PII ¼ 0.5–1.9), moderate (PII ¼ 2–4.9) or severe (PII ¼ 5–8) irritation24.
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Statistical analysis All the data are expressed as mean ± standard deviation (SD). All statistical analysis was performed using Sigma Stat Software, 3.5 (Systat Software Inc, San Jose, CA). Data were analyzed using the one-way analysis of variance (ANOVA) with the Holm–Sidak correction. p Values 50.05 were considered as statistically significant.
Results and discussion The aim of our study was to develop a topical MLX formulation to control the delivery of MLX across the skin. A preliminary assessment of NLC gel as drug carriers for MLX was undertaken and motivating results were obtained14. In this study, we further investigated the drug delivery potential and safety of MLX-NLC gel on topical application. Stability study Stability is mainly evaluated to ensure that the quality of the product will be maintained throughout its shelf life25. An important limitation of nanodispersion is their poor storage stability. To improve the storage stability, NLC dispersion is commonly transformed into semisolid gel. MLX-NLC gel stored at different temperature and humidity conditions (Table 1) was evaluated for any changes in particle size, PI, zeta potential and drug content at 30, 60, 90 days time points to assess the effect of storage conditions on the stability as a function of time. The particle size was monitored at regular time intervals during storage to assess particle aggregation. The nanoparticles are thermodynamically unstable system and for their stability, a zeta potential value between 30 mV and 60 mV is desirable to avoid aggregation of particles26. Thus, zeta potential measurement gives us the estimation of the storage stability of nanoparticles. Stability of MLX-NLC gel was evaluated in terms of their MLX entrapment efficiency. Log(E.E %) was plotted against time and the slopes (m) were calculated by linear regression. The slopes (m) were then substituted into the following equation for the determination of k values: k ¼ m 2:303 Shelf-life values (the time for 10% loss, i.e. t90) were then calculated by the following equation: t90 ¼ 0:105=k In case of MLX-NLC gel, no significant (p40.05) change of particle size, PI, zeta potential, drug entrapment efficiency was observed at 4 ± 2 C over the period of 90 days (Table 2). An increasing trend of particle size and PI, decreasing trend of zeta
Table 2. Effect of storage conditions on the particle size, polydispersity index, zeta potential and drug entrapment efficiency. Storage condition Parameter Particle size (nm)
Day
0 30 60 90 Polydispersity 0 index 30 60 90 Zeta potential 0 (mV) 30 60 90 Drug entrapment 0 efficiency (%) 30 60 90
4 C
25 ± 2 C and 60 ± 5% RH
40 ± 2 C and 75 ± 5% RH
210.66 ± 1.79 210.66 ± 1.79 210.66 ± 1.79 211.99 ± 2.80 229.45 ± 3.04 240.12 ± 3.18 213.33 ± 2.82 335.12 ± 4.43 513.54 ± 6.79 214.21 ± 2.83 414.65 ± 5.49 762.42 ± 10.09 0.213 ± 0.00600 0.213 ± 0.00600 0.213 ± 0.00600 0.215 ± 0.00284 0.291 ± 0.00385 0.318 ± 0.00420 0.218 ± 0.00288 0.309 ± 0.00409 0.365 ± 0.00482 0.220 ± 0.00291 0.333 ± 0.00441 0.410 ± 0.00542 27.33 ± 0.620 27.33 ± 0.620 27.33 ± 0.620 27.1 ± 0.358 25.23 ± 0.333 21.43 ± 0.283 26.95 ± 0.356 21.53 ± 0.284 17.89 ± 0.236 26.92 ± 0.355 19.43 ± 0.257 14.87 ± 0.196 85.61 ± 1.35 85.61 ± 1.35 85.61 ± 1.35 84.56 ± 1.12 81.01 ± 1.07 78.33 ± 1.04 83.98 ± 1.11 77.05 ± 1.02 72.54 ± 0.96 82.67 ± 1.09 71.32 ± 0.94 60.23 ± 0.80
The data reported are mean ± SD (n ¼ 3).
potential and drug entrapment efficiency were observed with storage time at 25 ± 2 C/60% ± 5% RH and 40 ± 2 C/75 ± 5% RH. The increase of particle size and polydispersity index, and diminution of zeta potential and drug entrapment efficiency were comparatively higher at 40 ± 2 C/75%±5% RH. This demonstrated the physical stability of the MLX-NLC gel at 4 ± 2 C over the period of 90 days. The results are in agreement with previous results27 suggesting the good stability of MLX-NLC gel. Upon storage at high temperature, the change in the particle size in MLX-NLC gel can be attributed to the increase in the kinetic energy of system at high temperature that may accelerate the collision between particles, and consequently can increase the chances of aggregation of particles28. This decrease in entrapment efficiency at high temperature could also be attributed to lipid transformation of the solid lipid used in NLC over time, leading to the formation of a highly ordered lipid structure resulting in drug expulsion. Shelf-life values, i.e. the time at which the drug concentration is lost by 10% were calculated. A good chemical stability of MLX-NLC gel (t90, 281.90 days) was observed when stored at 4 ± 2 C It is evident from the data that MLX-NLC gel had good long-term physical and chemical stability at 4 ± 2 C and the recommended storage temperature is 4 ± 2 C. Visualization of skin penetration in vivo Confocal laser scanning microscopy (CLSM) provides information about the localization and the permeation pathway of fluorescent model compound in the tissue. The major advantage of CLSM is that the distribution of the fluorescent model compound in the sample can be visualized without cryo-fixing or embedding the tissue29. The depth of Rhodamine 123 (lipophilic fluorescence marker) penetration into skin from NLC gel (Figure 1) was measured by CLSM. NLC gel was found to be effective in permeating Rhodamine 123 to a skin depth of 160 mm (Figure 1). Our results indicate that NLC gel is effective in improving the skin penetration of MLX and the findings are consistent with the in vitro skin permeation study14 supporting the high permeation of MLX to deeper layers of the skin from NLC gel. These characteristics are significant for its use as topical dosage form.
Nanostructured lipid carriers
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DOI: 10.3109/03639045.2014.950586
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Figure 1. Photomicrograhs (a) and fluorescence intensity (b) measured by confocal laser scanning microscopy of rat skin after 24 h treatment with Rhodamine 123 containing nanostructured lipid carriers gel.
Similar results were reported by Teeranachaideekul et al.30. They suggested that the high amount and deep penetration of dye into skin from NLC could be attributed to its high occlusion factor. In addition to the occlusion effect, the observed effect may be a consequence of the NLC components, such as CA (short chain fatty acid), Tween 80 and PG that could significantly reduce the barrier properties of SC by modifying the thermodynamic activity of the drug in the vehicle. Tween 80 and PG can change the diffusional driving force for solvent partition into the tissue and thereby can facilitate the drug permeation into skin31,32. Further, the permeation of nanoparticles also rely on the characteristic features like surface properties, zeta potential, flexibility and particle size of the system. Shah et al.33 investigated the effect of surface modification of fluorescent dye encapsulated NLC on topical delivery by in vivo confocal microscopy across the skin and suggested that the surface modification of NLC with a peptide containing 11 arginines (R11) improved transport of spantide II (SP) and ketoprofen (KP) across the deeper skin layers. Biochemical investigation It is recognized that the lipid composition of the skin plays a key role in determining the barrier functions34. The effect of the skin lipids content on the rate of skin permeation has been recognized by a strong relationship between the quantity of extracted skin lipids with acetone/methanol and the quantity of drug
permeation19. Skin lipids may affect drug permeation, as they can interact with the formulation. Thus to elucidate penetration enhancement mechanism of nanogel, biochemical studies were carried out to examine the changes in the lipid profile of skin as a result of the interaction with the nanogel formulation. Skin is the outermost largest organ and mainly provides barrier attributes to the skin and makes the topical/transdermal delivery challenging. It is composed of hypodermis, hydrophilic dermis and epidermis. Epidermis is divided into living hydrophilic layer and a hydrophobic horny layer composed of dead cells (stratum corneum, SC)35,36. The application of MLX-NLC gel caused changes in the lipid content of skin and as a result, the barrier property of skin. The percentage of cholesterol, triacylglycerol, free fatty acids and total lipids extracted from excised rat skin treated with MLX-NLC gel and delipidized (negative control) is shown in Table 3. These findings imply that the formulation induced an alteration in lipid content and suggest that acted via lipid interactions with skin that lead to the disruption of the barrier properties and enhanced the permeation of the drug across the skin. Our results support the reports that a decrease in skin lipid level may result in decrease of skin barrier property37,38. Analgesic effect Both MLX-NLC gel and MLX-gel produced appreciable analgesic activity with PIE of 42.86 ± 2.79 and 8.82 ± 0.57%,
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Table 3. Percentage of lipids extracted from excised rat skin after 24 h (s) of treatment. Skin stratum corneum sample
Cholesterol (%)
Triacylglycerol (%)
Free fatty acids (%)
Total lipid content
39.13 ± 2.54 98.98 ± 0.86
25.37 ± 1.02 99.40 ± 0.86
11.31 ± 1.49 98.97 ± 0.86
23.11 ± 2.47 99.45 ± 0.86
MLX-NLC gel treated Delipidized The data reported are mean ± SD (n ¼ 3).
35 Plasma concentration (µg/ml)
Figure 2. Plasma concentration–time profiles of meloxicam in rat plasma after topical and oral treatment with nanogel and oral solution respectively each at 10 mg/kg dose level. The data reported are mean ± SD (n ¼ 3).
MLX oral solution
30 25 20 15 10 5 0
0
5
10
15
20
Time (h)
Figure 3. Meloxicam concentration–time profiles in rat skin after topical and oral treatment with nanogel and oral solution respectively each at 10 mg/kg dose level. The data reported are mean ± SD (n ¼ 3).
30
MLX-NLC gel
MLX oral solution
25 Concentration (µg/g)
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MLX-NLC gel
20 15 10 5 0 1
3
6
12
24
Time (h)
respectively, after 24 h and also that the analgesic effect was more pronounced in MLX-NLC gel compared to MLX gel. Eventually; MLX-NLC gel could be appropriate for topical pain reduction. Pharmacokinetic study Figure 2 shows the plasma concentration–time profile of MLX in rat plasma after topical and oral treatment with nanogel and oral solution, respectively, each at a dose level of 10 mg/kg. The related pharmacokinetic parameters were determined and it was observed that the maximum concentration (Cmax) in plasma appeared at 3 h and 24 h after oral administration and topical treatment, respectively. Cmax value in blood plasma was found to be much lower after topical treatment with MLX-NLC gel compared to that obtained after oral administration of MLXsolution indicating the potential of MLX-NLC-gel to reduce high levels in plasma that might be associated with side effects. The plasma AUC0 t after topical treatment with MLX-NLC gel was 14.46 times lesser than that observed after oral administration of MLX-solution indicating that lower fraction of MLX were absorbed in the circulation after MLX-NLC gel application compared with MLX oral solution. This can probably be ascribed to the drug reservoir forming potential of skin. Further, the drug deposition in skin following treatment with MLX-NLC gel was higher than that following MLX-oral solution administration (Figure 3). Cmax and AUC0 t values were higher in skin after topical application of MLX-NLC gel as compared to
that obtained after oral administration of MLX-solution. The AUC0 t in skin after topical administration of MLX-NLC gel was 79.03-fold, higher than that obtained after oral administration of MLX-solution. Our results indicated the potential of MLXNLC gel to avoid systemic uptake and to form drug reservoir in the skin that could act as the force for delivering drug into local deeper tissues. The lower systemic exposure would avoid GIT irritation and reduce the risk of adverse systemic effects. In the final outcome, the site-specific drug delivery of MLX from the proposed drug delivery system and consequently lowered side effects will facilitate the design of an optimized drug delivery system, overwhelmingly superior to the existing oral administration. Our results are in agreement with literature39,40. The authors found the lower systemic exposure of the drug after topical administration of flurbiprofen (FB)-loaded NLC gel than after oral administration of FB solution. Toxicity study As reported earlier, MLX is associated with risk of arterial thrombotic events, a functional renal failure, liver dysfunction and skin reactions, particularly at high doses and on long-term treatment41–43. In view of this, it became necessary to establish the safety of developed NLC-based nanogel formulation against the local and systemic toxic effects. Repeated application of MLX-NLC gel did not show any unusual behavioral changes in
Nanostructured lipid carriers
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Figure 4. Photomicrogrphs (400) of hematoxylin–eosin stained skin (A), heart (B), liver (C), lungs (D), kidney (E) and spleen (F) sections of untreated mice in repeated dose toxicity study.
Figure 5. Photomicrogrphs (400) of hematoxylin–eosin stained skin (A), heart (B), liver (C), lungs (D), kidney (E) and spleen (F) sections of mice treated with MLX-NLC gel in repeated dose toxicity study at 32 mg/kg dose.
the treated animals. No mortality was observed in any of the animal group. On the site of application of the MLX-NLC gel, no reaction was seen. The animals belonging to the test substancetreated group did not show significant change in body weight as compared with control. All the animals showed normal weight gains and revealed no toxic effects during the observation period. Following a 21-day repeated administration, the values of all the hematological and biochemistry parameters were within the normal limits and demonstrated normal kidney, liver, pancreas function and absence of bone marrow toxicity. In order to check out the potential toxicity on internal organs frequently coupled with the oral COX-2 selective anti-inflammatory drugs, histopathological examination of skin, heart, liver, lungs, kidney and spleen tissues, of high-dose treated group was performed. Compared to control (Figure 4A–F), microscopic examinations of skin, heart, liver, lungs, kidney and spleen in treated animals (Figure 5A–F) did not show any abnormality in their natural architecture. In addition, no evidence of inflammation, cell lysis or lesions were seen in the histological evaluation of organ tissues of any of the treated animals when compared to control. These results clearly established that the developed gel was non-irritant to the skin as well as non-toxic to the internal organs and illustrated the absence of the local and systemic toxicities of MLX-NLC gel at high doses in mice. Thus, the biocompatibility and safety of the developed MLX-NLC gel was confirmed. The safety of MLX-NLC gel for skin application can be attributed to the fact that the primary fraction of MLX remained in the skin and extremely low amount of MLX was absorbed in the circulation subsequent to application of nanogel. This eventually could reduce the chances of drug-associated undesirable systemic effects. Furthermore, the lipids selected for NLC gel formulation were reported to be biocompatible, safe and effective in topical/ transdermal formulations. Skin irritation study In order to further demonstrate the skin tolerability of the MLX-NLC gel, skin irritation study was conducted. According to
the PII scores, the degree of irritation was classified as negligible (PII ¼ 0–0.4), slight (PII ¼ 0.5–1.9), moderate (PII ¼ 2–4.9) or severe (PII ¼ 5–8) irritation24. The study indicated that MLXNLC gel had no significant effect on the animal weights at all time points (p40.05). MLX-NLC gel showed no erythema or edema on the rabbit skin with PII 0.0. However, 20% SLS treatment resulted in moderate skin irritation with PII value 2.33 and 2.0 after 1 h and 24 h, respectively. The skin tolerability of MLX-NLC gel could be due to the entrapment of drug in NLC leading to avoidance of direct contact of drug with the skin. Thus, MLX-NLC gel formulation would prove to be highly advantageous for topical use with improved safety profile.
Conclusions It can be concluded that MLX-NLC gel has the potential to form drug reservoir in the skin that can act as the force for delivering drug into local deeper tissues, and hence offers MLX-NLC gel promising platform as topical drug delivery system. The results of the toxicity study further substantiate the role of NLC gel as a promising topical carrier for MLX in order to achieve better therapeutic efficacy without systemic side effects. Storage stability study confirmed good physical stability of MLX-NLC gel at 4 ± 2 C. These preclinical observations necessitate to be extrapolated to humans for establishing their efficacy in clinical use. Scale up and pilot plant studies will have to be conducted in order to establish the industrial feasibility of the proposed delivery system. We anticipate that there is a bright perspective on the future development of the NLC gel in controlled and targeted drug delivery.
Declaration of interest The authors report no declarations of interest.
References 1. Joshi M, Patravale V. Formulation and evaluation of nanostructured lipid carrier (NLC)-based gel of valdecoxib. Drug Dev Ind Pharm 2006;32:911–18.
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S. Khurana et al.
2. Muller RH, Radtke M, Wissing SA. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Adv Drug Deliv Rev 2002;54:S131–55. 3. Schafer-Korting M, Mehnert W, Korting HC. Lipid nanoparticles for improved topical application of drugs for skin diseases. Adv Drug Deliv Rev 2007;59:427–43. 4. Burke A, Smyth E, FitzGerald GA. Analgesic-anti pyretic and anti inflammatory agents; pharmacotherapy of gout. In: Bruntom LL, Lazo JS, Parker KL, eds. Goodman’s and Gilman’s the pharmacological basis of therapeutics. New York: McGraw Hill; 2006: 676–700. 5. Abramson SB, Yazici Y. Biologics in development for rheumatoid arthritis: relevance to osteoarthritis. Adv Drug Deliv Rev 2006;58: 212–25. 6. Quan LD, Thiele GM, Tian J, Wang D. The development of novel therapies for rheumatoid arthritis. Expert Opin Ther Pat 2008;18: 723–38. 7. Yuan Y, Li S, Mo F, Zhong D. Investigation of microemulsion system for transdermal delivery of meloxicam. Int J Pharm 2006;32: 1117–23. 8. Jantharaprapap R, Stagni G. Effects of penetration enhancers on invitro permeability of meloxicam gels. Int J Pharm 2007;343:26–33. 9. Ruiz Martinez MA, Lopez-Viota Gallardo J, de Benavides MM, et al. Rheological behavior of gels and meloxicam release. Int J Pharm 2007;333:17–23. 10. Yuan Y, Chen X, Zhong D. Determination of meloxicam in human plasma by liquid chromatography–tandem mass spectrometry following transdermal administration. J Chromatogr 2007;B852: 650–4. 11. Yuan Y, Li S, Yu L, et al. Physicochemical properties and evaluation of microemulsion systems for transdermal delivery of meloxicam. Chem Res Chin Univ 2007;23:81–6. 12. Kasliwal N, Derle D, Negi J, Gohil K. Effect of permeation enhancers on the release and permeation kinetics of meloxicam gel formulations through rat skin. Asian J Pharm Sci 2008;3:3193–9. 13. Ah YC, Choi JK, Choi YK, et al. A novel transdermal patch incorporating meloxicam: in vitro and in vivo characterization. Int J Pharm 2010;385:12–19. 14. Khurana S, Bedi PMS, Jain NK. Development and characterization of a novel controlled release drug delivery system based on nanostructured lipid carriers gel for meloxicam. Life Sci 2013;93: 763–72. 15. Utreja S, Khopade AJ, Jain NK. Lipoprotein-mimicking biovectorized systems for methotrexate delivery. Pharm Acta Helv 1999;73: 275–9. 16. Khurana S, Bedi PMS, Jain NK. Development of nanostructured lipid carriers (NLC) for controlled delivery of meloxicam. Int J Biomed Nanosci Nanotechnol 2010;1:247–66. 17. Khurana S, Bedi PMS, Jain NK. Development of nanostructured lipid carriers (NLC) for controlled delivery of mefenamic acid. Int J Biomed Nanosci Nanotechnol 2012;2:232–50. 18. Dubey V, Mishra D, Dutta T, et al. Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J Control Release 2007;123:148–54. 19. Stahl J, Niedorf F, Kietzmann M. Characterisation of epidermal lipid composition and skin morphology of animal skin ex vivo. Eur J Pharm Biopharm 2009;72:310–16. 20. Xi H, Cun D, Xiang R, et al. Intra-articular drug delivery from an optimized topical patch containing teriflunomide and lornoxicam for rheumatoid arthritis treatment: does the topical patch really enhance a local treatment? J Control Release 2013;169:73–81. 21. Organization for Economic Cooperation and Development (OECD). Guidelines for testing chemicals 410 repeated dose dermal toxicity: 21/28-day study. Paris: OECD; 1981.
Drug Dev Ind Pharm, Early Online: 1–8
22. Lee BJ, Lee TS, Cha BJ, et al. Percutaneous absorption and histopathology of a poloxamer-based formulation of capsaicin analog. Int J Pharm 1997;159:105–14. 23. Organization for Economic Cooperation and Development (OECD) Guidelines. Test guideline 404 acute dermal irritation/corrosion. Paris: OECD; 2002. 24. Manosroi A, Chankhampan C, Manosroi W, Manosroi J. Transdermal absorption enhancement of papain loaded in elastic niosomes incorporated in gel for scar treatment. Eur J Pharm Sci 2013;48:474–83. 25. ICH Stability testing of new drug substances and products. International Conference on Harmonization (ICH) Guidelines Q1A (R2). European Medicines Agency, CPMP/ICH/2736/99; 2003. 26. Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art. Eur J Pharm Biopharm 2000;50:161–77. 27. Junyaprasert VB, Teeranachaideekul V, Souto EB, et al. Q10-loaded NLC versus nanoemulsions: stability rheology and in vitro skin permeation. Int J Pharm 2009;377:207–14. 28. Freitas C, Muller RH. Stability determination of solid lipid nanoparticles (SLN) in aqueous dispersion after addition of electrolyte. J Microencapsul 1999;16:59–71. 29. Chen H, Chang, X, Du D, et al. Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. J Control Release 2006;110: 296–306. 30. Teeranachaideekul V, Boonme P, Souto EB, et al. Influence of oil content on physicochemical properties and skin distribution of Nile red-loaded NLC. J Control Release 2008;128:134–41. 31. Williams AC, Barry BW. Penetration enhancers. Adv Drug Del Rev 2004;56:603–18. 32. Fox LT, Gerber M, Plessis JD, Hamman JH. Transdermal drug delivery enhancement by compounds of natural origin. Mol 2011;16: 10507–40. 33. Shah PP, Desai PR, Patel AR, et al. Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs. Biomaterials 2012;33:1607–17. 34. Elias PM, Menon GK. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv Lipid Res 1991;24:1–26. 35. Babita K, Kumar V, Rana V, et al. Thermotropic and spectroscopic behavior of skin: relationship with percutaneous permeation enhancement. Curr Drug Deliv 2006;3:95–113. 36. Karadzovska D, Brooks JD, Monteiro-Rivierem NA, Riviere JE. Predicting skin permeability from complex vehicles. Adv Drug Deliv Rev 2013;65:265–77. 37. Elias PM, Cooper ER, Korc A, Brown BE. Percutaneous transport in relation to stratum corneum structure and lipid composition. J Invest Dermatol 1981;76:297–301. 38. Kostiuk NV, Zhigulina VV, Belyakova MB, et al. Effect of melatonin on lipid barrier in rats’ skin. Am J Biochem 2012;2: 67–73. 39. Han F, Yin R, Che X, et al. Nanostructured lipid carriers (NLC) based topical gel of flurbiprofen: design characterization and in vivo evaluation. Int J Pharm 2012;439:349–57. 40. Chen-yu G, Chun-fen Y, Qi-lu L, et al. Development of a quercetinloaded nanostructured lipid carrier formulation for topical delivery. Int J Pharm 2012;430:292–8. 41. Distel M, Mueller C, Bluhmkij E, Fries J. Safety of meloxicam: a global analysis of clinical trials. Br J Rheumatol 1996;35:68–77. 42. Lanes SF, Rodriguez LA, Hwang GE. Baseline risk of gastrointestinal disorders among new users of meloxicam ibuprofen diclofenac naproxen and indomethacin. Pharmacoepidemiol Drug Saf 2000;9: 113–17. 43. Villegas I, Alarcon de la, Lastra C, et al. Effects of food intake and oxidative stress on intestinal lesions caused by meloxicam and piroxicam in rats. Eur J Pharmacol 2001;414:79–86.
