Novel topical anandamide formulation for alleviating peripheral neuropathic pain.

Peripheral neuropathy (PN) is a condition of peripheral nerve damage leading to severe pain. The first line therapies are associated with adverse psychotropic effects (PSE) and second line therapies are not efficient enough to relieve pain. There is an unmet drug need for relieving pain effectively without PSE in PN. Anandamide, an endocannabinoid activates cannabinoid receptors to relieve the pain due to peripheral neuropathy (PN). Anandamide has a very short biological half-life as they are extensively metabolized by fatty acid amide hydrolase enzyme (FAAH). Regional delivery of safe FAAH inhibitor (FI) with anandamide would be beneficial for PN without PSE. The objective of the study is to identify a safe FI and deliver the anandamide in combination with the FI topically for the management of PN. The FAAH inhibition potential of silymarin constituents was evaluated by molecular docking and in vitro studies. The topical gel formulation was developed to deliver anandamide and FI. The formulation was assessed in chemotherapeutic agent-induced PN rat models to relieve mechanical-allodynia and thermal-hyperalgesia. The molecular docking studies demonstrated that the Prime MM-GBSA free energy of silymarin constituents were in the order of silybin > isosilybin > silychristin > taxifolin > silydianin. In in vitro studies, silybin 20 µM inhibited > 61.8% of FAAH activity and increased the half-life of anandamide. The developed formulation increased permeation of anandamide and silybin across the porcine skin. Furthermore, on the application of anandamide and anandamide-silybin gel to rat paws, there was a significant increase in the pain threshold for allodynic and hyperalgesic stimulus up to 1 h and 4 h, respectively. The topical anandamide with silybin delivery approach could serve to alleviate PN efficiently and thus could minimize unwanted CNS side effects of synthetic or natural cannabinoids in patients.

Development of silymarin topical formulation: In vitro and ex vivo dermal kinetics of silymarin.

Silymarin constituents are extensively investigated in the treatment of skin disorders. The main constituents of silymarin include taxifolin (TX), silychristin (ST), silydianin (SDN), silybin A (SA), silybin B (SB), isosilybin A (ISA) and isosilybin B (ISB). The objective of the present study was to determine in-vitro dermal kinetics of individual silymarin constituents in human skin models and to develop a silymarin topical formulation. In-vitro studies indicate human skin binding of silymarin was in the range of 2.09 to 12.3% and half-life of silymarin constituents was > 15.5 h in epidermal and dermal cells. Topical silymarin cream was prepared using sulfobutylether-ß-cyclodextrins as solubilizer and propylene glycol as permeation enhancer. The cream was subjected to ex-vivo human skin permeation studies. In ex-vivo studies, cumulative amount of TX, ST, SDN, SA, SB, ISA and ISB permeated across human cadaver skin at 24 h was 921 ± 13.5, 1992 ± 67.6, 345 ± 39.2, 1089 ± 45.0, 1770 ± 100, 1469 ± 81.5 and 1285 ± 33.1 ng/cm2, respectively. The amount TX, ST, SDN, SA, SB, ISA and ISB retained after 24 h was 60.7 ± 8.2, 376 ± 45.5, 72.3 ± 6.9, 66.4 ± 8.0, 208 ± 31.3, 154 ± 12.4 and 102 ± 6.3 ng/mg of human cadaver skin, respectively. The study results demonstrate silymarin topical formulation could deliver significant amount of silymarin constituents into skin. The developed silymarin formulation could be beneficial for treatment or management of a broad spectrum of dermatological disorders.

Effect of Lipid Vehicles on Solubility, Stability, and Topical Permeation of Delta-9-Tetrahydrocannabinol.

Delta-9-tetrahydrocannabinol (THC) is one of the most effective antinociceptive agents used in the treatment of peripheral neuropathy. THC is highly lipophilic and susceptible to thermal and oxidative degradation. Identifying appropriate solvents in which THC is stable as well as adequately solubilized is crucial in developing topical dosage forms. Lipid solvent systems are of utmost utility and relevance for formulating highly lipophilic drugs. Hence, the objective of this project was to screen the solubility of THC in lipidic excipients, monitor THC content in the selected vehicles during stability, and study the influence of these excipients on permeation of THC across skin. The solubility of THC in liquid lipid excipients was in the range of 421 to 500 mg/g. The solubility of THC in solid lipid excipients was in the range of 250 to 750 mg/g. THC in its neat form was poorly stable, but when dissolved in lipid-based excipients, its stability improved significantly. THC in lipid excipients was more stable at 4 ± 3°C compared to samples stored at 25 ± 2°C. The antioxidants (butylated hydroxytoluene and ascorbyl palmitate) used in the excipients further improved the stability of THC. The results demonstrated that the liquid and solid lipid excipients used in the study could solubilize THC freely and mitigate the degradation of THC significantly. The binary combination of lipid excipients enhanced THC skin permeation and retention, demonstrating the potential for topical formulation development of THC.

Polymer Coated Polymeric (PCP) Microneedles for Controlled Delivery of Drugs (Dermal and Intravitreal).

Microneedles are used to deliver drugs topically across the skin and mucous membranes. Dissolvable microneedles are made using soluble polymers, which disintegrates in the tissue and release the entire payload instantaneously including the polymer construct. Often, a slow release of drug into the tissue is desirable to overcome the severity of side effects at the site of administration as well as systemic adverse effects. In addition, controlled release of active pharmaceutical ingredient (API) only (not the excipients) is safe and effective particularly when the drug delivery is intended to sensitive organs like the eye. In this project, the feasibility of fabricating polymer coated polymeric (PCP) microneedles to achieve a gradual release of only the active ingredient from the device was investigated. The potential application of such PCP microneedles in the dermal and intravitreal drug delivery was also explored using animal tissue models. The PCP microneedles were found to be intact even after prolonged contact with the release medium. The time at which 50% (T50%) of dextran (10 K) was released in case of microneedles prepared using 20% of core polymer (PVP-K30) was about 15 min versus less than 5 min in the case of uncoated microneedles. Whereas when the core polymer concentration was increased to 50%, the T50% was increased to 90 min. The rate of release depended on the polymer molecular weight grade. The rate of drug release was not influenced by the total amount of concentration of dextran. The PCP microneedles of lidocaine hydrochloride could constantly release the drug for up to 9 h in the skin tissue. Likewise, the PCP microneedles infused voriconazole, intravitreally for 6 h.

Analysis of docosanol using GC/MS: Method development, validation, and application to ex vivo human skin permeation studies.

Docosanol is the only US Food and Drug Administration (FDA) approved over-the-counter topical product for treating recurrent oral-facial herpes simplex labialis. Validated analytical methods for docosanol are required to demonstrate the bioequivalence of docosanol topical products. A gas chromatography/selected ion monitoring mode mass spectrometry (GC/SIM-MS) method was developed and validated for docosanol determination in biological samples. Docosanol and isopropyl palmitate (internal standard) were separated on a high-polarity GC capillary column with (88% cyanopropy)aryl-polysiloxane employed as the stationary phase. The ions of m/z 83 and 256 were selected to monitor docosanol and isopropyl palmitate, respectively; the total run time was 20 min. The GC/SIM-MS method was validated in accordance with US FDA guidelines, and the results met the US FDA acceptance criteria. The docosanol calibration standards were linear in the 100-10000 ng/mL concentration range (R 2>0.994). The recoveries for docosanol from the receptor fluid and skin homogenates were >93.2% and >95.8%, respectively. The validated method was successfully applied to analyze ex vivo human cadaver skin permeation samples. On applying Abreva® cream tube and Abreva® cream pump, the amount of docosanol that penetrated human cadaver skin at 48 h was 21.5 ± 7.01 and 24.0 ± 6.95 ng/mg, respectively. Accordingly, we concluded that the validated GC/SIM-MS was sensitive, specific, and suitable for quantifying docosanol as a quality control tool. This method can be used for routine analysis as a cost-effective alternative to other techniques.

Development and Validation of HPLC Method for Efinaconazole: Application to Human Nail Permeation Studies.

Efinaconazole is the first azole derivative approved by FDA for the topical treatment of onychomycosis. The objective of present study was to develop and validate HPLC method for estimation of efinaconazole in ex vivo human nail permeation study samples. The chromatographic analysis was performed on a HPLC system equipped with diode array detector. The efinaconazole and internal standard (IS) were extracted from the human nail samples by using the protein precipitation method. The samples were injected on to 5 µm Polar C18 100Å, 4.6 mm × 150 mm column. The mobile phase consisted of 0.01 M potassium dihydrogen phosphate: acetonitrile (36:64) and eluent was monitored at 205 nm. The chromatographic separation of drug and analyte was achieved using isocratic elution at flow rate of 1 mL/min with a total run time of 15 min. The efinaconazole and IS were eluted at 6.4 ± 0.5 and 8.3 ± 0.5 min, respectively. The developed method was validated as per FDA guidelines, and the results met with acceptance criteria. The method developed was specific, and the analyte concentrations were linear at range of 50 to 10000 ng/mL (R2 ≥ 0.9981). The validated HPLC method was applied for quantifying efinaconazole in human nail permeation study samples. The permeation of efinaconazole was increased by twofolds with Labarfac CC (15135.4 ± 2233.9 ng/cm2) compared to formulations containing Transcutol P (6892.0 ± 557.6 ng/cm2) and Labrasol (7266.1 ± 790.6 ng/cm2). The study results demonstrate that developed efinaconazole HPLC method can be employed for formulation evaluation and clinical studies.

Chemotherapeutic Agent-Induced Vulvodynia, an Experimental Model.

Vulvodynia is a chronic clinical condition associated with vulvar pain that can impair the sexual, social, and psychological life of women. There is a need for more research to develop novel strategies and therapies for the treatment of vulvodynia. Vulvodynia in experimental animal models induced via infections, allergens, and diabetes are tedious and with lessor induction rate. The objective of the study was to explore the possibility of inducing vulvodynia using a chemotherapeutic agent in a rodent model. Paclitaxel is commonly used in treating breast and ovarian cancer, whose dose-limiting side effect is peripheral neuropathy. Studies have shown that peripheral neuropathy is one of the etiologies for vulvodynia. Following paclitaxel administration (2 mg/kg i.p.), the intensity of vulvar hypersensitivity was assessed using a series of von Frey filaments (0.008 to 1 g) to ensure the induction of vulvodynia. Vulvodynia was induced from day 2 and was well sustained for 11 days. Furthermore, the induced vulvodynia was validated by investigating the potentiation of a flinch response threshold, upon topical application and systemic administration of gabapentin, a commonly used medication for treating neuropathic pain. The results demonstrate that vulvodynia was induced due to administration of paclitaxel. The fact that chemotherapeutic agent-induced vulvodynia was responsive to topical and parenterally administered gabapentin provides validity to the model. The study establishes a new, relatively simple and reliable animal model for screening drug molecules for vulvar hypersensitivity.

Optimization of sulfobutyl-ether-ß-cyclodextrin levels in oral formulations to enhance progesterone bioavailability.

Progesterone oral dose regimens are indicated for the treatment of luteal phase deficiency and estrogen dominance. The poor aqueous solubility of progesterone leads to erratic oral absorption, resulting in suboptimal or excessive plasma levels. Developing a formulation to enhance the solubility of progesterone in the gastrointestinal tract would be beneficial to decrease drug absorption variability and increase bioavailability. The solubility of progesterone at 400 mM sulfobutyl-ether-ß-cyclodextrin (SBE-ß-CD) concentration was ~7000-fold greater than its intrinsic solubility, aided by the formation of SBE-ß-CD-progesterone complex. The complex was characterized using differential scanning colorimeter, Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy techniques. FTIR and NMR studies of the complex confirm the interaction between functional groups of SBE-ß-CD and progesterone to form an inclusion complex. Molecular modeling studies demonstrated progesterone binding poses with four probable SBE-ß-CD isomers and these results matched with NMR and FTIR data. The progesterone oral formulations were optimized by increasing the levels of SBE-ß-CD in the formulation to prevent the displacement of progesterone from the complex by gastrointestinal contents. The oral bioavailability of progesterone in rats was increased 5-fold when administered with the optimized formulation compared to administration with progesterone API capsules. Studies demonstrated that the optimized formulation prevents precipitation of progesterone in the intestinal tract and increases progesterone oral bioavailability in rats.

A Rapid Tool to Optimize Process Variables for Continuous Manufacturing of Metronidazole Ointment Using Melt Extrusion Technique.

The use of hot-melt extrusion (HME) technique in the preparation of semi-solid products offers several advantages over conventional processes. However, the optimization of the technique for preparation of semi-solid pharmaceuticals is challenging due to involvement of ingredients with different physical properties. Hence, a simple tool to optimize the mixing of ingredients that results in a target ratio and drug content uniformity is utmost important. In this study, a handheld colorimeter has been explored to optimize the process variables of twin screw processor for preparation of hydrophilic PEG-based ointment. The process parameters which were optimized with use of handheld colorimeter have been used for preparation of polyethylene glycol-based metronidazole ointment. The metronidazole ointment prepared by twin screw processor was compared with commercially available metronidazole gel for in vitro release testing and ex vivo permeation. The flux, ex vivo bioavailability, and Tmax of polyethylene glycol-based metronidazole ointment was found to be similar to that of marketed metronidazole gel.

Insight into hydroxyl radical-mediated cleavage of caged methylene blue: the role of Fenton's catalyst for antimalarial hybrid drug activation.

Methylene blue with a 10-N carbamoyl linkage was reported to be a hydroxyl radical triggered cleavable ligand. Probed by this platform, hemoproteins were demonstrated to be a much more efficient Fenton's catalyst than commonly used inorganic Fe(ii) salts. The applicability of this ligand was demonstrated through the capability of being triggered by elevated reactive oxygen species levels at diseased tissue, with malaria-parasitized erythrocytes as an in vitro model.

Role of Taurine Transporter in the Retinal Uptake of Vigabatrin.

Vigabatrin (VGB) is a first-line drug used for treatment of infantile spasms. On therapeutic dose, VGB accumulates in the retina causing permanent peripheral visual field constriction. The mechanism involved in retinal accumulation of VGB is ambiguous. In the present study, mechanism of VGB transport into retina was evaluated. VGB uptake into retina was studied in vitro using human adult retinal pigment epithelial (ARPE-19) cells as a model for outer blood retinal barrier. The VGB cell uptake studies demonstrated saturation kinetics with Km value of 13.1 mM and uptake was significantly increased at pH 7.4 and hyperosmolar conditions indicating involvement of carrier-mediated Na+-Cl--dependent transporter. In the presence of taurine transporter (TauT) substrates (taurine and GABA) and inhibitor guanidinoethyl sulfonate (GES), the uptake of VGB decreased significantly demonstrating contribution of TauT. The VGB retinal levels in rats were decreased by 1.5- and 1.3-folds on chronic administration of GES and taurine, respectively. In conclusion, this study demonstrated the TauT involvement in VGB uptake and accumulation in retina.

Topical Pilocarpine Formulation for Diagnosis of Cystic Fibrosis.

Cystic fibrosis is diagnosed in infants by estimating the levels of chloride ions present in the sweat induced by iontophoresis of pilocarpine solution. Elevated levels of chloride (≥60 mMol/L) in sweat are indicative of cystic fibrosis. However, the iontophoretic method of delivering pilocarpine is cumbersome and usually is associated with several side effects such as skin burn, skin rashes, erythema, and so forth. The objective of this study was therefore to develop a topical formulation that delivers adequate amount of pilocarpine. The drug delivery of formulation was compared with iontophoresis of aqueous solution of pilocarpine nitrate in vitro using porcine skin model. The pilocarpine levels in the skin exposed to topical pilocarpine solution under mild hyperthermia was 152.04 ± 52.23 µg/cm2 after 10 min of application, whereas it was 97.05 ± 27.93 µg/cm2 in the skin after 10 min of iontophoresis. The topical formulation was subjected to clinical evaluation to assess the efficacy of the product to induce sweat production. The average amount of the sweat secreted on application of topical formulation was found to be 77.28 ± 18.97 mg. Based on these results, it was found that the topical formulation was successful in delivering pilocarpine and to stimulate sweat secretion.

Methylene blue as a far-red light-mediated photocleavable multifunctional ligand.

Methylene blue (MB) with a 10-N-carbamoyl linkage was discovered and developed as a multifunctional far-red (660 nm) photocleavable ligand capable of rendering a series of MB-conjugated compounds with off-to-on fluorescence switch properties through the controlled release of MB.

Preparation and evaluation of cefuroxime axetil gastro-retentive floating drug delivery system via hot melt extrusion technology.

Cefuroxime Axetil (CA) is a poorly soluble, broad spectrum antibiotic which undergoes enzymatic degradation in gastrointestinal tract. The objective of the present study was to develop lipid-based gastro-retentive floating drug delivery systems containing CA using hot-melt extrusion (HME) to improve absorption. Selected formulations of CA and lipids were extruded using a twin screw hot-melt extruder. Milled extrudates were characterized for dissolution, floating strength, and micromeritic properties. Solid-state characterization was performed using differential scanning calorimetry (DSC), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and hot-stage microscopy. In vitro characterization demonstrated that the formulations exhibited a sustained drug release profile for 12 h. All formulations showed desired floating and flow properties. Solid-state characterization revealed no phase separation and no chemical interactions between the drug and excipients. Based on in vitro study results, an optimized formulation (F8) was further evaluated for in vivo performance. Oral bioavailability (Cmax and AUC0-24h) of F8 was significantly higher than that of pure CA. This study describes the use of lipid-based gastro-retentive floating drug delivery systems to achieve desired sustained release profile for more complete dissolution which could potentially reduce enzymatic degradation. This study also highlights the effectiveness of HME technology to improve dissolution and bioavailability.

Water Activity and Its Significance in Topical Dosage Forms.

Unique properties of thermodynamic activity of solvents in topical semisolids and its effects on in vitro product performance have not been fully understood. Mechanistic investigation was undertaken to demonstrate the significance of thermodynamic potential of solvents [water activity (aw) or solvent activity (as)] on in vitro performance of model topical formulations. Drug transport across synthetic membranes was found to decrease with decreasing water activity of formulations. Similarly, in vitro permeation of model permeant (caffeine) across porcine epidermis was found to decrease with decreasing water activity of formulations. Notably, relatively low water activity formulations (aw, 0.78) induced dehydration in porcine skin associated with significant structural changes like detachment of individual stratum corneum layers. Inclusion of hydrating agents (propylene glycol) in low water activity (aw, 0.78) formulations restored hydration levels and structural integrity of porcine skin. Most importantly, incremental inclusion of propylene glycol in low water activity formulations (aw, 0.78) enhanced in vitro permeation of model permeant (fluorescein sodium). Further investigation revealed that variability in processing conditions (high shear mixing during emulsification step) could modulate water activity in semisolid formulations despite their compositional sameness. In retrospect, water activity was found to be a critical quality attribute of topical semisolid products which impacts overall product performance and drug delivery.

RP-HPLC method for simultaneous estimation of vigabatrin, gamma-aminobutyric acid and taurine in biological samples.

Vigabatrin is used as first line drug in treatment of infantile spasms for its potential benefit overweighing risk of causing permanent peripheral visual field defects and retinal damage. Chronic administration of vigabatrin in rats has demonstrated these ocular events are result of GABA accumulation and depletion of taurine levels in retinal tissues. In vigabatrin clinical studies taurine plasma level is considered as biomarker for studying structure and function of retina. The analytical method is essential to monitor taurine levels along with vigabatrin and GABA. A RP-HPLC method has been developed and validated for simultaneous estimation of vigabatrin, GABA and taurine using surrogate matrix. Analytes were extracted from human plasma, rat plasma, retina and brain by simple protein precipitation method and derivatized by naphthalene 2, 3­dicarboxaldehyde to produce stable fluorescent active isoindole derivatives. The chromatographic analysis was performed on Zorbax Eclipse AAA column using gradient elution profile and eluent was monitored using fluorescence detector. A linear plot of calibration curve was observed in concentration range of 64.6 to 6458, 51.5 to 5150 and 62.5 to 6258 ng/mL for vigabatrin, GABA and taurine, respectively with r2 ≥ 0.997 for all analytes. The method was successfully applied for estimating levels of vigabatrin and its modulator effect on GABA and taurine levels in rat plasma, brain and retinal tissue. This RP-HPLC method can be applied in clinical and preclinical studies to explore the effect of taurine deficiency and to investigate novel approaches for alleviating vigabatrin induced ocular toxicity.