Gel meloksikama za topičku primjenu: In vitro i in vivo vrednovanje

Skin delivery of NSAIDs offers several advantages over the oral route associated with potential side effects. In the present investigation, topical gel of meloxicam (MLX) was formulated using N-methyl pyrrolidone (NMP) as a solubilizer and Carbopol Ultrez 10® as a gelling polymer. MLX gel was evaluated with respect to different physicochemical parameters such as pH, viscosity and spreadability. Irritation potential of MLX gel was studied on rabbits. Permeation of MLX gel was studied using freshly excised rat skin as a membrane. Anti-inflammatory activity of MLX gel was studied in rats and compared with the commercial formulation of piroxicam (Pirox® gel, 0.5 %, m/m). Accelerated stability studies were carried out for MLX gel for 6 months according to ICH guidelines. MLX gel was devoid of any skin irritation in rabbits. After 12 h, cumulative permeation of MLX through excised rat skin was 3.0 ± 1.2 mg cm2 with the corresponding flux value of 0.24 ± 0.09 mg cm2 h1. MLX gel exhibited significantly higher anti-inflammatory activity in rats compared to Pirox® gel. Physicochemically stable and non-irritant MLX gel was formulated which could deliver significant amounts of active substance across the skin in vitro and in vivo to elicit the anti-inflammatory activity.Primjena nesteroidnih protuupalnih lijekova na kožu ima nekoliko prednosti nad peroralnim načinom primjene uz koju se vežu određene nuspojave. U radu je opisana priprava gela meloksikama (MLX) za topičku primjenu. U pripravi gela korišten je N-metil pirolidon (NMP) kao otapalo i Carbopol ultrez 10® kao polimer za geliranje. Određivani su različiti fizikokemijski parametri kao što su pH, viskoznost i razmazljivost. Potencijalna iritacija MLX gela testirana je na kunićima, a svojstvo permeacije na svježim izrescima kože štakora. Protuupalno djelovanje praćeno je na štakorima i uspoređeno s registriranim pripravkom piroksikama (Pirox® gel, 0,5 % m/m). Testovi ubrzanog starenja MLX gela rađeni su tijekom 6 mjeseci prema ICH protokolu. Dobiveni rezultati ukazuju da MLX gel nimalo ne iritira kožu kunića. Kumulativna permeacija nakon 12 h bila je 3,0 ± 1,2 mg cm2, s odgovarajućem vrijednošću fluksa 0,24 ± 0,09 mg cm2 h1. MLX gel pokazao je značajno jače protuupalno djelovanje u odnosu na Pirox® gel. Pripravljeni gel je stabilan, ne iritira kožu, te in vitro i in vivo doprema kroz kožu ljekovitu tvar u dovoljnoj količini da ispolji protuupalno djelovanje

Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide--a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non-validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A-B-C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 M) and MeCN (60:40, v/v), B corresponds to NaClO₄ ·H₂O (pH 6.0; 0.2 M) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser-porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices

Development and validation of an analytical method for the quantification of cytochrome c in skin transport studies

A simple isocratic HPLC method for the quantification of Cytochrome c in skin permeation samples was developed and validated. The mobile phase comprised a 41 : 59 mixture of an organic phase A (0.1% trifluoroacetic acid in a 90 : 10 mixture of MeCN-H(2)O) and an aqueous phase B (0.1% trifluoroacetic acid in H(2)O). The Cytochrome c retention and run times were 2.62 and 8.0 min, respectively--much shorter than those for existing gradient methods. The response was accurate, precise and linear from 2.5 to 25 microg/mL. The mean recoveries for intra-day and inter-day analysis ranged from 88.5 to 103.8% and the RSD varied from 0.05 to 1.55%. The assay was used to quantify transport of Cytochrome c across intact and laser-microporated porcine skin in vitro. Cytochrome c permeation and the amount of protein retained within the membrane over 24 h were quantified as a function of the number of micropores. Although no Cytochrome c permeation was observed across intact skin, laser microporation enabled delivery of 22.9 +/- 3.3 and 56.0 +/- 15.9 microg/cm(2) of the protein across skin samples with 300 and 1800 micropores, respectively. In conclusion, the HPLC method provided a fast, efficient means to quantify Cytochrome c in samples from skin transport studies

Stability of triptorelin in the presence of dermis and epidermis

An important issue with respect to the transdermal delivery of peptides is their stability during transit through the epidermis and dermis before entry into the systemic circulation. The objective of the present study was to evaluate the effect of epidermal and dermal tissue on the stability of the luteinizing hormone releasing hormone superagonist, triptorelin. The decapeptide was dissolved in PBS (pH 7.4) and placed in contact with (i) heat separated epidermis (HSE), (ii) dermatomed skin (0.75 mm; DS) and (iii) full thickness skin (FTS) and the extent of peptide biotransformation monitored as a function of time by HPLC. The results showed that triptorelin was metabolized when in contact with each of the skin tissues. However, there were marked differences with respect to the extent of peptide degradation. Triptorelin was least stable in the presence of FTS. After 3 h exposure to HSE, DS and FTS, the extent of triptorelin degradation was 15.0+/-6.0%, 64.8+/-9.9% and 100%, respectively. After 24 h, further triptorelin degradation had occurred in the samples in contact with HSE and DS--with 51.3+/-6.0% and 87.8+/-4.4%, respectively, of the peptide being degraded. The chromatograms revealed the presence of a degradation peak at a higher retention time than the parent molecule--most probably the free acid

Using laser microporation to improve transdermal delivery of diclofenac: Increasing bioavailability and the range of therapeutic applications

The objective of the study was to investigate the effect of laser microporation, using P.L.E.A.S.E.® technology, on diclofenac delivery kinetics. Skin transport of diclofenac was studied from aqueous solution, propylene glycol and marketed formulations across intact and laser-porated porcine and human skins; cumulative permeation and skin deposition were quantified by HPLC. After 24h, cumulative diclofenac permeation across skins with 150, 300, 450 and 900 shallow pores (50-80 μm) was 3.7-, 7.5-, 9.2- and 13-fold superior to that across untreated skin. It was also found to be linearly dependent on laser fluence; Permeation (μg/cm(2))=11.35*Fluence (J/cm(2))+352.3; r(2)=0.99. After 24h, permeation was 539.6 ± 78.1, 934.5 ± 451.5, 1451.9 ± 151.3 and 1858.6 ± 308.5 μg/cm(2), at 22.65, 45.3, 90.6 and 135.9 J/cm(2), respectively. However, there was no statistically significant effect of laser fluence on skin deposition. Diclofenac delivery from marketed gel formulations was also significantly higher across laser-porated skins (e.g. for Solaraze, cumulative permeation after 24h across treated (900 pores/135.9 J/cm(2)) and untreated skin was 974.9 ± 368.8 and 8.2 ± 3.8 μg/cm(2), respectively. Diclofenac delivery from Solaraze across laser-porated porcine and human skins was also shown to be statistically equivalent. The results demonstrated that laser microporation significantly increased diclofenac transport from both simple and semi-solid formulations through porcine and human skin and that pore depth and pore number could modulate delivery kinetics. A similar improvement in topical diclofenac delivery in vivo may increase the number of potential therapeutic applications

Novel micelle formulations to increase cutaneous bioavailability of azole antifungals

Efficient topical drug administration for the treatment of superficial fungal infections would deliver the therapeutic agent to the target compartment and reduce the risk of systemic side effects. However, the physicochemical properties of the commonly used azole antifungals make their formulation a considerable challenge. The objective of the present investigation was to develop aqueous micelle solutions of clotrimazole (CLZ), econazole nitrate (ECZ) and fluconazole (FLZ) using novel amphiphilic methoxy-poly(ethylene glycol)-hexyl substituted polylactide (MPEG-hexPLA) block copolymers. The CLZ, ECZ and FLZ formulations were characterized with respect to drug loading and micelle size. The optimal drug formulation was selected for skin transport studies that were performed using full thickness porcine and human skin. Penetration pathways and micellar distribution in the skin were visualized using fluorescein loaded micelles and confocal laser scanning microscopy. The hydrodynamic diameters of the azole loaded micelles were between 70 and 165nm and the corresponding number weighted diameters (d(n)) were 30 to 40nm. Somewhat surprisingly, the lowest loading efficiency (13-fold higher than that from Pevaryl® cream (22.8±3.8 and 1.7±0.6μg/cm(2), respectively). A significant enhancement was also observed with human skin; the amounts of ECZ deposited were 11.3±1.6 and 1.5±0.4μg/cm(2), respectively (i.e., a 7.5-fold improvement in delivery). Confocal laser scanning microscopy images supported the hypothesis that the higher delivery observed in porcine skin was due to a larger contribution of the follicular penetration pathway. In conclusion, the significant increase in ECZ skin deposition achieved using the MPEG-dihexPLA micelles demonstrates their ability to improve cutaneous drug bioavailability; this may translate into improved clinical efficacy in vivo. Moreover, these micelle systems may also enable targeting of the hair follicle and this will be investigated in future studies

Effect of controlled laser microporation on drug transport kinetics into and across the skin

The objectives of this study were to investigate a novel laser microporation technology ( P.L.E.A.S.E. Painless Laser Epidermal System) and to determine the effect of pore number and depth on the rate and extent of drug delivery across the skin. In addition, the micropores were visualized by confocal laser scanning microscopy and histological studies were used to determine the effect of laser fluence (energy applied per unit area) on pore depth. Porcine ear skin was used as the membrane for both the pore characterization and drug transport studies. Confocal images in the XY-plane revealed that the pores were typically 150-200 microm in diameter. Histological sections confirmed that fluence could be used to effectively control pore depth - low energy application (4.53 and 13.59 J/cm(2)) resulted in selective removal of the stratum corneum (20-30 microm), intermediate energies (e.g., 22.65 J/cm(2)) produced pores that penetrated the viable epidermis (60-100 microm) and higher application energies created pores that reached the dermis (>150-200 microm). The effects of pore number and pore depth on molecular transport were quantified by comparing lidocaine delivery kinetics across intact and porated skin samples. After 24h, cumulative skin permeation of lidocaine with 0 (control), 150, 300, 450 and 900 pores was 107+/-46, 774+/-110, 1400+/-344, 1653+/-437 and 1811+/-642 microg/cm(2), respectively; there was no statistically significant difference between 300, 450 and 900 pore data - probably due to the effect of drug depletion since >50% of the applied dose was delivered. Importantly, increasing fluence did not produce a statistically significant increase in lidocaine permeation; after 24h, cumulative lidocaine permeation was 1180+/-448, 1350+/-445, 1240+/-483 and 1653+/-436 microg/cm(2) at fluences of 22.65, 45.3, 90.6 and 135.9 J/cm(2), respectively. Thus, shallow pores were equally effective in delivering lidocaine. Increasing lidocaine concentration in the formulation from 10 to 25mg/ml produced a corresponding increase in permeation (at 24h, 1650+/-437 and 4005+/-1389 microg/cm(2), respectively). The validity of the porcine skin model was confirmed as transport across porcine and human skins was shown to be statistically equivalent (at 24h, 1811+/-642 and 2663+/-208 microg/cm(2), respectively). The clinical potential of the technology and its capacity to provide significantly faster delivery than conventional passive administration was demonstrated in short duration experiments involving application of a marketed lidocaine cream (LMX4) to laser-porated skin; after only 5 min of formulation application, lidocaine deposition was measured at 61.3+/-7.5 microg/cm(2). In conclusion, the results demonstrate the ability of P.L.E.A.S.E.(R) (i) to create well-defined conduits in the skin, (ii) to provide a controlled enhancement of transdermal transport and (iii) to enable improvement in both the rate and extent of drug delivery

Using controlled laser-microporation to increase transdermal delivery of prednisone

The objective was to investigate whether laser microporation could enhance transdermal delivery of prednisone. Results demonstrated that transport was controlled by pore number and depth; it was substantially improved over delivery through intact skin. Prednisone delivery(permeation+deposition) across porated skin (1800 pores) after 24 h was 197.18±29.62 μg/cm2; in contrast, no transport was observed across intact skin. Increasing pore depth so that micropores reached the epidermis produced corresponding increases in prednisone transport

The Multi-kinase Inhibitor Debio 0617B Reduces Maintenance and Self-renewal of Primary Human AML CD34(+) Stem/Progenitor Cells

Acute myelogenous leukemia (AML) is initiated and maintained by leukemia stem cells (LSC). LSCs are therapy-resistant, cause relapse, and represent a major obstacle for the cure of AML. Resistance to therapy is often mediated by aberrant tyrosine kinase (TK) activation. These TKs primarily activate downstream signaling via STAT3/STAT5. In this study, we analyzed the potential to therapeutically target aberrant TK signaling and to eliminate LSCs via the multi-TK inhibitor Debio 0617B. Debio 0617B has a unique profile targeting key kinases upstream of STAT3/STAT5 signaling such as JAK, SRC, ABL, and class III/V receptor TKs. We demonstrate that expression of phospho-STAT3 (pSTAT3) inAML blasts is an independent prognostic factor for overall survival. Furthermore, phospho-STAT5 (pSTAT5) signaling is increased in primary CD34(+) AML stem/progenitors. STAT3/STAT5 activation depends on tyrosine phosphorylation, mediated by several upstream TKs. Inhibition of single upstream TKs did not eliminate LSCs. In contrast, the multi-TK inhibitor Debio 0617B reduced maintenance and self-renewal of primary human AML CD34(+) stem/progenitor cells in vitro and in xenotransplantation experiments resulting in long-term elimination of human LSCs and leukemia. Therefore, inhibition of multiple TKs upstream of STAT3/5 may result in sustained therapeutic efficacy of targeted therapy in AML and prevent relapses. (C)2017 AACR

Microemulsion-Based Vaginal Gel of Clotrimazole: Formulation, In Vitro Evaluation, and Stability Studies

The objective of the present investigation was to develop and evaluate microemulsion-based gel for the vaginal delivery of clotrimazole (CMZ). The solubility of CMZ in oils and surfactants was evaluated to identify components of the microemulsion. The ternary diagram was plotted to identify the area of microemulsion existence. Various gelling agents were evaluated for their potential to gel the CMZ microemulsion without affecting its structure. The bioadhesive potential and antifungal activity of the CMZ microemulsion-based gel (CMZ-MBG) was determined in comparison to the marketed clotrimazole gel (Candid-V® gel) by in vitro methods. The chemical stability of CMZ in CMZ-MBG was determined as per the International Conference on Harmonization guidelines. The CMZ microemulsion exhibited globule size of 48.4 nm and polydispersity index of 0.75. Carbopol® ETD 2020 could successfully gel the CMZ microemulsion without disturbing the structure. The CMZ-MBG showed significantly higher (P < 0.05) in vitro bioadhesion and antifungal activity as compared to that of Candid-V® gel. The stability studies indicated that CMZ undergoes acidic pH-catalyzed degradation at all the storage conditions at the end of 3 months