Stability and degradation profiles of Spantide II in aqueous solutions.

Spantide II is an 11 amino acid peptide that has been shown to be a potential anti-inflammatory agent. The stability and degradation profiles of Spantide II in aqueous solutions were evaluated with the long-term objective of developing topical formulations of this compound for various skin disorders. The stability profile of Spantide II at various temperature and pH conditions was monitored by high performance liquid chromatography (HPLC) and the resulting degradation products were identified by liquid chromatography-mass spectroscopy (LC-MS). Forced degradation of Spantide II was performed at extreme acidic (pH <2.0) and alkaline (pH >10.0) conditions and by addition of hydrogen peroxide (oxidizing agent). The degradation pattern of Spantide II followed pseudo first-order kinetics. The shelf life (T90%) of Spantide II in aqueous ethanol (50%) was determined to be 230 days at 25 degrees C. Spantide II was susceptible to degradation at pH <2 and pH >5 and showed maximum stability at pH 3-5. The stability under various pH conditions indicates that Spantide II was most stable at pH 3.0 with a half-life of 95 days at 60 degrees C. Spantide II degradation was attributed to hydrolysis of peptide bonds [Pro2-(pyridyl)Ala3, (nicotinoyl)Lys1-Pro2, Pro4-PheCl2(5), Trp7-Phe8, Phe8-Trp9, Nle11-NH2), racemization of the peptide fragments that resulted from hydrolysis, cleavage and formation of (nicotinoyl)Lys1-Pro2 diketopiperazine. In the presence of an oxidizing agent, Pro(2,4) residues degraded by ring opening to form glutamyl-semialdehyde and by bond cleavage at Pro4 to form 2-pyrrolidone, while Phe(5,8) degraded to form 2-hydroxyphenylalanine. Spantide II was found to be stable in aqueous medium with T90% of 230 days. The major degradation pathways of Spantide II were identified as hydrolysis, racemization, cleavage and formation of diketopiperazine.

Percutaneous absorption and anti-inflammatory effect of a substance P receptor antagonist: spantide II.

PURPOSE: There is accumulating evidence that neurogenic mediators such as substance P (SP) and alpha-melanocyte stimulating hormone (alpha-MSH) contribute to inflammation following chemical and thermal injuries or in disease conditions such as psoriasis and contact dermatitis. Spantide II is a peptide with a molecular weight of 1670.2 which binds to neurokinin-1 receptor (NKR-1) and blocks proinflammatory activities associated with SP. The aim of this study was to investigate in vitro permeation and distribution of spantide II through hairless rat skin and the anti-inflammatory effect of topically delivered spantide II in an allergic contact dermatitis (ACD) mouse model. METHODS: The in vitro permeation and distribution of spantide II with or without cysteine HCl (CH) as a penetration enhancer through hairless rat skin was studied using Franz diffusion cells. The anti-inflammatory effect of spantide II was studied by measuring the reduction of ACD in C57BL/6 mice after application of spantide II as a topical solution. RESULTS: The skin permeation experiments with or without cysteine HCl (as penetration enhancer) showed no detectable levels of spantide II permeation across rat skin over a period of 48 h. Cysteine HCl significantly increased the distribution of spantide II in skin layers; also, the reduction in ACD response was significantly higher with the formulation containing cysteine HCl (p < 0.05). Spantide II at different concentrations showed a dose-dependent reduction of ACD response in mice. CONCLUSIONS: The current study demonstrates that spantide II can effectively be delivered to epidermis and dermis to exert a significant anti-inflammatory activity on the reduction of inflammation in a mouse model of ACD.

Preformulation stability of Spantide II, a promising topical anti-inflammatory agent for the treatment of psoriasis and contact dermatitis.

Substance P is readily expressed in skin inflammatory disorders such as psoriasis and contact dermatitis. Spantide II is a peptide (MW 1668.76) that specifically binds to neurokinin-1 receptor (NKR-1) and blocks inflammation associated with substance P. The anti-inflammatory property of Spantide II makes it a suitable candidate to be studied as a topical formulation for the treatment of dermal inflammatory disorders. The objective of this study was to investigate the influence of pH, temperature, salt concentration and concentration on the aqueous stability of Spantide II. The stability of Spantide II was also assessed by circular dichroic (CD) spectroscopy and mass spectrometry (MS). The influence of various dermatological vehicles (ethanol, Transcutol, propylene glycol, N-methyl-2-pyrrolidone (NMP), ethyl oleate, isopropyl myristate and laurogylcol FCC (LFCC)) on the stability of Spantide II was investigated. A precise high-performance liquid chromatography (HPLC) assay was developed for analysis of Spantide II. At higher temperature (40 degrees C) the stability of Spantide II decreased with increase in pH (P < 0.05). Change in salt concentration did not appreciably affect the stability of Spantide II (P > 0.05). The concentration of Spantide II in the solution had no significant influence on its stability (P > 0.05). CD spectroscopy studies showed that Spantide II has a relatively stable alpha-helix structure in the liquid state. The stability of Spantide II was affected by the type of vehicle used in the study (P < 0.01) at different temperatures (P < 0.05). Spantide II at high temperature undergoes lysine-proline diketopiperazine degradation as evident in MS data. Spantide II was relatively more stable in ethyl oleate-ethanol, ethanol-water, ethanol and N-methyl-2-pyrrolidone. The results of this study indicate that ethyl oleate-ethanol (1:1) and ethanol-water (1:1) could be used as potential vehicles in the development of topical formulations of Spantide II.

Iontophoresis-based transdermal delivery systems.

Transdermal iontophoresis is the administration of ionic therapeutic agents through the skin by the application of a low-level electric current. This article presents an overview of transdermal iontophoretic delivery of drugs, including peptides and oligonucleotides. Recent advances in the area of iontophoretic delivery, including devices, hydrogel formulations, safety, clinical relevance and future prospects, are discussed. Electroporation, another method of electrically assisted drug delivery, is also briefly reviewed. Transdermal iontophoresis appears to be a promising technique for the delivery of a variety of compounds in a controlled and preprogrammed manner. Transdermal iontophoresis would be particularly useful in the delivery of hydrophilic drugs produced by biotechnology (peptides and oligonucleotides). However, because of the complex physicochemical properties of peptides, many factors must be carefully considered for the proper design of an iontophoretic drug delivery system for peptides. Iontophoresis has been successfully used in the delivery of small peptides, such as leuprolide and calcitonin analogues, in humans. However, it appears that transdermal iontophoresis may not be a suitable method for the systemic delivery of larger peptides (>7,000D). The combined use of iontophoresis and electroporation may be more effective in the delivery of peptides, proteins, genes and oligonucleotides. The long-term safety of iontophoresis, patient compliance with the technique and the commercial success of this technology are yet to be demonstrated. Iontophoretic delivery of drugs would be beneficial in the treatment of certain skin disorders such as skin cancer, psoriasis, dermatitis, venous ulcers, keloid and hypertrophic scars. Investigations on reverse iontophoresis may yield interesting results that would be useful in the noninvasive measurement of clinically important molecules in the body.

Inhalation delivery of anticancer agents via HFA-based metered dose inhaler using methotrexate as a model drug.

In the present study, the feasibility of delivering anticancer drugs via metered dose inhaler (MDI) was demonstrated using methotrexate (MTX) as a model anticancer drug. MDI formulations of MTX were prepared using hydrofluoroalkane-134a containing 0.67% MTX and 10% ethyl alcohol. The particle size of MTX was reduced by cryo milling with or without a surfactant (Pluronic F77) and the milled drug was employed for MDI formulations, which were subsequently evaluated for their medication delivery, mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). Further, the efficacy of aerosolized MTX was evaluated by determining the in-vitro cytotoxicity of MTX against HL-60 cells using a six-stage viable impactor and the induction of apoptosis in HL-60 cells by acridine orange staining. Our results indicate that MTX aerosols having an MMAD varying between 2.2 and 3.2 microm (GSD 2.6-3.7) with a respirable fraction varying between 14.2 and 17.1% could be obtained by using MTX, which was cryo milled either alone or in combination with Pluronic F77. Exposure of HL-60 cells plated in third, fourth, fifth, and sixth stages of viable impactor to two actuations of MDI showed a cell kill of greater than 50%. Further, aerosolized MTX was found to induce apoptosis in HL-60 cells, as assessed by the morphological examination of the cells with fluorescent and confocal microscopy. Our results demonstrate that it is possible to deliver cytotoxic concentrations of MTX in an in vitro system simulating the lower respiratory tract (by using a six-stage viable impactor) via MDI and the cytotoxicity of the aerosolized MTX could be further improved by the optimization of the aerodynamic size.

Effect of jet fuels on the skin morphology and irritation in hairless rats.

Jet A and JP-8 are the major jet fuels used in civilian and military (US Air Force) flights, respectively. JP-8+100 is a new jet fuel recently introduced by US Air Force in some of its locations. The purpose of this study was to investigate the effects of dermal exposure of jet fuels (Jet A, JP-8, and JP-8+100) on the skin morphology, barrier function, moisture content, blood flow, and skin irritation (erythema and edema) in hairless rats. Jet fuels were applied by both occlusive and unocclusive methods. The skin of treated and control (untreated) sites were excised and analyzed by magnetic resonance imaging (MRI) (500 MHz, 11.7 Tesla). Unocclusive application of JP-8, Jet A, and JP-8+100 increased the transepidermal water loss (TEWL) gradually and the values at 120 h were significantly greater than the baseline value (P<0.05). Both occlusive and unocclusive application of jet fuels decreased the skin moisture content significantly (P<0.05). Unocclusive application of JP-8, Jet A, and JP-8+100 increased the skin blood flow, though the values returned to the baseline levels within 24 h. Occlusive application of jet fuels (8 h/day for 2 days) caused a substantial increase in the skin blood flow and the values at 48 h were about 6-fold greater than the baseline value. Occlusive application of jet fuels caused a moderate to severe erythema and a moderate edema. MRI was used to obtain proton images and water self-diffusion maps of hairless rat skin exposed to jet fuel. Exposure to JP-8 showed the largest difference from the control with regards to visual observations of the stratum corneum and hair follicles, while JP-8+100 appeared to affect the hair follicle region. The results of the present study demonstrate that exposure to jet fuels can disrupt the skin barrier function, cause skin irritation, and alter the skin structure (stratum corneum and viable epidermis) and MRI can be used as a tool to investigate the alterations in the skin morphology after exposure to toxic chemicals.

Comparison of the transdermal absorption of nimesulide from three commercially available gel formulations.

Nimesulide is a non-steroidal anti-inflammatory drug (NSAID) applied topically for a variety of conditions characterized by pain and inflammation. One of the aims of this study was to compare the permeation profile of nimesulide from the commercially available transdermal gel formulations across dermatomed porcine and human skin. The in vitro transdermal absorption of nimesulide formulations across porcine skin and human skin was studiedfor 24 hr using a continuous flow-through diffusion cell. The three commercial gels used in this study were Nimulid, Nise Gel, and Orthobid. All gels contained 1% (w/w) nimesulide. An infinite dose of nimesulide gel (about 300mg) was applied on the skin over 0.636 cm2 surface area. The rank order for the drug permeation from these formulations using porcine skin was: Nimulid > Orthobid > Nise Gel. The rank order of the permeation across human skin was: Nimulid> Nise Gel> Orthobid. The permeation profiles followed zero-order kinetics without any significant lag time. The steady-state flux of nimesulide from Nimulid was significantly higher than that of Nise Gel and Orthobid in both porcine and human skin (p <.05). However, there were no significant differences in the delivery of nimesulide (24 hr) from Nise Gel and Orthobid across both human and porcine skins. The results suggest that the Nimulid gel may have a greater bioavailability of nimesulide compared to the other gels. In addition, permeation profiles of the various gels across porcine skin did show a positive profile behavior to human skin. However, the in vitro drug release of nimesulide gels across a synthetic membrane did not correlate with skin permeation profiles.