Comparison of the effects of chemical permeation enhancers on the lipoidal pathways of human epidermal membrane and hairless mouse skin and the mechanism of enhancer action.

Previously, the effects of chemical permeation enhancers upon the permeability of the lipoidal pathway of hairless mouse skin (HMS) were investigated and a quantitative structure enhancement relationship was established. The present study was to study the effects of these enhancers on human epidermal membrane (HEM) using the same experimental method employed in the previous HMS studies. The effects of enhancers on the permeability coefficients of the lipoidal pathways of HEM and HMS for corticosterone were found to be essentially the same. In the equilibrium uptake studies of the enhancers and beta-estradiol, it was found that the amounts of enhancers taken up and the partitioning of beta-estradiol into the HEM stratum corneum (SC) intercellular lipid under the E = 10 conditions were different from those of HMS. Despite these differences, the HEM data show a correlation between the intercellular lipid/PBS partition coefficients of the enhancers and the enhancer n-octanol/PBS partition coefficients. This correlation is consistent with the observed chemical microenvironment of the site of enhancer action in the HMS SC in previous studies. Therefore, provided with proper experimental protocols, HMS can be a reliable model for the evaluation of the effects of skin permeation enhancers on the lipoidal pathway of HEM.

Feasibility of biowaiver extension to biopharmaceutics classification system class III drug products: cimetidine.

BACKGROUND: The extension of biowaivers (drug product approval without a pharmacokinetic bioequivalence study) to drugs belonging to Class III of the Biopharmaceutics Classification System (BCS) is currently a subject of much discussion. OBJECTIVES: To assess the relationship between in vitro dissolution characteristics and in vivo absorption performance of immediate-release (IR) products containing cimetidine, a BCS Class III compound, in human subjects. To evaluate the feasibility and appropriateness of an extension of the biowaiver concept to BCS Class III compounds. STUDY DESIGN AND PARTICIPANTS: BCS-conform dissolution tests were carried out on ten marketed cimetidine products from Thailand and Germany, as well as cimetidine tablet formulations containing cimetidine 400mg manufactured by direct compression using methacrylate copolymer (Eudragit) RS PO) as a release-retarding agent to yield three batches with significantly different release profiles. Twelve healthy male subjects were enrolled in a randomised, open-label, single-dose schedule based on a five-way Williams' design balanced for carryover effects. Subjects received the following treatments, with 1-week washout periods between: (i) Tagamet 400mg tablet; (ii) 7.5% methacrylate copolymer cimetidine tablet; (iii) 15% methacrylate copolymer cimetidine tablet; (iv) 26% methacrylate copolymer cimetidine tablet; and (v) Tagamet (300 mg/ 2 mL) intravenous injection. The area under the plasma concentration-time curve from 0 to 12 hours (AUC(12)) and AUC from time zero to infinity (AUC(infinity)), peak plasma concentration (C(max)), absolute bioavailability (F) and mean residence time (MRT) were evaluated and statistically compared among formulations. In vitro-in vivo correlation (IVIVC) analysis was then applied to elucidate the overall absorption characteristics of each tablet formulation. RESULTS: The release properties of the ten marketed cimetidine products were shown to comply with current US FDA criteria for rapidly dissolving drug products. As expected, the in vitro dissolution profiles of the cimetidine tablets containing different percentages of methacrylate copolymer differed considerably from one another. However, in vivo results showed no significant difference in AUC(12), AUC(infinity), C(max) and F between the tablets manufactured with methacrylate copolymer and the innovator. The MRT values obtained from 26% methacrylate copolymer tablets were significantly longer than for the other two methacrylate copolymer formulations and the Tagamet tablets. Furthermore, IVIVC analysis showed that the 26% methacrylate copolymer tablets exhibited dissolution rate-limited absorption, whereas the other formulations showed permeability rate-limited absorption. CONCLUSION: The results of the present study indicated that the absorption of cimetidine from IR tablets is, in general, limited by permeability rather than dissolution. IVIVC analysis demonstrated that only when the release was deliberately retarded (tablets containing 26% methacrylate copolymer), did the dissolution represent the rate-limiting step to drug absorption. On the in vitro side, it seems that 85% dissolution within 30 minutes, as currently required by the US FDA Guidance, is more than sufficient to guarantee bioequivalence of IR cimetidine products. For cimetidine and other BCS Class III drugs with a similar intestinal absorption pattern, application of the biowaiver concept seems to present little risk of an inappropriate bioequivalence decision.

Assessment of the neurotoxicity of oral dihydroartemisinin in mice.

High doses of the oil-soluble antimalarial artemisinin derivatives artemether and arteether, given by intramuscular injection to experimental mammals, produce an unusual pattern of selective damage to brainstem centres predominantly involved in auditory processing and vestibular reflexes. We have shown recently, in adult Swiss albino mice, that constant exposure either from depot intramuscular injection of oil-based artemisinin derivatives, or constant oral intake carries relatively greater neurotoxic potential than other methods of drug administration. Using the same model, oral dihydroartemisinin suspended in water was administered once or twice daily at different doses ranging from 50 to 300 mg/kg/day for 28 days. The neurotoxic potential of the oral dihydroartemisinin was assessed and compared to that of oral artemether and artesunate. Oral artemether, artesunate, and dihydroartemisinin had similar neurotoxic effects with no significant clinical or neuropathological evidence of toxicity at doses below 200 mg/kg/day. These data indicate that once and twice daily oral administration of artemether, artesunate and dihydroartemisinin is relatively safe when compared to intramuscular administration of the oil-based compounds.

Comparison of skin transport and metabolism of ethyl nicotinate in various species.

The skin transport and metabolism characteristics of ethyl nicotinate (EN) in rabbit, rat, guinea-pig, pig, shed snake skin and human were compared. In vitro skin transport using excised skin and hydrolysis experiments using skin homogenate were carried out. Flux of EN, a metabolite, nicotinic acid (NA), and the total (EN + NA), as well as kinetic parameters (V(max) and K(m)) for hydrolysis of EN were determined and compared among various species. The enzymatic conversion of EN to NA was observed for all skin permeation experiments. Total flux from EN-saturated solution between rabbit, rat, guinea-pig and human was significantly different (P < 0.05). A great difference between species was observed in skin esterase activity. The NA/total flux ratio of human was significantly lower than that of rabbit, rat or guinea-pig but lower than that of shed snake skin (P < 0.05). There is no significant difference in skin permeation and metabolism between human and pig (P > 0.05). Total flux increased linearly with an increase in EN donor concentration for all species. For pig, shed snake skin and human, NA flux increased with an increase in EN donor concentration and reached a plateau, suggesting the metabolic saturation was taking place in the skin. NA flux at plateau and EN donor concentration in which the NA flux reached a plateau were also affected by species difference. These findings indicated that the discrepancy in transdermal profiles of EN among species tested was predominantly due to the difference in the esterase activity in the skin.

Mechanistic studies of branched-chain alkanols as skin permeation enhancers.

As part of a long-term effort to understand the structure/function relationship between chemical permeation enhancers and skin permeation enhancement, the present study examined the influence of hydrocarbon chain branching on the effectiveness of skin permeation enhancers of the type that possesses a polar group (e.g., the hydroxyl group) attached to a hydrocarbon chain(s). The effects of x-hexanol, x-heptanol, x-octanol, and x-nonanol (where x is the position of the hydroxyl group ranging from 1 up to 5) on the transport of a probe permeant, corticosterone, across hairless mouse skin (HMS) were investigated. Isoenhancement concentrations are defined as the aqueous concentrations for which different enhancers induce the same extent of permeant transport enhancement, E, across the lipoidal pathway of stratum corneum (SC). The isoenhancement concentrations of 2-alkanol, 3-alkanol, 4-alkanol, and 5-alkanol to induce E = 10 were approximately 1.9-, 2.6-, 3.1-, and 3.9-fold higher, respectively, than those of the 1-alkanols of the same molecular formula. This suggested that the branched-chain alkanols have lower enhancer potency than the 1-alkanols of the same molecular formula; the potency decreases as the hydroxyl group moves from the end of the chain towards the center of the enhancer alkyl chain. To further investigate the mechanism(s) of action of the branched-chain alkanols as skin permeation enhancers, the equilibrium uptake of the enhancers into the hairless mouse skin stratum corneum (HMS SC) from aqueous enhancer solutions of E = 10 was determined. The data from these experiments provided a direct measure of the "intrinsic" potency of the enhancer. In the same experiments, the equilibrium partitioning (distribution) of a surrogate permeant, estradiol (E2beta), into the HMS SC was also determined and compared to the partitioning from PBS (no enhancer present). The uptake amounts (micromole/mg SC) for 1-alkanols into the intercellular lipids of the SC were found to be essentially the same at their isoenhancement concentrations. However, at their isoenhancement concentrations, the uptake amounts of the branched-chain alkanols into the intercellular lipids of HMS SC were higher than those of the 1-alkanols. These results support the view that: (1) the intrinsic potencies of the 1-alkanols are essentially the same and independent of their 1-alkyl chain length at their isoenhancement concentrations, (2) the intrinsic potencies of the branched-chain alkanols are lower than those of the normal alkanols, and (3) branching of the alkyl chain reduces the ability of the enhancer to effect lipid fluidization in the SC lipid lamellae at the target site(s). The enhancement effects of the branched-chain alkanols and the 1-alkanols at their isoenhancement concentrations upon E2beta partitioning into the SC intercellular lipids were found to be approximately the same and in the range of five- to eight-fold enhancement. The constancy of this enhancement for E2beta partitioning suggests that the mechanism of enhancement action for the branched-chain alkanols and the 1-alkanols are the same. Additionally, a good correlation of the intercellular lipid/PBS partition coefficients of both the branched-chain alkanols and the 1-alkanols with the n-octanol/PBS partition coefficients was found. This supports the view that the chemical microenvironment of the polar head group and the alkyl group of the studied enhancers at the site of skin permeation enhancer action in the SC lipid lamellae can be represented by water-saturated n-octanol for both the branched-chain alkanols and the 1-alkanols.

Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity.

The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl glucoside (OG) followed by detergent removal. Biochemical analysis revealed that these virosomes contained both the haemagglutinin-neuraminidase protein (HN) and the fusion protein (F), with preserved biological activity. Acidic environment triggered the fusion between virosomes and chicken erythrocyte ghosts. Formation of ISCOMs was achieved by solubilizing phospholipids, cholesterol, envelope protein antigen and Quil A in Triton X-100. The ISCOM particles were formed by removal of the detergent. In each formulation the relative HN content correlated with the capability to agglutinate red blood cells. The immunogenicity of these lipid-based subunit vaccines was determined in chickens after subcutaneous immunization. The relative HN content of the subunit vaccines correlated with the haemagglutination-inhibition (HI) antibody titres. Virosomes prepared with Triton X-100 and ISCOMs offered high clinical protection (> 80%) upon challenge with virulent NDV. Virosomes prepared with OG yielded lower clinical protection despite high HI antibody titres. Virosomes with reduced antigen density showed poor immunogenicity and protection. In conclusion, ND virosomes and ISCOMs were found to be immunogenic and provided good protection.

Shed king cobra and cobra skins as model membranes for in-vitro nicotine permeation studies.

Shed king cobra skin (SKCS) and shed cobra skin (SCS) were investigated for use as barrier membranes, including some pre-hydration factors, for in-vitro nicotine permeation. Inter-specimen variations in nicotine fluxes using shed snake skin were compared with those using human epidermis. Nicotine in the form of 1% w/v aqueous buffer solution at pH 5 and transdermal patches (dose 14 mg day(-1)) were used. The nicotine fluxes across the shed snake skin were not significantly affected (P > 0.05) by temperature and duration of hydration pre-treatment. Scanning electron micrographs of SKCS and SCS revealed a remarkable difference in surface morphology, but the nicotine fluxes using both shed skins were not significantly different (P > 0.05). When compared with the results obtained using human epidermis, there were similarities in fluxes and permeation profiles of nicotine. Using nicotine solution, the nicotine permeation profiles of all membranes followed zero order kinetics. The amount of nicotine permeated provided good linearity with the square root of time over 24 h (R(2) > 0.98) when using nicotine patches. The nicotine fluxes using SKCS and SCS had less inter-specimen variation than those using human epidermis. The results suggest a potential use for SKCS or SCS as barrier membranes for in-vitro nicotine permeation studies.

Combination chemotherapy and photodynamic therapy with fab' fragment targeted HPMA copolymer conjugates in human ovarian carcinoma cells.

The biological activities of sequential combinations of anticancer drugs, SOS thiophene (SOS) and mesochlorin e 6 monoethylenediamine (Mce 6), in the form of free drugs, nontargeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates, P-GFLG-Mce 6 and P-GFLG-SOS (P is the HPMA copolymer backbone and GFLG is the glycylphenylalanylleucylglycine spacer), and Fab'-targeted HPMA copolymer-drug conjugates, P-(GFLG-Mce 6)-Fab' and P-(GFLG-SOS)-Fab' (Fab' from OV-TL16 antibodies complementary to CD47), were evaluated against human ovarian carcinoma OVCAR-3 cells. Mce 6, SOS, P-GFLG-Mce 6, P-GFLG-SOS, P-(GFLG-Mce 6)-Fab', and P-(GFLG-SOS)-Fab', when used as single agents or in binary combination, exhibited cytotoxic activities against OVCAR-3 cells, as determined using a modified MTT assay. The binding and internalization of P-(GFLG-Mce 6)-Fab' and P-(GFLG-SOS)-Fab' by OVCAR-3 cells were visualized by confocal microscopy and flow cytometry. The results confirmed an enhanced biorecognition by OVCAR-3 cells of Fab'-targeted HPMA copolymer conjugates over nontargeted conjugates. The median-effect analysis and the determination of the combination index (CI) were used to describe the drug interaction and quantify the synergism, antagonism, or additivity in anticancer effects. The sequential combinations of SOS+Mce 6 and P-GFLG-SOS+P-GFLG-Mce 6 displayed very strong synergism to synergism in the entire range of cell inhibition levels ( f a = 0.5 - 0.95). The P-(GFLG-SOS)-Fab'+P-(GFLG-Mce 6)-Fab' exhibited a strong synergism for f a values up to about 0.85, but showed synergistic effect and nearly additive effect at f a = 0.9 and 0.95, respectively. These observations support the continuation of in vivo investigations of these conjugates for the treatment of ovarian cancer.

Characterization of "Yaa Chud" Medicine on the Thailand-Myanmar border: selecting for drug-resistant malaria and threatening public health.

Multidrug-resistant Plasmodium falciparum malaria is a severe public health problem on the Thailand-Myanmar border. Many villagers buy packets of 4-5 mixed medicines ("yaa chud") from shops without medical assessment as their first-line malaria treatment. In 2000-2001 a local researcher purchased 50 yaa chud from 44 shops around Mae Sot, Thailand and Myawaddy, Myanmar (Burma), for his wife who was said to be pregnant with fever and drowsiness. The tablets/capsules were provisionally identified by appearance and active ingredients determined in a subset by using mass and atomic spectrometry. The most frequently detected active ingredients were acetaminophen (22%), chlorpheniramine (13.4%), chloroquine (12.6%), tetracycline/doxycycline (11.4%), and quinine (5.1%). Only seven bags contained potentially curative medicine for malaria. A total of 82% of the bags contained medicines contraindicated in pregnancy. Inappropriate, ineffective antimalarial drugs on the Thailand-Myanmar border are likely to increase malaria morbidity, mortality and health costs and engender the emergence and spread of antimalarial drug resistance.

Effect of poly(lactide-co-glycolide) molecular weight on the release of dexamethasone sodium phosphate from microparticles.

The objective of this study was to investigate the effect of poly(lactide-co-glycolide) (PLGA) molecular weight (Resomer RG 502H, RG 503H, and RG 504H) on the release behavior of dexamethasone sodium phosphate-loaded microparticles. The microparticles were prepared by three modifications of the solvent evaporation method (O/W-cosolvent, O/W-dispersion, and W/O/W-methods). The encapsulation efficiency of microparticles prepared by the cosolvent- and W/O/W-methods increased from approximately 50% to >90% upon addition of NaCl to the external aqueous phase, while the dispersion method resulted in lower encapsulation efficiencies. The release of dexamethasone sodium phosphate from PLGA microparticles (>50 microm) was biphasic. The initial burst release correlated well with the porosity of the microparticles, both of which increased with increasing polymer molecular weight (RG 504H > 503H > 502H). The burst was also dependent on the method of preparation and was in the order of dispersion method > WOW method > consolvent method. In contrast to the higher molecular weight PLGA microparticles, the release from RG 502H microparticles prepared by cosolvent method was not affected by volume of organic solvent (1.5-3.0 ml) and drug loading (4-13%). An initial burst of approximately 10% followed by a 5-week sustained release phase was obtained. Microparticles with a size <50 microm released in a triphasic manner; an initial burst was followed by a slow release phase and then by a second burst.

Convenient and rapid determination of cimetidine in human plasma using perchloric acid-mediated plasma protein precipitation and high-performance liquid chromatography.

This study demonstrates the analysis of cimetidine in human plasma with HPLC using a simplified sample preparation by protein precipitation with perchloric acid. Plasma cimetidine concentration was determined by plotting peak height ratio of cimetidine to ranitidine (internal standard, IS) against cimetidine concentrations in plasma. The cimetidine and ranitidine peaks were completely separated and no interference from plasma was observed. The lower limit of quantification (LLOQ) of the method was established at 0.1 microg/mL with a precision of 4.3% and a relative error of 1.9%. The average analytical recovery was >90% over the range of cimetidine concentrations (0.1-15.0 microg/mL). The linearity of calibration curve was excellent (r(2) > 0.999). The within- and between-day precision and accuracy, expressed as the coefficients of variation and relative error, were found to be less than 5%. Compared with previously reported methods, the analytical technique for cimetidine determination in human plasma presented here demonstrates comparable accuracy and precision, an acceptable analysis time, shorter and simpler sample preparation, and a reduced need for complicated equipment. The method presented here is simple and rapid, and the precision and sensitivity are appropriate for the determination of cimetidine in plasma in pharmacokinetic studies.

Acrylic matrix type nicotine transdermal patches: in vitro evaluations and batch-to-batch uniformity.

Nicotine transdermal patches (NTPs) were fabricated using an acrylic pressure sensitive adhesive emulsion to form a transparent matrix film. An automated thin layer chromatography (TLC) plate scraper was used to control the thickness of the cast nicotine matrix film. The in vitro release behavior and permeation of nicotine across abdominal human epidermis (HE) from the NTPs was studied using United States Pharmacopeia (USP) dissolution apparatus 5 (paddle over disk) and modified Franz-diffusion cell, respectively. The release of nicotine from the NTPs showed a good linear correlation with the square root of time (R2 > 0.99). This indicated a matrix diffusion controlled-release mechanism. The surface morphology of the matrix of the NTP was uniform and nonporous before and after release, indicating that the dried adhesive nicotine matrix was a homogeneous single-phase film. Neither the nicotine content in the range 4.70-8.41% w/w nor the film thicknesses of the NTPs affected the apparent diffusion coefficient of nicotine in the acrylic matrix. A good relationship between the amount of nicotine permeated across the HE and the square root of time was also observed with R2 > 0.98. This study also showed that the NTPs provided a good delivery system with more than 65% of the nicotine delivery being controlled by the device. Moreover, the release of nicotine from six production batches met the criteria of USP 24. This finding presented a good potential of this method for upscaling to industrial manufacturing.