Computer-based formulation design and optimization using Hansen solubility parameters to enhance the delivery of ibuprofen through the skin.

Trial-and-error approach to formulation development is long and costly. With growing time and cost pressures in the pharmaceutical industry, the need for computer-based formulation design is greater than ever. In this project, emulgels were designed and optimized using Formulating for Efficacy™ (FFE) for the topical delivery of ibuprofen. FFE helped select penetration enhancers, design and optimize emulgels and simulate skin penetration studies. pH, viscosity, spreadability, droplet size and stability of emulgels were evaluated. Franz cell studies were performed to test in vitro drug release on regenerated cellulose membrane, drug permeation in vitro on Strat-M® membrane and ex vivo on porcine ear skin, a marketed ibuprofen gel served as control. Emulgels had skin compatible pH, viscosity and spreadability comparable to a marketed emulgel, were opaque and stable at 25 °C for 6 months. Oleyl alcohol (OA), combined with either dimethyl isosorbide (DMI) or diethylene glycol monoethyl ether (DGME) provided the highest permeation in 24 h in vitro, which was significantly higher than the marketed product (p < 0.01). OA + DGME significantly outperformed OA ex vivo (p < 0.05). The computer predictions, in vitro and ex vivo penetration results correlated well. FFE was a fast, valuable and reliable tool for aiding in topical product design for ibuprofen.

Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria.

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.

Water soluble complexes of C14 and C16 fatty acids and alcohols in media for cultivation of leprosy-derived psychrophilic mycobacteria.

Host-grown Mycobacterium leprae cell suspensions oxidized water-soluble complexes of palmitic acid, myristic acid, cetyl alcohol, and myristyl alcohol prepared with randomly methylated-beta-cyclodextrin as host molecules. Gas chromatography analysis showed that the water-soluble complexes retained their chemical structure following sterilization in the autoclave. Bioavailability of the two long-chain fatty acids and the corresponding long-chain alcohols was confirmed by Warburg manometric techniques with host-grown M. leprae cell suspensions. Inoculated with host-grown M. leprae cells in chemically well-defined, simple liquid and agar media, acid-fast bacilli were cultivable in primary cultures and subcultures at 10 degrees C with (NH4)2SO4 as the N source and water-soluble palmitic acid, myristic acid, cetyl alcohol or myristyl alcohol as the C and potent energy sources. M. phlei oxidized the complexed palmitic acid and myristic acid but not cetyl alcohol or myristyl alcohol. On agar media with any of these four carbon sources and (NH4)2SO4 but not ammonium thioglycolate as the N source, M. phlei grew abundantly at 36 degrees C. In liquid media only myristyl alcohol supported growth of M. phlei without any growth with palmitic acid, cetyl alcohol or myristic acid. The leprosy-derived, cold-loving cultures ("M. psychrophilum") were not fully tested for classification and identification. The cells are strongly acid-fast facultative psychrophiles, adapted in subcultures to mesophilic growth. They grow in chemically well-defined media with 14 and 16 C long-chain fatty acids or alcohols as the C and energy sources. None of the cultures grow on Low-enstein or 7H9 media. Heat-killed suspensions of the 4th and 6th subcultures provoke Mitsuda-type late skin reactions in tuberculoid, borderline and borderline-tuberculoid but not in lepromatous leprosy volunteers. When grown with (NH4)2SO4 as the N source (but not with the reducing agent ammonium thioglycolate) the subcultures multiplied abundantly in the foot pads of mice. It became evident that leprosy-derived, facultative psychrophilic mycobacteria really exist. Mycobacteria of this cluster do not distinguish between 14 or 16 C long chains with COOH or CH2OH as terminal bindings. Cells are quite aerophilic and grow preferentially on agar slant surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Further stereochemical studies of phthiocerol and phenol phthiocerol in mycobacteria.

Mycobacterium tuberculosis, M. marinum and some other pathogenic species elaborate waxes A, based on a long-chain beta-diol (phthiocerol and companion compounds) and polymethyl-branched fatty acids. The stereochemical studies conducted on waxes A showed that those of M. tuberculosis, M. leprae and M. kansasii differ from waxes A isolated from M. marinum and M. ulcerans by the absolute configuration of the methyl-branched chiral centres occurring in both the long-chain beta-diols and the fatty acyls. Furthermore, the two mycobacterial groups also differ in the stereochemistry of the beta-diol chiral centres.

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans.

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.

Two-dimensional gas chromatography with electron capture detection for the sensitive determination of specific mycobacterial lipid constituents.

A method was developed for determining two characteristic mycobacterial lipid constituents, tuberculostearic acid (as its pentafluorobenzyl ester) and 2-eicosanol (as its pentafluorobenzoyl ester), by using gas chromatography with electron capture detection. A microprocessor-controlled column-switching system (two-dimensional gas chromatography) facilitated sample preparation and increased specificity. The usefulness of the technique was illustrated by its ability to reveal picogram amounts of tuberculostearate in a suspension of Mycobacterium leprae isolated from a naturally infected armadillo. Two-dimensional gas chromatography with electron capture detection may in some instances provide a convenient alternative to gas chromatography-mass spectrometry for use in demonstrating the presence of mycobacteria in a complex environment.

Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria.

Among 28 mycobacterial species studied, only Mycobacterium tuberculosis, M. bovis, M. africanum, M. marinum, M. kansasii, M. gastri and M. ulcerans produced waxes yielding long-chain beta-diol components (called phthiocerol and companions) and polymethyl-branched fatty acids on saponification. The same mycobacterial species also produced diesters of phenol phthiocerol and companions. Fatty acids esterifying these fatty alcohols in M. marinum and M. ulcerans were found to belong to the phthioceranic series (dextrorotatory fatty acids), in contrast to those of the other species (laevorotatory fatty acids called mycocerosic acids), both groups having the same chain length and methyl-branched positions. M. kansasii and M. gastri contained the same waxes with identical structures, as did M. tuberculosis, M. bovis and M. africanum. Neither the type strain of M. tuberculosis, nor that of M. bovis or M. marinum accumulated the strain-specific phenolic glycolipids.

Use of gas chromatography to differentiate Mycobacterium leprae from cultivable armadillo-derived mycobacteria, M. avium/intracellulare, and M. lepraemurium by analysis of secondary alcohols.

Two long-chain secondary alcohols, 2-octadecanol and 2-eicosanol, were demonstrated by gas chromatography in hydrolysates of Mycobacterium avium/intracellulare, in cultivable, armadillo-derived mycobacteria, and in M. lepraemurium grown in vivo, but they were not found in purified suspensions of M. leprae isolated from experimentally infected armadillos. Gas chromatographic analysis of these alcohols constitutes a method for rapid detection and quantification of contaminating mycobacteria in preparations of M. leprae intended, for example, for vaccine use. The technique may also be of value for critical evaluation of cultures of "in vitro-grown" M. leprae.

Further specific extracellular phenolic glycolipid antigens and a related diacylphthiocerol from Mycobacterium leprae.

Mycobacterium leprae in infected armadillo tissue produces extracellular phthiocerol-containing lipids in amounts well in excess of the bacterial mass. The principal component (1.38 mg in 1 g of liver, wet weight, containing 3.7 X 10(10) M. leprae bacilli) consists of a mixture of two phthiocerol homologs, 3-methoxyl-4-methyl-9, 11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9, 11-dihydroxytriacontane, (formula: see text); in which the hydroxyl functions are acylated by a mixture of three 'mycocerosic acids': 2,4,6,8-tetramethylhexacosanoate, 2,4,6,8-tetramethyloctacosanoate, and 2,4,6,8-tetramethyltriacontanoate. The structures were established by saponification of the native lipid, direct probe electron impact- or chemical ionization-mass spectrometry of the phthiocerol or its permethylated derivative, and gas-liquid chromatography-electron impact-mass spectrometry of the methyl esters of the fatty acids. In addition to the previously reported M. leprae-specific triglycosylphenolicdiacyl phthiocerol (Hunter, S. W., Fujiwara, T., and Brennan, P. J. (1982) J. Biol. Chem. 257, 15072-15078), the extracellular products contain small amounts (about 60 micrograms/g of infected liver, wet weight) of two other phenolic glycolipids, one of which (Phenolic Glycolipid III) has been structurally elucidated, (formula: see text); assuming certain enantiomeric configurations for the sugar substituents; the R-acyl functions are identical with those in the diacylphthiocerol. Phenolic Glycolipid-III reacts in enzyme-linked immunosorbent assays with sera from patients with leprosy and with rabbit antisera raised against whole M. leprae. The phthiocerol-containing lipids may be synonymous with the electron transparent capsules of M. leprae, and their unreactive state may confer on them the role of passive protectors of the bacillus.