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We have performed fully atomistic molecular dynamics simulations of the intracellular domain of a model of the GABA

We present a general method called atom-wise free energy perturbation (AFEP), which extends a conventional molecular dynamics free energy perturbation (FEP) simulation to give the contribution to a free energy change from each atom. AFEP is derived from an expansion of the Zwanzig equation used in the exponential averaging method by defining that the system total energy can be partitioned into contributions from each atom. A partitioning method is assumed and used to group terms in the expansion to correspond to individual atoms. AFEP is applied to six example free energy changes to demonstrate the method. Firstly, the hydration free energies of methane, methanol, methylamine, methanethiol, and caffeine in water. AFEP highlights the atoms in the molecules that interact favorably or unfavorably with water. Finally AFEP is applied to the binding free energy of human immunodeficiency virus type 1 protease to lopinavir, and AFEP reveals the contribution of each atom to the binding free energy, indicating candidate areas of the molecule to improve to produce a more strongly binding inhibitor. FEP gives a single value for the free energy change and is already a very useful method. AFEP gives a free energy change for each "part" of the system being simulated, where part can mean individual atoms, chemical groups, amino acids, or larger partitions depending on what the user is trying to measure. This method should have various applications in molecular dynamics studies of physical, chemical, or biochemical phenomena, specifically in the field of computational drug discovery.

Evaluating solvation entropies directly and combining with direct energy calculations is one way of calculating free energies of solvation and is used by Inhomogeneous Fluid Solvation Theory (IFST). The configurational entropy of a fluid is a function of the interatomic correlations and can thus be expressed in terms of correlation functions. The entropies in this work are directly calculated from a truncated series of integrals over these correlation functions. Many studies truncate all terms higher than the solvent-solute correlations. This study includes an additional solvent-solvent correlation term and assesses the associated free energy when IFST is applied to a fixed Lennard-Jones particle solvated in neon. The strength of the central potential is varied to imitate larger solutes. Average free energy estimates with both levels of IFST are able to reproduce the estimate made using the Free energy Perturbation (FEP) to within 0.16 kcal/mol. We find that the signal from the solvent-solvent correlations is very weak. Our conclusion is that for monatomic fluids simulated by pairwise classical potentials the correction term is relatively small in magnitude. This study shows it is possible to reproduce the free energy from a path based method like FEP, by only considering the endpoints of the path. This method can be directly applied to more complex solutes which break the spherical symmetry of this study.

This study reports on the perceptions of recent graduates of their undergraduate pharmacy course. Questionnaires were sent to 952 graduates undertaking their preregistration training in the period 1992–3. Of the total response of 695 (73 per cent), 55 per cent were in community pharmacy, 38 per cent in hospital and the remainder in various joint posts. Their opinions on the usefulness of course topics indicated that pharmacognosy and some topics within pharmaceutical chemistry were significantly regarded as of little use (P The study suggests that changes in course design should not include undergraduate specialisation, the graduates' branch of pharmacy having no influence on their opinion of the usefulness of clinical pharmacy and pharmacy practice topics. It also suggests that some science-based topics need to be made more relevant to pharmacy and that clinical and practice-based topics should have a major role in an extended course.

The loading and relase of ibuprofen, Ketoprofen and mefenamic acid from a range of strong anionic exchange resins, including cholestyramine, is decribed. Release rates into simulated gastric fluid increase with stirring speed up to 300 rpm and decrease as either the particle size of the resin beads of the degree of cross-linking increase. An increase in the temperature of loading enhances the capacity of the resin towards the drug and reduce its relase rate. Coating of the resing also enables suppression of drug release to be achieved. The samll particle size of cholestyramine enables a rapid relese of drug from the resin to be achieved. This rate is significantly greater than that obtained by monitoring dissolution from a drug-lactose dispersion and may indicate that ion-exchange technology may provide and opportunity to overcome poor dissolution characteristicsf for weekly ionic compounds.

To investigate the transdermal delivery of a model macromolecule by passive and iontophoretic means following pretreatment with C12-penetration enhancers and to visualise transport across human stratum corneum (SC) in real time.Transport studies of dextran, labelled with fluorescent Cascade Blue (D-CB: M(R) = 3 kDa) across human stratum corneum, were conducted during passive and iontophoretic modes of delivery following pretreatment with either dodecyltrimethylammonium bromide (DTAB), sodium dodecyl sulphate (SDS) or Azone. Size-exclusion chromatography was used to assess maintenance of dextran structural integrity throughout experimental lifetime. Two-photon excitation microscopy was employed to visualise real-time dextran transport during current application.The positively charged C12-enhancer DTAB elevated passive D-CB steady-state flux (J(ss)) and was the only enhancer to do so above control during iontophoresis. The negatively charged SDS had the least effect during both stages. On-line macromolecular transport was visualised, indicating both inter- and intra-cellular pathways across SC during current application. No transport was visible across untreated SC during passive transport.Use of a positively charged enhancer may improve J(ss) of anionic macromolecular penetrants during passive and iontophoretic delivery. On-line visualisation of iontophoresis across SC was possible and can provide mechanistic insight into SC transport pathways.

To investigate the effect of ultrasound on the histological integrity and permeability properties of whole rat skin in vitro.A defined, field-free source of ultrasound was used to irradiate excised rat skin prior to in vitro transport studies in Franz-type cells using sucrose, mannitol, hydrocortisone, 5-fluorouracil and aminopyrine.High intensity ultrasound irradiation (1 to 2 W cm-2) irreversibly damaged cutaneous structures and increased the percutaneous transport rate of permeants. In contrast, skin integrity was largely maintained with low intensity ultrasound (0.1 to 1 W cm-2) which merely discharged sebum from the sebaceous glands so as to fill much of the hair follicle shafts. This effect caused the transfollicular absorption pathway to be blocked for hydrophilic molecules that penetrate via this route and reduced the transport rate significantly.This phenomenon may be used as a probe to elucidate the relative follicular contribution to total penetration for hydrophilic permeants. It was demonstrated that the shunt pathway was responsible for virtually all mannitol and sucrose penetration, perhaps half of hydrocortisone transport but negligible aminopyrine and 5-fluorouracil penetration.

The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line, that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense, mechanism affecting receptor tyrosine kinase activity.

Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells.We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides.The cellular association of both an internally [3H]-labelled and a 5'end [32P]-labelled PS oligonucleotide (3.0% at 0.4 microM extracellular concentration) was similar and was an order of magnitude greater than that of the 5'end [32P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 microM whereas that of PO was less susceptible to competition (0.2% at 5 microM, 0.1% at 200 microM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37 degrees C to 17 degrees C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase, removed approximately 50% of the cell-associated oligonucleotide for both backbone types.Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.

Leucine enkephalin (YGGFL) undergoes rapid degradation in sheep nasal mucosa to yield GGFL which is further degraded to FL. The activity of the nasal mucosal homogenate against YGGFL and GGFL (t1/2 12 and 7 min) was significantly greater than that observed with a nasal wash fluid (t1/2 40 and 13 min). The effect of cyclodextrins on the rate of degradation of FGG and YGGFL by leucine aminopeptidase (LAP) and of GGF by carboxypeptidase A (CPA) was monitored. Little effect was observed with FGG (with LAP) but the half-life of YGGFL (with LAP) was extended from approximately 44 min to approximately 75 min in the presence of a 25-fold excess of beta-cyclodextrin. The stability of GGF (with CPA) was also enhanced; an effect was observable with a 5-fold excess of cyclodextrin and the half-life could be extended by 40-75%. An equation is presented which allows the estimation of the concentration of free peptide in the peptide-cyclodextrin solutions.

Circular high-performance thin-layer chromatography (HPTLC) has been used to differentiate a series of twelve tricyclic neuroleptics, using both normal phase and reverse phase procedures. The use of normal phase systems also allows the resolution of geometric isomers of chlorprothixene, clopenthixol and flupenthixol. Thirteen sulphonamides and Trirnethoprim may also be distinguished using HPTLC.

Summary The effect of a legislation-induced slowing of product turnover on the quality of some aspirin and phenacetin containing products is studied. A new reversed phase high pressure liquid chromatographic method for assaying salicylic acid in compound preparations is described and the results are discussed with reference to the U.K. compendial limit tests for salicylic acid in aspirin-containing products. Comment is made on the calculation of the results.

The pyrolysis gas chromatography mass spectrometry of Ibuprofen, Fenoprofen, Naproxen and Ketoprofen, a series of anti-inflammatory propionic acid derivatives, is shown to proceed via decarboxylation and elimination to yield characteristic ethyl and vinyl fragments. The pyrogram enables the identification of the drug to be achieved as the pure compound, in a formulated dosage form or excreted in urine. The presence of metabolites derived from Ibuprofen causes four new fragments to be observed in the urine pyrogram. The identification of 16 components of the control urine pyrogram is presented.

Comparison of fragmentation pathways and high resolution measurements has revealed that the molecular ions of 1-(2-hydroxy-2-phenylethyl)imidazoles undergo elimination of benzaldehyde. The parent ions of trans-1-styrylimidazoles also cyclise in a manner similar to their photochemical reactivity. A rearrangement involving OH or OMe transfer between the trans-1-styryl and 5-acid or 5-methyl ester group of an imidazole is also described.

High-performance liquid chromatographic assays for the controlled-release penicillin complexes benzathine-cloxacillin, benzathine-penicillin V, procaine-penicillin G, benethamine -penicillin G and benzathine-penicillin G are described. Reversed-phase ion-paired chromatography using related aqueous acetonitrile mobile phases enables both components of the complex to be determined simultaneously. The utility of the methods is demonstrated by application to various formulations including fortified injections, which contain two complexes, and a suspension which also contains four preservatives.

The reaction between nitrones and copper acetylides in pyridine yields cis- and trans-azetidinones. Deuteriation and isomerisation studies indicate that the trans-compound is produced from the initially formed cis-isomer and that the amount of cis-product may be increased by using non-basic solvents. Lactams with aromatic, aliphatic, and ethoxycarbonyl substituents may be obtained from appropriately substituted acetylenes, and the use of cyclic nitrones leads to bicyclic compounds.

The reaction between the potassium salt of dimethyl imidazole-4,5-dicarboxylate and styrene oxide is shown to produce 4-methoxycarbonyl-1-styrylimidazole-5-carboxylic acid or methyl 5,6-dihydro-8-oxo-6-phenyl-8H-imidazo[5,1-c][1,4]oxazin-1-carboxylate. The subsequent cleavage of this lactone and the use of styrene [18O]oxide indicate the intermediacy of the lactone in the formation of the styryl acid.

The reaction between ethyl 2-quinolylacetate and phenacyl bromide has been re-investigated and is now shown to proceed via C-alkylation to yield 2-(1-benzoyl-2-ethoxycarbonylethyl)quinoline. Treatment of this compound with acetic anhydride yielded 2-benzoyl-3-ethoxycarbonyl-1-methylpyrrolo[1,2-a]quinoline and 3-ethoxycarbonyl-1-phenylpyrrolo[1,2-a]quinoline.

The photoisomerisation of trans-1-styrylimidazole and derivatives to the cis-isomers is described. The cis-compounds undergo a stereospecific photodehydrocyclisation involving the 2-position of the imidazole ring to give imidazo[2,1-a]isoquinoline, benzimidazo[2,1-a]isoquinoline, and derivatives. The reverse mode of cyclisation, involving 2-styrylbenzimidazoles in C–N bond formation, is also demonstrated; benzimidazo[1,2-a]quinoline and its 2,3,10-trimethyl derivative have been obtained from the corresponding 2-styryl compounds.

Bromination of 3,6-diphenylpyrrolo[1,2-c]pyrimidine with N-bromosuccinimide in chloroform solution yields 5- and 7-monobromo- and 5,7-dibromo-derivatives; with use of increasing amounts of N-bromosuccinimide the 5,7-dibromo-compound becomes the sole product isolated. Bromination with an excess of bromine in chloroform at room temperature rapidly yields 5,7-dibromo-3,6-diphenylpyrrolo[1,2-c]pyrimidine perbromide hydrobromide in high yield. The perbromide is easily converted into the dibromo-derivative by heat or treatment with base but in the presence of methanol or ethanol 4-alkoxy-5,7-dibromo-derivatives are obtained, together with traces of 4,5,7-tribromo-3,6-diphenylpyrrolo[1,2-c]pyrimidine. Similar treatment of 5,7-dibromo-3-methyl-6-phenylpyrrolo-[1,2-c]pyrimidine perbromide hydrobromide with ammonia yielded the 5,7-dibromo-derivative and treatment with alcohols gave the 4,5,7-tribromo-derivative. Structures of products were assigned on the basis of n.m.r. and mass spectra.