A mechanistic physiologically based model to assess the effect of study design and modified physiology on formulation safe space for virtual bioequivalence of dermatological drug products.

Physiologically based pharmacokinetic (PBPK) models are widely accepted tools utilised to describe and predict drug pharmacokinetics (PK). This includes the use of dermal PBPK models at the regulatory level including virtual bioequivalence (VBE) studies. The current work considers the Topicort® Spray formulation, which contains 0.25% desoximetasone (DSM), as an example formulation. Quantitative formulation composition and in vitro permeation testing (IVPT) data were obtained from the public literature to develop a mechanistic model using the multi-phase, multi-layer (MPML) MechDermA IVPT module in the Simcyp Simulator. In vitro-in vivo extrapolation functionality was used to simulate in vivo PK for various scenarios and predict a 'safe space' for formulation bioequivalence using the VBE module. The potential effect of vasoconstriction, impaired barrier function, and various dosing scenarios on the formulation safe space was also assessed. The model predicted 'safe space' for formulation solubility suggesting that a 50% change in solubility may cause bio-in-equivalence, whereas viscosity could deviate by orders of magnitude and the formulation may still remain bioequivalent. Evaporation rate and fraction of volatile components showed some sensitivity, suggesting that large changes in the volume or composition of the volatile fraction could cause bio-in-equivalence. The tested dosing scenarios showed decreased sensitivity for all formulation parameters with a decreased dose. The relative formulation bioequivalence was insensitive to vasoconstriction, but the safe space became wider with decreased barrier function for all parameters, except viscosity that was unaffected.

Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells.

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (approximately 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti(-)[insulin receptor substrate 1 (IRS1)] and anti-alpha-p85 antibodies show the same level of insulin-stimulated activity, approximately 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of alpha-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon.

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.

Aluminum inhibits calpain-mediated proteolysis and induces human neurofilament proteins to form protease-resistant high molecular weight complexes.

We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits, respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50 = 200 microM) and by aluminum lactate (IC50 = 400 microM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 microM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 microM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50 = 1.2 and 2.1 mM for aluminum lactate and AlCl3, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.

Aluminum salts induce the accumulation of neurofilaments in perikarya of NB2a/dl neuroblastoma.

NB2a/dl neuroblastoma cells were exposed to aluminum chloride or aluminum lactate (0.1-1 mM) for 3 and 6 days. Additional cultures were exposed to aluminum salts as the cells were stimulated to elaborate axonal neurites by dibutyryl cyclic AMP. By phase-contrast microscopy, aluminum salts had no effect on the morphology of undifferentiated (NB2a(-] or differentiated (NB2a(+] cells, or on neuritic elaboration and maintenance. Silver straining by the Bielschowsky method, however, demonstrated argyrophilic accumulations in perikarya of many NB2a(-) and NB2a(+) cells treated with aluminum salts. At the ultrastructural level, whorls of intermediate filaments were the most prominent abnormalities in neuronal perikarya. Although phosphorylated high-molecular weight neurofilament subunits (NF-H) are normally detected by immunocytochemical analyses only within axonal neurites of NB2a/dl cells, aluminum salt treatment caused the detection of phosphorylated epitopes of NF-H within perikaryal of NB2a(-) and NB2a(+) cytoskeletons, suggesting that the argyrophilic filamentous accumulations are composed at least partly of phosphorylated NF-H.

Serological survey for Toxoplasma infections in sheep.

A factorial-design survey was performed to determine the prevalence of specific antibody against Toxoplasma in young and adult sheep from 6 areas in 3 different geoclimatic zones in South Australia. Serum samples obtained from 1,159 sheep belonging to 59 flocks were tested by a conventional indirect haemagglutination test (IHAT) as well as by an enzyme-linked immunosorbent assay (ELISA) which used class-specific conjugates to detect both IgG and IgM against Toxoplasma. Titres greater than 64 were detected in 7.4%, 9.2% and 25.2% of the sheep by the IHAT, IgG-ELISA and IgM-ELISA respectively. A significant positive correlation was found between the results of the IgG-ELISA and the IHAT with identical results obtained for 1,050 samples. Antibody detected by all 3 tests was more prevalent and higher in titre in adult sheep than in lambs as well as in sheep from Kangaroo Island than in those from mainland South Australia. Although the regional differences in prevalence suggested a relationship with climate, no significant correlations were detected between the prevalence results and any single climatic factor; namely, average annual rainfall, average annual evaporation or mean temperature range.

The tokamak: model T fusion reactor.

During the past several years there have been significant scientific and technological advances related to the tokamak magnetic confinement scheme. These are summarized in the context of a recent tokamak reactor design study which emphasizes reduced size, higher power density, and enhanced plant reliability and maintainability relative to earlier tokamak reactor design studies. The direct plant cost of the proposed reactor is estimated to be in the range $1000 to $1500 per electrical kilowatt. A three-phase strategy for demonstrating tokamak fusion power generation at a committed site is outlined. It is estimated that implementation of the three-phase program would require about 20 years and a total escalated expenditure $10 billion to $15 billion. The tokamak power plant described here is not viewed as definitive but rather as a point of departure in the development of a plan to demonstrate tokamak power generation.

Gas chromatographic determination of some chlorothyl phthalimide compounds.

The use of small-bore glass columns packed with low liquid-phase-loaded insert supports has provided the capability for the separation and quantitative determination of a series of chloroethyl phthalimides. Compounds such as N-(2-chlorovinyl) and N-(1,2-DICHLOROETHYL) PHTHALIMIDE HAVE BEEN MEASURED WITH A RELATIVE STANDARD DEVIATION OF NO MORE THAN 1.4%. The N-(1-chloroethyl) phthalimide compound is not stable even in thisinert system, but it can be stabilized by the room temperature preparation of the N-(1-ethoxyethyl) phthalimide derivative. Methods of standards preparation, calibration, and analysis of N-(1,2-dichloroethyl) phthalimide reaction mixtures are presented.