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Biomedical subjects

B H Stewart

Publications and source records attributed to B H Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1996

Absorption of Cam-2445, and NK1 neurokinin receptor antagonist: in vivo, in situ, and in vitro evaluations.

Cam-2445 is a selective, high-affinity NK1 receptor antagonist that is a potentially useful treatment for arthritis, asthma, migraine, anxiety, psychosis, and emesis. Cam-2445 exhibits low aqueous solubility and high lipophilicity and has a molecular weight of 470. Cam-2445 has poor oral bioavailability and the purpose of this research was to examine the potential barriers to the oral bioavailability of Cam-2445. Cam-2445 was relatively stable at 37 degrees C in 0.1 N HCl, 5 microM alpha-chymotrypsin, rat intestinal perfusate, and in rat jejunal brush border membrane suspension. High permeability was observed from CACO-2 cells and from rat single-pass intestinal perfusions. Cam-2445 was administered as a solution to rats by intravenous (i.v.), oral (p.o.), intraduodenal (i.d.), and intraportal (i.p.v.) routes. The total oral bioavailability was poor at 1.4%. Absorption appeared to be rapid after i.d. dosing; bioavailability was 26%, and 54% of the dose was absorbed intact into the portal system. After i.p.v. dosing, 48% of the dose was available to the systemic circulation. The elimination t1/2 after i.d. dosing (2.91 h) was comparable to that i.v. dosing (2.93 h), whereas it was significantly longer after p.o. dosing (12.4 h). The p.o. dose apparently precipitated in the gastrointestinal (GI) tract, resulting in low oral bioavailability. These results indicated that neither stability in the GI tract nor membrane transport were major obstacles to the absorption of Cam-2445. While hepatic extraction of 52% was significant, the low aqueous solubility of Cam-2445, as well as the differences noted between p.o. and i.d. studies, strongly support GI dissolution and/or precipitation as the limiting factor for the oral bioavailability of the compound.

Animals↗
PubMed1995

Evaluation of physicochemical parameters important to the oral bioavailability of peptide-like compounds: implications for the synthesis of renin inhibitors.

A series of radiolabeled compounds related to renin inhibitor structures was synthesized to represent a range of physicochemical properties. These compounds were tested in assays for intestinal absorption and hepatic clearance in order to define parameters conducive to optimizing bioavailability. In general, compounds with higher lipophilicity were better absorbed from the intestine. Absorption may also be dependent on molecular charge, as compounds with ionizable functionality were less well-absorbed than neutral compounds. Neutral compounds showed some dependency on molecular weight, with smaller compounds exhibiting better absorption. While uptake into hepatic cells was rapid regardless of partition coefficient or molecular weight, rate of appearance in bile was dependent on the molecular weight of the compounds.

Administration, Oral↗
PubMed1995

Comparison of intestinal permeabilities determined in multiple in vitro and in situ models: relationship to absorption in humans.

In vitro and in situ experimental models that are descriptive of drug absorption in vivo are valuable tools in the discovery of new chemical entities that are bioavailable after oral administration. The specific objective of the study was to compare the intestinal permeabilities obtained in the three absorption models for consistency, and to assess the utility of the models in predicting the fraction of dose absorbed in human studies. The intestinal absorption models that were compared are widely used: the rat in situ single-pass intestinal perfusion system, the rat everted intestinal ring method, and monolayers of human colon adenocarcinoma cell line (CACO-2). The models were compared using small molecular reference compounds, as well as a series of peptidomimetic (PM) analogs. Each model had strong potential for estimating the fraction absorbed. For small organic molecules, excellent correlation was observed when permeabilities from CACO-2 cells and perfusions, or everted rings and perfusions, were compared. Weaker correlation was observed between everted rings and CACO-2 cells. Permeabilities for the set of reference compounds and PMs were positively correlated between any two of the three systems. Variance between correlations for reference compounds and PMs are likely due to structural features and physicochemical properties that are unique to the latter class of compounds. The results support caution in extrapolating correlations based on findings with small organic molecules to the behavior of complex peptidomimetics. Corroboration of permeabilities with two methods of determination is a useful cross-validation of experimental systems, as well as producing a reliable permeability assessment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1994

Effect of interferon-gamma, interleukin-2 and interleukin-4 on cyclosporin-A-mediated inhibition of anti-CD3-induced T-lymphocyte proliferation.

It is generally believed that cyclosporin A (CsA) inhibits T-cell activation largely by blocking interleukin (IL)-2 production, although CsA also inhibits the secretion of other growth-promoting lymphokines. To investigate the importance of downregulated synthesis of IL-4 and interferon (IFN)-gamma, in addition to IL-2, in CsA-mediated inhibition of T-lymphocyte proliferation, exogenous IL-2, IL-4, and IFN-gamma were added to murine T-cells stimulated with anti-CD3 monoclonal antibody in the presence of an inhibitory concentration of CsA. Either IL-2 or IL-4 alone were able to partially counteract the inhibitory effect of CsA on anti-CD3-induced T-lymphocyte proliferation, whereas IFN-gamma had no discernable effect. IL-2 and IL-4, in combination, were able to largely reverse the immunosuppressive activity of CsA. These results indicate that (1) CsA fails to block T-cell signal transduction pathways coupled to IL-2 and IL-4 receptors, and (2) IL-2 and IL-4 have an additive effect in promoting the proliferation of a heterogenous T-cell population stimulated with anti-CD3 monoclonal antibody.

Animals↗
PubMed1993

A saturable transport mechanism in the intestinal absorption of gabapentin is the underlying cause of the lack of proportionality between increasing dose and drug levels in plasma.

Gabapentin (1-(aminomethyl)cyclohexaneacetic acid) is a neuroprotective agent with antiepileptic properties. The structure is small (molecular weight less than 200), is zwitterionic, and resembles an amino acid with the exception that it does not contain a chiral carbon and the amino group is not alpha to the carboxylate functionality. Gabapentin is not metabolized by humans, and thus, the amount of gabapentin excreted by the renal route represents the fraction of dose absorbed. Clinical trials have reported dose-dependent bioavailabilities ranging from 73.8 +/- 18.3 to 35.7 +/- 18.3% when the dose was increased from 100 to 1600 mg. The permeability of gabapentin in the rat intestinal perfusion system was consistent with carrier-mediated absorption, i.e., a 75 to 80% decrease in permeability when the drug concentration was increased from 0.01 to 50 mM (0.46 +/- 0.05 to 0.12 +/- 0.04). Excellent agreement was obtained between the actual clinical values and the predicted values from in situ results for the fraction of dose absorbed calculated using the theoretically derived correlation, Fabs = 1 - exp(-2Peff) by Amidon et al. (Pharm. Res. 5:651-654, 1988). The permeability values obtained for gabapentin correspond to 67.4 and 30.2% of the dose absorbed at the low and high concentrations, respectively. In the everted rat intestinal ring system, gabapentin shared an inhibition profile similar to that of L-phenylalanine. Characteristics of gabapentin uptake included cross-inhibition with L-Phe, sensitivity to inhibition by L-Leu, stereoselectivity as evidenced by incomplete inhibition by D-Phe, and lack of effect by Gly.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1992

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.

Animals↗
PubMed1991

Purification of an equine apotransferrin variant (thyromedin) essential for thyroid hormone dependent growth of GH1 rat pituitary tumor cells in chemically defined culture.

Pituitary tumor cells require thyroid hormones for growth in vivo [Sorrentino, J. M., Kirkland, W. L., & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158]. In vitro, GH1 rat pituitary tumor cells were studied in a serum-free defined medium (PCM-10) formulated with Ham's F12 and Dulbecco's modified Eagle's media (1:1, v/v) supplemented with 2.2 g/L sodium bicarbonate, 15 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (pH 7.2), 10 micrograms/mL human transferrin, 50 microM ethanolamine, 10 micrograms/mL insulin, 10 ng/mL selenous acid, 0.1 nM 3,5,3'-triiodothyronine (T3) and 500 micrograms/mL bovine serum albumin and in the same medium without T3 (PCM-0). The cells only grew in PCM-10 when low concentrations of horse serum were added. Attempts to replace the serum factor requirement with known growth factors and adhesion proteins were unsuccessful. The Mr 65,000-72,000 serum factor regulating T3-induced growth (thyromedin) was purified to homogeneity and identified as equine transferrin R and/or D by amino acid sequencing. The ED50 in PCM-10 was 17-40 micrograms/mL (260-620 nM) while in PCM-0 half-maximum growth was not achieved at 200 micrograms/mL. Concentrations of 75 micrograms/mL in PCM-10 caused 80% of serum-stimulated growth rate. Removal of iron from thyromedin, and assay in iron salts reduced PCM-10, increased the specific activity 110-270-fold to ED50 150 ng/mL (2.3 nM); at 1.0 micrograms/mL, growth in PCM-10 was 16-fold greater than in PCM-0. Iron saturation of thyromedin caused total loss of biological activity. We conclude that the horse transferrin variant isolated in this report is active as apotransferrin.

Amino Acid Sequence↗
PubMed1990

Regulatory control of the terminal complement proteins at the surface of human endothelial cells: neutralization of a C5b-9 inhibitor by antibody to CD59.

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.

Antibodies, Monoclonal↗
PubMed1989

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids.

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.

Adenosine Triphosphate↗
PubMed1989

Rat pituitary tumor cells in serum-free culture. I. Selection of thyroid hormone-responsive and autonomous cells.

The growth of GH4C1, GH3, GH1, and GH3C15 rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1:1 (vol/vol) mixture of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 micrograms/ml gentamicin supplemented with 10 micrograms/ml bovine insulin, 10 micrograms/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3'-triiodothyronine (T3), 50 microM ethanolamine (Etn), and 500 micrograms/ml bovine serum albumin. Of the lines evaluated, only the GH1 failed to grow in TRM-1. Passage of the GH4C1 and GH3 lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations of T3 (1.0 to 10 nM). After adaptation of the GH4C1 line in TRM-1 for greater than or equal to 20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 micrograms/ml gentamicin, 10 micrograms/ml Tf, 10 nM T3, and 50 microM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was lost progressively. When T3 was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only slightly stimulated by T3. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary cell populations that a) required both serum-factor(s) and T3 for optimum growth, b) required supraphysiologic concentrations of T3 without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented only with Tf and nutrients without necessity of other serum factor(s) or T3.

Animals↗
PubMed1986

Oral absorption of 21-corticosteroid esters: a function of aqueous stability and intestinal enzyme activity and distribution.

The intestinal absorption of hydrocortisone and prednisolone are compared with three water-soluble derivatives (succinate, phosphate, and lysinate) in experiments at two levels of biological system complexity. Rates of absorption are compared by measuring permeabilities from rat intestinal perfusions of drugs and derivatives in solution. Extents of absorption are compared over a 10-fold dose range of parent steroid and with the steroid derivatives by measuring plasma levels from solid oral dosage in dogs. While the parent steroids are well absorbed over the entire length of the intestinal tract, variability in plasma levels is observed at higher doses. Limited solubility and resultant dissolution rate variability are likely to be playing a role in the early erratic blood level profiles found at higher doses. While the soluble prodrugs have a dissolution rate advantage which results in a greater concentration gradient, their absorption is limited by their aqueous luminal stability, their polarity and resultant passive membrane permeability, and the distribution and activity of enzyme reconversion sites in the intestinal tract. The unstable lysinate ester, targeted for aminopeptidase, has an absorption profile and permeability similar to that of the parent steroid. The absorption of the moderately stable succinate ester is limited by its polarity and the activity of intestinal esterases. The stable phosphate derivative is well absorbed in the upper intestine, where high levels of alkaline phosphatase exist, while the prodrug polarity and drop-off of enzyme activity limit its absorption from the lower gastrointestinal (GI) tract.

Adrenal Cortex Hormones↗
PubMed1986

Uptake of prodrugs by rat intestinal mucosal cells: mechanism and pharmaceutical implications.

The in vitro intestinal ring uptake of prednisolone (11 beta,17,21-trihydroxypregna-1,4-diene-3,20-dione), prednisolone 21-succinate, and prednisolone 21-phosphate was compared in rings prepared from rat jejunum and colon. An HPLC assay was developed to determine whether the drug or intact prodrug was taken up by the tissue. In jejunum and colon, the uptake of prednisolone was limited by its solubility (0.84 mM, 37 degrees C). The freely soluble succinate and phosphate esters were well absorbed in the jejunum. The only species detected in jejunal tissue after incubation with prednisolone 21-phosphate was prednisolone, indicating hydrolysis prior to absorption. This implication was verified by light microscopy. Incubation of tissue from the jejunum with prednisolone 21-succinate resulted in uptake of a mixture of prodrug and parent drug, with the latter form predominating. Prednisolone 21-succinate was also absorbed well in the colon, where the predominant species taken up by the tissue was the intact ester. The half-life of the succinate ester in the tissue was approximately 1 h postincubation, implicating enzyme mediation. Uptake of the phosphate ester by the colon was less than 20% of that observed in the jejunum, with the species absorbed still being primarily the parent alcohol. Light microscopy techniques confirmed that prednisolone 21-phosphate and hydrocortisone 21-phosphate are good substrates for brush border membrane alkaline phosphatase in the jejunum, and that lack of this enzyme in colon tissue was most likely responsible for poor uptake in the colon.

Alkaline Phosphatase↗
PubMed1984

Radical cystectomy without radiation therapy for carcinoma of the bladder.

A review of a 20-year experience of radical cystectomy identified 99 patients who had not undergone preoperative radiation therapy. The 5-year survival rate of patients with clinical high stage lesions (T3, T4a) was 40 per cent. Life-table analyses showed that survival rate in this group was comparable with that reported for other groups receiving preoperative radiation therapy. The pelvic recurrence rate was 9 per cent and was frequently related to either urethral or nodal disease. Unquantifiable patient selection factors may well limit the reliability of these results.

Actuarial Analysis↗
PubMed1983

Surgical treatment of renal artery aneurysms.

Twenty-seven patients have undergone surgical treatment of renal artery aneurysms and the classification, radiographic features and complications of these aneurysms are reviewed. Aortorenal bypass is the preferred method of in situ revascularisation. Extracorporeal microvascular branch arterial reconstruction is reserved for patients with complex intrarenal aneurysms. Removal of the aneurysm with preservation of the involved renal unit is possible in most cases.

Adolescent↗
PubMed1982

A critical review of immunotherapy of disseminated renal adenocarcinoma.

Sixty patients with renal adenocarcinoma have been treated with five different immunotherapy trials consisting of 1) Transfer Factor (TF), 2) TF and Bacillus Calmette-Guerin (BCG), 3) TF, BCG, Chloroethyl-cyclohexy-nitrosurea (CCNU) and megestrol acetate (Megase), 4) BCG, CCNU, and Megase, or 5) BCG. Using strict response criteria for measurable disease, objective responses were seen in 14-22% of cases. While this nonspecific immunotherapy of renal adenocarcinoma has been associated with documented regression of metastases, response rates are similar to that obtained with hormonal therapy alone. Objective responses support the concept of further trials in this disease with more sophisticated immunotherapy.

Adenocarcinoma↗