Douglas D. Glover

The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit

The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal≈ nasal ≥ bronchial ≥ tracheal > vaginal ≥ rectal > corneal > buccal > skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.

Regiospecific expression of cytochrome P4501A1 and 1B1 in human uterine tissue

The goal of this study was to investigate the expression of these isoforms in different regions of the human uterus. Expression was determined in the endometrium (ENDO), endocervix (CERV) and squamous region (SR) from six non-smoking women by using RT-PCR. The transcripts encoding for CYP1A1 were significantly higher (P < 0.05) in the SR compared to the other areas. However, the expression of CYP1B1 was significantly higher (P < 0.05) in the ENDO. CYP1B1 expression appeared to be extremely low in a woman in the secretory stage of the menstrual phase, relative to the ENDO of the other patients who were all in the proliferative stage at hysterectomy. CYP1B1 mRNA was expressed in only two out of six patients in the SR and in three out of five patients in the CERV. CYP1A1 was also uniformly expressed in the ENDO of all except one patient, whereas expression was minimal in the other regions. It is likely that variability in the expression of these isoforms may be responsible for the differential susceptibility to cancer in women.

Relationship of Fungal Vaginitis Therapy to Prior Antibiotic Exposure

Objective: To address the putative association of antibiotic use and subsequent yeast vaginitis in a population of non-pregnant women. Methods: Three hundred and sixteen women who received medical care in rural family medicine clinics enrolled in this study. Participants were pre-menopausal and non-pregnant and were followed until they used a course of antifungal therapy for vaginitis, became pregnant or moved from the catchment area. At entry subjects were free of vaginitis symptoms and had taken no antibiotics for 30 days. Patients were followed by repeated review of clinic records, hospital records and telephone or personal interviews. Data collection included documentation of episodes of antifungal treatment for vulvovaginal candidiasis and confirmed antibiotic treatment or credible history of antibiotic use prior to the use of antifungal therapy. Physician-reported uses of antibiotic and antifungal as well as patient-reported uses of these were recorded. Results: There were four reported cases of antifungal therapy following within a month of antibiotic use, in contrast to 484 antibiotic uses not followed by antifungal use. If time of observation was extended to 6 months from antibiotic use, there were 13 uses of antifungal therapy after antibiotics and 475 uses of antibiotics not followed by antifungal therapy. Conclusion: Our results cast doubt on the association of antibiotics as a putative cause of yeast vulvovaginitis.

Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase

The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies.

The effects of diabetes on placental aromatase activity

Diabetes complicates 2-3% of all pregnancies and is associated with an increase in both perinatal morbidity and mortality, though reasons for these adverse outcomes are unknown. Estrogen biosynthesis is a critical factor during pregnancy and is carried out in the placenta via aromatase (cytochrome P450 19A1), which catalyzes the conversion of C-19 androgens to C-18 estrogens. Previous studies have shown that hormones such as insulin-like growth factors and insulin regulate aromatase activity when studied in vitro. Interestingly, levels of these hormones are altered in patients with diabetes. Thus, we hypothesized that the presence of maternal diabetes may alter placental aromatase activity and thus estrogen biosynthesis, possibly serving as one factor in the adverse outcomes of babies born to mothers with diabetes. To this end, we measured the production of 19-hydroxyandrostenedione, 19-oxoadrostenedione and estrone in 30 placental tissues from diabetic patients, using [7-3H]androst-4-ene-3,17-dione as a model substrate for aromatase (P450 19A1). A statistical difference was detected in the percentage of 19-oxoandrostenedione formed between the overt and control groups (P < 0.05). Additionally, NADPH P450-reductase levels were measured in these same tissues to determine whether alterations in this enzyme necessary for aromatase activity could be affected by diabetes. No differences in reductase levels were detected among the patient groups. However, a statistical correlation was found between NADPH P450-reductase activity and the formation velocities of all three estrogen products (P < 0.05). Thus, it appears that the presence of diabetes does not affect placental aromatase activity.

Effects of gestational and overt diabetes on placental cytochromes P450 and glutathione S-transferase

Objective: Animal and in vivo human studies have observed that diabetes alters the expression of hepatic metabolizing cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes. The placenta has the ability to metabolize a number of xenobiotic and endogenous compounds by processes similar to those seen in the liver. Our objective was to compare placental xenobiotic metabolizing activity in diabetics to matched non-diabetic controls to determine if the presence of diabetes alters placental xenobiotic metabolizing activity.Methods: The catalytic activities of 7-ethoxyresorufin-O-deethylation [EROD] (CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation with glutathione (GST) from placentas of diet controlled (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared to matched controls.Results: No differences in EROD activity were observed among overt or gestational diabetics and their respectively matched controls. CYP2E1, 2D6, and 3A4 enzyme activity were not detected in human placentas. In contrast, GST activity was significantly reduced by 30% (P <.05) in overt diabetics as compared to their matched controls and gestational diabetics.Conclusion: Pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in exposure of the fetus to harmful reactive electrophilic metabolites.

Serum Erythromycin Levels in Pregnancy

Erythromycin is the recommended therapy for pregnant women with chlamydial infection. Despite the fact that this drug has been available since 1952, relatively little is known about its pharmacokinetic behavior in pregnant women, and no investigations have been conducted in women who were in the third trimester of pregnancy. In this study, 10 women were treated with erythromycin for chlamydial infection during pregnancy; 7 of these women were in the third trimester of pregnancy, and 3 were in the second trimester. Serum samples were obtained at 0.5, 1, 2, 3, and 4 hours after a 500-mg oral dose of erythromycin base and were analyzed to determine absorption and peak serum levels of erythromycin. Results indicated that absorption was delayed and serum levels were diminished in comparison with values reported in the literature for patients in the second trimester of pregnancy. In 2 women, erythromycin serum levels were not detectable at any time during the 4 hours of the study; these 2 women also experienced the most severe gastrointestinal symptoms. Although the size of our study population was small and certainly not definitive, the data suggest that in patients in the third trimester of pregnancy, severe adverse gastrointestinal events may forewarn of subtherapeutic plasma concentrations of erythromycin, which could have consequences for the treatment outcome.

Ampicillin Vs. Penicillin for In Utero Therapy

The pharmacokinetics of penicillin G and ampicillin are reviewed as they pertain to their potential use in in vitro therapy.

Antibiotics and Pregnancy

Antibiotherapy during pregnancy has to be adapted according two factors: pregnancy related pharmacodynamic changes and potentially negative side effects for the developing foetus of some antibiotics. Pregnancy induce a weight gain, an increase of intravascular volume, of glomerular filtration, and of hepatic metabolism. The foetoplacental unit create a new volume in which antibiotics penetrates increasingly during gestation. As a consequence, during pregnancy, maternal plasmatic levels of antibiotics reaches values 10-50% under the ranges observed with the same dosages administered to non pregnant women. Tobramycin of which clearance diminish during pregnancy is the only exception for this rule. In term pregnancy, placental transfer of antibiotics is very high, except for macrolides. Thalidomide, a well-known responsible of phocomely, is still used in the treatment of leprae. No other antibiotic as a well demonstrated teratogenic effect in human foetuses. Nevertheless, tetracyclin use in pregnancy must be avoided according a high risk of enamel dysplasia, with brownish discoloration of deciduous teeth. No teratogenic effect has been recorded for betalactams and for erythromycin, the first choice antibiotics during pregnancy. Some antibiotherapy are specific of obstetrical conditions. Antibiotics interferes with estrogens metabolism, inducing a fall in circulating level of estriol.