Pieter van der Bijl

Permeation of 17β-estradiol through human vaginal and buccal mucosa

Because of the relative scarcity of fresh human oral mucosa specimens for permeability studies, we investigated the use of human vaginal mucosa as a model of the former. In a previous study we demonstrated the comparable diffusion rate of water across human vaginal and buccal mucosa and proposed the use of the former as a suitable model of the latter for in vitro drug permeability studies. To further evaluate the human vaginal/buccal mucosa model, we decided to compare these two tissues with respect to their permeability to 17beta-estradiol. Clinically healthy human vaginal and buccal mucosa specimens were obtained during vaginal hysterectomies and different oral surgical procedures. The permeability of each tissue specimen to 17beta-estradiol was determined through the use of a continuous flow-through diffusion system. Specimens were examined histologically before and after experiments. Mean flux values for 17beta-estradiol across human buccal mucosa tended to be slightly higher than those observed for vaginal tissue, but no statistically significant differences could be demonstrated. The results from this study further support our hypothesis that human vaginal mucosa may be a suitable model of human buccal mucosa for in vitro drug permeability studies.

Comparative Permeability of Human and Rabbit Corneas to Cyclosporin and Tritiated Water

Diffusion of cyclosporin A (CsA) and water through fresh and frozen (liquid nitrogen, -85 degrees C) human and rabbit corneas was compared to establish the appropriateness of using the latter tissue as a model for its human counterpart for in vitro permeability studies. Permeation of CsA in the presence of three penetration enhancers, 0.01% benzalkonium chloride (BZCl), 20% dimethylsulfoxide (DMSO), 10% and 20% Cremophor-EL was also studied. Permeability was determined using a flow-through diffusion apparatus (20 degrees C, 24 hours). ANOVA, Duncans multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. No statistically significant differences in flux values of CsA and water could be detected between fresh and frozen/thawed human and rabbit corneas. The diffusion of water was significantly lower across frozen/thawed rabbit cornea than across the same human tissue. CsA flux rates across frozen/thawed rabbit cornea in the presence of all three enhancers increased significantly when compared to the same tissues without enhancer, except in the presence of 20% Cremophor-EL after 18 hours. The rabbit cornea appears to be an appropriate in vitro model for studying human transcorneal penetration of drugs. Smaller M(w) substances, however, may have higher diffusion rates across frozen/thawed human corneal tissue.

Comparative in vitro permeability of human vaginal, small intestinal and colonic mucosa

Our previous experience with a continuous flow-through perfusion system has demonstrated its usefulness for studying diffusion kinetics of drugs across small intestinal mucosa for bioavailability/bioequivalence (BA/BE) studies. During the last decade, delivery of drugs to the colon for systemic absorption as well as for local delivery in certain colonic diseases, has been extensively investigated. For this reason, we sought to assess the in vitro comparative permeability of human vaginal, small intestinal and colonic mucosa using a flow-through perfusion method. It was clear from our studies that human colonic epithelium was statistically significantly (P 300 Da may necessitate using other epithelial membranes, e.g. vaginal mucosa, as alternative barriers for in vitro BA/BE studies. We also concluded that the flow-through mucosal perfusion system used in our laboratory is therefore also potentially useful for determining the permeability of a therapeutic agent from the colon for registration purposes.

Penetration of human vaginal and buccal mucosa by 4.4-kd and 12-kd fluorescein-isothiocyanate-labeled dextrans

In a previous study we demonstrated that human vaginal mucosa was as permeable to water as was buccal mucosa. Water, however, is a very small molecule with a molecular weight of 18 d. To further explore similarities between these two types of mucosa with respect to permeability, it was decided to investigate the passage of two large, hydrophilic molecules across these epithelia. Specimens of fresh, clinically healthy human vaginal and buccal mucosa were taken from excised tissue obtained during vaginal hysterectomies and various oral surgical procedures. Seven biopsy materials from each specimen were mounted in flow-through diffusion cells (exposed area, 0.039 cm2), and their permeability to 4.4- and 12-kd fluorescein-isothiocyanate-labeled dextrans was determined through use of a continuous flow-through perfusion system. Dextran was detected by means of a fluorospectrophotometric method at excitation and emission wave lengths of 498 and 520 nm, respectively. Specimens were examined histologically before and after permeability experiments, and similarities between vaginal and buccal tissues were verified. No statistically significant differences between the flux values of the 4.4-kd dextran across vaginal and buccal mucosa were found. However, for the 12-kd dextran the flux rate across buccal mucosa was significantly higher than the rate across vaginal mucosa. These results demonstrate that human vaginal mucosa is for practical purposes as permeable as buccal mucosa to 4.4-kd hydrophilic molecules. This further supports the hypothesis that vaginal mucosa may be a useful model for studying the passage across buccal mucosa of chemical compounds and therapeutic agents that are less than approximately 4.4 kd in molecular mass. For a 12-kd dextran the flux rate across buccal mucosa is significantly higher than the flux rate across vaginal mucosa, and the model becomes inaccurate.

Comparative diffusion of drugs through bronchial tissue

The purpose of the study was to investigate the molecular diffusion of drugs across porcine bronchial tissue. Using an in vitro flow-through diffusion system, a series of model compounds were tested. These included theophylline, caffeine, theobromine, enprofylline, salbutamol, ipratropium bromide, and trimethoprim. All drugs were assayed by HPLC in conjunction with UV/vis or MS/MS detection. The results indicated that the mean flux value of theophylline was higher than that of all the other drugs listed above. Within the log10P range from -2.21 (ipratropium bromide) to 1.364 (trimethoprim), a sigmoidal relationship was found to exist between the apparent permeability coefficients (Papp) and the octanol/water partition coefficients across the bronchial tissue. The diffusion of ipratropium bromide (Papp 1.6 x 10(-8)cm/s) across bronchial tissue was similar to that of salbutamol (Papp 1.5 x 10(-8)cm/s). The data obtained in this study indicate that although lipophilicity is a main determinant in the diffusion of drug compounds across bronchial tissue, the number and position of alkyl groups also reflect the ability of the latter to cross membrane barriers.

Penetration of benzo[a]pyrene through human buccal and vaginal mucosa.

Abstract Objective. The aim of this study was to compare buccal and vaginal mucosa with respect to their permeability to a potent carcinogen, benzo[a]pyrene. Study design. Six clinically healthy vaginal mucosa specimens (mean patient age ± standard deviation, 52 ± 13.4 years; age range, 37-69 years) and 6 buccal mucosa specimens (from 5 male patients and 1 female patient: mean patient age ± standard deviation, 32 ± 5.2 years; age range, 24-39 years) were obtained during surgery. In vitro flux rates of benzo[a]pyrene across specimens were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t test were used to determine steady state kinetics and flux differences over time intervals. Results. No statistically significant differences were observed between the overall mean flux values of benzo[a]pyrene across the 2 kinds of mucosa. Conclusions. The findings further support the hypothesis that human vaginal mucosa can be used as a model for buccal mucosa in studies of permeability to various chemical compounds. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:452-5)

Development of a Reproducible Procedure for Plasmid DNA Encapsulation by Red Blood Cell Ghosts

AbstractObjective: The binding and encapsulation of [3H] pGL3 luciferase reporter plasmid DNA by red blood cell (RBC) ghosts, intended as a vehicle for transfection and ultimately for gene therapy, were studied using two methods for DNA compaction. Methods and Results: In the first approach, DNA was compacted through binding electrostatically to poly-L-lysine. Complexes were constructed to have a slight negative charge. Experimentally, it was found that a high percentage of binding was to the outside of the resealed RBC ghosts.An alternative approach using polyethylene glycol6000 at a final concentration of 15% (weight/volume) was used to collapse [3H] pGL3 DNA in the presence of 0.025M MgCl2. Addition of the reagents, premixed with DNA, to a pelleted suspension of RBC ghosts followed by a short incubation and then addition of 1.5M NaCl to restore tonicity, resulted in resealing of the ghosts. Uptake of [3H] pGL3 DNA by the ghosts was approximately 20% of the input amount of DNA. Further work showed that 60–70% of the DNA was inside the resealed ghosts and largely present in the supercoiled form. At no stage was any freezing and thawing used. Conclusion: Transfection studies have demonstrated that pGL3 DNA carrying the luciferase gene is successfully transferred from RBC ghosts to recipient HeLa cells in culture under mild fusion conditions.

Permeability of human intestinal mucosa using a continuous flow-through perfusion system.

Continued interest in in vitro methods for performing bioavailability/bioequivalence (BA/BE) studies for drug registration purposes, prompted us to investigate the suitability of a continuous flow-through perfusion system to determine diffusion of a wide variety of permeants, through human intestinal mucosa. Permeability of fresh and frozen intestinal mucosa towards water, 17beta-estradiol, sumatriptan, arecoline and vasopressin was compared. Furthermore, diffusion studies of water, sumatriptan, arecoline, arecaidine, estradiol, cyclosporin and vasopressin across frozen/thawed intestinal mucosa specimens (-85 degrees C) were performed. No statistically significant differences between the flux values of the five compounds tested across fresh and frozen intestinal tissue, were found. Furthermore, it was demonstrated that the flux rates of the various compounds across these tissues decreased with increasing molecular size. However, the flux rates across frozen intestinal mucosa for compounds with molecular weights >300 Da, were low. Flux rates for the compounds studied across frozen/thawed human vaginal and buccal mucosa were 36-160% higher than those across frozen intestinal mucosa. We concluded that the continuous flow-through perfusion system used shows promise as an in vitro method for permeability determination through intestinal mucosa. However, other human mucosa e.g. vaginal mucosa, may have to be considered as alternatives to intestinal mucosa if therapeutic agents with molecular weights >500 Da are to be compared for in vitro BA/BE purposes, and further studies in this respect are warranted.

Redefining effusive-constrictive pericarditis with echocardiography

Background Effusive-constrictive pericarditis (ECP) is traditionally diagnosed by using the expensive and invasive technique of direct pressure measurements in the pericardial space and the right atrium. The aim of this study was to assess the diagnostic role of echocardiography in tuberculous ECP. Methods Intrapericardial and right atrial pressures were measured pre- and post-pericardiocentesis, and right ventricular and left ventricular pressures were measured post-pericardiocentesis in patients with tuberculous pericardial effusions. Echocardiography was performed post-pericardiocentesis. Traditional, pressure-based diagnostic criteria were compared with post-pericardiocentesis systolic discordance and echocardiographic evidence of constriction. Results Thirty-two patients with tuberculous pericardial disease were included. Sixteen had ventricular discordance (invasively measured), 16 had ECP as measured by intrapericardial and right atrial invasive pressure measurements and 17 had ECP determined echocardiographically. The sensitivity and specificity of pressure-guided measurements (compared with discordance) for the diagnosis of ECP were both 56%. The positive and negative predictive values were both 56%. The sensitivity of echocardiography (compared with discordance) for the diagnosis of ECP was 81% and the specificity 75%, while the positive and the negative predictive values were 76% and 80%, respectively. Conclusion Echocardiography shows a better diagnostic performance than invasive, pressure-based measurements for the diagnosis of ECP when both these techniques are compared with the gold standard of invasively measured systolic discordance.

QTc prolongation prior to angiography predicts poor outcome and associates significantly with lower left ventricular ejection fractions and higher left ventricular end-diastolic pressures

Background QT prolongation on the surface ECG is associated with sudden cardiac death. The cause of QT prolongation in ischaemic heart disease (IHD) patients remains unknown, but may be due to a complex interplay between genetic factors and impaired systolic and/or diastolic function through as yet unexplained mechanisms. It was hypothesised that QT prolongation before elective coronary angiography is associated with an increased mortality at six months. Methods Complete records of 321 patients who underwent coronary angiography were examined for QT interval corrected for heart rate (QTc), left ventricular ejection fraction (LVEF), left ventricular end-diastolic pressure (LVEDP) and known ischaemic heart disease risk factors. Patients were designated long QTc (LQTc) when they had prolonged QTc intervals or normal QTc (NQTc) when the QTc interval was normal. Patients with atrial fibrillation, bundle branch blocks, no ECG in the 24 hours before angiography, or a creatinine level > 200 μmol/l were excluded. Survival was determined telephonically at six months. Results Twenty-eight per cent of the total population had LQTc. During follow up, 15 patients (4.7%) died suddenly, 73% of whom had a LQTc. LQTc was significantly associated with mortality (LQTc 12% vs NQTc 1.7%; p < 0.01), and with lower but normal LVEF (LQTc 52.9 ± 15.4% vs NQTc 61.6 ± 13.6%; p < 0.01), higher LVEDP at LVEF > 45% (LQTc 19.2 ± 9.0 mmHg vs NQTc 15.95 ± 7.5 mmHg; p < 0.05), hypercholesterolaemia and a negative family history of IHD. Conclusion In patients with sinus rhythm and normal QRS width, QTc prolongation before coronary angiography predicted increased mortality at six months. QTc also associated strongly with left ventricular systolic and diastolic dysfunction, hypercholesterolaemia and a negative family history of IHD.