Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Substrate- and species-dependent inhibition of P-glycoprotein-mediated transport: implications for predicting in vivo drug interactions.

P-glycoprotein (P-gp)-based drug interactions are a major concern in the clinic and in preclinical drug development, especially with respect to the intestinal absorption of drugs and distribution of drugs across the blood-brain barrier. Thus, there is significant interest in developing in vitro (e.g., cell culture) and in vivo models (e.g., rodents) to predict such interactions. In order to generate accurate predictions from these models, however, an understanding of the magnitude of substrate- and species-dependent differences in P-gp inhibition is required. We have used a sensitive flow cytometry assay to measure the ability of various drugs to inhibit the initial rate of accumulation of two fluorescent drug analogs (probe substrates), 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -indacene (BODIPY)-verapamil and BODIPY-prazosin, into Lewis lung carcinoma-porcine kidney 1 (LLC-PK1) cells expressing human or rat P-gp. The inhibition of P-gp-mediated efflux of these two fluorescent substrates by several drugs, including quinidine and itraconazole, was found to be substrate- and/or species-dependent. These data suggest that to provide accurate prediction of clinically significant P-gp drug interactions, multiple P-gp substrates will need to be used in both in vitro and in vivo (including human) drug interaction studies. In addition, extrapolation of P-gp-based drug interaction in rodents to humans must be conducted with caution.

The mechanism of action of antidepressants: a unitary hypothesis based on transport-p.

Endogenous depression is a common mental illness which is associated with significant morbidity and mortality. Tricyclic antidepressants and their newer derivatives are the main treatment for this disease. However, there are serious deficiencies in the use of existing antidepressants for the treatment of depressive illness. An obstacle in the development of better antidepressants is that the mechanism of the therapeutic action of these compounds is unknown. The prevailing view is that antidepressants exert their therapeutic effect by inhibiting the pre-synaptic re-uptake of the neurotransmitter amines, noradrenaline and serotonin. However, there are objections to this hypothesis. Transport-P is a new factor in this field; it is an antidepressant-sensitive, proton-dependent, V-ATPase linked uptake process for amines in peptidergic neurones. It differs from other uptake processes in its anatomical location in post-synaptic (peptidergic) neurones, in its functional properties and in the structure of its ligands. Therapeutic concentrations of antidepressants are active at Transport-P. This review describes a hypothesis which postulates that antidepressants exert a therapeutic effect by an action on Transport-P [1]. According to this hypothesis, Transport-P accumulates antidepressants in acidified vesicles in post-synaptic neurones. The normal function of the vesicles is to degrade internalised post-synaptic receptors. As their amine groups are basic, the antidepressants tend to neutralise the acidity of the vesicles. This slows the rate of degradation of post-synaptic receptors, and makes post-synaptic neurones more responsive to the excitatory actions of neurotransmitter amines. This hypothesis resolves the problems with the pre-synaptic re-uptake hypothesis and offers a unitary explanation for hitherto inexplicable observations. If the hypothesis is correct, compounds which act as potent and selective ligands for Transport-P would have a more rapid onset of action and would represent an advance in the treatment of depressive illness. The data on Transport-P which are described in this article are derived entirely from the work of the author who is not aware of any other research groups working on Transport-P. Therefore, the amount of work which has been done so far is relatively limited. The evidence on which the hypothesis is based is derived from work on alpha(1) adrenoceptors in hypothalamic, peptidergic neurones. There are large gaps in the evidence which would be required to support a mechanistic hypothesis: for example, the serotonergic system, which is likely to be involved in depressive illness, has not been investigated. Further, no attempt has been made so far to address the applicability of the phenomena which were observed in the hypothalamus to other brain regions which may be involved in depressive illness. Nevertheless, the hypothesis, as it stands at present, appears to solve problems which have been inexplicable on the basis of the pre-synaptic re-uptake hypothesis. More work is required to determine the validity of the solutions which are proposed in this review.

Terazosin, doxazosin, and prazosin: current clinical experience.

Lower urinary tract symptoms (LUTS) suggestive of benign prostatic obstruction are common in aging men. Nearly 25% of men >40 years of age have LUTS. Medical therapy with alpha-blockade is the most common method of medical therapy for benign prostatic obstruction. Multiple methods of minimally invasive surgical therapies have been introduced in the last decade. These methods include balloon dilatation, temporary and permanent urethral stents, various laser techniques, microwave thermotherapy, transurethral needle ablation, electrovaporization, and high-intensity focused ultrasound. alpha-Receptor blockers to reduce the sympathetic tone of the prostate are considered as first-line therapy to relieve the symptoms of benign prostatic hyperplasia. Selective alpha(1)-receptor blockers relax prostatic smooth muscle, relieve bladder outlet obstruction, and enhance urine flow with fewer side effects. In addition, it was determined that treating patients with alpha-blockers increases prostatic apoptosis. Pharmacokinetic activity, mode of action, clinical efficacy, and side effects of the selective alpha(1)-receptor blockers terazosin, doxazosin, and prazosin are reviewed.

Combination of nifedipine and doxazosin in essential hypertension.

Pharmacodynamic and pharmacokinetic interactions have been reported when an alpha 1-antagonist is combined with a calcium antagonist. We evaluated the clinical usefulness of the combination of nifedipine (20 mg twice daily, b.i.d.) and doxazosin (2 mg once daily, o.d.) in hypertensive patients in whom blood pressure (BP) control was suboptimal after doxazosin (group A) or nifedipine (group B) as monotherapy and investigated the underlying kinetic and dynamic interactions, including changes in vascular responsiveness to i.v. infusions of angiotensin II (ANGII) and phenylephrine (PE). The combination was well tolerated and associated with further significant reductions in BP. After 4 weeks of combined therapy, average supine BP over 8 h was 122/77 in group A and 137/80 in group B as compared with 140/86 and 150/88 mm Hg, respectively, during monotherapy + placebo. The combination attenuated both phenylephrine and ANG-induced pressor responses: e.g., the mean PD15 values (dose of agonist required to increase systolic BP by 15 mm Hg) for group A at 1.5-3 h were 3.5 micrograms/kg/min for PE and 7.5 ng/kg/min for ANGII as compared with 2.9 and 2.3, respectively, during treatment with doxazosin and placebo. There was no evidence of a significant kinetic interaction between the two drugs and, in particular, addition of nifedipine had no effect on the steady-state kinetics of doxazosin. In conclusion, doxazosin and nifedipine are an effective antihypertensive combination in patients who require treatment with more than one drug.

Effect of pregnancy and sex steroids on alpha 1-adrenoceptor mechanisms in the guinea-pig uterine vascular bed.

Adrenoceptor mechanisms in the extrinsic uterine arteries from late pregnant guinea-pigs were characterized pharmacologically and compared with contractile responses of uterine arteries from non-pregnant and oophorectomized control, progesterone and oestrogen treated animals. Pregnancy caused an increase in diameter of the arteries, more pronounced the more distal they were. There was no change in potassium-evoked maximum contractions during pregnancy. Noradrenaline (10 nM to 1 mM), in the presence of cocaine (1 microM) and propranolol (0.1 microM), induced concentration-dependent contractions of the arterial segments, with approximately similar pD2 values. The maximum responses (Emax) were significantly increased during pregnancy and hormone supplementation. Prazosin (10 nM to 1 microM), but not rauwolscine (10 nM to 1 microM), antagonized noradrenaline-evoked contractions of the arteries. Isoprenaline (10 nM to 1 microM), in the presence of prazosin (0.1 microM) and cocaine (1 microM), had no relaxant effect on arteries contracted submaximally by prostaglandin F2a (5 microM). Neither cocaine nor normetanephrine modified noradrenaline-evoked contractions of the uterine artery. The results indicate that guinea-pig uterine vasoconstriction is mediated by alpha 1-adrenoceptors while relaxant beta-adrenoceptor effects, neuronal and extraneuronal uptake mechanisms are of minor importance. The observed increase in Emax may be due either to an increase in the number of alpha 1-adrenoceptors or to an enhanced pharmaco-mechanical coupling, which conceivably is regulated by sex steroids since this response was reproduced in castrated animals by hormone supplementation.