Serotonin and Pulmonary Hypertension; Sex and Drugs and ROCK and Rho.

Serotonin is often referred to as a "happy hormone" as it maintains good mood, well-being, and happiness. It is involved in communication between nerve cells and plays a role in sleeping and digestion. However, too much serotonin can have pathogenic effects and serotonin synthesis is elevated in pulmonary artery endothelial cells from patients with pulmonary arterial hypertension (PAH). PAH is characterized by elevated pulmonary pressures, right ventricular failure, inflammation, and pulmonary vascular remodeling; serotonin has been shown to be associated with these pathologies. The rate-limiting enzyme in the synthesis of serotonin in the periphery of the body is tryptophan hydroxylase 1 (TPH1). TPH1 expression and serotonin synthesis are elevated in pulmonary artery endothelial cells in patients with PAH. The serotonin synthesized in the pulmonary arterial endothelium can act on the adjacent pulmonary arterial smooth muscle cells (PASMCs), adventitial macrophages, and fibroblasts, in a paracrine fashion. In humans, serotonin enters PASMCs cells via the serotonin transporter (SERT) and it can cooperate with the 5-HT1B receptor on the plasma membrane; this activates both contractile and proliferative signaling pathways. The "serotonin hypothesis of pulmonary hypertension" arose when serotonin was associated with PAH induced by diet pills such as fenfluramine, aminorex, and chlorphentermine; these act as indirect serotonergic agonists causing the release of serotonin from platelets and cells through the SERT. Here the role of serotonin in PAH is reviewed. Targeting serotonin synthesis or signaling is a promising novel alternative approach which may lead to novel therapies for PAH. © 2022 American Physiological Society. Compr Physiol 12: 1-16, 2022.

MSG-10: a Phase 2 study of oral ibrexafungerp (SCY-078) following initial echinocandin therapy in non-neutropenic patients with invasive candidiasis.

OBJECTIVES: To evaluate the safety and efficacy of two dosing regimens of oral ibrexafungerp (formerly SCY-078), a novel orally bioavailable ß-glucan synthase inhibitor, in subjects with invasive candidiasis versus the standard of care (SOC) and to identify the dose to achieve target exposure (15.4 µM·h) in >80% of the intended population. METHODS: In a multinational, open-label study, patients with documented invasive candidiasis were randomized to receive step-down therapy to one of three treatment arms: two dosing regimens of novel oral ibrexafungerp or the SOC treatment following initial echinocandin therapy. Plasma samples were collected to evaluate exposure by population pharmacokinetic (PK) modelling. Safety was assessed throughout the study and global response at the end of treatment. RESULTS: Out of 27 subjects enrolled, 7 received ibrexafungerp 500 mg, 7 received ibrexafungerp 750 mg and 8 received the SOC. Five did not meet criteria for randomization. Population PK analysis indicated that an ibrexafungerp 750 mg regimen is predicted to achieve the target exposure in ∼85% of the population. The rate of adverse events was similar among patients receiving ibrexafungerp or fluconazole. Similar favourable response rates were reported among all groups: 86% (n = 6) in the ibrexafungerp 750 mg versus 71% (n = 5) in both the fluconazole and ibrexafungerp 500 mg treatment arms. The one subject treated with continued micafungin had a favourable global response. CONCLUSIONS: The oral ibrexafungerp dose estimated to achieve the target exposure in subjects with invasive candidiasis is 750 mg daily. This dose was well tolerated and achieved a favourable global response rate, similar to the SOC.

Topical Treatment for Cutaneous Leishmaniasis: Dermato-Pharmacokinetic Lead Optimization of Benzoxaboroles.

Cutaneous leishmaniasis (CL) is caused by several species of the protozoan parasite Leishmania, affecting an estimated 10 million people worldwide. Previously reported strategies for the development of topical CL treatments have focused primarily on drug permeation and formulation optimization as the means to increase treatment efficacy. Our approach aims to identify compounds with antileishmanial activity and properties consistent with topical administration. Of the test compounds, five benzoxaboroles showed potent activity (50% effective concentration [EC50] < 5 µM) against intracellular amastigotes of at least one Leishmania species and acceptable activity (20 µM < EC50 < 30 µM) against two more species. Benzoxaborole compounds were further prioritized on the basis of the in vitro evaluation of progression criteria related to skin permeation, such as the partition coefficient and solubility. An MDCKII-hMDR1 cell assay showed overall good permeability and no significant interaction with the P-glycoprotein transporter for all substrates except LSH002 and LSH031. The benzoxaboroles were degraded, to some extent, by skin enzymes but had stability superior to that of para-hydroxybenzoate compounds, which are known skin esterase substrates. Evaluation of permeation through reconstructed human epidermis showed LSH002 to be the most permeant, followed by LSH003 and LSH001. Skin disposition studies following finite drug formulation application to mouse skin demonstrated the highest permeation for LSH001, followed by LSH003 and LSH002, with a significantly larger amount of LSH001 than the other compounds being retained in skin. Finally, the efficacy of the leads (LSH001, LSH002, and LSH003) against Leishmania major was tested in vivo LSH001 suppressed lesion growth upon topical application, and LSH003 reduced the lesion size following oral administration.

Assessment of a pretomanid analogue library for African trypanosomiasis: Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides.

A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.

Species-Selective Pyrimidineamine Inhibitors of Trypanosoma brucei S-Adenosylmethionine Decarboxylase.

New therapeutic options are needed for treatment of human African trypanosomiasis (HAT) caused by protozoan parasite Trypanosoma brucei. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the polyamine pathway of T. brucei. Previous attempts to target this enzyme were thwarted by the lack of brain penetration of the most advanced series. Herein, we describe a T. brucei AdoMetDC inhibitor series based on a pyrimidineamine pharmacophore that we identified by target-based high-throughput screening. The pyrimidineamines showed selectivity for T. brucei AdoMetDC over the human enzyme, inhibited parasite growth in whole-cell assay, and had good predicted blood-brain barrier penetration. The medicinal chemistry program elucidated structure-activity relationships within the series. Features of the series that were required for binding were revealed by determining the X-ray crystal structure of TbAdoMetDC bound to one analog. The pyrimidineamine series provides a novel starting point for an anti-HAT lead optimization.

Synthesis and evaluation of analogs of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811, or AbeAdo) - An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity.

We describe our efforts to improve the pharmacokinetic properties of a mechanism-based suicide inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), essential for the survival of the eukaryotic parasite Trypanosoma brucei responsible for Human African Trypanosomiasis (HAT). The lead compound, 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (1, also known as MDL 73811, or AbeAdo), has curative efficacy at a low dosage in a hemolymphatic model of HAT but displayed no demonstrable effect in a mouse model of the CNS stage of HAT due to poor blood-brain barrier permeation. Therefore, we prepared and evaluated an extensive set of analogs with modifications in the aminobutenyl side chain, the 5'-amine, the ribose, and the purine fragments. Although we gained valuable structure-activity insights from this comprehensive dataset, we did not gain traction on improving the prospects for CNS penetration while retaining the potent antiparasitic activity and metabolic stability of the lead compound 1.

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay.

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (Tb). Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.

Preclinical Pharmacokinetics and Pharmacodynamic Target of SCY-078, a First-in-Class Orally Active Antifungal Glucan Synthesis Inhibitor, in Murine Models of Disseminated Candidiasis.

SCY-078 (MK-3118) is a novel, semisynthetic derivative of enfumafungin and represents the first compound of the triterpene class of antifungals. SCY-078 exhibits potent inhibition of ß-(1,3)-d-glucan synthesis, an essential cell wall component of many pathogenic fungi, including Candida spp. and Aspergillus spp. SCY-078 is currently in phase 2 clinical development for the treatment of invasive fungal diseases. In vitro disposition studies to assess solubility, intestinal permeability, and metabolic stability were predictive of good oral bioavailability. Preclinical pharmacokinetic studies were consistent with once-daily administration to humans. After intravenous delivery, plasma clearance in rodents and dogs was low, representing <15% and <25% of hepatic blood flow, respectively. The terminal elimination-phase half-life was 5.5 to 8.7 h in rodents, and it was ∼9.3 h in dogs. The volume of distribution at steady-state was high (4.7 to 5.3 liters/kg), a finding suggestive of extensive tissue distribution. Exposure of SCY-078 in kidney tissue, a target organ for invasive fungal disease such as candidiasis, exceeded plasma by 20- to 25-fold for the area under the concentration-time curve from 0 h to infinity (AUC0-∞) and Cmax SCY-078 achieved efficacy endpoints following oral delivery across multiple murine models of disseminated candidiasis. The pharmacokinetic/pharmacodynamic indices Cmax/MIC and AUC/MIC correlated with outcome. Target therapeutic exposure, expressed as the plasma AUC0-24, was comparable across models, with an upper value of 11.2 µg·h/ml (15.4 µM·h); the corresponding mean value for free drug AUC/MIC was ∼0.75. Overall, these results demonstrate that SCY-078 has the oral and intravenous (i.v.) pharmacokinetic properties and potency in murine infection models of disseminated candidiasis to support further investigation as a novel i.v. and oral treatment for invasive fungal diseases.

Benzoxaboroles: a new class of potential drugs for human African trypanosomiasis.

Human African trypanosomiasis, caused by the kinetoplastid parasite Trypanosoma brucei, affects thousands of people across sub-Saharan Africa, and is fatal if left untreated. Treatment options for this disease, particularly stage 2 disease, which occurs after parasites have infected brain tissue, are limited due to inadequate efficacy, toxicity and the complexity of treatment regimens. We have discovered and optimized a series of benzoxaborole-6-carboxamides to provide trypanocidal compounds that are orally active in murine models of human African trypanosomiasis. A key feature of this series is the presence of a boron atom in the heterocyclic core structure, which is essential to the observed trypanocidal activity. We also report the in vivo pharmacokinetic properties of lead compounds from the series and selection of SCYX-7158 as a preclinical candidate.

SCYX-7158, an orally-active benzoxaborole for the treatment of stage 2 human African trypanosomiasis.

BACKGROUND: Human African trypanosomiasis (HAT) is an important public health problem in sub-Saharan Africa, affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. We have discovered and optimized a novel class of small-molecule boron-containing compounds, benzoxaboroles, to identify SCYX-7158 as an effective, safe and orally active treatment for HAT. METHODOLOGY/PRINCIPAL FINDINGS: A drug discovery project employing integrated biological screening, medicinal chemistry and pharmacokinetic characterization identified SCYX-7158 as an optimized analog, as it is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense, is efficacious in both stage 1 and stage 2 murine HAT models and has physicochemical and in vitro absorption, distribution, metabolism, elimination and toxicology (ADMET) properties consistent with the compound being orally available, metabolically stable and CNS permeable. In a murine stage 2 study, SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD×7 days). In vivo pharmacokinetic characterization of SCYX-7158 demonstrates that the compound is highly bioavailable in rodents and non-human primates, has low intravenous plasma clearance and has a 24-h elimination half-life and a volume of distribution that indicate good tissue distribution. Most importantly, in rodents brain exposure of SCYX-7158 is high, with C(max) >10 µg/mL and AUC(0-24 hr) >100 µg*h/mL following a 25 mg/kg oral dose. Furthermore, SCYX-7158 readily distributes into cerebrospinal fluid to achieve therapeutically relevant concentrations in this compartment. CONCLUSIONS/SIGNIFICANCE: The biological and pharmacokinetic properties of SCYX-7158 suggest that this compound will be efficacious and safe to treat stage 2 HAT. SCYX-7158 has been selected to enter preclinical studies, with expected progression to phase 1 clinical trials in 2011.

SAR of 2-amino and 2,4-diamino pyrimidines with in vivo efficacy against Trypanosoma brucei.

A series of 2,4-diaminopyrimidines was investigated and compounds were found to have in vivo efficacy against Trypanosoma brucei in an acute mouse model. However, in vitro permeability data suggested the 2,4-diaminopyrimidenes would have poor permeability through the blood brain barrier. Consequently a series of 4-desamino analogs were synthesized and found to have improved in vitro permeability.

Method to screen substrates of apical sodium-dependent bile acid transporter.

Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.

Enfuvirtide cerebrospinal fluid (CSF) pharmacokinetics and potential use in defining CSF HIV-1 origin.

BACKGROUND: Enfuvirtide is a potent inhibitor of systemic HIV-1 replication, but its penetration into the human central nervous system (CNS) has not been analysed. Here, we define cerebrospinal fluid (CSF) enfuvirtide pharmacokinetics and present a case illustrating the use of enfuvirtide as a probe to trace the origins of CSF HIV-1 quasispecies. METHODS: Enfuvirtide CSF pharmacokinetics were assessed in 18 CSF and plasma sample pairs from four HIV-1-infected individuals. Enfuvirtide levels were measured by liquid chromatography tandem mass spectrometry using known standards and controls that included spiked CSF samples from untreated, HIV-negative individuals. A segment of the gp41 coding region encompassing the heptad repeat HR-1 and HR-2 domains was amplified from selected CSF and plasma samples and independent clones sequenced to assess resistance-associated mutations. RESULTS: CSF and plasma samples obtained between 2 and 20 h after enfuvirtide injection showed plasma concentrations similar to previous reports (mean 3.687 SD +/- 1.828 mg/ml) with prolonged decay. By contrast, enfuvirtide in all CSF samples was below the assay detection limit of 0.025 mg/ml. In one individual, who developed a transient increase in CSF HIV-1 RNA, seven of seven CSF and plasma clones had identical enfuvirtide resistance-associated V38A mutations, suggesting that the CSF quasispecies derived from that of blood. CONCLUSIONS: Enfuvirtide penetration into CSF is negligible; thus, in clinical settings, where direct CNS drug exposure is crucial, this drug Is not likely to directly contribute to the local therapeutic effect. Enfuvirtide can be used as a tool to dissect the origin of the CNS virus.

Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus.

Enfuvirtide (ENF), the first approved fusion inhibitor (FI) for HIV, is a 36-aa peptide that acts by binding to the heptad repeat 1 (HR1) region of gp41 and preventing the interaction of the HR1 and HR2 domains, which is required for virus-cell fusion. Treatment-acquired resistance to ENF highlights the need to create FI therapeutics with activity against ENF-resistant viruses and improved durability. Using rational design, we have made a series of oligomeric HR2 peptides with increased helical structure and with exceptionally high HR1/HR2 bundle stability. The engineered peptides are found to be as much as 3,600-fold more active than ENF against viruses that are resistant to the HR2 peptides ENF, T-1249, or T-651. Passaging experiments using one of these peptides could not generate virus with decreased sensitivity, even after >70 days in culture, suggesting superior durability as compared with ENF. In addition, the pharmacokinetic properties of the engineered peptides were improved up to 100-fold. The potent antiviral activity against resistant viruses, the difficulty in generating resistant virus, and the extended half-life in vivo make this class of fusion inhibitor peptide attractive for further development.

Interaction of native bile acids with human apical sodium-dependent bile acid transporter (hASBT): influence of steroidal hydroxylation pattern and C-24 conjugation.

PURPOSE: The human apical sodium-dependent bile acid transporter (hASBT) is a potential target for drug delivery, but an understanding of hASBT substrate requirements is lacking. The objective of this study was to characterize hASBT interaction with its native substrates, bile acids, and to evaluate C-24 conjugation and steroidal hydroxylation on transport affinity and inhibition potency. METHODS: Transport and inhibition kinetics of 15 bile acids were evaluated (cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate, and lithocholate, including their glycine and taurine conjugates) with an hASBT-Madin-Darby canine kidney (MDCK) monolayer assay. Samples were analyzed via liquid chromatography-mass spectrometry (LC-MS) or chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). RESULTS: C-24 conjugation improved the inhibitory potency of all native bile acids. There was an inverse association between number of steroidal hydroxyl groups and inhibitory potency. Glycolithocholate and taurolithocholate were the most potent inhibitors. Results from transport studies followed trends from inhibition studies. Conjugated dihydroxy and monohydroxy bile acids exhibited the highest hASBT-mediated transport (i.e., lower Kt and higher Jmax). Across the 15 bile acids, Kt generally followed Ki. Additionally, Jmax correlated with Ki, where greater inhibition potency was associated with higher transport capacity. CONCLUSION: C-24 conjugation and steroidal hydroxylation pattern modulated native bile acid interaction with hASBT, with C-24 effect dominating steroidal hydroxylation effect. Results indicate that bile acid binding to hASBT may be the rate-limiting step in the apical transport of bile acids.

Influence of charge and steric bulk in the C-24 region on the interaction of bile acids with human apical sodium-dependent bile acid transporter.

The human apical sodium-dependent bile acid transporter (hASBT) is a potential target for drug delivery, but an understanding of hASBT substrate requirements is limited. The objective of this study was to evaluate the influence of ionic character and steric bulk in the C-24 region of bile acid conjugates in governing interaction with hASBT. Ionic character was studied using chenodeoxycholate (CDCA) conjugates of glutamic acid and lysine, which varied in charge (monoanionic, dianionic, cationic, neutral, and zwitterionic) and location of charge (proximal or distal to C-24). Steric effects were evaluated using ester conjugates that varied in ester substituent size (methyl, benzyl, and tert-butyl) and location (proximal and/or distal). Conjugate interaction with hASBT was assessed via transport and inhibition studies, using a hASBT-MDCK monolayer. Monoanionic, cationic, and neutral conjugates of CDCA exhibited high inhibitory potency (Ki<10 microM). High inhibition potency of neutral and cationic conjugates indicated that a negative charge is not essential for hASBT binding. Dianionic conjugates exhibited low inhibition potency (Ki>100 microM). Conjugates with a single bulky ester substituent proximal or distal to the C-24 region exhibited high inhibition potency. However, two bulky substituents practically abolished interaction. In transport studies, monoanionic conjugates were high affinity hASBT substrates. Meanwhile, cationic and zwitterionic conjugates were not substrates for hASBT. Overall, C-24 ionic character influenced interaction with hASBT. Although the presence of a single negative charge was not essential for interaction with hASBT, monoanionic conjugates were favored for hASBT-mediated transport compared to cationic and zwitterionic conjugates.

Enfuvirtide plasma levels and injection site reactions using a needle-free gas-powered injection system (Biojector).

OBJECTIVES: To assess the use of the Biojector B2000 needle-free gas-powered injection system for subcutaneous administration of enfuvirtide in HIV-infected patients and to compare this system with standard needles and syringes with respect to ease of use, severity of injection site reactions (ISR), and enfuvirtide plasma levels. DESIGN: An observational study among 32 treatment-experienced HIV clinic patients receiving enfuvirtide. METHODS: Adult patients were assessed before and after switching from standard needles to the Biojector for enfuvirtide administration. Patients used the Biojector for up to 24 weeks and rated ease of use from 0 (easy) to 3 (difficult). ISR were graded from 0 to 31 for signs and symptoms (erythema, induration, pruritus, nodules/cysts, ecchymosis), duration of individual lesions, and number of lesions. Plasma was collected pre-dose and 1 h post-dose for enfuvirtide measurement. The high-pressure liquid chromatography with tandem mass spectrometry method used was specific for enfuvirtide over its known plasma metabolite. Wilcoxon rank sum tests were used to compare needle-based and Biojector outcomes. RESULTS: The Biojector was rated as being significantly easier to use (P < 0.001) and reduced the occurrence of ISR compared with standard needles (P < 0.001). Enfuvirtide plasma levels were not statistically different between the two administration methods at either pre-dose trough (P = 0.41) or 1 h post-dose (P = 0.74). CONCLUSIONS: The Biojector needle-free injection system was easy to use for enfuvirtide administration and was associated with a decreased severity of ISR. Plasma enfuvirtide levels pre-dose and 1 h post-dose were comparable when injecting with standard needles or the Biojector.

Steady-state brain concentrations of antihistamines in rats: interplay of membrane permeability, P-glycoprotein efflux and plasma protein binding.

The purpose of this study was to measure the in vivo brain distribution of antihistamines and assess the influence of in vitro permeability, P-glycoprotein (Pgp) efflux, and plasma protein binding. Six antihistamines (acrivastine, chlorpheniramine, diphenhydramine doxylamine, fexofenadine, terfenadine) were selected based on previously reported in vitro permeability and Pgp efflux properties and dosed intravenously to steady-state plasma concentrations of 2-10 micromol/l in rats. Plasma and brain concentrations were measured by LC/MS/MS, and protein binding determined by ultrafiltration. Doxylamine, diphenhydramine and chlorpheniramine had brain-to-plasma concentration ratios of 4.34 +/- 1.26, 18.4 +/- 2.35 and 34.0 +/- 9.02, respectively. These drugs had high passive membrane permeability (>310 nm/s), moderate protein binding (71-84%) and were not Pgp substrates; features that yield high CNS penetration. In contrast, acrivastine and fexofenadine had low brain-to-plasma ratios of 0.072 +/- 0.014 and 0.018 + 0.002, consistent with low passive membrane permeability for both compounds (16.2 and 66 nm/s, respectively) and Pgp efflux. Finally, terfenadine had a brain-to-plasma ratio of 2.21 +/- 1.00 even though it underwent Pgp-mediated efflux (in vitro ratio = 2.88). Terfenadine's high passive permeability (285 nm/s) overcame the Pgp-mediated efflux to yield brain-to-plasma ratio >1. The brain-to-unbound plasma ratio was 22-fold higher suggesting that protein binding (96.3% bound) limited terfenadine's brain distribution. In conclusion, passive membrane permeability, Pgp-mediated efflux and/or high plasma protein binding influence the in vivo brain distribution of antihistamine drugs.

Predicting P-glycoprotein substrates by a quantitative structure-activity relationship model.

A quantitative structure-activity relationship (QSAR) model has been developed to predict whether a given compound is a P-glycoprotein (Pgp) substrate or not. The training set consisted of 95 compounds classified as substrates or non-substrates based on the results from in vitro monolayer efflux assays. The two-group linear discriminant model uses 27 statistically significant, information-rich structure quantifiers to compute the probability of a given structure to be a Pgp substrate. Analysis of the descriptors revealed that the ability to partition into membranes, molecular bulk, and the counts and electrotopological values of certain isolated and bonded hydrides are important structural attributes of substrates. The model fits the data with sensitivity of 100% and specificity of 90.6% in the jackknifed cross-validation test. A prediction accuracy of 86.2% was obtained on a test set of 58 compounds. Examination of the eight "mispredicted" compounds revealed two distinct categories. Five mispredictions were explained by experimental limitations of the efflux assay; these compounds had high permeability and/or were inhibitors of calcein-AM transport. Three mispredictions were due to limitations of the chemical space covered by the current model. The Pgp QSAR model provides an in silico screen to aid in compound selection and in vitro efflux assay prioritization.

Passive permeability and P-glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs.

Membrane permeability and P-glycoprotein (Pgp) can be limiting factors for blood-brain barrier penetration. The objectives of this study were to determine whether there are differences in the in vitro permeability, Pgp substrate profiles, and physicochemical properties of drugs for central nervous system (CNS) and non-CNS indications, and whether these differences are useful criteria in selecting compounds for drug development. Apparent permeability (P(app)) and Pgp substrate profiles for 93 CNS (n = 48) and non-CNS (n = 45) drugs were determined by monolayer efflux. Calcein-AM inhibition assays were used to supplement the efflux results. The CNS set (2 of 48, 4.2%) had a 7-fold lower incidence of passive permeability values <150 nm/s compared with the non-CNS set (13 of 45, 28.9%). The majority of drugs (72.0%, 67 of 93) were not Pgp substrates; however, 49.5% (46 of 93) were positive in the calcein-AM assay when tested at 100 microM. The CNS drug set (n = 7 of 48, 14.6%) had a 3-fold lower incidence of Pgp-mediated efflux than the non-CNS drug set (n = 19 of 45, 42.2%). Analysis of 18 physicochemical properties revealed that the CNS drug set had fewer hydrogen bond donors, fewer positive charges, greater lipophilicity, lower polar surface area, and reduced flexibility compared with the non-CNS group (p < 0.05), properties that enhance membrane permeability. This study on a large, diverse set of marketed compounds clearly demonstrates that permeability, Pgp-mediated efflux, and certain physicochemical properties are factors that differentiate CNS and non-CNS drugs. For CNS delivery, a drug should ideally have an in vitro passive permeability >150 nm/s and not be a good (B --> A/A --> B ratio <2.5) Pgp substrate.