Amides as modifiable directing groups in electrophilic borylation.

Amide directed C-H borylation using ≥two equiv. of BBr3 forms borenium cations containing a R2N(R')C[double bond, length as m-dash]O→B(Ar)Br unit which has significant Lewis acidity at the carbonyl carbon. This enables reduction of the amide unit to an amine using hydrosilanes. This approach can be applied sequentially in a one-pot electrophilic borylation-reduction process, which for phenyl-acetylamides generates ortho borylated compounds that can be directly oxidised to the 2-(2-aminoethyl)-phenol. Other substrates amenable to the C-H borylation-reduction sequence include mono and diamino-arenes and carbazoles. This represents a simple method to make borylated molecules that would be convoluted to access otherwise (e.g. N-octyl-1-BPin-carbazole). Substituent variation is tolerated at boron as well as in the amide unit, with diarylborenium cations also amenable to reduction. This enables a double C-H borylation-reduction-hydrolysis sequence to access B,N-polycyclic aromatic hydrocarbons (PAHs), including an example where both the boron and nitrogen centres contain functionalisable handles (N-H and B-OH). This method is therefore a useful addition to the metal-free borylation toolbox for accessing useful intermediates (ArylBPin) and novel B,N-PAHs.

Synthesis of Electrophiles Derived from Dimeric Aminoboranes and Assessing Their Utility in the Borylation of π Nucleophiles.

Dimeric aminoboranes, [H2BNR2]2 (R = Me or CH2CH2) containing B2N2 cores, can be activated by I2, HNTf2 (NTf2 = [N(SO2CF3)2]), or [Ph3C][B(C6F5)4] to form isolable H2B(µ-NR2)2BHX (for X = I or NTf2). For X = [B(C6F5)4]- further reactivity, presumably between [H2B(µ-NMe2)2BH][B(C6F5)4] and aminoborane, forms a B3N3-based monocation containing a three-center two electron B-(µ-H)-B moiety. The structures of H2B(µ-NMe2)2BH(I) and [(µ-NMe2)BH(NTf2)]2 indicated a sterically crowded environment around boron, and this leads to the less common O-bound mode of NTf2 binding. While the iodide congener reacted very slowly with alkynes, the NTf2 analogues were more reactive, with hydroboration of internal alkynes forming (vinyl)2BNR2 species and R2NBH(NTf2) as the major products. Further studies indicated that the B2N2 core is maintained during the first hydroboration, and that it is during subsequent steps that B2N2 dissociation occurs. In the mono-boron systems, for example, i Pr2NBH(NTf2), NTf2 is N-bound; thus, they have less steric crowding around boron relative to the B2N2 systems. Notably, the monoboron systems are much less reactive in alkyne hydroboration than the B2N2-based bis-boranes, despite the former being three coordinate at boron while the latter are four coordinate at boron. Finally, these B2N2 electrophiles are much more prone to dissociate into mono-borane species than pyrazabole [H2B(µ-N2C3H3)]2 analogues, making them less useful for the directed diborylation of a single substrate.

Two Directing Groups Used for Metal Catalysed Meta-C-H Functionalisation Only Effect Ortho Electrophilic C-H Borylation.

Two templates used in meta-directed C-H functionalisation under metal catalysis do not direct meta-C-H borylation under electrophilic borylation conditions. Using BCl3 only Lewis adduct formation with Lewis basic sites in the template is observed. While combining BBr3 and the template containing an amide linker only led to amide directed ortho C-H borylation, with no pyridyl directed meta borylation. The amide directed borylation is selective for the ortho borylation of the aniline derived unit in the template, with no ortho borylation of the phenylacetyl ring - which would also form a six membered boracycle - observed. In the absence of other aromatics amide directed ortho borylation on to phenylacetyl rings can be achieved. The absence of meta-borylation using two templates indicates a higher barrier to pyridyl directed meta borylation relative to amide directed ortho borylation and suggests that bespoke templates for enabling meta-directed electrophilic borylation may be required.

Preparation and Characterization of P2 BCh Ring Systems (Ch=S, Se) and Their Reactivity with N-Heterocyclic Carbenes.

Four-membered rings with a P2 BCh core (Ch=S, Se) have been synthesized by the reaction of phosphinidene chalcogenide (Ar*P=Ch) and phosphaborene (Mes*P=BNR2 ). The mechanistic pathways towards these rings are explained by detailed computational work that confirmed the preference for the formation of P-P, not P-B, bonded systems, which seems counterintuitive given that both phosphorus atoms contain bulky ligands. The reactivity of the newly synthesized heterocycles, as well as that of the known (RPCh)n rings (n=2, 3), was probed by the addition of N-heterocyclic carbenes, which revealed that all investigated compounds can act as sources of low-coordinate phosphorus species.

Disposition of loratadine in healthy volunteers.

The absorption, metabolism and excretion of carbon-14-labeled loratadine (LOR, SCH 29851, Claritin) administered orally to healthy male volunteers were evaluated. Following a single oral 10-mg dose of [(14)C]LOR ( approximately 102 microCi), concentrations of LOR and desloratadine (DL; a pharmacologically active descarboethoxy metabolite of LOR) were determined in plasma. Metabolites in plasma, urine and feces were characterized using a liquid chromatography-mass spectrometry system (LC-MS) connected in line with a flow scintillation analyzer (FSA). Maximum plasma LOR and DL concentrations were achieved at 1.5 h and 1.6 h, respectively; thus, LOR was rapidly absorbed but also rapidly metabolized as indicated by these similar t(max) values. Metabolite profiles of plasma showed that LOR was extensively metabolized via descarboethoxylation, oxidation and glucuronidation. Major circulating metabolites included 3-hydroxy-desloratadine glucuonide (3-OH-DL-Glu), dihydroxy-DL-glucuronides, and several metabolites resulting from descarboethoxylation and oxidation of the piperidine ring. LOR was completely metabolized by 6 h post-dose. LOR-derived radiocarbon was excreted almost equally in the urine (41%) and feces (43%). About 13% of the dose was eliminated in the urine as 3-OH-DL-Glu. DL accounted for less than 2% of the dose recovered in the urine and only trace amounts of LOR were detected. 3-OH-DL was the major fecal metabolite ( approximately 17% of the dose). The combined amount of 5- and 6-hydroxy-DL contributed to an additional 10.7% of the dose in feces. Approximately 5.4% and 2.7% of the dose were excreted in the feces as unchanged drug and DL, respectively.

Disposition of desloratadine in healthy volunteers.

The absorption, metabolism and excretion of desloratadine (DL, Clarinex) were characterized in six healthy male volunteers. Subjects received a single oral 10-mg dose of [(14)C]DL ( approximately 104 microCi). Blood, urine and feces were collected over 240 h. DL was well absorbed; drug-derived radioactivity was excreted in both urine (41%) and feces (47%). With the exception of a single subject, DL was extensively metabolized; the major biotransformation pathway consisted of hydroxylation at the 3 position of the pyridine ring and subsequent glucuronidation (3-OH-DL-glucuronide or M13). In five of the six subjects, DL was slowly eliminated (mean t((1/2)) = 19.5 h) and persisted in the plasma for 48-120 h post-dose. This is in contrast to a t((1/2)) of approximately 110 h and quantifiable plasma DL concentrations for the entire 240-h sampling period in one subject, who was identified phenotypically as a poor metabolizer of DL. This subject also exhibited correspondingly lower amounts of M13 in urine and 3-OH-DL (M40) in feces. Disposition of DL in this subject was characterized by slow absorption, slow metabolism and prolonged elimination. Further clinical studies confirmed the lack of safety issues associated with polymorphism of DL metabolism (Prenner et al. 2006, Expert Opinion on Drug Safety, 5: 211-223).

Quantitative distinctions of active site molecular recognition by P-glycoprotein and cytochrome P450 3A4.

The bulk of characterized xenobiotic defense and disposition is conferred by the abundant enzymes cytochrome P450 3A4 and P-glycoprotein. Although expressed in many tissues, these enzymes are most abundant in the liver and intestine and seem to share most substrates and inhibitors, with the apparent synergy between these two promiscuous enzymes asserted because of their extensive overlap of substrates and shared tissue location. Since the broad-spectrum tolerance to lipophilic compounds of various sizes naturally results in a similar pattern of substrate/inhibitor recognition, the cause or mechanism of many drug/drug and drug/herb interactions can be difficult to determine. These two seemingly indiscriminate enzymes, however, do not share some unique inhibitor selectivity. Particularly, we show various potent CYP3A4 inhibitors that do not affect P-gp active transport function. Remarkably, we have also identified several compounds-valinomycin, norverapamil, reserpine, nobiletin, emetine, gallopamil, fluphenazine-that uniquely inhibit P-gp function with affinities comparable to benchmark P-gp inhibitors despite a lack of effect on CYP3A4 function at physiologically relevant concentrations. Indeed, valinomycin inhibits P-gp with an IC(50) similar to cyclosporin A yet apparently does not affect CYP3A4 function, and emetine and nobiletin are also specific for interaction with P-gp. Additionally, norverapamil and reserpine have, respectively, a 60- and 40-fold preference for inhibition of P-gp over CYP3A4. Some striking structural analogies among these compounds are discussed. These distinguishing qualities of substrate recognition between CYP3A4 and P-gp should reveal nuances of active-site architecture unique to each and could serve as tools to probe for the specific discernment of P-gp-mediated drug/drug or drug/herb interactions. Learning more about binding distinctions and quantitative activity relationships of substrate/inhibitor interactions with these two enzymes and the differences between them may indicate how they recognize such a wide variety of molecules as substrates (and/or inhibitors). Moreover, identification of specific inhibitors will allow the determination of which enzyme is responsible for drug interactions and/or the extent of contribution in a multiple exposure situation.

Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors.

With P-glycoprotein (P-gp) continuing to have prominence among the ABC transporters for its ability to remove various xenobiotics from many cell types, accurate and robust methods for estimating the exposure of drug, carcinogen, toxicant, pesticide, and even some endobiotics to tissues and cells affected by P-gp are valuable. The inhibition of P-gp active transport of molecules, therefore, has often been quantified by concentration dependence of inhibitor effect on fluorescent substrate marker efflux mediated by this enzyme, with much evidence indicating two asymmetric yet interdependent substrate binding sites on P-gp. A uniqueness in the pair of binding sites could result in distinct effects of an inhibitor on the transport of certain substrates, thus leading to differences in fluorescent substrate responsiveness or sensitivity. Seven different fluorescent substrates of P-gp were quantitatively tested for their responsiveness to inhibition by a wide range of P-gp substrates/inhibitors. Interesting differences were observed in the IC(50) values caused by each of the inhibitors employed, in part exemplified by DNR and LDS being generally more sensitive to inhibition effects than any other fluorescent marker. However, no clear trend emerged to designate any fluorochrome marker as the most or least responsive to inhibition. Furthermore, LDS is more sensitive to some P-gp inhibitors than the substrate marker DNR, generally the most responsive. These results support the assertion of two unequal substrate binding sites that are allosterically dependent on each other. Therefore, an inhibitor that favors binding to the site opposite from that favored by a particular marker may have significant transduced effects through the protein between the two binding sites. Nevertheless, although either DNR or LDS is generally the fluorescent substrate most responsive to inhibition, there may be other substrates yet even more sensitive.

The farnesyl protein transferase inhibitor SCH66336 is a potent inhibitor of MDR1 product P-glycoprotein.

P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs, particularly cancer chemotherapeutics. Multidrug resistance (MDR) is caused largely by the efflux of therapeutics out of the tumor cell by Pgp, resulting in reduced efficacy of chemotherapy. SCH66336, a farnesyl transferase inhibitor in development for cancer therapy, was examined in the present study for its ability to inhibit Pgp. In a test system consisting of a NIH-G185 cell line presenting an overexpressed amount of the human transporter Pgp, known Pgp inhibitors, such as cyclosporin A, paclitaxel, verapamil, tamoxifen, and vinblastine, were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin. SCH66336 significantly inhibited daunorubicin transport with an IC50 of about 3 microM and similarly affected the transport of rhodamine 123 with a potency similar to cyclosporin A. Additionally, by an ATP-hydrolysis assay, SCH66336 was shown to decrease Pgp-mediated ATP hydrolysis by >70% with a Km of 3 microM. This observation indicates that SCH66336 directly interacts with the substrate binding site of Pgp, a quality unique to SCH66336 and its analogues, although not inherent to farnesyl transferase inhibitors in general. Moreover, low concentrations of SCH66336 exhibit synergy with the Pgp substrate/inhibitors paclitaxel, tamoxifen, and vinblastine respectively by significantly potentiating their inhibition of Pgp. Treatment with SCH66336 would be predicted to be synergistic with coadministered cancer therapeutics that are substrates of Pgp. A further benefit of coadministration of SCH66336 could be reduced chemotherapy dosage, hence, lower exposure to normal cells and, therefore, less undesired toxicity.

In vitro characterization of the inhibition profile of loratadine, desloratadine, and 3-OH-desloratadine for five human cytochrome P-450 enzymes.

The purpose of this study was to evaluate loratadine, desloratadine, and 3-OH-desloratadine as inhibitors of certain human liver cytochrome P-450 enzymes. Pooled human liver microsomes were used to determine whether loratadine, desloratadine, and 3-OH-desloratadine were inhibitors of cytochrome P-450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4. Loratadine did not inhibit CYP1A2 or CYP3A4 at concentrations up to 3829 ng/ml, which is approximately 815-fold greater than the expected maximal human plasma concentration (4.7 +/- 2.7 ng/ml) following the recommended dose of 10 mg/day. Loratadine inhibited CYP2C19 and CYP2D6 with IC(50) values of approximately 0.76 microM [291 ng/ml; K(i) congruent with 0.61 microM (234 ng/ml)] and 8.1 microM [3100 ng/ml; K(i) congruent with 2.7 microM (1034 ng/ml)], respectively, which are approximately 62 and 660 times the expected loratadine therapeutic exposure concentration. Neither desloratadine nor 3-OH-desloratadine inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 greater than 25% at concentrations of 3108 or 3278 ng/ml, respectively. These results suggest that loratadine and the active metabolites desloratadine and 3-OH-desloratadine are unlikely to affect the pharmacokinetics of coadministered drugs which are metabolized by these five cytochrome P-450 enzymes.

Evaluation of the interaction of loratadine and desloratadine with P-glycoprotein.

The absorption of many drugs is affected by their interaction with ATP-binding cassette (ABC) transporters. The most extensively studied of these ABC transporters is the proein product of MDR1 (multidrug resistance) that encodes a 170-kDa integral plasma membrane phosphorylated glycoprotein known as P-glycoprotein (P-gp). The purpose of this study was to determine, using two different methods, whether the nonsedating antihistamine loratadine (L) and its active metabolite desloratadine (DL) interact with P-gp. MDR cells presenting human P-gp were incubated with the fluorescent P-gp substrate daunorubicin with or without L, DL, and several positive controls. The IC(50) of loratadine (approximately 11 microM) was approximately 160 times the maximum observed plasma concentration (C(max)) following a dose of 10 mg. The IC(50) of desloratadine (approximately 43 microM) was approximately 880 times the C(max) following a dose of 5 mg. The positive control, cyclosporin A, had an IC(50) of approximately 1 microM. ATP hydrolysis activity was measured in the membrane fraction prepared from MDR cells presenting P-gp, which were exposed to various concentrations of test compounds. Known substrates of P-gp demonstrated clear, repeatable, concentration-dependent increases in ATP hydrolysis activity. L caused an increase in ATPase activity above basal levels. L had a V(max) about 200% basal activity and K(m) of approximately 3 microM for P-gp. In contrast, DL had no significant effect on baseline ATP hydrolysis. L inhibited human P-gp much less than verapamil or cyclosporin A. DL inhibited human P-gp significantly less than L (4 times). DL therefore is not a significant inhibitor of P-gp and should not cause clinical drug interactions with agents that are P-gp substrates.

Inhibition of P-glycoprotein transport function by grapefruit juice psoralen.

PURPOSE: The grapefruit juice component bergamottin is known to inactivate cytochrome P450 3A4, with grapefruit juice consumption causing increased absorption and enhanced oral bioavailability of many cytochrome P450 3A4 substrates. Many of these substrates are also recognized by the efflux transporter P-glycoprotein. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. METHODS: Using a whole ceil assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of grapefruit juice components to inhibit the function of this transporter. RESULTS: In a cell line presenting an overexpressed amount of the human transporter, the enzyme exhibited a 40 microM IC50 for inhibition by bergamottin. Additionally, using the ATP-hydrolysis assay, we showed that bergamottin increases P-gp-mediated ATP hydrolysis by approximately 2.3 fold with a Km of 8 microM. The concentration for this interaction is similar to that for CYP3A4 inactivation. CONCLUSIONS: These results suggest that observed grapefruit juice drug pharmacokinetic clinical interactions may be due to P-gp inhibition rather than or in addition to CYP3A4 inhibition. Inhibition of P-gp by citrus psoralens could present ways both to enhance bioavailability of therapies without increasing the dose and to diminish drug resistance in refractory cells.

HMG-CoA reductase inhibitors (statins) characterized as direct inhibitors of P-glycoprotein.

PURPOSE: HMG-CoA reductase inhibitors (statins) are commonly prescribed for lipid lowering to treat hypercholesterolemia. Although they are well tolerated, their pharmacokinetic interactions with other drugs can lead to some adverse clinical consequences. The avenue of interaction has been asserted to be CYP3A4 because most (or all) known interactions are with CYP3A4 inhibitors, and statin oxidative metabolism is mediated by CYP3A4 as well as other CYP enzymes. However, these same drugs that exert a clinical pharmacokinetic effect on statin disposition are generally also P-gp substrates/inhibitors; hence, this transporter may be, or may contribute to, the mechanism of interaction. METHODS: This study shows directly, as well as quantifies, the inhibition of P-gp-mediated transport of a fluorescent marker substrate. RESULTS: Lovastatin and simvastatin are very potent and effective inhibitors of P-gp transport with IC50's of 26 and 9 microM, respectively, for the human enzyme. Atorvastatin is also an effective P-gp inhibitor, but at higher concentrations. Uniquely, pravastatin, whose functional groups render it an inferior inhibitor of P-gp in the whole cell, had no effect in this assay. This result is consistent with known clinical interactions. The effect of these statins on ATP consumption by P-gp was also assessed, and the Km results were congruent with the IC50 observations. CONCLUSIONS: Therefore, the clinical interactions of statins with other drugs may be due, in part or all, to inhibition of P-gp transport.

Evaluation of a four-channel multiplexed electrospray triple quadrupole mass spectrometer for the simultaneous validation of LC/MS/MS methods in four different preclinical matrixes.

A four-channel multiplexed electrospray interface on a triple quadrupole mass spectrometer was evaluated for the simultaneous validation of LC/MS/MS methods for the quantitation of loratadine and its metabolite, descarboethoxyloratadine, in four different biological matrixes. The assays were performed in rat, rabbit, mouse, and dog plasma from 1 to 1000 ng/mL using 96-well solid-phase extraction for sample preparation. The limit of quantitation of 1 ng/mL corresponded to 5.56 pg of each analyte injected on-column. For the drug, quality control samples (n = 6 at four concentrations) had precision ranging from 0.967 to 16.0% and accuracy ranging from -8.44 to 10.5% across all four species. For the metabolite, the precision ranged from 0.684 to 11.0% and the accuracy was between 6.36 and -9.06%. Intersprayer cross talk for the multiplexed electrospray ion source was evaluated as a function of analyte concentration and was less than 0.08% at concentrations as high as 1000 ng/mL. These results demonstrate the feasibility of using parallel analysis to reduce the time required for method validation and to increase sample throughput in drug development studies.

Mechanism-based inactivation of CYP2D6 by 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine.

SCH 66712 [5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine] caused a time- and NADPH-dependent loss of CYP2D6 activity. The inactivation of human liver (HL) microsomal dextromethorphan O-demethylase activity, a prototype marker for CYP2D6, was characterized by a K(I) of 4.8 microM and a maximal rate constant of inactivation (k(inact)) of 0.14 min(-1). The inactivation of the recombinant CYP2D6 in Supersomes (r-CYP2D6) was characterized by a K(I) of 0.55 microM and a k(inact) of 0.32 min(-1). Extensive dialysis of the SCH 66712-inhibited enzyme failed to restore the activity to control levels (dialyzed reaction mixture lacking SCH 66712) for both HL microsomes and r-CYP2D6. Addition of glutathione, superoxide dismutase, or mannitol to the reaction mixture failed to protect CYP2D6 against SCH 66712-NADPH-catalyzed inactivation. Addition of quinidine, a reversible inhibitor of CYP2D6, to a preincubation mixture consisting of SCH 66712, HL microsomes, or Supersomes and NADPH partially protected CYP2D6 from inactivation. SCH 66712 also inhibited HL microsomal CYP3A4, CYP2C9, and CYP2C19; however, the concentrations required to inhibit those isoforms were 5- to 10-fold higher than those required to inhibit CYP2D6. These results demonstrate that SCH 66712 is a potent and fairly selective mechanism-based inhibitor of CYP2D6.

Bioavailability and metabolism of mometasone furoate following administration by metered-dose and dry-powder inhalers in healthy human volunteers.

These studies were conducted to assess the systemic bioavailability of mometasone furoate (MF) administered by both the dry-powder inhaler (DPI) and the metered-dose inhaler with an alternate propellant (MDI-AP). The pharmacokinetics of single doses (400 micrograms) of MF administered by intravenous (i.v.) and inhalation routes was assessed in a randomized, three-way crossover study involving 24 healthy volunteers. In a separate study, 6 healthy subjects were administered a single dose of tritiated (3H-) MF by DPI, and the radioactivity in blood, urine, feces, and expired air was determined. Following i.v. administration, MF was detected in all subjects for at least 8 hours postdose. The half-life (t1/2) following i.v. administration was 4.5 hours. In contrast, following DPI administration, plasma MF concentrations were below the limit of quantification (LOQ, 50 pg/mL) for many subjects (10 of 24), and the systemic bioavailability by this route was estimated to be less than 1%. Only two plasma samples following MDI-AP administration had plasma concentrations of MF above the LOQ indicating no detectable systemic bioavailability in 92% of the subjects. A separate study with 6 healthy male subjects administered a single dose of 3H-MF (200 microCi) by DPI revealed that much of the dose (approximately 41%) was excreted unchanged in the feces (0-72 hours), while that which was absorbed was extensively metabolized. These results indicate that inhaled MF has negligible systemic bioavailability and is extensively metabolized and should therefore be well tolerated in the chronic treatment of asthma.

Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity.

P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.

Cholesterol interaction with the daunorubicin binding site of P-glycoprotein.

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp.

A dose-ranging study of pegylated interferon alfa-2b and ribavirin in chronic hepatitis C. The Hepatitis C Intervention Therapy Group.

The objectives of this study were to assess the safety, pharmacokinetics, and efficacy of pegylated interferon alfa-2b (PEG-Intron) plus ribavirin in patients with chronic hepatitis C. A total of 72 patients (35 men/37 women, age range 20-68 years) with clinically compensated chronic hepatitis C virus (HCV) were enrolled into this open-label, randomized, active controlled study. Patients received either PEG-Intron 0.35, 0.7, or 1.4 microg/kg subcutaneously weekly for 24 weeks alone, or in combination with ribavirin 600, 800, or 1,000 to 1,200 mg orally daily. Patients were evaluated during treatment and after a 24-week follow-up period for safety and efficacy. Detailed pharmacokinetic assessments were performed at weeks 1 and 4. PEG-Intron alone produced expected dose-related reductions in white cells, neutrophils and platelets. Addition of ribavirin reduced hemoglobin levels in a dose-related manner, did not further reduce PEG-Intron-induced decreases in neutrophil or white cell count, and increased platelet counts. Neutrophil function tests (C5a and FMLP migration, killing curves) were unaltered. Reported adverse events (flu-like symptoms, asthenia) were qualitatively similar in all dose groups. Anti-HCV activity, as measured by loss of detectable serum HCV RNA (i.e. <100 copies/mL) at the end of treatment (week 24) and after 24 weeks of follow-up (week 48) showed dose-response trends for PEG-Intron. At each PEG-Intron dose level, anti-HCV activity was higher in patients coadministered ribavirin than in patients treated with PEG-Intron monotherapy. There was no evidence of pharmacokinetic interactions with either drug. We conclude that the safety and tolerability of combined PEG-Intron/ribavirin and PEG-Intron alone were comparable. Combined PEG-Intron/ribavirin showed dose-related synergistic anti-HCV effects, which were numerically superior to those obtained with PEG-Intron monotherapy.

Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence.

The ATP-dependent transport enzyme known as P-glycoprotein (P-gp) confers multidrug resistance (MDR) against many unrelated drugs and xenobiotics. To understand better the broad substrate specificity of the enzyme as well as the mechanism of substrate transport out of the cell, it is critical to characterize the substrate binding sites. Since approximately 1 ATP is hydrolyzed per transport event, phosphate release rate provides a steady-state kinetics assay. Notably, the substrate H33342 causes a decrease in the baseline hydrolysis of ATP (probably due to competition for transport with an endogenous membrane lipid substrate) providing an excellent tool for a comprehensive graphical kinetic analysis of the interaction of substrate pairs at the transport site(s) allowing the determination of inhibition type and hence characterization of transport binding sites. The substrate H33342 interacted with quinidine, progesterone, and propranolol in a non-competitive manner, indicating that binding of H33342 precludes active transport of these other substrates at a distinct site. Compounds such as TPP+ and verapamil, and perhaps also nicardipine, interacted with H33342 as mixed-type inhibitors. This type of interaction results from a reduced affinity at the opposing active site by a factor of alpha and sometimes a partial activity of a fraction beta. Indeed, H33342 binding caused a roughly four-fold reduced affinity for TPP+. Using this definitive approach to inhibition kinetics, we were able to establish traits of a second transport site in P-gp. Therefore, the sites are unequal; however, the performance at one site is contingent on the other being unoccupied, and transport is also sometimes mitigated when the other site is occupied.