Insertion of constant region domains of human IgG1 into CD4-PE40 increases its plasma half-life.

CD4-PE40 is a recombinant toxin containing the binding domain of CD4 and a mutant form of Pseudomonas exotoxin A from which the cell binding domain has been removed. To increase the serum half-life of CD4-PE40, we have inserted various portions of the constant domain of human IgG1 into CD4-PE40. The constructs made include CD4-CH2-PE40, CD4-CH3-PE40, CD4-CH1-CH2-PE40 and CD4-CH2-CH3-PE40. The fusion proteins were expressed and purified from E. coli. Insertion of various domains from the constant region of IgG1 did not alter the cytotoxic activity of CD4-PE40; all these molecules were equally cytotoxic to cells expressing gp120 on their surface. However, there was a marked increase in the serum mean residence time of CD4-CH2-PE40 which was 115 min as compared to 47 min for CD4-PE40. Insertion of other domains also increased the half-life of CD4-PE40, however, CD4-CH2-PE40 was found to have the longest mean residence time in the circulation. One possible explanation for the increase in plasma half-life is diminished susceptibility of proteins to proteolysis. It was found that CD4-CH2-PE40 was much more resistant to proteolysis by trypsin than CD4-PE40. We proposed that insertion of the CH2 domain into CD4-PE40 covers up the protease sensitive sites in the molecule, thereby making the molecule less susceptible to degradation. The increase in size and reduced sensitivity to proteases could both be responsible for the increased plasma half-life of CD4-CH2-PE40.

Synthesis and structure-activity relationships of the (alkylamino)piperidine-containing BHAP class of non-nucleoside reverse transcriptase inhibitors: effect of 3-alkylpyridine ring substitution.

Development of resistance to currently approved HIV therapies has continued to fuel research efforts to improve the metabolic stability and spectrum of activity of the (alkylamino)piperidine-containing bis(heteroaryl)piperazine (AAP-BHAP) class of non-nucleoside reverse transcriptase inhibitors (NNRTIs). The synthesis of analogues in which the usual 3-alkylamino substituent on the pyridine ring is replaced by a 3-alkyl substituent led to compounds which retained activity against recombinant P236L and wild-type (WT) reverse transcriptase (RT), while inhibition of the Y181C mutant RT was reduced relative to the activity of the 3-alkylamino-substituted congeners. Testing of representative analogues in an in vitro liver microsome assay indicated that the alkyl substituent would not appreciably improve the metabolic stability of the AAP-BHAP template. In vivo pharmacokinetic evaluation of three compounds confirmed these results in that high systemic clearances were observed. Nevertheless, one compound (13), PNU-103657, possessed oral bioavailability in rats approaching that of the structurally related NNRTI drug delavirdine which is currently on the market for the treatment of HIV infection.

Synthesis and bioactivity of novel bis(heteroaryl)piperazine (BHAP) reverse transcriptase inhibitors: structure-activity relationships and increased metabolic stability of novel substituted pyridine analogs.

The major route of metabolism of the bis(heteroaryl)piperazine (BHAP) class of reverse transcriptase inhibitors (RTIs), atevirdine and delavirdine, is via oxidative N-dealkylation of the 3-ethyl- or 3-isopropylamino substituent on the pyridine ring. This metabolic pathway is also the predominant mode of metabolism of (alkylamino)piperidine BHAP analogs (AAP-BHAPs), compounds wherein a 4-(alkylamino)piperidine replaces the piperazine ring of the BHAPs. The novel AAP-BHAPs possess the ability to inhibit non-nucleoside reverse transcriptase inhibitor (NNRTI) resistant recombinant HIV-1 RT and NNRTI resistant variants of HIV-1. This report describes an approach to preventing this degradation which involves the replacement of the 3-ethyl- or 3-isopropylamino substituent with either a 3-tert-butylamino substituent or a 3-alkoxy substituent. The synthesis, bioactivity and metabolic stability of these analogs is described. The majority of analogs retain inhibitory activities in enzyme and cell culture assays. In general, a 3-ethoxy or 3-isopropoxy substituent on the pyridine ring, as in compounds 10, 20, or 21, resulted in enhanced stabilities. The 3-tert-butylamino substituent was somewhat beneficial in the AAP-BHAP series of analogs, but did not exert a significant effect in the BHAP series. Lastly, the nature of the indole substitution sometimes plays a significant role in metabolic stability, particularly in the BHAP series of analogs.

Targeting delavirdine/atevirdine resistant HIV-1: identification of (alkylamino)piperidine-containing bis(heteroaryl)piperazines as broad spectrum HIV-1 reverse transcriptase inhibitors.

A novel class of bis(heteroaryl)piperazine (BHAP) analogs which possesses the ability to inhibit NNRTI (non-nucleoside reverse transcriptase inhibitor) resistant recombinant HIV-1 reverse transcriptase (RT) and NNRTI resistant variants of HIV-1 has been identified via targeted screening. Further investigation of the structure-activity relationships of close congeners of these novel (alkylamino)piperidine BHAPs (AAP-BHAPs) led to the synthesis of several compounds possessing the desired phenotype (e.g., activity against recombinant RTs carrying the Y181C and P236L substitutions). Further structural modifications were required to inhibit metabolism and modulate solubility in order to obtain compounds with the desired biological profile as well as appropriate pharmaceutical properties. The AAP-BHAPs with the most suitable characteristics were compounds 7, 15, and 36.

Identification of dioxin-responsive genes in Hep G2 cells using differential mRNA display RT-PCR.

Differential mRNA display RT-PCR (DD RT-PCR) offers a tool to identify genes which are regulated or responsive to certain receptors or chemicals such as dioxin (TCDD). Treatment of Hep G2 cells with TCDD followed by DD analysis of a gel with series of different primers revealed a significantly different pattern from the control for a number of mRNAs. The differentially displayed mRNAs were isolated and reamplified. A GenBank search of four mRNAs revealed two known and two unknown sequences. Northern blot analysis revealed that two known sequences, fibrinogen gamma chain and plastin mRNAs were down regulated by TCDD in a time-dependent manner, whereas two unknown mRNAs were induced by TCDD treatment. The function of these genes in TCDD toxicity is not known; however, the application of DD RT-PCR in the studies of TCDD-induced responses could be very useful in the discovery of other unknown genes important for TCDD toxicity.

Interaction of delavirdine with human liver microsomal cytochrome P450: inhibition of CYP2C9, CYP2C19, and CYP2D6.

Delavirdine, a non-nucleoside inhibitor of HIV-1 reverse transcriptase, is metabolized primarily through desalkylation catalyzed by CYP3A4 and CYP2D6 and by pyridine hydroxylation catalyzed by CYP3A4. It is also an irreversible inhibitor of CYP3A4. The interaction of delavirdine with CYP2C9 was examined with pooled human liver microsomes using diclofenac 4'-hydroxylation as a reporter of CYP2C9 catalytic activity. As delavirdine concentration was increased from 0 to 100 microM, the K(M) for diclofenac metabolism rose from 4.5+/-0.5 to 21+/-6 microM, and V(max) declined from 4.2+/-0.1 to 0.54+/-0.08 nmol/min/mg of protein, characteristic of mixed-type inhibition. Nonlinear regression analysis revealed an apparent K(i) of 2.6+/-0.4 microM. There was no evidence for bioactivation as prerequisite to inhibition of CYP2C9. Desalkyl delavirdine, the major circulating metabolite of delavirdine, had no apparent effect on microsomal CYP2C9 activity at concentrations up to 20 microM. Several analogs of delavirdine showed similar inhibition of CYP2C9. Delavirdine significantly inhibited cDNA-expressed CYP2C19-catalyzed (S)-mephenytoin 4'-hydroxylation in a noncompetitive manner, with an apparent K(i) of 24+/-3 microM. Delavirdine at concentrations up to 100 microM did not inhibit the activity of CYP1A2 or -2E1. Delavirdine competitively inhibited recombinant CYP2D6 activity with a K(i) of 12.8+/-1.8 microM, similar to the observed K(M) for delavirdine desalkylation. These results, along with previously reported experiments, indicate that delavirdine can partially inhibit CYP2C9, -2C19, -2D6, and -3A4, although the degree of inhibition in vivo would be subject to a variety of additional factors.

Metabolism of delavirdine, a human immunodeficiency virus type-1 reverse transcriptase inhibitor, by microsomal cytochrome P450 in humans, rats, and other species: probable involvement of CYP2D6 and CYP3A.

The metabolism of delavirdine was examined using liver microsomes from several species with the aim of comparing metabolite formation among species and characterizing the enzymes responsible for delavirdine metabolism. Incubation of 10 microM [14C]delavirdine with either an S9 fraction from human jejunum or liver microsomes from rat, human, dog, or monkey followed by high pressure liquid chromatography analysis showed qualitatively similar metabolite profiles among species with the formation of three significant metabolites. The major metabolite was desalkyl delavirdine; however, the identity of MET-7 and MET-7a (defined by high pressure liquid chromatography elution) could not be unambiguously established, but they seem to be related pyridine hydroxy metabolites, most likely derived from 6'-hydroxylation of the pyridine ring. The apparent KM for delavirdine desalkylation activity ranged from 4.4 to 12.6 microM for human, rat, monkey, and dog microsomes, whereas Vmax ranged from 0.07 to 0.60 nmol/min/mg protein, resulting in a wide range of intrinsic clearance (6-135 microL/min/mg protein). Delavirdine desalkylation by microsomes pooled from several human livers was characterized by a KM of 6.8 +/- 0.8 microM and Vmax of 0. 44 +/- 0.01 nmol/min/mg. Delavirdine desalkylation among 23 human liver microsomal samples showed a meaningful correlation (r = 0.96) only with testosterone 6beta-hydroxylation, an indicator of CYP3A activity. Among ten human microsomal samples selected for uniform distribution of CYP3A activity, formation of MET-7 was strongly correlated with CYP3A activity (r = 0.95) and with delavirdine desalkylation (r = 0.98). Delavirdine desalkylation was catalyzed by cDNA-expressed CYP2D6 (KM 10.9 +/- 0.8 microM) and CYP3A4 (KM 5.4 +/- 1.4 microM); however, only CYP3A4 catalyzed formation of MET-7 and MET-7a. Quinidine inhibited human liver microsomal delavirdine desalkylation by about 20%, indicating a minor role of CYP2D6. These findings suggest the potential for clinical interaction with coadministered drugs that are metabolized by or influence the activity of CYP3A or CYP2D6.

Microsomal metabolism of delavirdine: evidence for mechanism-based inactivation of human cytochrome P450 3A.

Administration of delavirdine, an HIV-1 reverse transcriptase inhibitor, to rats or monkeys resulted in apparent loss of hepatic microsomal CYP3A and delavirdine desalkylation activity. Human CYP3A catalyzes the formation of desalkyl delavirdine and 6'-hydroxy delavirdine, an unstable metabolite, while CYP2D6 catalyzes only desalkyl delavirdine. CYP2D6 catalyzed desalkyl delavirdine formation was linear with time (up to 30 min) but when catalyzed by cDNA expressed CYP3A4 or human liver microsomes the reaction rate declined progressively with time. Coincubation with triazolam showed that delavirdine caused a time- and NADPH-dependent loss of CYP3A4 activity in human liver microsomes as measured by triazolam 1'-hydroxylation. The catalytic activity loss was saturable and was characterized by a Ki of 21.6 +/- 8.9 microM and a kinact of 0.59 +/- 0.08 min-1. An apparent partition ratio of 41 was determined with cDNA expressed CYP3A4, based on the substrate depletion method. Incubation of [14C]delavirdine with microsomes from several species resulted in irreversible association with an approximately 50 kDa protein, as demonstrated by SDS-PAGE/autoradiography. Binding to the protein was NADPH dependent, glutathione insensitive, proportional to the level of CYP3A expression and was inhibited by ketoconazole, a specific CYP3A inhibitor. NADPH-dependent irreversible binding to human and rat total microsomal protein was demonstrated following exhaustive extraction of microsomal protein. Binding was decreased in the presence of glutathione and appeared to be related to expression level of CYP3A. These results suggest that delavirdine can inactivate CYP3A and has the potential to slow the metabolism of coadministered CYP3A substrates.

Absorption, distribution, metabolism, and excretion of atevirdine in the rat.

Atevirdine mesylate (U-87201E) is a highly specific nonnucleoside inhibitor of human immunodeficiency virus type 1 reverse transcriptase. The absorption, metabolism, and excretion of atevirdine were investigated in male and female Sprague-Dawley rats after oral administration of nonradiolabeled atevirdine mesylate at doses of 20 mg/kg/day or 200 mg/kg/day for 8 days, with [14C]atevirdine mesylate single doses of 10 mg/kg or 100 mg/kg on study days 1 and 10. The distribution of [14C]atevirdine mesylate was also evaluated by whole-body autoradiography in male and female Sprague-Dawley, pregnant Sprague-Dawley, and male Long-Evans rats after a single 10 mg/kg oral dose. Plasma levels of atevirdine and its N-desethyl and O-desmethyl metabolites were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection, urine and feces were profiled for atevirdine and metabolites by HPLC with radiochemical detection, major metabolites in urine were isolated and identified by nuclear magnetic resonance and mass spectrometry, and minor urinary metabolites were identified by liquid chromatography/mass spectrometry. Atevirdine was rapidly absorbed. The pharmacokinetics of atevirdine were nonlinear. Gender differences in the pharmacokinetics and metabolism of atevirdine were observed, consistent with the involvement of cytochrome P450 3A. Atevirdine effectively crossed the blood-brain barrier and had a high rate of maternal-fetal transfer. At the low doses, <2% of the dose was excreted as unchanged parent drug, while atevirdine constituted 9%-25% of the dose at the high doses. The metabolism of atevirdine was extensive in the rat and involved N-deethylation, O-demethylation, hydroxylation at the C-6 position of the indole ring, and hydroxylation of the pyridine ring.