Loss to follow-up and mortality among HIV-infected adolescents receiving antiretroviral therapy in Pune, India.

OBJECTIVES: India has the highest number of HIV-infected adolescents in Asia, but little is known about their treatment outcomes. We assessed rates and factors associated with loss to follow-up (LTFU) and mortality among Indian adolescents. METHODS: The analysis included adolescents (10-19 years old) starting antiretroviral therapy (ART) between 2005 and 2014 at BJ Government Medical College, Pune, India. LTFU was defined as missing more than three consecutive monthly visits. The competing-risks method was used to calculate subdistribution hazard ratios (SHRs) of predictors for LTFU, with death as the competing risk. Cox proportional hazard models were used to identify predictors of mortality. RESULTS: Of 717 adolescents starting ART, 402 with complete data were included in the analysis. Of these, 61% were male and 80% were perinatally infected, and the median baseline CD4 count was 174 cells/µL. LTFU and mortality rates were 4.4 and 4.9/100-person years, respectively. Cumulative LTFU incidence increased from 6% to 15% over 6 years. Age ≥ 15 years [adjusted SHR (aSHR) 2.44; 95% confidence interval (CI) 1.18-5.02] was a risk factor for LTFU. Cumulative mortality increased from 9.5% to 17.9% over 6 years. World Health Organization (WHO) stages III and IV [adjusted hazard ratio (aHR) 2.26; 95% CI: 1.14-4.48] and an increase in CD4 count by 100 cells/µL (aHR: 0.59; 95% CI: 0.43-0.83) were associated with mortality. CONCLUSIONS: A third of adolescents had been lost to follow-up or died by follow-up year 6. Older age was a risk factor for LTFU and advanced clinical disease for death. Strategies to improve retention counselling for older adolescents and closer clinical monitoring of all adolescents must be considered.

Isoniazid hair concentrations in children with tuberculosis: a proof of concept study.

Assessing treatment adherence and quantifying exposure to anti-tuberculosis drugs among children is challenging. We undertook a 'proof of concept' study to assess the drug concentrations of isoniazid (INH) in hair as a therapeutic drug monitoring tool. Children aged <12 years initiated on a thrice-weekly treatment regimen including INH (10 mg/kg) for newly diagnosed tuberculosis were enrolled. INH concentrations in hair were measured using liquid chromatography-tandem mass spectrometry at 1, 2, 4 and 6 months after initiating anti-tuberculosis treatment. We found that INH hair concentrations in all children on thrice-weekly INH were detectable and displayed variability across a dynamic range.

Studies on the monoamine oxidase-B-catalyzed biotransformation of 4-azaaryl-1-methyl-1,2,3,6-tetrahydropyridine derivatives.

The substrate properties of a series of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridinyl (MPTP) analogues in which the C-4 phenyl group has been replaced with various 4-azaaryl moieties have been examined in an effort to evaluate the contribution of electronic, polar, and steric parameters to the MAO-B-catalyzed oxidation of this type of cyclic tertiary allylamine to the corresponding dihydropyridinium metabolite. No significant correlation could be found with the calculated energy of the C-H bond undergoing cleavage. A general trend, however, was observed between the magnitude of the log P value with the magnitude of kcat/Km. The results indicate that the placement of a polar nitrogen atom in the space occupied by the phenyl group of MPTP leads to a dramatic decrease in substrate properties. Enhanced substrate properties, however, were observed when benzoazaarenes replaced the corresponding five-membered azaarenes. These results are consistent with our previously published molecular model of the active site of MAO-B.

Design, synthesis, and evaluation of azapeptides as substrates and inhibitors for human rhinovirus 3C protease.

A series of azapeptides was prepared and assessed as inhibitors of the human rhinovirus 3C protease. Boc-VLFaQ-OPh was a slow-turnover substrate that gave transient (ca. 1-2 h) inhibition as it underwent hydrolysis. Boc-VLFaG-OPh gave very slow but essentially irreversible inhibition.

Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication.

Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3Cpro) which is highly selective for -Q approximately GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3Cpro). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3Cpro with submicromolar IC50 values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3Cpro inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3Cpro, which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael acceptors lacking a P4 residue have greatly reduced or negligible activity against 3Cpro, consistent with previously established structure-activity relationships for 3Cpro substrates. Hydrolysis of the P1 vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.

Synthesis and monoamine oxidase B catalyzed oxidation of C-4 heteroaromatic substituted 1,2,3,6-tetrahydropyridine derivatives.

The monoamine oxidase B (MAO-B) catalyzed oxidation of amines has been proposed to proceed via a polar pathway, an initial single-electron transfer pathway and an initial hydrogen atom transfer pathway. Results from previous studies on selected N-cyclopropyl-4-substituted-1,2,3,6-tetrahydropyridine derivatives have led us to consider a mechanism for these cyclic tertiary allylamines which may not necessarily involve the aminyl radical cation as required by an initial single-electron transfer step. The studies summarized in this paper were undertaken to explore further the structural features that determine the MAO-B substrate and/or inactivator properties of various 1,4-disubstituted tetrahydropyridine derivatives. We report here the results of our studies on the synthesis and MAO-B catalyzed oxidation of 1-methyl- and 1-cyclopropyl-1,2,3,6-tetrahydropyridine derivatives bearing a variety of heteroaromatic groups at C-4. All of the N-cyclopropyltetrahydropyridine analogs were time and concentration dependent inhibitors of MAO-B while all of the N-methyltetrahydropyridine analogs and the N-cyclopropyl-4-(1-methyl-2-pyrryl)tetrahydropyridine analog were substrates. The substrate properties (Kcat/KM) covered a range of 6 to 1800 min-1 mM-1 while the range for the inactivator properties for which Kinact/KI values could be obtained was 0.1-1.0 min-1 mM-1. The partition ratios for the N-cyclopropyl analogs varied from 4 to 17 except for the 4-(1-methyl-2-pyrryl) analog, which had a partition ratio of 400. These results are discussed in terms of the putative allylic radical intermediate and in the context of the hydrogen atom transfer and single-electron transfer based mechanisms.

Probing the mechanism of bioactivation of MPTP type analogs by monoamine oxidase B: structure-activity studies on substituted 4-phenoxy-, 4-phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridines.

Previous studies have shown that 4-benzyl-1-cyclopropyl-1,2,3,6-tetrahydropyridine is an excellent monoamine oxidase B (MAO-B) substrate (kappa cat/KM = 1538 min-1 mM-1) although the corresponding 4-phenyl analog displays MAO-B inactivating properties only. This behavior led us to speculate that the pathway for the MAO-B catalyzed oxidation of these tetrahydropyridines may not necessarily proceed via an initial single electron transfer step as proposed by others but rather through an initial alpha-carbon hydrogen atom abstraction step. In the present studies we have examined the interactions of various 4-phenoxy-, 4-phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridine derivatives, some of which bear substituents on the phenyl ring. The 4-thiophenoxy- and all of the 4-phenoxytetrahydropyridine derivatives proved to be substrates but not inactivators of MAO-B, while several of the 4-phenyltetrahydropyridine derivatives were inactivators but not substrates. A case of particular interest was 1-cyclopropyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine, which displayed only substrate properties. The results are discussed in terms of two catalytic pathways, one of which involves partitioning of the proposed cyclopropylaminyl radical cation intermediate between cyclopropyl ring opening and proton loss while the second involves partitioning of the parent amine between an initial single electron transfer step, leading to cyclopropylaminyl radical cation formation and enzyme inactivation, and an initial alpha-carbon hydrogen atom abstraction step, leading to an allylic radical and dihydropyridinium product formation.

Characteristics of the growth inhibition and cytotoxicity of long-chain (sphingoid) bases for Chinese hamster ovary cells: evidence for an involvement of protein kinase C.

Long-chain bases are potent inhibitors of protein kinase C and cellular processes mediated by this enzyme. However, when added to cells they usually cause some degree of growth inhibition and cytotoxicity and it is unclear whether this reflects inhibition of protein kinase C or nonspecific detergent effects of these amphipathic compounds. This study examined the effects of sphinganine on Chinese hamster ovary (CHO) cells to gain more insight into these possibilities. Sphinganine concentrations between 0.75 and 4 microM resulted in a combination of growth inhibition and cytotoxicity that correlated with protein kinase C inhibition by five criteria: (1) the effective concentrations were comparable to those for protein kinase C inhibition in vitro and in other intact cells; (2) the structural specificity for the long-chain base moiety paralleled the potency of protein kinase C inhibition; (3) sphinganine blocked changes in protein phosphorylation patterns that occurred in response to phorbol 12-myristate 13-acetate (and vice versa); whereas (4) a mutant cell line that exhibited increased resistance to sphinganine cytotoxicity lacked both phorbol ester- and sphinganine-induced phosphorylation changes and differed somewhat in the behavior of protein kinase C assayed in vitro; and (5) sphinganine did not appear to be acting as a detergent (except at higher concentrations) nor as a lysosomotrophic agent. While the complexity of this cellular behavior mandates caution in interpreting these results, they suggest that the cytotoxicity and growth inhibition may be a consequence of protein kinase C inhibition.

Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds.

Sphingosine, sphinganine, and other long-chain (sphingoid) bases inhibit protein kinase C in vitro and block cellular responses to agonists that are thought to act via this enzyme. To gain further insight into the mechanism of this inhibition, a series of long-chain analogues differing in alkyl chain length (11-20 carbon atoms), stereochemistry, and headgroup were examined for (a) inhibition of protein kinase C activity in vitro, (b) the neutrophil respiratory burst in response to phorbol myristate acetate (PMA), (c) the PMA-induced differentiation of HL-60 cells, and (d) the growth of Chinese hamster ovary cells. In every instance, the effects were maximal with the 18-carbon homologues, which are the same length as the predominant naturally occurring long-chain base (sphingosine). The lower potency of the shorter chain homologues was partially due to decreased uptake by cells. Small differences were obtained with the four stereoisomers of sphingosine (i.e., D and L forms of erythro- and threo-sphingosine), with N-methyl derivatives of the different sphingosine homologues, and with simpler alkylamines (e.g., stearylamine). The potency of the different headgroup analogues may be affected by the degree of protonation at the assay pH. The pKa of sphingosine was measured to be 6.7; the pKa varied among the analogues. These findings establish that the major structural features required for inhibition of protein kinase C and cellular processes dependent on this enzyme are the presence of a free amino group and an aliphatic side chain and that other groups have more subtle effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of free sphingosine in liver by high-performance liquid chromatography.

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.