Long-acting cabotegravir (CAB) extends antiretroviral drug (ARV) administration from daily to monthly. However, dosing volumes, injection site reactions, and health care oversight are obstacles towards broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18, and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB, and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics (PK), and biodistribution. PK studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. The NM2CAB, compared to NMCAB and NM3CAB, demonstrated prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg/kg intramuscular injection. These prodrug modifications could significantly improve CAB’s effectiveness.

: There are currently approximately 16 million people taking NNRTI-based first-line treatment in low-income and middle-income countries. Most of these patients are using the combination of tenofovir (TDF), lamivudine (3TC) and efavirenz (EFV). The integrase inhibitor dolutegravir (DTG) has shown an improved safety profile compared with EFV in randomized studies. DTG also has a high barrier to development of drug resistance. New co-formulated tablets with TDF/3TC/DTG are being introduced into LMICs, for a median price of $75 per person-year. The prodrug of TDF, tenofovir alafenamide (TAF) is cheaper to manufacture than TDF. A combined pill with TAF/3TC/DTG is also being launched in LMICs, at a similar low price. However, the clinical development programmes for DTG and TAF did not include extensive analysis of several key populations: pregnant women, people with HIV-tuberculosis (TB) coinfection taking rifampicin-based treatment, and treatment-naive or pretreated patients with NRTI drug resistance. An observational study in Botswana has shown an increased risk of neural tube defects when dolutegravir is used in early pregnancy. In LMICs, only 50% of patients have access to regular viral load testing, and genotypic resistance testing is rarely performed. There is currently no clinical data to support switching patients from TDF/3TC/EFV directly to TDF/3TC/DTG if their viral load is either detectable or unknown. New clinical trials and observational studies will be needed to better understand the consequences of this switch of treatment in LMICs. Clinical trials of new antiretrovirals in key populations should be conducted earlier in their development. This will ensure that new treatments can be introduced into LMICs soon after their launch in high-income countries.

Protein self-assembly is critical for numerous biological processes. Yet, assembly is rarely targeted by therapeutic agents, in part because it is hard to identify molecules that interfere with proteinprotein interactions. Here we describe a simple fluorescence-based screen for self-association and its application to the assembly of hepatitis B virus capsids. These data are analyzed to identify kinetic and thermodynamic effects -both of which are critical for the viral lifecycle and for understanding the mechanism of assembly effectors. Suggestions are made for modification of this protocol so that it can be applied to other self-assembling systems. With manual pipetting, setting up a plate takes about two hours, the initial reading takes one hour, and the endpoint reading the following day takes about 5 minutes.

The formation of tyrosine from phenylalanine catalyzed by rat liver phenylalanine hydroxylase is coupled to the generation of a 4a-hydroxy adduct from the requisite tetrahydropterin cofactor. As indicated by its circular dichroism (CD) spectrum, the optical activity of the adduct generated from racemic 6-methyltetrahydropterin requires stereoselectivity of the oxygenation. The absolute configuration of this new stereocenter is 4a(S)-hydroxy-6(RS)-methyltetrahydropterin by analogy to the CD spectrum of one of the four stereoisomers of 5-deaza-4a-hydroxy-6-methyltetrahydropterin. The source of the 4a-hydroxy oxygen is O2, as demonstrated by the observation of a 18O-induced 13C shift in the 13C NMR spectrum of the adduct when generated from [4a-13C]-6-methyltetrahydropterin and 18O2.

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The synthesis and biological evaluation of cAMP-specific phosphodiesterase (PDE IV) inhibitors is described. The PDE IV inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724, 2) was used as a template from which to design a set of rigid oxazolidinones, imidazolidinones, and pyrrolizidinones that mimic Ro 20-1724 but differ in the orientation of the carbonyl group. The endo isomer of each of these heterocycles was more potent than the exo isomer in an enzyme inhibition assay and a cellular assay, which measured TNF alpha secretion from activated human peripheral blood monocytes (HPBM). Imidazolidinone 4a inhibited human PDE IV with a Ki of 27 nM and TNF alpha secretion from HPBM with an IC50 of 290 nM. By comparison, Ro 20-1724 is significantly less active in these assays with activities of 1930 and 1800nM, respectively.

The association and metabolism of exogenously-derived lysophosphatidylcholine (lysoPC) with cultured mammalian cells from a variety of sources was studied, and a mechanism was defined by computer modeling for Vero cells. Cell monolayers were incubated with radiolabeled lysoPC, and the kinetics of disappearance from the medium, association with the cells, and metabolism by the cells of lysoPC were monitored both in the absence and in the presence of fetal bovine serum. Exogenously-supplied lysoPC first associated with cell membranes, followed by an almost complete conversion to phosphatidylcholine (PC). The kinetics of partitioning and metabolism were identical regardless of whether the exogenously-supplied lysoPC was labeled with [methyl-3H]choline or with [1-14C]palmitate. A two-step mechanism, consisting of a reversible partitioning of exogenous lysoPC into the cell membrane followed by enzymatic reacylation of PC, was found to adequately describe the observed kinetics in the presence of 0 or 0.5% fetal bovine serum. The effect of temperature on the individual rate constants and on the overall process was examined. An Arrhenius plot indicated an acute temperature sensitivity between 15 and 23 degrees C, consistent with a dependence on the lipid phase of the membrane and a regional phase transition temperature characteristic of mammalian cells. The acute temperature sensitivity was almost entirely due to the temperature dependence of reacylation. A multistep mechanism was established by combining the kinetic constants determined under conditions of low exogenous protein with the binding constant between lysoPC and serum protein. This mechanism accurately predicts the rates of uptake of exogenously-derived lysoPC with cultured cells in the presence of serum concentrations between 0 and 10%. A survey of a variety of cultured cells indicated that the kinetics of association and metabolism of exogenously-derived lysoPC is cell-type specific.

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