Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy.

Parkinson's disease (PD) is a neurodegenerative aging disorder in which postmortem PD brain exhibits neuroinflammation, as well as synucleinopathy-associated protein phosphatase 2A (PP2A) enzymatic activity loss. Based on our translational research, we began evaluating the PD-repurposing-potential of an anti-inflammatory, neuroprotective, and PP2A stimulatory oral drug that is FDA-approved for multiple sclerosis, FTY720 (fingolimod, Gilenya®). We also designed two new FTY720 analogues, FTY720-C2 and FTY720-Mitoxy, with modifications that affect drug potency and mitochondrial localization, respectively. Herein, we describe the metabolic stability and metabolic profiling of FTY720-C2 and FTY720-Mitoxy in liver microsomes and hepatocytes. Using mouse, rat, dog, monkey, and human liver microsomes the intrinsic clearance of FTY720-C2 was 22.5, 79.5, 6.0, 20.2 and 18.3 µL/min/mg; and for FTY720-Mitoxy was 1.8, 7.8, 1.4, 135.0 and 17.5 µL/min/mg, respectively. In hepatocytes, both FTY720-C2 and FTY720-Mitoxy were metabolized from the octyl side chain, generating a series of carboxylic acids similar to the parent FTY720, but without phosphorylated metabolites. To assess absorption and distribution, we gave equivalent single intravenous (IV) or oral doses of FTY720-C2 or FTY720-Mitoxy to C57BL/6 mice, with two mice per time point evaluated. After IV delivery, both FTY720-C2 and FTY720-Mitoxy were rapidly detected in plasma and brain; and reached peak concentrations at the first sampling time points. After oral dosing, FTY720-C2 was present in plasma and brain, although FTY720-Mitoxy was not orally bioavailable. Brain-to-plasma ratio of both compounds increased time-dependently, suggesting a preferential partitioning to the brain. PP2A activity in mouse adrenal gland increased ~2-fold after FTY720-C2 or FTY720-Mitoxy, as compared to untreated controls. In summary, FTY720-C2 and FTY720-Mitoxy both (i) crossed the blood-brain-barrier; (ii) produced metabolites similar to FTY720, except without phosphorylated species that cause S1P1-mediated-immunosuppression; and (iii) stimulated in vivo PP2A activity, all of which encourage additional preclinical assessment.

Desvenlafaxine and venlafaxine exert minimal in vitro inhibition of human cytochrome P450 and P-glycoprotein activities.

OBJECTIVE: Identification of potential pharmacokinetic drug-drug interactions is an important step in clinical drug development.We assessed and compared the drug-drug interaction potential of desvenlafaxine and venlafaxine, based on their inhibitory potency on human cytochrome P450 (CYP) and P-glycoprotein (P-gp) activities in vitro. METHODS: Reversible inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and mechanism-based inhibition of CYP2C9, CYP2C19, CYP2D6, and CYP3A activity by desvenlafaxine and venlafaxine were determined in human liver microsomes.Whether these drugs were substrates for efflux or inhibitors of P-gp were determined in Caco-2 monolayers. RESULTS: Desvenlafaxine and venlafaxine showed little or no reversible inhibition of various CYP enzymes (concentration that inhibits 50% [IC50] or inhibition constant [Ki] ~ or >100 muM). In addition, neither drug acted as a mechanism-based inhibitor of CYP2C9, CYP2C19, CYP2D6, or CYP3A as they did not reduce the IC50 value for any of these enzymes in the presence of preincubations with or without a nicotinamide adenine dinucleotide phosphate-regenerating system. Desvenlafaxine and venlafaxine showed little inhibition of P-gp activity (IC50 values >250 muM) and did not act as substrates (efflux ratios <2) for efflux in Caco-2 monolayers. CONCLUSIONS: Considering in vitro and available clinical data, desvenlafaxine and venlafaxine appear to have low potential for pharmacokinetic drug-drug interactions via inhibiting the metabolic clearance of concomitant drugs that are substrates of various CYP enzymes, in particular CYP2D6. In addition, these data suggest that desvenlafaxine and venlafaxine exhibit little potential for pharmacokinetic interactions with concomitant drugs that are substrates or inhibitors of P-gp.

Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds.

Pleconaril is a broad-spectrum antirhinovirus and antienterovirus compound that binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating. When crystals of human rhinovirus 16 (HRV16) and HRV14 are incubated with pleconaril, drug occupancy in the binding pocket is lower than when pleconaril is introduced during assembly prior to crystallization. This effect is far more marked in HRV16 than in HRV14 and is more marked with pleconaril than with other compounds. These observations are consistent with virus yield inhibition studies and radiolabeled drug binding studies showing that the antiviral effect of pleconaril against HRV16 is greater on the infectivity of progeny virions than the parent input viruses. These data suggest that drug integration into the binding pocket during assembly, or at some other late stage in virus replication, may contribute to the antiviral activity of capsid binding compounds.

VP1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds.

Rhinoviruses are the most common infectious agents of humans. They are the principal etiologic agents of afebrile viral upper-respiratory-tract infections (the common cold). Human rhinoviruses (HRVs) comprise a genus within the family Picornaviridae. There are >100 serotypically distinct members of this genus. In order to better understand their phylogenetic relationship, the nucleotide sequence for the major surface protein of the virus capsid, VP1, was determined for all known HRV serotypes and one untyped isolate (HRV-Hanks). Phylogenetic analysis of deduced amino acid sequence data support previous studies subdividing the genus into two species containing all but one HRV serotype (HRV-87). Seventy-five HRV serotypes and HRV-Hanks belong to species HRV-A, and twenty-five HRV serotypes belong to species HRV-B. Located within VP1 is a hydrophobic pocket into which small-molecule antiviral compounds such as pleconaril bind and inhibit functions associated with the virus capsid. Analyses of the amino acids that constitute this pocket indicate that the sequence correlates strongly with virus susceptibility to pleconaril inhibition. Further, amino acid changes observed in reduced susceptibility variant viruses recovered from patients enrolled in clinical trials with pleconaril were distinct from those that confer natural phenotypic resistance to the drug. These observations suggest that it is possible to differentiate rhinoviruses naturally resistant to capsid function inhibitors from those that emerge from susceptible virus populations as a result of antiviral drug selection pressure based on sequence analysis of the drug-binding pocket.