Comparative Study of Dermal Pharmacokinetics Between Topical Drugs Using Open Flow Microperfusion in a Pig Model.

PURPOSE: Accurate methods to determine dermal pharmacokinetics are important to increase the rate of clinical success in topical drug development. We investigated in an in vivo pig model whether the unbound drug concentration in the interstitial fluid as determined by dermal open flow microperfusion (dOFM) is a more reliable measure of dermal exposure compared to dermal biopsies for seven prescription or investigational drugs. In addition, we verified standard dOFM measurement using a recirculation approach and compared dosing frequencies (QD versus BID) and dose strengths (high versus low drug concentrations). METHODS: Domestic pigs were topically administered seven different drugs twice daily in two studies. On day 7, drug exposures in the dermis were assessed in two ways: (1) dOFM provided the total and unbound drug concentrations in dermal interstitial fluid, and (2) clean punch biopsies after heat separation provided the total concentrations in the upper and lower dermis. RESULTS: dOFM showed sufficient intra-study precision to distinguish interstitial fluid concentrations between different drugs, dose frequencies and dose strengths, and had good reproducibility between studies. Biopsy concentrations showed much higher and more variable values. Standard dOFM measurements were consistent with values obtained with the recirculation approach. CONCLUSIONS: dOFM pig model is a robust and reproducible method to directly determine topical drug concentration in dermal interstitial fluid. Dermal biopsies were a less reliable measure of dermal exposure due to possible contributions from drug bound to tissue and drug associated with skin appendages.

The Comparison of Machine Learning and Mechanistic In Vitro-In Vivo Extrapolation Models for the Prediction of Human Intrinsic Clearance.

Accurate prediction of human pharmacokinetics (PK) remains one of the key objectives of drug metabolism and PK (DMPK) scientists in drug discovery projects. This is typically performed by using in vitro-in vivo extrapolation (IVIVE) based on mechanistic PK models. In recent years, machine learning (ML), with its ability to harness patterns from previous outcomes to predict future events, has gained increased popularity in application to absorption, distribution, metabolism, and excretion (ADME) sciences. This study compares the performance of various ML and mechanistic models for the prediction of human IV clearance for a large (645) set of diverse compounds with literature human IV PK data, as well as measured relevant in vitro end points. ML models were built using multiple approaches for the descriptors: (1) calculated physical properties and structural descriptors based on chemical structure alone (classical QSAR/QSPR); (2) in vitro measured inputs only with no structure-based descriptors (ML IVIVE); and (3) in silico ML IVIVE using in silico model predictions for the in vitro inputs. For the mechanistic models, well-stirred and parallel-tube liver models were considered with and without the use of empirical scaling factors and with and without renal clearance. The best ML model for the prediction of in vivo human intrinsic clearance (CLint) was an in vitro ML IVIVE model using only six in vitro inputs with an average absolute fold error (AAFE) of 2.5. The best mechanistic model used the parallel-tube liver model, with empirical scaling factors resulting in an AAFE of 2.8. The corresponding mechanistic model with full in silico inputs achieved an AAFE of 3.3. These relative performances of the models were confirmed with the prediction of 16 Pfizer drug candidates that were not part of the original data set. Results show that ML IVIVE models are comparable to or superior to their best mechanistic counterparts. We also show that ML IVIVE models can be used to derive insights into factors for the improvement of mechanistic PK prediction.

Prediction of Human Brain Penetration of P-glycoprotein and Breast Cancer Resistance Protein Substrates Using In Vitro Transporter Studies and Animal Models.

Four P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates with human cerebrospinal fluid (CSF) concentrations and preclinical neuropharmacokinetics were used to assess in vitro-in vivo extrapolation of brain penetration in preclinical species and the ability to predict human brain penetration. Unbound brain (Cb,u), unbound plasma (Cp,u), and CSF compound concentrations (CCSF) were measured in rats and nonhuman primates (NHPs), and the unbound partition coefficients (Cb,u/Cp,u and CCSF/Cp,u) were used to assess brain penetration. The results indicated that for P-gp and BCRP dual substrates, brain penetration was severally impaired in all species. In comparison, for P-gp substrates that are weak or non-BCRP substrates, improved brain penetration was observed in NHPs and humans than in rats. Overall, NHP appears to be more predictive of human brain penetration for P-gp substrates with weak or no interaction with BCRP than rat. Although CCSF does not quantitatively correspond to Cb,u for efflux transporter substrates, it is mostly within 3-fold higher of Cb,u in rat and NHP, suggesting that CCSF can be used as a surrogate for Cb,u. Taken together, a holistic approach including both in vitro transporter and in vivo neuropharmacokinetics data enables a better estimation of human brain penetration of P-gp/BCRP substrates.

The discovery and characterization of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor potentiator N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242).

A unique tetrahydrofuran ether class of highly potent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiators has been identified using rational and structure-based drug design. An acyclic lead compound, containing an ether-linked isopropylsulfonamide and biphenyl group, was pharmacologically augmented by converting it to a conformationally constrained tetrahydrofuran to improve key interactions with the human GluA2 ligand-binding domain. Subsequent replacement of the distal phenyl motif with 2-cyanothiophene to enhance its potency, selectivity, and metabolic stability afforded N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242, 3), whose preclinical characterization suggests an adequate therapeutic index, aided by low projected human oral pharmacokinetic variability, for clinical studies exploring its ability to attenuate cognitive deficits in patients with schizophrenia.

Enhancing ketamine translational pharmacology via receptor occupancy normalization.

Ketamine is used preclinically and clinically to study schizophrenia and depression. Accordingly, it is imperative to understand the temporal relationship between the central concentrations and N-methyl-d-aspartate receptor (NMDAR) interactions of both ketamine and norketamine, its primary active metabolite, across species to assess the translatability of animal models to humans and the back-translation of clinical observations to the preclinical realm. However, such an interspecies normalization of ketamine and norketamine exposures at different clinical and preclinical doses (and their different routes and regimens) is lacking. This work defines the NMDAR occupancy (RO) time course following single doses of ketamine in rats, nonhuman primates (nhp) and humans to allow direct interspecies comparisons of specific ketamine-mediated pharmacodynamics via RO normalization. Total plasma concentration (Cp)-time profiles of ketamine and norketamine were generated from rats and nhp following a single, memory-impairing dose of ketamine; neuropharmacokinetics were determined in rats. [(3)H]MK-801-displacement studies in rats determined estimated mean (95% confidence interval) unbound plasma concentrations (Cp,u) for ketamine and norketamine producing 50% RO (IC50) of 1420 (990, 2140) nM and 9110 (5870, 13700) nM, respectively. Together, these datasets transformed Cp,u-time data to predicted RO (ROpred)-time profiles for rats, nhp and humans at behaviorally relevant ketamine doses. Subsequently, this approach helped determine an infusion paradigm in rats producing a ROpred-time profile mirroring that for a clinically antidepressant infusion. The described indication-independent methodology allows normalization to RO at any time following any ketamine dose (regardless of route or regimen) in any species by simply quantifying the Cp of ketamine and norketamine. Matching temporal RO relationships in animals and humans should allow direct comparisons of specific ketamine-dependent NMDAR-based pharmacodynamics.

Diphenhydramine has similar interspecies net active influx at the blood-brain barrier.

In rats, oxycodone, diphenhydramine, and [4-chloro-5-fluoro-2-(3-methoxy-2-methyl-phenoxy)-benzyl]-methylamine (CE-157119) undergo net active influx at the blood-brain barrier (BBB) based on significantly greater interstitial fluid compound concentrations (CISF ) than unbound plasma compound concentrations (Cp,u ). Oxycodone and diphenhydramine have CISF :Cp,u of 3.0 and 5.5, respectively, while CE-157119 has an unbound brain compound concentration (Cb,u ):Cp,u of 3.90; Cb,u is a high-confidence CISF surrogate. However, only CE-157119 has published dog and nonhuman primate (nhp) neuropharmacokinetics, which show similar Cb,u :Cp,u (4.61 and 2.04, respectively) as rats. Thus, diphenhydramine underwent identical interspecies neuropharmacokinetics studies to determine if its net active BBB influx in rats replicated in dogs and/or nhp. The single-dose-derived rat Cb,u :Cp,u (3.90) was consistent with prior steady-state-derived CISF :Cp,u and similar to those in dogs (4.88) and nhp (4.51-5.00). All large animal interneurocompartmental ratios were ≤1.8-fold different than their rat values, implying that diphenhydramine has constant and substantial Cb,u -favoring disequilibria in these mammals. Accordingly, the applied Cb,u -forecasting methodology accurately predicted [estimated mean (95% confidence interval) of 0.84 (0.68, 1.05)] Cb,u from each measured Cp,u in large animals. The collective datasets suggest these Cb,u -preferring asymmetries are mediated by a species-independent BBB active uptake system whose identification, full characterization, and structure-activity relationships should be prioritized for potential exploitation.

The effect of ketamine-contaminated control plasma on small-molecule plasma protein binding.

Due to macroeconomic factors, the pharmaceutical industry has progressively outsourced larger amounts of its internal research efforts. One such example is the purchase of large volumes of control nonhuman primate (nhp) plasma from external vendors. During the bioanalysis of plasma samples collected during a Cynomolgus monkey PK study with racemic (±) ketamine, it was discovered that the vendor control nhp plasma used for standard curve samples contained ketamine (32 µM). This ketamine concentration correlated to an anesthetic dose of ketamine (10-15 mg/kg, IM), which was not cited in the vendor's plasma harvesting protocol. This study evaluated the effect of ketamine in control nhp plasma on the in vitro-derived unbound plasma fraction of small molecules, which would cause erroneous nhp efficacy and safety values.

Positive allosteric modulation of AMPA receptors from efficacy to toxicity: the interspecies exposure-response continuum of the novel potentiator PF-4778574.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) positive allosteric modulation (i.e., "potentiation") has been proposed to overcome cognitive impairments in schizophrenia, but AMPAR overstimulation can be excitotoxic. Thus, it is critical to define carefully a potentiator's mechanism-based therapeutic index (TI) and to determine confidently its translatability from rodents to higher-order species. Accordingly, the novel AMPAR potentiator N-{(3R,4S)-3-[4-(5-cyano-2-thienyl)phenyl]tetrahydro-2H-pyran-4-yl}propane-2-sulfonamide (PF-4778574) was characterized in a series of in vitro assays and single-dose animal studies evaluating AMPAR-mediated activities related to cognition and safety to afford an unbound brain compound concentration (Cb,u)-normalized interspecies exposure-response relationship. Because it is unknown which AMPAR subtype(s) may be selectively potentiated for an optimal TI, PF-4778574 binding affinity and functional potency were determined in rodent tissues expected to express a native mixture of AMPAR subunits and their associated proteins to afford composite pharmacological values. Functional activity was also quantified in recombinant cell lines stably expressing human GluA2 flip or flop homotetramers. Procognitive effects of PF-4778574 were evaluated in both rat electrophysiological and nonhuman primate (nhp) behavioral models of pharmacologically induced N-methyl-d-aspartate receptor hypofunction. Safety studies assessed cerebellum-based AMPAR activation (mouse) and motor coordination disruptions (mouse, dog, and nhp), as well as convulsion (mouse, rat, and dog). The resulting empirically derived exposure-response continuum for PF-4778574 defines a single-dose-based TI of 8- to 16-fold for self-limiting tremor, a readily monitorable clinical adverse event. Importantly, the Cb,u mediating each physiological effect were highly consistent across species, with efficacy and convulsion occurring at just fractions of the in vitro-derived pharmacological values.

An evaluation of using rat-derived single-dose neuropharmacokinetic parameters to project accurately large animal unbound brain drug concentrations.

Previous publications suggest that interstitial fluid compound concentrations (C(ISF)) best determine quantitative neurotherapeutic pharmacology relationships, although confirming large animal C(ISF) remains elusive. Therefore, this work primarily evaluated using respective acute dose, rat-derived unbound brain compound concentration-to-unbound plasma compound concentration ratios (C(b,u)/C(p,u)) to project accurately dog and nonhuman primate (nhp) C(b,u), a C(ISF) surrogate, from measured C(p,u) for the highly permeable non-P-glycoprotein substrates N-{(3R,4S)-3-[4-(5-cyano-2-thienyl)phenyl]tetrahydro-2H-pyran-4-yl}propane-2-sulfonamide (PF-4778574) and [4-chloro-5-fluoro-2-(3-methoxy-2-methyl-phenoxy)-benzyl]-methylamine (CE-157119) and the P-glycoprotein substrates risperidone and 9-hydroxyrisperidone. First, in rats, it was determined for eight of nine commercial compounds that their single-dose-derived C(b,u)/C(p,u) were ≤2.5-fold different from their steady-state values; for all nine drugs, their C(b,u)/C(p,u) were ≤2.5-fold different from their steady-state C(ISF)/C(p,u) (Drug Metab Dispos 37:787-793, 2009). Subsequently, PF-4778574, CE-157119 and risperidone underwent rat, dog, and nhp neuropharmacokinetics studies. In large animals at each measured C(p,u), the methodology adequately predicted [estimated mean (95% confidence interval) of 1.02 (0.80, 1.29)] the observed C(b,u) for PF-4778574 and CE-157119 but underpredicted [0.17 (0.12, 0.22)] C(b,u) for risperidone and 9-hydroxyrisperidone. The data imply that forecasting higher species C(b,u) from a measured C(p,u) and rat acute dose-determined C(b,u):C(p,u) is of high confidence for nonefflux transporter substrates that show net passive diffusion (PF-4778574) or net active influx (CE-157119) at the blood-brain barrier in rats. However, this methodology appears ineffective for correctly predicting large animal C(b,u) for P-glycoprotein substrates (risperidone and 9-hydroxyrisperidone) because of their apparently much greater C(p,u)-favoring C(b,u):C(p,u) asymmetry in rats versus dogs or nhp. Instead, for such P-glycoprotein substrates, large animal-specific cerebrospinal fluid compound concentrations (C(CSF)) seemingly best represent C(b,u).

Quantitative pharmacokinetic/pharmacodynamic analyses suggest that the 129/SVE mouse is a suitable preclinical pharmacology model for identifying small-molecule γ-secretase inhibitors.

Alzheimer's disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of ß- or γ-secretase, key enzymes for the production of amyloid ß (Aß), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aß species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aß lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aß across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aßx-42 and CSF Aßx-40 in the 129/SVE mouse is indicative of that for human CSF Aß. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aß data are established.

A novel series of [3.2.1] azabicyclic biaryl ethers as alpha3beta4 and alpha6/4beta4 nicotinic receptor agonists.

We report the synthesis of a series of [3.2.1]azabicyclic biaryl ethers as selective agonists of alpha3- and alpha6-containing nicotinic receptors. In particular, compound 17a from this series is a potent alpha3beta4 and alpha6/4beta4 receptor agonist in terms of both binding and functional activity. Compound 17a also shows potent in vivo activity in CNS-mediated animal models that are sensitive to antipsychotic drugs. Compound 17a may thus be a useful tool for studying the role of alpha3beta4 and alpha6/4beta4 nicotinic receptors in CNS pharmacology.

Manipulation of the vertebrate host's testosterone does not affect gametocyte sex ratio of a malaria parasite.

Gametocyte sex ratio of the malaria parasite Plasmodium mexicanum is variable in its host, the western fence lizard (Sceloporus occidentalis), both among infections and within infections over time. We sought to determine the effect of host physiological quality on the gametocyte sex ratio in experimentally induced infections of P. mexicanum. Adult male lizards were assigned to 4 treatment groups: castrated, castrated + testosterone implant, sham implant, and unmanipulated control. No significant difference in gametocyte sex ratio was found among the 4 treatment groups. Two other analyses were performed. A surgery stress analysis compared infection sex ratio of castrated, castrated + testosterone implant, and sham implant groups with the unmanipulated control group. A testosterone alteration analysis compared infection sex ratio of the castrated and castrated + testosterone implant groups with the sham implant and unmanipulated control groups. Again, no significant difference was observed for these 2 comparisons. Thus, physiological changes expected for experimentally induced variation in host testosterone and the stress of surgery were not associated with any change in the gametocyte sex ratio. Also, theex-periment suggests testosterone is not a cue for shaping the sex ratio of gametocytes in P. mexicanum. These results are related to the evolutionary theory of sex ratios as applied to malaria parasites.

Gametocyte sex ratio of a malaria parasite: experimental test of heritability.

The gametocyte sex ratio of Plasmodium mexicanum, a malaria parasite of western fence lizards, was studied in a modified garden experiment. Each of 6 naturally infected lizards was used to initiate 20 replicate-infections in naive western fence lizards. A significant donor effect was observed for the sex ratios of recipient infections at their maximal parasitemia, and this effect was associated with the sex ratio of the donor infection. In 20 infections in which sex ratio was followed during the course of the infection, 9 revealed constant sex ratios and 11 showed an increase in proportion of males over time. Recipient sex ratio was correlated with another life-history trait, a composite of rate of asexual replication and peak parasitemia, such that higher Rate-Peak scores were associated with infections with less female-biased sex ratios. These results are placed into the context of sex ratio theory that concludes that the degree of selfing of parasite genotypes (number of parasite clones) within the vector will influence the evolution of gametocyte sex ratio. The theory predicts that the sex ratio should be under some genetic control and thus be heritable as observed in the experiment. Clonal diversity should also influence the life-history trait, Rate-Peak, which was found to be correlated with sex ratio.