Susan Cole

Prediction of Human Pharmacokinetics From Preclinical Information: Comparative Accuracy of Quantitative Prediction Approaches

Quantitative prediction of human pharmacokinetics is critical in assessing the viability of drug candidates and in determining first‐in‐human dosing. Numerous prediction methodologies, incorporating both in vitro and preclinical in vivo data, have been developed in recent years, each with advantages and disadvantages. However, the lack of a comprehensive data set, both preclinical and clinical, has limited efforts to evaluate the optimal strategy (or strategies) that results in quantitative predictions of human pharmacokinetics. To address this issue, the authors conducted a retrospective analysis using 50 proprietary compounds for which in vitro, preclinical pharmacokinetic data and oral single‐dose human pharmacokinetic data were available. Five predictive strategies, involving either allometry or use of unbound intrinsic clearance from microsomes or hepatocytes, were then compared for their ability to predict human oral clearance, half‐life through predictions of systemic clearance, volume of distribution, and bioavailability. Use of a single‐species scaling approach with rat, dog, or monkey was as accurate as or more accurate than using multiple‐species allometry. For those compounds cleared almost exclusively by P450‐mediated pathways, scaling from human liver microsomes was as predictive as single‐species scaling of clearance based on data from rat, dog, or monkey. These data suggest that use of predictive methods involving either single‐species in vivo data or in vitro human liver microsomes can quantitatively predict human in vivo pharmacokinetics and suggest the possibility of streamlining the predictive methodology through use of a single species or use only of human in vitro microsomal preparations.

Simulation of Human Intravenous and Oral Pharmacokinetics of 21 Diverse Compounds Using Physiologically Based Pharmacokinetic Modelling

AbstractBackground: The importance of predicting human pharmacokinetics during compound selection has been recognized in the pharmaceutical industry. To this end there are many different approaches that are applied. Methods: In this study we compared the accuracy of physiologically based pharmacokinetic (PBPK) methodologies implemented in GastroPlus™ with the one-compartment approach routinely used at Pfizer for human pharmacokinetic plasma concentration-time profile prediction. Twenty-one Pfizer compounds were selected based on the availability of relevant preclinical and clinical data. Intravenous and oral human simulations were performed for each compound. To understand any mispredictions, simulations were also performed using the observed clearance (CL) value as input into the model. Results: The simulation results using PBPK were shown to be superior to those obtained via traditional one-compartment analyses. In many cases, this difference was statistically significant. Specifically, the results showed that the PBPK approach was able to accurately predict passive distribution and absorption processes. Some issues and limitations remain with respect to the prediction of CL and active transport processes and these need to be improved to further increase the utility of PBPK modelling. A particular advantage of the PBPK approach is its ability to accurately predict the multiphasic shape of the pharmacokinetic profiles for many of the compounds tested. Conclusion: The results from this evaluation demonstrate the utility of PBPK methodology for the prediction of human pharmacokinetics. This methodology can be applied at different stages to enhance the understanding of the compounds in a particular chemical series, guide experiments, aid candidate selection and inform clinical trial design.

The Human Specific CCR1 Antagonist CP-481,715 Inhibits Cell Infiltration and Inflammatory Responses in Human CCR1 Transgenic Mice

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-γ and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.

The anti-tumor activity of anti-CTLA-4 is mediated through its induction of IFNγ

Abstract The T-cell-specific receptor, CTLA-4, has been demonstrated to be a potent negative regulator of lymphocyte activation, the functional significance of which has been demonstrated in murine tumor models using blocking antibodies. However, the mechanism(s) involved in enhancing tumor regression has not been identified. In this study, we determined whether IFNγ was playing a role in this activity. In vitro, anti-CTLA-4 enhanced IFNγ production by lymph node cells obtained from tumor-bearing mice (351 pg/ml vs 77 pg/ml). Additionally, fibrosarcoma-challenged animals treated with anti-CTLA-4 had elevated levels of the IFN-inducible enzyme 2-5-OAS in draining lymph nodes (850 pM vs 260 pM for controls) and an increased amount of IFNγ in tumor lysates (at day 7, 620 pg/100 μg vs 160 pg/100 μg in controls). The importance of IFNγ was demonstrated by the ability of neutralizing antibodies to completely abrogate the anti-tumor effects of anti-CTLA-4. Moreover, fibrosarcoma cells were shown to be exquisitely sensitive to IFNγ-mediated class I upregulation and histological examination of tumors from anti-CTLA-4-treated mice revealed a trend toward increased tumor cell apoptosis and decreased angiogenesis. These studies have demonstrated that one mechanism for the anti-tumor effects of anti-CTLA-4 relates to its ability to augment IFNγ production, resulting in an increased expression of class I on the tumor, enhanced apoptosis, and a decrease in blood vessel growth.

Full efficacy with no CNS side-effects: unachievable panacea or reality? DMPK considerations in design of drugs with limited brain penetration

Optimising drug properties can be an important strategy to limit penetration into the CNS and offers advantages in reducing the risk of undesirable neurological effects When considering the design of these drugs it is important to consider the relative influx and efflux rates at the relevant biological membranes The highest degree of restriction at the brain is probably achievable by utilising active transport to exclude compounds from the brain Affinity for the efflux transporters Pgp and BCRP has been achieved in two in-house chemistry programmes by increasing polar surface area, which resulted in highly orally bioavailable low CNS penetrant compounds in preclinical species

PD168077, a D4 receptor agonist, reverses object recognition deficits in rats: potential role for D4 receptor mechanisms in improving cognitive dysfunction in schizophrenia:

This study investigated the effects of the dopamine D4 receptor agonist, PD168077, on recognition memory using a novel object recognition task, which detects disruption and improvement of recognition memory in rats by measuring their ability to discriminate between familiar and novel objects. When acquisition and test were 6 h apart (experiment 1), control rats failed to discriminate between familiar and novel objects at test. Rats given low doses of PD168077 (0.3; 1.0 mg/kg) also failed to discriminate between the objects, while rats given higher doses (3.0; 10.0 mg/kg) explored the novel object more than the familiar object, indicating retained memory of the familiar object. Thus, at higher doses, PD168077 improved recognition memory in rats. Experiment 2 tested whether PD168077 would attenuate deficits in novel object recognition induced by sub-chronic phencyclidine. Testing was 1 min after acquisition, such that vehicle pre-treated rats differentiated between the novel and familiar objects: however, sub-chronic phencyclidine-treated rats failed to discriminate between the two, indicating disruption of recognition memory. PD168077 (10 mg/kg) restored the ability of phencyclidine-treated rats to differentiate between the novel and familiar objects, indicating improved recognition memory. The results suggest that D4 receptor activation can improve cognitive dysfunction in an animal model relevant to schizophrenia.

Discovery of a Selective Small-Molecule Melanocortin-4 Receptor Agonist with Efficacy in a Pilot Study of Sexual Dysfunction in Humans

The relevance of the melanocortin system to sexual activity is well established, and nonselective peptide agonists of the melanocortin receptors have shown evidence of efficacy in human sexual dysfunction. The role of the MC4 receptor subtype has received particular scrutiny, but the sufficiency of its selective activation in potentiating sexual response has remained uncertain owing to conflicting data from studies in preclinical species. We describe here the discovery of a novel series of small-molecule MC4 receptor agonists derived from library hit 2. The addition of methyl substituents at C3 and C5 of the 4-phenylpiperidin-4-ol ring was found to be markedly potency-enhancing, enabling the combination of low nanomolar potencies with full rule-of-five compliance. In general, the series shows only micromolar activity at other melanocortin receptors. Our preferred compound 40a provided significant systemic exposure in humans on both sublingual and oral administration and was safe and well tolerated up to the maximum tested dose. In a pilot clinical study of male erectile dysfunction, the highest dose of 40a tested (200 mg) provided a similar level of efficacy to sildenafil.

Evaluation of metaquant microdialysis for measurement of absolute concentrations of amphetamine and dopamine in brain: A viable method for assessing pharmacokinetic profile of drugs in the brain

Direct measurement of absolute brain concentration of amphetamine and dopamine were obtained using metaquant (MQ) microdialysis, which achieves near 100% recovery, in the caudate nucleus. Conventional microdialysis monoprobes were also implanted in the caudate nucleus in the contralateral side of the same animals to compare the brain concentrations obtained from these two probe types. In addition plasma concentrations of amphetamine were obtained simultaneously from the same animals. The distribution of amphetamine in the plasma and of amphetamine and dopamine in both probe types followed same profile at each time interval. The basal dialysate concentration of dopamine in the caudate nucleus measured by MQ, was 9.40+/-0.60 nM, while measured by conventional microdialysis it was 6.35+/-0.36 nM. This study demonstrates that MQ microdialysis is an appropriate method for determination of true extracellular levels of drugs and neurotransmitters in the brain, under dynamic conditions. Since these measurements, together with measurements of plasma concentrations of the drug, can be made in a single animal, the method can be used to study pharmacokinetic-pharmacodyamics profile of psychoactive agents.

Human lymphocyte activation assay: an in vitro method for predictive immunotoxicity testing.

Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell–Dutton assay (in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4+ and CD8+ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment.

The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics

This paper describes our recent efforts to design and synthesise potent and selective PDE5 inhibitors and the use of in vitro predictors of clearance, absorption and permeability to maximise the potential for dose-proportional pharmacokinetics and good oral bioavailability in man. Optimisation of the preclinical profile resulted in the identification of UK-369003 (19a) and its nomination as a clinical candidate. The clinical pharmacokinetic and safety profile has enabled us to progress the compound to test its efficacy in patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) and a paper describing its efficacy has recently been published.