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A key element of the drug discovery process is target selection. Although the topic is subject to much discussion and experimental effort, there are no defined quantitative rules around optimal selection. Often 'rules of thumb', that have not been subject to rigorous exploration, are used. In this paper we explore the 'rule of thumb' notion that the molecule that initiates a pathway signal is the optimal target. Given the multi-factorial and complex nature of this question, we have simplified an example pathway to its logical minimum of two steps and used a mathematical model of this to explore the different options in the context of typical small and large molecule drugs. In this paper, we report the conclusions of our analysis and describe the analysis tool and methods used. These provide a platform to enable a more extensive enquiry into this important topic.

PF-05105679 is a moderately potent TRPM8 blocker which has been evaluated for the treatment of cold pain sensitivity. The TRPM8 channel is responsible for the sensation of cold environmental temperatures and has been implicated in regulation of core body temperature. Consequently, blockade of TRPM8 has been suggested to result in lowering of core body temperature. As part of the progression to human studies, the effect of PF-05105679 on core body temperature has been investigated in animals. Safety pharmacology studies showed that PF-05105679 reduced core body temperature in a manner that was inversely related to body weight of the species tested (greater exposure to PF-05105679 was required to lower temperature by 1°C in higher species). Based on an allometric (body weight) relationship, it was hypothesized that PF-05105679 would not lower core body temperature in humans at exposures that could exhibit pharmacological effects on cold pain sensation. On administration to humans, PF-05105679 was indeed effective at reversing the cold pain sensation associated with the cold pressor test in the absence of effects on core body temperature.

Mathematical models have been widely used for understanding the dynamics of the hepatitis C virus (HCV). We propose a method to predict final clinical outcome for 24 HIV–HCV - coinfected patients with the help of a mathematical model based on the first two weeks of PEG-IFN therapy. Applying a pharmacokinetic-pharmacodynamic (PKPD) approach, together with mixture models, to the adapted model of viral dynamics developed by Neumann et al., we have analyzed the influence of PEG-IFN on the kinetics and interaction of target cells, infected cells and virus mRNA. It was found that PEG-IFN pharmacokinetic parameters were similar in sustained virological responders and nonresponders, while the plasma PEG-IFN concentration that decreases HCV production by 50% (EC50) and the rate of infected cell death were different. The treatment outcome depended mainly on the initial viral mRNA concentration and the rate of infected cell death. The population PKPD approach with a mixture model enabled the determination of individual PKPD parameters and showed high sensitivity (93.5%) and specificity (97.4%) for the prediction of the treatment outcome.

In an effort to uncover systematic learnings that can be applied to improve compound survival, an analysis was performed on data from Phase II decisions for 44 programs at Pfizer. It was found that not only were the majority of failures caused by lack of efficacy but also that, in a large number of cases (43%), it was not possible to conclude whether the mechanism had been tested adequately. A key finding was that an integrated understanding of the fundamental pharmacokinetic/pharmacodynamic principles of exposure at the site of action, target binding and expression of functional pharmacological activity (termed together as the 'three Pillars of survival') all determine the likelihood of candidate survival in Phase II trials and improve the chance of progression to Phase III.

In this paper we analyse the impact of protein-, lipid- and receptor-binding on receptor occupancy in a two-compartment system, with proteins in both compartments and lipids and receptors in the peripheral compartment only. We do this for two manners of drug administration: a bolus administration and a constant rate infusion, both into the central compartment. We derive explicit approximations for the time-curves of the different compounds valid for a wide range of realistic values of rate constants and initial concentrations of proteins, lipids, receptors and the drug. These approximations are used to obtain both qualitative and quantitative insight into such critical properties as the distribution of the drug over the two compartments, the maximum receptor occupancy and the area under the drug-receptor complex curve. In particular we focus on assessing the impact of the dissociation constants, K(P), K(L) and K(R) of the drug with, respectively, the proteins, the lipids and the receptors, the permeability and the surface area of the membrane between compartments, and the rate the drug is eliminated from the system.

In this paper we analyse the dynamics of an inhibitor I which can either bind to a receptor R or to a plasma protein P. Assuming typical association and dissociation rates, we find that after an initial dose of inhibitor, there are three time scales: a short one, measured in fractions of seconds, in which the inhibitor concentration and the plasma-protein complex jump to quasi-stationary values, a medium one, measured in seconds in which the receptor complex rises to an equilibrium value and a large one, measured in hours in which the inhibitor–receptor complex slowly drops down to zero. We show that the average receptor occupancy, the pharmacologically relevant quantity, taken over, say, 24 h reaches a maximal value for a specific value of the plasma-protein binding constant. Potentially, understanding and exploiting this optimum could be of great interest to those involved in drug discovery and development.

In spite of the extensive use of long-acting β2-agonist (LABA) bronchodilators in asthma, the actual mechanism of their in vivo duration of action is not well understood, primarily due to limitations of standard pharmacokinetic–pharmacodynamic (PKPD) analysis methodologies. We have developed a novel method of analysing lung efficacy vs. time profiles for LABAs that can be used to provide comparative information on the lung PK. We hypothesised that for compounds that do not differ in their PK at the site of PD action, but differ in their in vivo potencies, the relationship between the area under the effect curve (AUEC) and the observed maximum effect (OME) at different doses is described by the same sigmoid curve. We have illustrated this property for standard PKPD models by obtaining analytical solution and through simulations. Anaesthetised dog in vivo effect vs. time profiles were gathered for six inhaled LABA candidates that differ in their in vitro potencies. Neither lung nor systemic PK was available for any compound. Analysis of the AUEC vs. OME data, derived from the efficacy profiles, using nonlinear mixed effects modelling indicated that for four compounds, the observed differences in in vivo duration of action was due to differences in their in vivo potencies and not because of lung PK differences. Therefore, it was concluded that for these compounds, characterisation of lung PK was unlikely to differentiate their PKPD characteristics. Thus, the proposed approach helped focus resources during translational research leading to lead candidate selection.

A series of substituted glutaramides were synthesised using Candoxatrilat 1 as a lead and evaluated for potency against neutral endopeptidase (NEP) as a potential treatment for female sexual arousal disorder (FSAD). In this paper, we describe studies in which we were able to increase NEP activity substantially over the levels reported for previous compounds from this programme by appropriate substitution in both the P(1)(') and P(2)(') regions. Optimisation led to the 4-chlorophenpropylamide S-30 which was selected as a candidate for further study.

Classical models of G protein coupled receptor (GPCR) signalling assume that each receptor functions as a single unit. However, evidence is increasing that GPCRs may form functional assemblies of dimeric or oligomeric units. There are several methods that can be used to give evidence of GPCR oligomerisation that will be discussed in this review. These include co-immunoprecipitation and Western blotting, resonance energy transfer methods and transactivation / complementation of partially functional receptors. One definitive method currently does not exist and there are various advantages and disadvantages to each method depending upon the system considered. Although co-immunoprecipitation and Western blot studies require disruption of the cellular environment and require specific antibodies, they are a good starting point to show that receptor oligomerisation occurs in native systems. Resonance energy transfer techniques provide evidence that receptors are in close proximity, are measured in living cells and some formats may be used for imaging applications. Transactivation / complementation requires extensive modification of the GPCR, but provides evidence that the receptors are in physical contact. Despite great advances being made using these techniques, future challenges involve the development of other methodologies to determine the role of receptor complexes in the pharmacology and physiology of native systems.

A rapid, sensitive and enantioselective HPLC assay for the simultaneous determination of the reference 5-HT1A receptor agonists, R-(+)- and S-(-)-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT and S-8-OH-DPAT, respectively), in rat blood is presented. A selective extraction procedure was developed using a preliminary sample clean-up followed by isolation of R- or S-8-OH-DPAT on mixed-mode NARC-2 solid-phase columns. Separation of the enantiomers was performed by high-performance liquid chromatography using a Chiracel OD-R column. Detection was obtained using an electrochemical detector set at a voltage of 0.63 V. The mobile phase consisted of a 50 mM phosphate buffer (pH 5.5)-acetonitrile (80:20, v/v) mixture. At a flow-rate of 1 ml min(-1), the total run time was approximately 14 min. The limit of detection for R- and S-8-OH-DPAT was 0.5 ng ml(-1). In the concentration range between 50 ng ml(-1) and 1000 ng ml(-1) intra- and inter-day relative standard deviations were less than 12%. The assay was applied to a pharmacokinetic-pharmacodynamic study in rats in which decrease of body temperature was used as a measure of 5-HT1A receptor-mediated effect. Values for clearance, volume of distribution at steady state and terminal elimination rate constant were 22+/-2 ml min(-1), 1969+/-473 ml and 156+/-34 min for R-8-OH-DPAT and 16+/-1 ml min(-1), 3353+/-347 ml and 334+/-36 min for S-8-OH-DPAT, respectively. No enantiomeric interconversion was observed in vivo from R-8-OH-DPAT to S-8-OH-DPAT or vice versa.

Inhaled capsaicin elicits cough reproducibly in human subjects and is widely used in the study of cough and antitussive therapies. However, the traditional end points C2 and C5 (the concentrations of capsaicin inducing at least 2 or 5 coughs, respectively) display extensive overlap between health and disease and therefore might not best reflect clinically relevant mechanisms.We sought to investigate capsaicin dose responses in different disease groups.Two novel capsaicin cough challenges were compared in patients with chronic cough (CC; n = 20), asthmatic patients (n = 18), and healthy volunteers (HVs; n = 20). Increasing doubling doses of capsaicin (0.48-1000 μmol/L, 4 inhalations per dose) were administered in challenge 1, whereas the order of the doses was randomized in challenge 2. A nonlinear mixed-effects model compared dose-response parameters by disease group and sex. Parameters were also correlated with objective cough frequency.The model classified subjects based on maximum cough response evoked by any concentration of capsaicin (Emax) and the capsaicin dose inducing half-maximal response (ED50). HVs and asthmatic patients were not statistically different for either parameter and therefore combined for analysis (mean ED50, 38.6 μmol/L [relative SE, 28%]; mean Emax, 4.5 coughs [relative SE, 11%]). Compared with HVs/asthmatic patients, patients with CC had lower ED50 values (14.7 μmol/L [relative SE, 28%], P = .008) and higher Emax values (8.6 coughs [relative SE, 11%], P.0001). Emax values highly correlated with 24-hour cough frequency (r = 0.71, P.001) and were 37% higher in female compared with male subjects, regardless of disease group (P.001).Nonlinear mixed-effects modeling demonstrates that maximal capsaicin cough responses better discriminate health from disease and predict spontaneous cough frequency and therefore provide important insights into the mechanisms underlying CC.

In a recent TiPS article [1xThe ligand paradox between affinity and efficacy: can you be there and not make a difference?. Kenakin, T. and Onaran, O. Trends Pharmacol. Sci. 2002; 23: 275–280Abstract | Full Text | Full Text PDF | PubMed | Scopus (98)See all References][1], Kenakin and Onaran presented a probabilistic model of protein conformation that describes efficacy and affinity as a function of agonist- and G-protein-induced perturbations of receptor states.In a simulation, the authors demonstrated that the model predicts a negative correlation between affinity and efficacy for different ligands acting in the same system, although homoscedacity in the model parameters allows for different agonists having identical efficacies with different affinities and identical affinities with different efficacies. The authors comment that the model contains too many parameters to use it to fit experimental data. Additionally, it seems very difficult to verify their simulated example experimentally because: (1) it would be practically impossible to test each compound in the same assay; and (2) a very large number of compounds would have to be synthesized and screened to reveal a correlation that is not overshadowed by the homoscedacity in the relationship as a result of between-assay variability. An alternative, perhaps more feasible, approach would be to use the inherent between-assay variability to test the predictions of the probabilistic model of receptor function with a single ligand. There are several examples in the literature indicating that it is indeed possible to reveal correlations between affinity and efficacy for a single agonist. For example, our analysis [2xAnalysis of inactivation experiments with the operational model of agonism yields correlated estimates of agonist affinity and efficacy. Van der Graaf, P.H. and Stam, W.B. J. Pharmacol. Toxicol. Meth. 1999; 41: 117–125Crossref | PubMed | Scopus (6)See all References][2] with the operational model of agonism [3xOperational models of pharmacological agonism. Black, J.W. and Leff, P. Proc. R. Soc. Lond. B. 1983; 220: 141–162Crossref | PubMedSee all References][3] of noradrenaline responses in rat small mesenteric arteries before and after phenoxybenzamine treatment revealed a highly significant, negative correlation between operational affinity (pKA) and efficacy (log τ), consistent with the predictions of the probability model. A similar conclusion can be inferred [2xAnalysis of inactivation experiments with the operational model of agonism yields correlated estimates of agonist affinity and efficacy. Van der Graaf, P.H. and Stam, W.B. J. Pharmacol. Toxicol. Meth. 1999; 41: 117–125Crossref | PubMed | Scopus (6)See all References][2] from data reported for the effects of carbachol and pilocarpine in guinea-pig ileum [4xGraphical assessment of the operational model of agonist action. Henry, A. et al. FASEB J. 1992; 6: A1561See all References][4], 5-HT in rabbit aorta [5xEstimation of agonist affinity and efficacy by direct, operational model-fitting. Leff, P. et al. J. Pharmacol. Meth. 1990; 23: 225–237Crossref | PubMed | Scopus (61)See all References][5], 5-methylfurmethide in guinea-pig trachea [6xApplication of the operational model of agonism to establish conditions when functional antagonism may be used to estimate agonist dissociation constants. Leff, P. et al. Br. J. Pharmacol. 1985; 85: 655–663Crossref | PubMedSee all References][6] and phenylephrine in rat tail artery [7xEndothelial modulation of α1-adrenoceptor contractile responses in the tail artery of spontaneous hypertensive rats. Tabernero, A. et al. Br. J. Pharmacol. 1996; 119: 765–771Crossref | PubMedSee all References][7]. Although it cannot be excluded that such observations are due to a statistical rather than a pharmacological phenomenon and reflect the issue of over-parameterization inherent to mechanistic models of agonism [2xAnalysis of inactivation experiments with the operational model of agonism yields correlated estimates of agonist affinity and efficacy. Van der Graaf, P.H. and Stam, W.B. J. Pharmacol. Toxicol. Meth. 1999; 41: 117–125Crossref | PubMed | Scopus (6)See all References][2], it is tempting to suggest that they are examples of experimental evidence for the conformational selection hypothesis of agonist action.