The relationship between receptor-effector unit heterogeneity and the shape of the concentration-effect profile: Pharmacodynamic implications

The apparent concentration-effect relationship is the ensemble of many effector units (such as individual cells or channels) that do not always exhibit a uniform stimulus-effect relationship. This concept is substantiated by many observations of heterogeneity in receptor-effector populations including hormone secreting cells, response to hormonal stimuli, activity pattern of second messengers, stimulus-evoked synaptic currents, and single ion channels. The relationship between drug concentration and magnitude of pharmacologic response is commonly described by the sigmoidalE _max model which was derived from the Hill equation. The sigmoidicity factor (N) in this model is assumed to be a pure mathematical parameter without physiological connotations. This work demonstrates that the numerical value ofN (measured empirically) is the product of two factors: (i) the degree of heterogeneity of the effector subunits, i.e., the elemental component that upon drug stimulus contributes its pharmacological effect independently and does not interact with other subunits (it could range from a single receptor up to a whole tissue), and (ii) value ofN ^*—the shape factor of the subunits' concentration-effect relationship. A special case of this approach occurs whenN ^*>5, which is an on-off case. HereN is determined by the distribution (density equation) of the subunit values. In case of heterogeneity of the microparameters of the effector subunits the apparentN will always have a lower value thanN ^*. According to this theory it can be concluded that without knowledge of the distribution of the microparameters no mechanistic interpretation can be deduced from the apparentN value. If in the futureN ^* can be determined by theoretical or experimental methods, the distribution function relatingN ^* toN can be calculated. The relevance of this theory is increased in view of the progress being made in advanced research techniques which may enable us to determine the concentration-effect relationship at the level of the individual effector unit.     

Implications for clinical pharmacodynamic studies of the statistical characterization of an in vitro antiproliferation assay.

Modeling of nonlinear pharmacodynamic (PD) relationships necessitates the utilization of a weighting function in order to compensate for the heteroscedasticity. The structure of the variance was studied for concentration-effect data generated in an in vitro 96-well plate cell growth inhibition assay, where data are numerous (480 data points per experiment) and replication is easy. From the five candidate models that were considered, the power function S2Y = phi 2Y phi 3, where Y is the sample mean and S2Y is the sample variance, was shown to be the most appropriate to describe the nonuniformity of the variance along the range of measured effect for 253 sets of (Y; S2Y) data. The Hill model was fit to the concentration-effect data with weighted nonlinear regression, where the weights were equal to the reciprocal of the predicted variance. The examination of the distribution of the 253 sets of parameters of the PD model showed that IC50 was lognormally distributed whereas the distribution of gamma was normal. The characterization of the appropriate variance function and concentration-effect function in a simple in vitro experimental setting with a large number of experiments, with each experiment including a large number of data points, will be useful for guiding similar in vitro concentration-effect studies where data are plentiful and for guiding PD modeling in complex clinical settings in which extensive data for model characterization is impossible to obtain.     

Local Delivery of Indomethacin to Arthritis-Bearing Rats through Polymeric Micelles Based on Amphiphilic Polyphosphazenes

Purpose Preparation, in vitro and in vivo evaluation of indomethacin-loaded polymeric micelles based on amphiphilic polyphosphazene. Methods Amphiphilic polyphosphazenes (PNIPAAm/EAB-PPPs) with poly (N-isopropylacrylamide) (PNIPAAm) and ethyl 4-aminobenzoate (EAB) as side groups were synthesized through thermal ring-opening polymerization and subsequent substitution reactions. Indomethacin (IND) loaded polymeric micelles based on PNIPAAm/EAB-PPPs were prepared by dialysis procedure. In vitro IND release kinetics was investigated in 0.1 M PBS (pH 7.4), while in vivo pharmacokinetics was performed in Sprague–Dawley rats. In vivo pharmacodynamic study was carried out based on two animal models, i.e. carrageenan-induced acute paw edema and complete Freund’s adjuvant (CFA) induced ankle arthritis model. Results Drug loading capacity of micelles based on this type of amphiphilic copolymers was mainly determined by copolymer composition and the chemical structure of drug. In addition to the compatibility between drug and micellar core, hydrogen bonding interaction between drug and hydrophilic corona may significantly influence drug loading as well. In vitro drug release in PBS suggested that there was no significant difference in release rate between micelles based on copolymers with various EAB content. Compared with the rats administered with free IND aqueous solution, IND concentration in rats’ plasma showed a prolonged maintenance in experimental group treated with IND-loaded polymeric micelles. In vivo pharmacodynamic study indicated that sustained therapeutic efficacy could be achieved through topical injection of the aqueous solution of IND-loaded micelles. Local delivery of IND can avoid the severe gastrointestinal stimulation, which was frequently associated with oral administration as evidenced by ulceration evaluation. Conclusions The promising results of current preliminary study suggest that this type of amphiphilic copolymers could be used as injectable drug carriers for hydrophobic drugs.     

Propranolol pharmacodynamic modeling using unbound and total concentrations in healthy volunteers

In an attempt to evaluate the propranolol (P) concentration-effect relationship, percentage reduction in exercise heart rate was modeled as a function of unbound and total P concentrations using the linear, E _max ,and sigmoid E _max models. Nine volunteers underwent repeated treadmill exercise tests over 48 hr during a control period, after receiving 160 mg of P orally and again after receiving 160 mg once daily for 7 days. Beta blockade was assessed as the percentage reduction in exercise heart rate compared to control. Total serum P concentrations were determined by HPLC and unbound fractions by equilibrium dialysis. Using nonlinear least-squares regression, the E _max model was best in describing the concentration-effect relationship in each subject. Mean parameters for combined single dose and steady state were E _max 33.6±4.5% and EC₅₀ 18.2±15.6ng/ml for total P and E _max 33.5±4.3% and EC₅₀ 1.66±1.56 for unbound P. Model fits were not significantly better for unbound versus total P and EC₅₀ values showed similar intersubject variability. The observed unbound EC₅₀ values are consistent with reported receptor dissociation constants. Therefore the large intersubject variability in EC₅₀ could not be accounted for by variability in P protein binding.     

Experience with darunavir in HIV-infected adults enrolled in a US expanded access program: results from a single center

ABSTRACT

Objective: A multicenter, international, expanded access program (EAP) was initiated in October 2005 for patients failing multiple antiretroviral regimens. The primary objective was to provide early access to darunavir (DRV) (Prezista) co-administered with low-dose ritonavir (DRV/r) for antiretroviral-experienced patients who failed multiple regimens and had limited treatment options; the secondary objective was to gather additional DRV/r safety information.

Methods: Following initiation of DRV/r 600/100?mg bid, patients were evaluated at baseline, Weeks 4 and 12, and every 12 weeks thereafter for changes in CD4 cell counts and HIV-1 RNA levels. Safety and tolerability were also evaluated.

Results: Results from patients treated at a single US EAP center in Houston, Texas (N = 38; mean age 47 years; 92% male; 60% Caucasian, 24% Black, 16% Hispanic) are presented. At time of analysis, 38 and 23 patients completed 12 and 24 (± 1) weeks of therapy, respectively. At Weeks 12 and 24, the mean change in viral load (VL) from baseline was –1.96?log₁₀?copies/mL (n = 38) and –2.17?log₁₀?copies/mL (n = 22), and 54% and 50% of patients achieved HIV-1 RNA < 50?copies/mL, respect­ively. Mean CD4 cell count increased by 109?cells/mm³ from baseline to Week 24. DRV/r was generally safe and well tolerated. Most adverse events (AEs) were mild to moderate in severity (nine events considered possibly related to DRV); neither of the two serious AEs was considered related to DRV/r.

Conclusions: Although this study reflects results from only a small cohort of patients at a single center, among this community-based population of highly treatment-experienced patients, DRV/r 600/100?mg bid provided clinically meaningful decreases in VL, an undetectable rate similar to that seen in the POWER studies, and was well tolerated with infrequent AEs.     

Population pharmacokinetics and association between A77 1726 plasma concentrations and disease activity measures following administration of leflunomide to people with rheumatoid arthritis

AIMS: To investigate the concentration-effect relationship and pharmacokinetics of leflunomide in patients with rheumatoid arthritis (RA). METHODS: Data were collected from 23 RA patients on leflunomide therapy (as sole disease modifying antirheumatic drug (DMARD)) for at least 3 months. Main measures were A77 1726 (active metabolite of leflunomide) plasma concentrations and disease activity measures including pain, duration/intensity of morning stiffness, and SF-36 survey. A population estimate was sought for apparent clearance (CL/F) and volume of distribution was fixed (0.155 l kg(-1)). Factors screened for influence on CL/F were weight, age, gender and estimated creatinine clearance. RESULTS: Significantly higher A77 1726 concentrations were seen in patients with less swollen joints and with higher SF-36 mental summary scores than in those with measures indicating more active disease (P < 0.05); concentration-effect trends were seen with five other disease activity measures. Statistical analysis of all disease activity measures showed that mean A77 1726 concentrations in groups with greater control of disease activity were significantly higher than those in whom the measures indicated less desirable control (P < 0.05). There was large between subject variability in the dose-concentration relationship. A steady-state infusion model best described the pharmacokinetic data. Inclusion of age as a covariate decreased interindividual variability (P < 0.01), but this would not be clinically important in terms of dosage changes. Final parameter estimate (% CV interindividual variability) for CL/F was 0.0184 l h(-1) (50%) (95% CI 0.0146, 0.0222). Residual (unexplained) variability (% CV) was 8.5%. CONCLUSIONS: This study of leflunomide in patients using the drug clinically indicated a concentration-effect relationship. From our data, a plasma A77 1726 concentration of 50 mg l(-1) is more likely to indicate someone with less active disease than is a concentration around 30 mg l(-1). The marked variability in pharmacokinetics suggests a place for individualized dosing of leflunomide in RA therapy.     

Solubility Enhancement of Indomethacin with Poly(amidoamine) Dendrimers and Targeting to Inflammatory Regions of Arthritic Rats

This work includes investigation on solubility enhancement of indomethacin (IND) in the presence of poly(amidoamine) (PAMAM) dendrimers and passive targeting of the PAMAM/IND complex so formed to the inflamed regions in an animal model. The complex formation was confirmed by infrared and ¹H nuclear magnetic resonance spectroscopy methods. Solubility of IND in aqueous G4-PAMAM followed Higuchi's A_N curve depending on pH of the solubilizing medium. The solubility was decreased upon addition of dendrimer to the IND saturated solution at various pH, indicating aggregation behavior of the PAMAM/IND complex and conforming to the Higuchi's A_N solubility profile. The in vitro release of IND from the PAMAM/IND complex through a cellophane membrane, from a Franz diffusion cell, showed 79±3.2% drug release in 24 h. The drug release was further retarded in the presence of human serum albumin (HSA) suggesting the significance of complex HSA binding in altering in vivo behavior of the complex. Intravenous administration of the PAMAM/IND complex formulation in rats showed a two-compartment pharmacokinetic profile. Enhanced effective IND concentrations in the inflamed regions were obtained for the prolonged time period with the PAMAM/IND complex compared to the free drug in arthritic rats indicating preferred accumulation of IND to the inflamed region. The targeting efficiency of PAMAM/IND complex was 2.29 times higher compared to free drug. In contrast to the previous investigations, two interesting findings reported here are: (a) solubility behavior of IND in G4-PAMAM dendrimer deviates from linearity with increasing concentrations of dendrimer at acidic to neutral pH values and (b) inspite of lymphatic drainage, retention of PAMAM/IND complexes occurs at the inflammatory site.     

Expression of Concern: Preventive effect of nerolidol on isoproterenol induced myocardial damage in Wistar rats: Evidences from biochemical and histopathological studies

The present study aimed at investigating the protective effects of nerolidol (NRD) against myocardial infarction (MI) induced by isoproterenol (ISO) in Wistar rats. The rats were randomly divided into five groups, each group consisting of six rats. Group I were treated as control rats, group II received NRD (200 mg/kg b.w.) by intragastric intubation for 21 days, group III received ISO (60 mg/kg b.w) subcutaneously (s.c) for two consecutive days on 22nd and 23rd day, group IV and V received NRD (100 and 200 mg/kg b.w) as in group II and additionally ISO was given for two consecutive days (22nd and 23rd). On 24th day all the rats were sacrificed by cervical dislocation and the blood and heart samples were collected. In the present study, ISO-induced myocardial damage was indicated by the changes in body weight, heart weight and the cardiac and hepatic marker enzymes such as creatine kinase (CK), creatine kinase-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and troponin T and I (cTnT, cTnI) in the serum. In addition, the levels of lipid peroxidation products such as thiobarbituric acid reactive substances (TBARS), conjugated dines (CD), and lipid hydroperoxides (LHPs) increased significantly in the plasma and heart tissue. Activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) in erythrocytes and heart tissue and the levels of nonenzymatic antioxidants like vitamin C, vitamin E, and reduced glutathione (GSH) in plasma and heart tissue were decreased in ISO-induced rats. Histopathological observations were also supported with the biochemical parameters. Pretreatment with NRD at different doses (100 and 200 mg/kg b.w) for 21 days prevented the above changes induced by ISO. The 200 mg/kg b.w of NRD was more pronounced than the other dose and brought back all the above parameters near to normalcy.     

Effects of indomethacin, with vitamin A or β-carotene, on the rat gastric mucosa: Extra-and intracellular membrane-bound ATP mechanisms

Background. Indomethacin (IND) is a widely used non-steroidal anti-inflammatory agent in the treatment of various inflammatory disorders, which causes gastrointestinal injury in humans and animal experiments. Vitamin A and β-carotene prevent the IND-induced gastric mucosal injury. These compounds modify the membrane-bound ATP-dependent energy systems. The aims of this investigation were: (1) To study the IND-induced gastric mucosal damage and its prevention by vitamin A and β-carotene; (2) to measure the biochemical compounds of the gastric mucosa ATP, ADP, ATP/ADP, AMP, ATP+ADP+AMP, ‘energy charge’ (ATP + 0.5 ADP)/(ATP+ADP+AMP), cAMP, lactate under the circumstances mentioned above; (3) to analyze the extra- and intracellular regulatory mechanisms between the membrane-bound ATP-dependent energy systems. Methods. The observations were carried out with CFY (Sprague-Dawstrein rats, weighing 180-210 g). The gastric mucosal damage was produced by IND (20 mg/kg sc. administration) and it was prevented by vitamin A (or β-carotene), given in doses of 0.01-0.1 to 1.0-10.0 mg/kg ig. Different biochemical compounds (ATP, ADP, AMP, cAMP, lactate) and parameters (ATP/ADP, adenylate pool, ‘energy charge’) were measured and calculated. Results. (1) Vitamin A and β-carotene prevented dose-dependently the IND-induced gastric mucosal damage; (2) the extent of ATP-ADP transformation was increased significantly, while the ATP-cAMP decreased in the gastric mucosa after IND-treatment; (3) vitamin A and β-carotene enhanced the extent of ATP-cAMP transformation, while the ATP-ADP transformation was inhibited (the actions were dose-dependent responses); (4) No change was found in ‘energy charge’ by IND, while its value decreased significantly with vitamin A and β-carotene. Conclusions. (1) A very complex extra- and intracellular feedback mechanism system exists in the gastric mucosa during IND, IND + vitamin A, and IND + β-carotene treatments; (2) The gastric mucosal preventive effect of vitamin A and β-carotene only partly depend on their scavenger properties.     

Novel oral delivery system of indomethacin by solidified mPEG-PDLLA micelles: in vivo study

To administer indomethacin (IND) orally using polymeric micelles, IND loaded solidified polymeric micelles (IND-SPM) were prepared and evaluated for their in vitro and in vivo characteristics. IND and methoxy-poly(ethylene glycol) poly(d, l-lactide) copolymer (mPEG-PDLLA) were dissolved in acetone followed by the addition of an equivalent amount of polyplasdone XL-10 and stirred to obtain a suspension. Afterwards, acetone was completely evaporated. It was found that IND-SPM generates small polymeric micelles of 18.1 nm. Moreover, the solubility of IND at pH 6.8 increased 4.6-folds, and more than 90% of IND in 20 mg of IND-SPM was dissolved in 250 mL SIF pH 6.8 within 30 min. Pharmacokinetic parameters in fasted rats showed that IND-SPM 1:3 resulted in 3-folds increase of AUC and C_max compared to commercial IND. mPEG-PDLLA micelles were found to be efficient carriers for oral administration of IND as solid dosage forms by adsorption on polyplasdone XL-10.     

Pharmacodynamic model of the haemodynamic effects of pinacidil in normotensive volunteers

Summary The concentration-effect relationships of pinacidil, a peripheral vasodilator, have been measured in 12 healthy adults who received placebo or pinacidil 25 mg daily for 1 week in a cross-over experiment. Diastolic blood pressure (DBP) and heart rate (HR) were recorded and blood samples were taken on days 1 and 7. Plasma drug concentration-time data were fitted by a biexponential function with zero-order input. The pharmacokinetic model was incorporated into a combined pharmacokinetic-dynamic model (PK-PD) using the Hill equation, which has three parameters: n, the sigmoidicity parameter, E_max the maximum effect and EC₅₀ the concentration which gives 50% of E_max. For DBP, the parameter medians were estimated as n=5, EC₅₀=44.6 ng · ml^–1 and E_max=13.5 mm Hg.A hysteresis loop was found when HR was plotted against concentration, which could be fitted by a linear effect compartment model. Simulations showed that experimental DBP points on Day 7 could be predicted from a simulated curve computed by the model using parameters estimated on Day 1.Using the simulation, it was possible to suggest an optimal dosage regimen for pinacidil tablets.     

Description of the time course of the prolactin suppressant effect of the dopamine agonist CQP201-403 by an integrated pharmacokinetic-pharmacodynamic model

Six male volunteers (mean age 24 years) received a single oral dose of 0.025 mg CQP201-403 and placebo in a randomised double-blind crossover design. Fifteen plasma samples were collected over 48 h and were assayed by radioimmunoassay for drug substance and prolactin (PRL). Three of the samples were drawn during sleep on the first study day. The pharmacological effect (E%) of CQP201-403 was expressed as reduction in plasma PRL levels. The pharmacokinetic (PK)-pharmacodynamic (PD) model consisted of two kinetic compartments and an effect compartment linked to the central compartment. A sigmoid Emax model (Hill equation) described the relationship between the drug concentration in the effect compartment and E%. Curve-fitting of PK and PD data provided individual parameter estimates which served to generate computer-simulated PK and PD profiles after single and multiple doses in order to: investigate the in vivo concentration-effect relationship; evaluate the consequence of dosage reduction on the steady-state PD profile; and study the robustness of the response to changes in drug potency and bioavailability.     

Protective effect of compound Danshen (Salvia miltiorrhiza) dripping pills alone and in combination with carbamazepine on kainic acid-induced temporal lobe epilepsy and cognitive impairment in rats

Context: Temporal lobe epilepsy (TLE) is resistant to antiepileptic drugs (AEDs) and is associated with cognitive impairment. The modern Chinese medicine, compound Danshen dripping pills (CDDP), is clinically effective in treating epilepsy and improving cognitive impairment.

Objective: This study evaluated the protective effects of CDDP alone and in combination with carbamazepine (CBZ) on kainic acid-induced TLE and cognitive impairment in rats.

Materials and methods: Sprague–Dawley rats were randomly divided into five groups: control (sham operated), model, CDDP, CBZ and combined. A TLE model was then created via bilateral intrahippocampal injection of 0.35?μg kainic acid (KA). Rats received CDDP (85?mg/kg), CBZ (100?mg/kg) or combined (85?mg/kg CDDP +100?mg/kg CBZ) via intragastric administration for 90?d, respectively. Seizure intensity, apoptosis and glial cell line-derived neurotrophic factor (GDNF) were measured. Furthermore, the improvement in cognitive impairment and hippocampal neuronal damage was evaluated.

Results: CDDP combined with CBZ significantly decreased seizure severity and frequency (p?p?p?p?p?Conclusion: These findings support the use of CDDP as an adjuvant drug for the treatment of TLE and cognitive deficit. Its mechanism might be related to an anti-apoptosis effect and up-regulation of GDNF.     

Pharmacokinetic-pharmacodynamic modeling of risperidone effects on electroencephalography in healthy volunteers

Rationale: CNS-active drugs produce specific electroencephalographic changes and the concentration-effect relationship of antipsychotics may be elucidated by adopting electroencephalography (EEG) as an effect measurement tool. Objective: The purpose of the present study was to determine the concentration-effect relationship of risperidone by assessing the EEG effect after oral administrations of single dose risperidone in healthy young males. Methods: Nine healthy male volunteers received a 1 mg single oral dose of risperidone according to a placebo controlled crossover design. Plasma levels of risperidone and its active metabolite 9-hydroxyrisperidone were measured by radioimmunoassay. Quantitative EEG parameters were obtained for each of four frequency bands through spectral EEG analysis. The difference in the absolute power in the delta frequency band for the F3 lead between risperidone and placebo was used as a drug effect parameter. For pharmacokinetic-pharmacodynamic modeling, the hypothetical effect compartment kinetically linked to plasma by a first-order process was postulated. All curve fittings were done with the non-linear curve-fitting program NONLIN. Results: Our results showed that absolute powers in delta and theta frequency bands were higher for risperidone administration than for placebo at all EEG leads, and the maximum effects were detected at about 3 h after administration of the drug. The hysteresis loop was observed in the plot of plasma concentration of risperidone or sum of risperidone and 9-hydroxyrisperidone (C_p) versus EEG effect for each subject. A linear model adequately described the relationship between the effect compartment concentrations (C_e) and EEG effects, and the two limbs of hysteresis in the C_p-effect plot were collapsed in the C_e-effect plot for risperidone or risperidone plus 9-hydroxyrisperidone. Conclusion: The increases of absolute power for delta and theta frequency bands of EEG were induced by single oral administration of risperidone. The linear PK-PD model fit well with the relationship between effect compartment concentrations (C_e) and EEG effects of risperidone. Received: 1 October 1998/ Final version: 12 January 1999     

FDA“IND安全性报告的安全性评估指导原则”介绍

美国食品药品管理局（FDA）于2015年12月发布了“IND安全性报告的安全性评估指导原则（草案）”,包括前言、背景、安全评估组织结构、安全性评估实践、前瞻性计划等5个部分。该指导原则为按新药临床研究（IND）开发的人用药物和生物制品IND安全性报告的系统方法提供指导,对IND安全性报告的安全性评估从程序、组织架构、具体操作等方面提供了较为详细的描述。我国目前尚无这类指导原则,了解该指导原则对于新药研究者对临床试验严重不良事件和不良反应的评估和判断有所帮助,简介其主要内容。     

Initial blood pressure as a predictor of the response to antihypertensive therapy.

1. The relationship between fall in systolic blood pressure and initial systolic blood pressure has been investigated in 255 mixed normotensive and hypertensive subjects given placebo or one of five types of antihypertensive drug (ACE inhibitors, calcium antagonists, direct vasodilators, alpha-adrenoceptor blocker, beta-adrenoceptor blocker). 2. In all cases there was a significant correlation between the change in blood pressure and initial blood pressure. When Oldham's transformation was used (replacing the initial blood pressure by the mean of the initial and minimum pressures) the correlation coefficients were all reduced, although five out of six were still statistically significant. 3. In a subset of 43 hypertensive subjects given four antihypertensive agents, concentration-effect analysis was carried out. For three of the agents a linear model was used to relate effect to concentration; for the remaining agent a Langmuir type model was used. 4. For all four sets of data for which concentration-effect analysis was carried out, there was a significant correlation between the sensitivity of response and the initial blood pressure. 5. The observed relationships between initial blood pressure, change in blood pressure and sensitivity of response can be qualitatively explained by postulating a general form of dose-response relationship for all antihypertensive agents.     

Propranolol pharmacodynamic modeling using unbound and total concentrations in healthy volunteers

In an attempt to evaluate the propranolol (P) concentration-effect relationship, percentage reduction in exercise heart rate was modeled as a function of unbound and total P concentrations using the linear, Emax, and sigmoid Emax models. Nine volunteers underwent repeated treadmill exercise tests over 48 hr during a control period, after receiving 160 mg of P orally and again after receiving 160 mg once daily for 7 days. Beta blockade was assessed as the percentage reduction in exercise heart rate compared to control. Total serum P concentrations were determined by HPLC and unbound fractions by equilibrium dialysis. Using nonlinear least-squares regression, the Emax model was best in describing the concentration-effect relationship in each subject. Mean parameters for combined single dose and steady state were Emax 33.6 +/- 4.5% and EC50 18.2 +/- 15.6 ng/ml for total P and Emax 33.5 +/- 4.3% and EC50 1.66 +/- 1.56 for unbound P. Model fits were not significantly better for unbound versus total P and EC50 values showed similar intersubject variability. The observed unbound EC50 values are consistent with reported receptor dissociation constants. Therefore the large intersubject variability in EC50 could not be accounted for by variability in P protein binding.