Quantifying and Communicating Uncertainty in Preclinical Human Dose-Prediction.

Human dose-prediction is fundamental for ranking lead-optimization compounds in drug discovery and to inform design of early clinical trials. This tutorial describes how uncertainty in such predictions can be quantified and efficiently communicated to facilitate decision-making. Using three drug-discovery case studies, we show how several uncertain pieces of input information can be integrated into one single uncomplicated plot with key predictions, including their uncertainties, for many compounds or for many scenarios, or both.

The direct thrombin inhibitor melagatran and its oral prodrug H 376/95: intestinal absorption properties, biochemical and pharmacodynamic effects.

UNLABELLED: Suboptimal gastrointestinal absorption is a problem for many direct thrombin inhibitors. The studies presented herein describe the new oral direct thrombin inhibitor H 376/95, a prodrug with two protecting residues added to the direct thrombin inhibitor melagatran. Absorption properties in vitro: H 376/95 is uncharged at intestinal pH while melagatran is charged. H 376/95 is 170 times more lipophilic (octanol water partition coefficient) than melagatran. As a result, the permeability coefficient across cultured epithelial Caco-2 cells is 80 times higher for H 376/95 than for melagtran. Pharmacokinetic studies in healthy volunteers: H 376/95 is converted to melagatran in man. Oral bioavailability, measured as melagatran in plasma, is about 20% after oral administration of H 376/95, which is 2.7-5.5 times higher than after oral administration of melagatran. The variability in the area under the drug plasma concentration vs. time curve (AUC) is much smaller with oral H 376/95 (coefficient of variation 20%) than with oral melagatran (coefficient of variation 38%). Pharmacodynamic properties: H 376/95 is inactive towards human alpha-thrombin compared with melagatran [inhibition constant (K(i)) ratio, 185 times], a potential advantage for patients with silent gastrointestinal bleeding. In an experimental thrombosis model in the rat, oral H 376/95 was more effective than the subcutaneous low molecular weight heparin dalteparin in preventing thrombosis. CONCLUSION: By the use of the prodrug principle, H 376/95 endows the direct thrombin inhibitor melagatran with pharmacokinetic properties required for oral administration without compromising the promising pharmacodynamic properties of melagatran.

Pharmacokinetics and arteriovenous differences in clevidipine concentration following a short- and a long-term intravenous infusion in healthy volunteers.

BACKGROUND: Clevidipine is an ultra-short-acting calcium antagonist developed for reduction and control of blood pressure during cardiac surgery. The objectives of the current study were to determine the pharmacokinetics of clevidipine after 20-min and 24-h intravenous infusions, and to determine the relation between the arterial and venous concentrations and the hemodynamic responses to clevidipine in healthy volunteers. METHODS: Four volunteers received clevidipine for 20 min, and eight subjects were administered clevidipine intravenously for 24 h at two different dose rates. Arterial and venous blood samples were drawn for pharmacokinetic evaluation, and blood pressure and heart rate were recorded. RESULTS: A triexponential disposition model described the pharmacokinetics of clevidipine. The mean arterial blood clearance of clevidipine was 0.069l/kg-1/min-1 and the mean volume of distribution at steady state was 0.19 l/kg. The duration of the infusion had negligible effect on the pharmacokinetic parameters, and the context-sensitive half-time for clevidipine, simulated from the mean pharmacokinetic parameters derived after 24 h infusion at the highest dose, was less than 1 min. The arterial blood levels reached steady state within 2 min of the start of infusion and were about twice as high as those in the venous blood at steady state. The peak response preceded the peak venous concentration and was slightly delayed from the peak arterial blood concentration. CONCLUSION: Clevidipine is a high clearance drug with a small volume of distribution, resulting in extremely short half-lives in healthy subjects. The initial rapid increase in the arterial blood concentrations and the short equilibrium time between the blood and the biophase suggest that clevidipine can be rapidly titrated to the desired effect.

Stereoselective metabolism by human liver CYP enzymes of a substituted benzimidazole.

H 259/31 is a substituted benzimidazole developed as a structural analog of omeprazole. Metabolites of H 259/31 formed in human liver microsomes were identified by using the synthetic reference compounds and liquid chromatography/mass spectrometry. The predominant metabolic pathways found include oxidation of the sulfoxide to sulfone, oxidative O-dealkylation of the cyclopropylmethoxy group to the corresponding pyridone and aromatic hydroxylation to give the phenolic derivative. Stereoselectivity in the metabolism of the enantiomers of H 259/31 was demonstrated in human liver microsomes. The sum of the formation intrinsic clearances of all three metabolites was higher for the S-enantiomer than that of the R-form, indicating that the S-enantiomer is eliminated more rapidly. It was also shown in the present study that the sulfone metabolite is subject to additional metabolism, which should be taken into account when determining the intrinsic clearance for formation of metabolites and when the relative importance of metabolic pathways is determined. Expressed enzymes indicate major involvement of cytochrome P-450 (CYP) 2C19 in the formation of the hydroxy derivative as well as in pyridone formation from the enantiomers of H 259/31. CYP3A4 and CYP2C9 seem to contribute as low-affinity enzymes in both reactions. The sulfone metabolite was formed mainly from CYP3A4. Stereoselectivity in CYP3A4-, CYP2C19-, and CYP2C9-mediated metabolic pathways was demonstrated.

Animal pharmacokinetics of inogatran, a low-molecular-weight thrombin inhibitor with potential use as an antithrombotic drug.

Pharmacokinetics, excretion, and metabolism of inogatran, a low-molecular-weight thrombin inhibitor, were studied in the rat, dog, and cynomolgus monkey. After intravenous administration the half-life was short in all three animal species, due to a small volume of distribution and a relatively high clearance. At doses of 0.1-5 mumol kg-1, the mean residence time was about 10 min in the rat, 35 min in the dog, and 20 min in the cynomolgus monkey. The oral bioavailability of inogatran was incomplete, presumably due to a low membrane permeability, and dose dependent. The bioavailability was 4.8% at 20 mumol kg-1 and 32-51% at 500 mumol kg-1 in rats, 14% at 10 mumol kg-1 and 34-44% at 150 mumol kg-1 in dogs, and 2.1% at 1 mumol kg-1 in cynomolgus monkeys. The radioactivity excreted in urine and faeces was predominantly unchanged inogatran. After intravenous administration the percentage of the radioactivity recovered in faeces was about equal to or higher than the urinary recovery, which indicates biliary excretion of inogatran. After oral dosing, most of the dose was excreted in faeces, as expected from the estimates of oral bioavailability. The plasma protein binding of inogatran in rat, dog, and human plasma, was 20-28%. The blood-plasma concentration ratio was 0.39-0.56, indicating limited distribution into red blood cells.

The protective action of chlormethiazole against ischaemia-induced neurodegeneration in gerbils when infused at doses having little sedative or anticonvulsant activity.

1. The effect of chlormethiazole administration on delayed neuronal death in gerbil hippocampus following transient global ischaemia has been examined. Chlormethiazole was administered either intraperitoneally or by intravenous infusion with either the dose or the time of infusion varied. 2. Chlormethiazole (600 mumol kg-1, i.p.) given 60 min after ischaemia produced substantial (> 60%) neuroprotection when damage was assessed 5, 14 or 21 days later, indicating the drug does not merely delay cell death. 3. Infusion protocols were developed which would result in sustained and defined plasma concentrations. Chlormethiazole (930 mumol kg-1) was then infused intravenously for 30 min, 76.5 min or 110 min in ways resulting in sustained plasma concentrations of 200, 100 and 50 nmol ml-1 respectively. When treatment was initiated 30 min after the ischaemic episode all protocols provided effective neuroprotection. There was a dose-dependent decline in protection when plasma chlormethiazole concentrations of 50, 30 and 10 nmol ml-1 were sustained for 110 min with no protection observed at 10 nmol ml-1. 4. In contrast, when a plasma concentration of 10 nmol ml-1 was sustained by infusion for 24 h, almost total neuroprotection against the ischaemic damage was achieved. This plasma concentration produced no sedative or anticonvulsant activity. 5. These data suggest that neuroprotection depends on both dose and duration of chlormethiazole administration and that excellent neuroprotection is possible in the absence of the sedative and anticonvulsant effects of the drug.

A comparison between the semisimultaneous and the stable isotope techniques for bioavailability estimation of terbutaline in humans.

A recently proposed bioavailability estimation procedure, the "semisimultaneous" method, in which the test and reference dose administrations are separated by a short time interval and total concentrations are analyzed, was compared with the stable isotope method for precision and accuracy. By administering isotope-labeled (reference) and unlabeled (test) terbutaline in a semisimultaneous fashion, the bioavailability could be determined with both methods at the same time. The extent and rate of bioavailability of oral terbutaline was determined in eight healthy volunteers by use of both model fitting, AUC methods, and deconvolution. The AUC ratio and the deconvolution methods, by use of the separate isotope data, gave bioavailability estimates of 14.5% +/- 4.1% and 12.2% +/- 3.9%, respectively. According to the semisimultaneous method, bioavailability estimates with the same data sets were 11.8% +/- 4.5% obtained from fitting a model to the data and 11.0% +/- 3.7% by use of a combined model fitting-deconvolution procedure. An excellent agreement between the semisimultaneous and the stable isotope methods was also obtained in the estimation of the rate of absorption.

Mathematical description of the concentration of oxytetracycline and penicillin-G in tissue cages in calves as related to the serum concentration.

A mathematical model based on Fick's laws of diffusion describing the concentration of drug in tissue cage models was elaborated. The model takes into account differences in protein binding, tissue cage geometry and serum pharmacokinetics. The validity of the model was tested against experimental data obtained from a tissue cage model in calves by simultaneous fitting to serum and tissue cage fluid (TCF) data in a non-linear least-squares regression computer program. Concentrations of penicillin-G (pen-G) in serum and TCF following intravenous (i.v.) administration of potassium pen-G were adequately described by the mathematical model. Concentrations in TCF after intramuscular (i.m.) administration of the same drug and of procaine pen-G could be predicted by the mathematical model. Concentrations of oxytetracycline (OTC) in serum and TCF following i.v. administration and continuous i.v. infusions were also adequately described by the model, and TCF concentrations after i.m. administration of the same drug could be roughly predicted. The results indicate that pen-G and OTC have the same permeability coefficient for transport from serum to TCF.

In vivo evaluation of the semi-simultaneous method for bioavailability estimation using controlled intravenous infusion as an 'extravascular' route of administration.

A recently proposed method for bioavailability estimation, called the semi-simultaneous method, was evaluated in vivo in rats using methysergide as a test substance. In this method the test and the reference dose are administered with a short time interval and a model including the bioavailability parameters is fitted to the concentration-time profile. In the present study, in order to control the true bioavailability, intravenous infusion was used to mimic extravascular administration and various input profiles were produced. Mono-, bi- and triexponential disposition functions with the true and also various erroneous input models were fitted to the individual data sets. The models were also fitted to truncated data sets to mimic a situation where a long duration of sampling is precluded. A combined fitting-deconvolution procedure was also applied. The simi-simultaneous method gave precise and accurate estimates of the bioavailability in most groups and a robustness in the estimate concerning the model fitted was noted. The true input model could be identified for all data sets using common goodness-of-fit criteria. In the groups where a 'flip-flop' situation was created (slower input than elimination) a poorer precision and accuracy and a higher sensitivity concerning the model fitted was observed. The model fitting and the fitting-deconvolution procedure generally gave very similar results.

Pharmacokinetics of methysergide and its metabolite methylergometrine in the rat.

The pharmacokinetics of methysergide (MS) and its metabolite methylergometrine (MEM) was studied in male Sprague-Dawley rats. MS was administered iv in doses of 0.71 (0.25 mg/kg) or 2.8 mumol/kg (1.0 mg/kg). The metabolite MEM was administered as iv doses of 0.74 (0.25 mg/kg) or 2.9 mumol/kg (1.0 mg/kg). The steady state characteristics of these compounds were also studied after constant rate iv infusion of MS at two different rates, 0.70 and 14.0 nmol/min per kg. Plasma protein binding and blood/plasma partitioning for MS were determined over a range of concentrations. Plasma and blood concentrations of MS and MEM were measured by HPLC with fluorescence detection. The plasma clearance of MS was high and ranged from 74.2-102 ml/min per kg. The two iv doses of MS were not equivalent after dose correction; clearance, volume of distribution at steady-state and terminal half-life were significantly greater for the higher dose. Plasma clearance from the two iv infusions of MS were in accordance with that from the lower iv dose. Protein binding as well as the plasma/blood partitioning, of MS was constant over the range of concentrations observed in the disposition studies, averaging 84.2% and 1.67%, respectively. The metabolite MEM had a plasma clearance five to six times lower than that of the parent drug but a similar volume of distribution at steady state. The formation of MEM after MS administration was relatively low and appeared to be saturable since the formation clearance of MEM decreased significantly from 3.5 to 1.9 ml/min per kg for the low and the high rate of iv infusion of MS, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioavailability estimation by semisimultaneous drug administration: a Monte Carlo simulation study.

The performance of a novel method for determination of drug absorption characteristics was evaluated by Monte Carlo simulations. In bioavailability studies with use of this method, the test and the reference doses are administered within a time interval of hours. Estimates of bioavailability are obtained by fitting an appropriate model to the concentration-time profile, which in its terminal portion is thus the summed concentration of the two doses. Drugs with different properties, mimicked by varying the kinetic rate constants (ka, lambda 1 and lambda 2), and experimental designs with different sets of conditions regarding the interval between doses, dose ratio, dose order, and duration of sampling, were simulated to determine what factors govern parameter estimation. The absorption characteristics of the simulated drugs could be adequately determined in experiments lasting for 12 hr or less, provided that a proper design was used. Fitting of a simpler or a more complex disposition model produced estimates with similar accuracy and precision to those noted with the true model. For some conditions the use of an improper absorption model resulted in slightly reduced accuracy, but as these fits were poor there was a clear need to try other models. In another set of simulations the use of the proposed method to assess the relative availability of two extravascular doses was evaluated. The relative rate and extent of absorption could be estimated with good precision for two formulations exhibiting a rapid to moderate rate of absorption.

Pharmacokinetics of methylergometrine in the rat: evidence for enterohepatic recirculation by a linked-rat model.

The pharmacokinetics of methylergometrine were investigated in the rat, with emphasis on the role of biliary excretion and enterohepatic recirculation in the overall disposition of the drug. A linked-rat model, where the bile from a rat receiving a constant rate of iv infusion of methylergometrine was allowed to flow into the duodenum of another rat, was used for the estimation of the degree of enterohepatic recirculation (EHC). The excretion of unchanged methylergometrine in the bile was estimated separately. Plasma protein binding and plasma-to-whole blood partitioning were also determined. Plasma clearance in control rats was 17.4 +/- 0.7 ml/min x kg for iv bolus and 15.4 +/- 0.7 ml/min x kg for iv infusion. The corresponding values in the bile-cannulated rats were significantly lower, 7.7 +/- 0.4 and 8.7 +/- 0.1 ml/min x kg, respectively. The lower clearance in the bile-cannulated rats was caused mainly by a lower free fraction in plasma, fu (0.11 +/- 0.01), in this group compared with the control group (0.19 +/- 0.0.03). The unbound volume of distribution at steady state (Vssu) was only 6.5 liters/kg in the bile-cannulated rats, compared to 14.7 liters/kg in control rats, suggesting that under steady-state conditions, more than 50% of the methylergometrine is conjugated or residues in the hepatobiliary loop, either as a conjugate or unchanged. The fraction of unchanged methylergometrine excreted in the bile was less than 0.3% of the given dose, while the fraction of the dose being reabsorbed during one cycle (freabs) was 8.4 +/- 6.3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of bioavailability on a single occasion after semisimultaneous drug administration.

A method for determination of absorption rate and bioavailability was developed to reduce the influence of intraindividual variability and applied to the absolute intraperitoneal availability of lithium in the rat. In this method the test and the reference doses are given with a few hours' interval, and the resulting concentration-time data are analyzed by nonlinear regression. The bioavailability estimation by this approach was compared to that of the traditional method, with test and reference doses given on separate days. The mean estimates of availability were 1.035 +/- 0.109 (N = 7) and 0.984 +/- 0.052 (N = 11) for the traditional and the alternative method, respectively. Thus, the precision was better in the latter. Major influences of dose- or time-dependent kinetics of lithium on the availability estimates were excluded by the design used. The estimation of bioavailability was robust with respect to the choice of absorption and disposition model and the duration of sampling. The plasma clearance of lithium was 169 +/- 15 ml/hr/kg, with a terminal half-life of 5.0 +/- 0.5 hr (N = 5).

A new human retrovirus isolate of West African origin (SBL-6669) and its relationship to HTLV-IV, LAV-II, and HTLV-IIIB.

A new human retrovirus of West African origin (SBL-6669) has been isolated from a patient with immunological and clinical signs of immunodeficiency. Using radioimmunoprecipitation assays (RIPA) and Western blot (WB) tests with human sera, the new virus isolate has been compared with HTLV-IV, LAV-II, and the HTLV-IIIB prototype strain of the human immunodeficiency virus (HIV). The West African isolates appeared to be members of the same virus group since their glycoproteins were antigenically indistinguishable. West African sera showed no detectable cross reaction with HTLV-IIIB glycoproteins. The external glycoprotein in the different virus strains only showed minor variations in size. The size of the transmembranous protein was not unambiguously defined. In the West African virus isolates a 30-35 kD protein was seen similar to the protein previously described possibly to represent this component. However, in SBL-6669 a distinct 41 kD protein was also identified. There were interstrain variations in the size of several viral proteins among the West African virus isolates. Only minor differences were seen between SBL-6669 and LAV-II. The variations were most pronounced in two core proteins corresponding to the 19 kD and 24 kD proteins of HTLV-IIIB. In addition, West African human retroviruses appear to differ in pathogenicity. LAV-II and SBL-6669 are associated with immunodeficiency, whereas HTLV-IV was isolated from healthy individuals. Since further spread of these viruses to other parts of the world is imminent, it is necessary to consider their antigenic and immunogenic properties in serodiagnosis of HIV infections and in planning for immunoprophylactic interventions.

Pharmacokinetics of methysergide and its metabolite methylergometrine in man.

Five healthy men were given 1.0 mg methysergide maleate intravenously and 2.7 mg methysergide maleate orally in a cross-over study. The systemic availability of methysergide was only 13%, most probably due to a high degree of first-pass metabolism to methylergometrine. We also found evidence of extrahepatic clearance of methysergide. After oral administration the plasma concentrations of the metabolite were considerably higher than those of the parent drug and the area under the plasma concentration curve (AUC) for methylergometrine was more than ten times greater than for methysergide. Our findings may be relevant to the treatment of migraine if methylergometrine contributes to the effect of methysergide. Methylergometrine had a significantly longer elimination half-life than methysergide (223 +/- 43 min vs 62.0 +/- 8.3 min and 174 +/- 35 min vs 44.8 +/- 8.1 min in the oral and intravenous studies respectively).