Extent Of Plasma Protein Binding Research Articles

Development, testing, parameterisation, and calibration of a human PBPK model for the plasticiser, di-(2-ethylhexyl) terephthalate (DEHTP) using in silico, in vitro and human biomonitoring data.

A physiologically based pharmacokinetic model for di-(2-ethylhexyl) terephthalate (DEHTP) based on a refined model for di-(2-propylheptyl) phthalate (DPHP) was developed to interpret the metabolism and biokinetics of DEHTP following a single oral dose of 50mg to three male volunteers. In vitro and in silico methods were used to generate parameters for the model. For example, measured intrinsic hepatic clearance scaled from in vitro to in vivo and plasma unbound fraction and tissue:blood partition coefficients (PCs) were predicted algorithmically. Whereas the development and calibration of the DPHP model was based upon two data streams, blood concentrations of parent chemical and first metabolite and the urinary excretion of metabolites, the model for DEHTP was calibrated against a single data stream, the urinary excretion of metabolites. Despite the model form and structure being identical significant quantitative differences in lymphatic uptake between the models were observed. In contrast to DPHP the fraction of ingested DEHTP entering lymphatic circulation was much greater and of a similar magnitude to that entering the liver with evidence for the dual uptake mechanisms discernible in the urinary excretion data. Further, the absolute amounts absorbed by the study participants, were much higher for DEHTP relative to DPHP. The in silico algorithm for predicting protein binding performed poorly with an error of more than two orders of magnitude. The extent of plasma protein binding has important implications for the persistence of parent chemical in venous blood-inferences on the behaviour of this class of highly lipophilic chemicals, based on calculations of chemical properties, should be made with extreme caution. Attempting read across for this class of highly lipophilic chemicals should be undertaken with caution since basic adjustments to PCs and metabolism parameters would be insufficient, even when the structure of the model itself is appropriate. Therefore, validation of a model parameterized entirely with in vitro and in silico derived parameters would need to be calibrated against several human biomonitoring data streams to constitute a data rich source chemical to afford confidence for future evaluations of other similar chemicals using the read-across approach.

Application of empirical scalars to enable early prediction of human hepatic clearance using IVIVE in drug discovery: an evaluation of 173 drugs.

The utilization of in vitro data to predict drug pharmacokinetics (PK) in vivo has been a consistent practice in early drug discovery for decades. However, its success is hampered by mispredictions attributed to uncharacterized biological phenomena/experimental artifacts. Predicted drug clearance (CL) from experimental data (i.e. hepatocyte intrinsic clearance: CLint, fraction unbound in plasma: fu,p) is often systematically underpredicted using the well-stirred model (WSM). The objective of this study was to evaluate using empirical scalars in the WSM to correct for CL mispredictions. Drugs (N=28) were used to generate numerical scalars on CLint (α), and fu,p (β) to minimize the error (AAFE) for CL predictions. These scalars were validated using an additional dataset (N=28 drugs) and applied to a non-redundant AstraZeneca (AZ) dataset available in the literature (N=117 drugs) for a total of 173 compounds. CL predictions using the WSM were improved for most compounds using an α value of 3.66 (~64%<2-fold) compared to no scaling (~46%<2-fold). Similarly, using a β value of 0.55 or combination of α and β scalars (values of 1.74 and 0.66, respectively) resulted in a similar improvement in predictions (~64%<2-fold and ~65%<2-fold, respectively). For highly bound compounds (fu,p{less than or equal to}0.01), AAFE was substantially reduced across all scaling methods. Using the β scalar alone or a combination of α and β appeared optimal; and produce larger magnitude corrections for highly-bound compounds. Some drugs are still disproportionally mispredicted, however the improvements in prediction error and simplicity of applying these scalars suggests its utility for early-stage CL predictions. Significance Statement In early drug discovery, prediction of human clearance using in vitro experimental data plays an essential role in triaging compounds prior to in vivo studies. These predictions have been systematically underestimated. Here we introduce empirical scalars calibrated on the extent of plasma protein binding that appear to improve clearance prediction across multiple datasets. This approach can be used in early phases of drug discovery prior to the availability of pre-clinical data for early quantitative predictions of human clearance.

Evaluation of quantitative structure property relationship algorithms for predicting plasma protein binding in humans

The extent of plasma protein binding is an important compound-specific property that influences a compound’s pharmacokinetic behavior and is a critical input parameter for predicting exposure in physiologically based pharmacokinetic (PBPK) modeling. When experimentally determined fraction unbound in plasma (fup) data are not available, quantitative structure–property relationship (QSPR) models can be used for prediction. Because available QSPR models were developed based on training sets containing pharmaceutical-like compounds, we compared their prediction accuracy for environmentally relevant and pharmaceutical compounds. Fup values were calculated using Ingle et al., Watanabe et al. and ADMET Predictor (Simulation Plus). The test set included 818 pharmaceutical and environmentally relevant compounds with fup values ranging from 0.01 to 1. Overall, the three QSPR models resulted in over-prediction of fup for highly binding compounds and under-prediction for low or moderately binding compounds. For highly binding compounds (0.01 ≤ fup ≤ 0.25), Watanabe et al. performed better with a lower mean absolute error (MAE) of 6.7% and a lower mean absolute relative prediction error (RPE) of 171.7% than other methods. For low to moderately binding compounds, both Ingle et al. and ADMET Predictor performed better than Watanabe et al. with superior MAE and RPE values. The positive polar surface area, the number of basic functional groups and lipophilicity were the most important chemical descriptors for predicting fup.This study demonstrated that the prediction of fup was the most uncertain for highly binding compounds. This suggested that QSPR-predicted fup values should be used with caution in PBPK modeling.

Canine orosomucoid (alpha-1 acid glycoprotein) variants and their influence on drug plasma protein binding.

Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed-breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n=12). Free and total drug plasma concentrations were quantified by liquid chromatography-mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n=1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n=21). Free drug fractions did not differ significantly (p>.05; Mann-Whitney U) between plasma collected from dogs with c.70AA (n=4) and those with c.70GG (n=8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.

Absorption, Distribution and Excretion of [14C] Zamicastat after Single Oral and Intravenous Administration in Rats and Dogs

BackgroundZamicastat is a dopamine‐β‐hydroxylase inhibitor (1), previously showed to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.ObjectivesThe purpose of this study was to characterize the absorption, distribution and elimination of 14C‐zamicastat in rat and dog, after single oral (p.o.) or intravenous (i.v.) administration.MethodsWistar rats and Beagle dogs were administered with 14C‐zamicastat at 30 mg/kg orally or intravenously with 2.5 mg/kg (rat) and 0.1 mg/kg (dog). Plasma, urine and faeces samples were collected at different time‐points to measure the plasma exposure and the recovery of total radioactivity up to 168 h post‐dose. Whole body phosphor imaging (WBPI) was evaluated in rat after p.o. administration of 30 mg/kg of 14C‐zamicastat. The extent of plasma protein binding was determined by equilibrium dialysis and blood cell binding was determined, using centrifugation, on pooled male samples from each species.ResultsAfter single oral dosing of 14C‐zamicastat, more than 95% of total radioactivity was recovered at five and seven days from rats and dogs, respectively. The radioactivity peaked in plasma between 6 to 8 h and 2 to 4 h post‐dosing in rats and dogs, respectively, followed by a slow elimination over time. Zamicastat related radioactivity was mainly recovered in faeces with very low levels (less than 20% of total radioactivity) excreted in urine in both species. Following i.v. administration more than 80% of radioactivity was recovered in both species mainly via feaces excretion. The WBPI study indicates that radioactivity was distributed throughout the body with the majority of tissues achieving a tissue:blood ratio greater than 2. Elimination of radioactivity progressed over time with most tissues reaching the low limit of quantification at 168h. The highest tissue:blood ratios were noted in thyroid gland, lung and harderian gland at, respectively, 168h, 8h and 8h post‐dosing. Protein binding data indicates that zamicastat is preferentially bound to plasma proteins in a concentration independent manner, with 98% and 96% bound to rat and dog plasma proteins, respectively.ConclusionZamicastat is a highly plasma protein bound compound after oral administration and it is extensively distributed to tissues. After oral administration to rats and dogs, radioactivity was almost completely recovered with most of zamicastat‐related radioactivity excreted through faeces.

Determination of plasma protein binding for sympathomimetic drugs by means of ultrafiltration

Ephedrine and its diastereomer pseudoephedrine have long been used in therapy and are part of over-the-counter combination drugs against colds or allergies. Nonetheless, there is scarcely any information on their plasma protein binding in literature. Plasma protein binding is an important parameter from a pharmacokinetic and pharmacodynamic point of view and can play a crucial role in therapy. The aim of this study was to determine the extent of plasma protein binding using ultrafiltration and different types of plasma proteins like human (HSA) and bovine serum albumin (BSA) and human serum. Two orthogonal methods – continuous and discontinuous ultrafiltration - were used to confirm our findings. To get some structural and stereochemical insights into binding affinity towards plasma proteins, all four stereoisomers of ephedrine and pseudoephedrine were included in this study. Since all four stereoisomers exhibit a low affinity towards albumin, other sympathomimetic drugs of structural similarity were tested to investigate the influence of the basic character of the Ephedra alkaloids in their binding to plasma proteins.

Ex vivo binding of the immunosuppressant mycophenolic acid to dog and cat plasma proteins and the effect of co-incubated dexamethasone and prednisolone.

Mycophenolic acid (MPA) has been shown to be promising for the treatment of autoimmune diseases in dogs and cats. In humans, MPA is highly bound to plasma proteins (~97%). It has been recommended to monitor free drug plasma concentrations because the free MPA correlates with its immunosuppressive effect. However, it is unknown if MPA is highly bound to plasma proteins in dogs and cats. The objectives of this study were to determine the extent of plasma protein binding of MPA and evaluate the effect of prednisolone and dexamethasone on the extent of protein binding of MPA in dogs and cats. The extent of plasma protein binding of MPA was determined in plasma collected from clinically healthy adult cats (n=13) and dogs (n=14) by combining high-throughput dialysis and ultra-high-liquid chromatography. This study reveals that MPA is highly bound to plasma proteins (>90%) in dogs and cats, mean extent of binding of MPA at 15μg/ml to plasma proteins being 96% (range, 95%-97%) and 92% (range, 90%-93%) for dogs and cats, respectively. In dog plasma, MPA is primarily bound to albumin. In vitro, prednisolone increased the unbound MPA in dogs (p<.01) but not in cats (p=.07) while dexamethasone had no effect on MPA plasma binding in either species (p>.05). Results of this study provide valuable information for designing future pharmacokinetic and pharmacodynamic studies and also therapeutic monitoring programs for dogs and cats.

Non-clinical pharmacokinetic profiles of rovatirelin, an orally available thyrotropin-releasing hormone analogue

1. The non-clinical pharmacokinetic profiles of rovatirelin, a novel thyrotropin-releasing hormone (TRH) analogue, were investigated in vivo and in vitro.2. Rovatirelin orally administered to rats and dogs was rapidly absorbed and bioavailability was estimated to be 7.3 and 41.3%, respectively. The extent of plasma protein binding of rovatirelin in rats, dogs, and humans was low in all species (∼15%). The permeability of rovatirelin from blood to brain (permeability-surface area) ranged from 1.04 ± 0.14 to 1.29 ± 0.28 μL/min/g in rats, and rovatirelin was stable in rat plasma and brain homogenates.3. The metabolite pattern was qualitatively similar in vitro and in vivo. In animals, rovatirelin aminopentanoic acid (rovatirelin-acid), rovatirelin aminopentanone (rovatirelin-ketone), rovatirelin pyrrolidine (4S)-hydroxy (rovatirelin-OH), (thiazoylalanyl)methylpyrrolidine (TAMP), 3-(4-thiazoyl)-l-alanine (TA), and unknown metabolites were observed. In human hepatocytes, TAMP was mainly formed and no unique human metabolite was observed.4. The radioactivity from administered [14C]rovatirelin was predominantly excreted in faeces in rats and dogs, and almost all radioactivity was recovered 168 h after administration. Absorption, brain penetration, and stability of rovatirelin in the brain were greater than for taltirelin.5. Thus, orally administered rovatirelin is a potentially improved treatment for spinocerebellar degeneration compared with taltirelin.

Screening of pharmacokinetic properties of fifty dihydropyrimidin(thi)one derivatives using a combo of in vitro and in silico assays

The heterocycles dihydropyrimidin(thi)ones have been under intensive pharmacological research, but their pharmacokinetic properties remain almost unknown. Herein, fifty dihydropyrimidin(thi)ones were submitted to in vitro screening tests using parallel artificial membrane permeability assays (PAMPA) to evaluate their apparent permeability (Papp) through intestinal membrane and blood-brain barrier models, and cell-based assays to assess their interference on the efflux transporter P-glycoprotein (P-gp). Moreover, a set of kinetic and toxicological parameters was also estimated employing a new computational tool, the pkCSM. The in vitro results suggested that 82% of the test compounds have good intestinal permeability (Papp>1.1×10−6cm/s), and 66% of these are also expected to exhibit good permeability through blood-brain barrier (Papp>2.0×10−6cm/s); these findings are consistent with a high transport rate by passive transcellular pathway. In both PAMPA models, thiourea derivatives presented higher Papp values than the respective urea analogues, which were further corroborated by in silico predictions. The in vitro results also suggested a low extent of plasma protein binding for all compounds (Papp<1.0×10−5cm/s), and these findings were also supported by in silico data (unbound fraction ranging from 0.13 to 0.59). In addition, although approximately half of the compounds did not modulate P-gp at the tested concentrations (10 and 50μM), nine of them presented a trend to induce P-gp and particularly the chlorinated compounds exhibited a marked P-gp inhibition at 50μM. Furthermore, the in silico predictions suggested that half of the compounds have hepatotoxic potential. Overall, within this group of compounds, the thiourea derivatives containing an unsubstituted or a monosubstituted (NO2, CH3, OCH3) phenyl ring attached to the position 4 of the dihydropyrimidine ring represented the most promising structures and should be considered in the subsequent studies of the development of new structurally related drug candidates.

Estimation of residue depletion of cyadox and its marker residue in edible tissues of pigs using physiologically based pharmacokinetic modelling

Physiologically based pharmacokinetic (PBPK) models are powerful tools to predict tissue distribution and depletion of veterinary drugs in food animals. However, most models only simulate the pharmacokinetics of the parent drug without considering their metabolites. In this study, a PBPK model was developed to simultaneously describe the depletion in pigs of the food animal antimicrobial agent cyadox (CYA), and its marker residue 1,4-bisdesoxycyadox (BDCYA). The CYA and BDCYA sub-models included blood, liver, kidney, gastrointestinal tract, muscle, fat and other organ compartments. Extent of plasma-protein binding, renal clearance and tissue-plasma partition coefficients of BDCYA were measured experimentally. The model was calibrated with the reported pharmacokinetic and residue depletion data from pigs dosed by oral gavage with CYA for five consecutive days, and then extrapolated to exposure in feed for two months. The model was validated with 14 consecutive day feed administration data. This PBPK model accurately simulated CYA and BDCYA in four edible tissues at 24–120 h after both oral exposure and 2-month feed administration. There was only slight overestimation of CYA in muscle and BDCYA in kidney at earlier time points (6–12 h) when dosed in feed. Monte Carlo analysis revealed excellent agreement between the estimated concentration distributions and observed data. The present model could be used for tissue residue monitoring of CYA and BDCYA in food animals, and provides a foundation for developing PBPK models to predict residue depletion of both parent drugs and their metabolites in food animals.

Rifabutin reduces systemic exposure of an antimalarial drug 97/78 upon co-administration in rats: An in-vivo &in-vitro analysis.

To determine the potential drug-drug interactions between anti-malarial candidate 97/78 and anti-tubercular drug rifabutin in-vivo in rats followed by in-vitro investigation of the underlying mechanisms of drug interaction. Single oral dose study was conducted in male and female rats at 40mg/kg and 70mg/kg for 97/78 and rifabutin respectively. It was reported that rifabutin co-administration altered pharmacokinetics of 97/63 (active metabolite of 97/78). A significant decrease was reported in the systemic exposure of 97/63 by a factor of 3-4. The AUC0-last values were (4.03±0.60) and (5.44±1.15)μghmL(-1) upon 97/78 administration alone, while the values were decreased to (1.13±0.10) and (1.23±1.13)μghmL(-1) upon rifabutin co-administration in male and female rats respectively. Statistically significant differences were also reported in Cmax and Tmax values upon rifabutin co-administration. In-vitro drug metabolism study in rat liver microsomes has shown that the metabolism of 97/63 was increased by 10%-12% upon rifabutin co-incubation. The extent of plasma protein binding of 97/63 was found to be decreased from 54%-55% to 6%-8% upon rifabutin addition. It was concluded that rifabutin co-administration altered PK parameters of 97/63 in SD rats. However, no intersex influences were reported in the interaction pattern. The results obtained in the in-vivo study were well correlated with the in-vitro findings and can further be applied to explore other aspects of potential drug interactions between these two drugs.

Comparative Assessment of In Vitro–In Vivo Extrapolation Methods used for Predicting Hepatic Metabolic Clearance of Drugs

The purpose of this study was to perform a comparative analysis of various in vitro--in vivo extrapolation (IVIVE) methods used for predicting hepatic metabolic clearance (CL) of drugs on the basis of intrinsic CL data determined in microsomes. Five IVIVE methods were evaluated: the "conventional and conventional bias-corrected methods" using the unbound fraction in plasma (fu(p) ), the "Berezhkovskiy method" in which the fu(p) is adjusted for drug ionization, the "Poulin et al. method" using the unbound fraction in liver (fu(liver) ), and the "direct scaling method," which does not consider any binding corrections. We investigated the effects of the following scenarios on the prediction of CL: the use of preclinical or human datasets, the extent of plasma protein binding, the magnitude of CL in vivo, and the extent of drug disposition based on biopharmaceutics drug disposition classification system (BDDCS) categorization. A large and diverse dataset of 139 compounds was collected, including those from the literature and in house from Genentech. The results of this study confirm that the Poulin et al. method is robust and showed the greatest accuracy as compared with the other IVIVE methods in the majority of prediction scenarios studied here. The difference across the prediction methods is most pronounced for (a) albumin-bound drugs, (b) highly bound drugs, and (c) low CL drugs. Predictions of CL showed relevant interspecies differences for BDDCS class 2 compounds; the direct scaling method showed the greatest predictivity for these compounds, particularly for a reduced dataset in rat that have unexpectedly high CL in vivo. This result is a reflection of the direct scaling method's natural tendency to overpredict the true metabolic CL. Overall, this study should facilitate the use of IVIVE correlation methods in physiologically based pharmacokinetics (PBPK) model.

In-vitro binding of propiverine hydrochloride and some of its metabolites to serum albumin in man.

The distribution and pharmacological action of propiverine, a bladder spasmolytic agent, are affected by the extent of plasma-protein binding. Because attempts to assess the albumin-binding of propiverine have produced conflicting results, the binding parameters of the drug and some of its metabolites to serum albumin in man have been re-evaluated. In man propiverine is bound to serum albumin at a single site with high affinity (KA1 = 1.45 x 10(4) L mol-1) and at least two sites with low affinity (KA2 = 2.5 x 10(2) L mol-1). The metabolites of propiverine, namely M2 (dealkylated propiverine), M5 (the N-oxide of propiverine) and M6 (the N-oxide of M2), are less firmly bound to serum albumin; this is considered to be non-specific binding. Binding experiments with human serum revealed that there are additional binding proteins. At therapeutic plasma levels the extent of binding was calculated to be 90, 15, 60, and 20% for propiverine and the metabolites M2, M5, and M6, respectively. The strong binding of propiverine to serum proteins controls its availability to the liver. Because the metabolites are not tightly bound to serum proteins, after metabolism of propiverine its metabolites are easily eliminated.

Pharmacokinetics and Bioavailability of Moxifloxacin in Calves following Different Routes of Administrations

Background: Moxifloxacin is a new fourth-generation 8-methoxy fluoroquinolone developed primarily for the treatment of community-acquired pneumonia and upper respiratory tract infections. The aim of the study was to investigate the plasma pharmacokinetics characteristic of moxifloxacin in calves, after intravenous, intramuscular and subcutaneous administration of a single dose. Meanwhile, plasma protein binding and bioavailability of moxifloxacin were also estimated. Methods: Plasma concentrations of moxifloxacin were measured using a modified HPLC method, and the extent of plasma protein binding was determined in vitro using ultrafiltration. Results: Following intravenous administration, the half life of elimination, the volume of distribution at steady state and the area under the curve were 3.29 h, 0.94 l/kg and 24.72 µg·h/ml, respectively. After intramuscular and subcutaneous administration of moxifloxacin at the same dose, the peak plasma concentrations were 2.41 and 2.20 µg/ml and were obtained at 1.54 and 1.59 h, respectively. The systemic bioavailabilities were 87.19 and 75.94%, respectively. The in vitro plasma protein binding of moxifloxacin in plasma of calves was 27%. Conclusion: A high peak plasma concentration, area under the curve, rapid absorption and bioavailability following intramuscular and subcutaneous administration characterize the pharmacokinetics of moxifloxacin in calves.

The use of Cav rather than AUC in safety assessment

Toxicokinetic data have traditionally been presented as maximum observed plasma concentrations ( C max) and area under the concentration time curve (AUC) values. These values have been used to compare exposures across studies and species to provide valuable interpretation of drug safety data. Increasingly, questions are asked of toxicology studies to more accurately describe the concentration effect relationships in terms of compound affinity for target and off-target receptors. C max values can immediately be referenced to known pharmacological activities, particularly when the extent of plasma protein binding is taken into account. This provides a measure of the more pharmacologically relevant free drug exposure. AUC values on the other hand contain the component of time, which means that direct comparison to pharmacological activity values are not immediately possible. Conversion of AUC to average plasma concentration ( C av) provides a simple and convenient means to allow such a comparison without losing any information imparted by AUC values. In this paper, the benefit and advantage of applying C av values is illustrated using examples taken from the literature.

Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans

99mTc recombinant bitistatin (rBitistatin) is a radioligand for alphaIIbbeta3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [99mTc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. [99mTc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [99mTc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [99mTc]rBitistatin as part of a Phase I clinical trial. The main organs that accumulated [99mTc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [99mTc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [99mTc]rBitistatin did not affect platelet counts in humans or dogs. [99mTc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [99mTc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute thrombi.

The Use of Microdialysis for the Study of Drug Kinetics: Central Nervous System Pharmacokinetics of Diphenhydramine in Fetal, Newborn, and Adult Sheep

The central nervous system (CNS) pharmacokinetics of the H(1) receptor antagonist diphenhydramine (DPHM) were studied in 100- and 120-day-old fetuses, 10- and 30-day-old newborn lambs, and adult sheep using in vivo microdialysis. DPHM was administered i.v. at five infusion rates, with each step lasting 7 h. In all ages, cerebrospinal fluid (CSF) and extracellular fluid (ECF) concentrations were very similar to each other, which suggests that DPHM between these two compartments is transferred by passive diffusion. In addition, the brain-to-plasma concentration ratios were >or=3 in all age groups, suggesting the existence of a transport process for DPHM into the brain. Both brain and plasma DPHM concentrations increased in a linear fashion over the dose range studied. However, the ECF/unbound plasma and CSF/unbound plasma DPHM concentration ratios were significantly higher in the fetus and lambs (approximately 5 to 6) than in the adult (approximately 3). The factors f(CSF) and f(ECF), the ratios of DPHM areas under the curves (AUCs) in CSF and ECF to the plasma DPHM AUC, respectively, decreased with age, indicating that DPHM is more efficiently removed from the brain with increasing age. The extent of plasma protein binding of the drug increased with age. This study provides evidence for a transporter-mediated mechanism for the influx of DPHM into the brain and also for an efflux transporter for the drug, whose activity increases with age. Moreover, the higher brain DPHM levels in the fetus and lamb compared with the adult may explain the greater CNS effects of the drug at these ages.

Pharmacokinetics of cefepime administered by i.v. and i.m. routes to ewes

The pharmacokinetics of cefepime were studied following i.v. and i.m. administration of 20 mg/kg in 10 ewes. Following i.v. administration of a single dose, the plasma concentration-time curves of cefepime were best fitted using a two-compartment open model. The elimination half-life (t(1/2beta)) was 1.76 +/- 0.07 h, volume of distribution at steady-state [V(d(ss))] was 0.32 +/- 0.01 L/kg and total body clearance (Cl(B)) was 2.37 +/- 0.05 mL/min.kg. Following i.m. administration, the drug was rapidly absorbed with an absorption half-life (t(1/2ab)) of 0.49 +/- 0.05 h, maximum plasma concentration (Cmax) of 31.9 +/- 1.5 mug/mL was attained at (tmax) 1.1 +/- 0.2 h and the drug was eliminated with an elimination half-life (t(1/2el)) of 2.06 +/- 0.11 h. The systemic bioavailability (F) after i.m. administration of cefepime was 86.8 +/- 7.5%. The extent of plasma protein binding measured in vitro was 14.8 +/- 0.54%. The drug was detected in urine for 36 h postadministration by both routes.

Indinavir protein-free concentrations when used in indinavir/ritonavir combination therapy

To describe the in vivo protein-binding characteristics of indinavir (IDV) in the presence of ritonavir (RTV) relative to total IDV plasma concentrations. The ACTG protocol 5055 was a multicenter study comparing the safety and pharmacokinetics of IDV/RTV at doses of 800/200 and 400/400 mg twice daily in HIV-infected adults. Forty-four patients underwent a 12-h intensive pharmacokinetic assessment after 2 weeks of therapy. Three plasma samples from 35 patients at Cmax, 6 and 12 h post dose were used to determine the unbound IDV concentrations. Unbound IDV was separated in plasma samples using ultra-filtration and measured using high-performance liquid chromatography with UV detection. Mean IDV protein-bound fraction across all time points in the 800/200 and 400/400 arm were 53.4 and 51.8%, respectively. In the 800/200 arm, percentage binding at Cmax was 50% compared with 56% at 12 h (P = 0.008). In the 400/400 arm, percentage binding at Cmax was 49% compared with 54% at 12 h (P = 0.008). The extent of plasma protein binding of IDV in this study was less than in previously published data with IDV alone. Although IDV concentrations differed across the arms, the percentage of IDV protein binding at all time points was not different between the 800/200 and 400/400 arms. However, the percentage of IDV protein binding at Cmax was significantly lower compared with 12 h in each arm, possibly suggesting that IDV protein binding is concentration-dependent. These data suggest that RTV affects IDV protein-binding characteristics and IDV also exhibits concentration dependent binding when administered with RTV.

Disposition Kinetics of Difloxacin in Rabbit after Intravenous and Intramuscular Injection of Dicural

This study investigated the pharmacokinetic behaviour of difloxacin following a single intravenous (i.v.) bolus and intramuscular (i.m.) administration of 5 mg/kg body weight (bw) to rabbits (n = 6). Plasma concentrations were determined in triplicate by agar plate diffusion using E. coli (ATCC 25922) as the test organism. Difloxacin was assayed in plasma to determine its concentrations, kinetic behaviour and systemic availability. Plasma concentration-time data generated in the present study were analysed by non-compartmental methods based on statistical moment theory. Difloxacin was rapidly distributed to the tissues with a steady-state volume of distribution (Vdss) of 1.51 L/kg and the total body clearance (Cltot) was 0.59 L/kg/h. The elimination half-lives after i.v. and i.m. administration were 3.25 h and 3.82 h, respectively. After i.m. administration, difloxacin was rapidly absorbed, with mean peak plasma concentration (Cmax of 3.85 microg/ml achieved at 1.61 h (Tmax) post administration. The extent of plasma protein binding of difloxacin in rabbits was 21.45% and the systemic bioavailability was 95.29%.