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Erythropoietin (EPO) and thrombopoietin (TPO) have long been known to promote erythropoiesis and megakaryopoiesis, respectively. However, the fate-changing role of EPO and TPO on megakaryocyte-erythroid progenitors (MEPs) to develop along the erythroid versus megakaryocyte lineage remains unclear. We have previously shown that EPO may have a fate-changing role because EPO treatment could induce progenitor cells depletion and result in EPO resistance. Therefore, we hypothesize that a combination of romiplostim, a TPO receptor agonist that could stimulate the expansion of progenitors, with EPO can treat EPO resistance. Using rats with anemia due to chronic kidney disease, we demonstrated that romiplostim synergized with EPO to promote red blood cells production whereas EPO inhibited platelet production in a dose-dependent manner to reduce the risk of thrombosis. Corroborating findings from in vivo, in vitro experiments demonstrated that romiplostim expanded hematopoietic stem cells and stimulated megakaryopoiesis whereas EPO drove the progenitors toward an erythroid fate. We further developed a novel pharmacokinetic-pharmacodynamic model to quantify the effects of EPO and romiplostim on megakaryopoiesis and erythropoiesis simultaneously. The modeling results demonstrated that EPO increased the differentiation rate of MEPs into burst-forming unit-erythroid cells up to 22-fold, indicating that the slight increase of MEPs induced by romiplostim could be further amplified and recruited by EPO to promote erythropoiesis. The data herein support that romiplostim in combination with EPO can treat EPO resistance. SIGNIFICANCE STATEMENT: This study clarified that erythropoietin (EPO) drives the fate of megakaryocyte-erythroid progenitors (MEPs) toward the erythroid lineage, thus reducing their megakaryocyte (MK) lineage commitment, whereas romiplostim, a thrombopoietin receptor agonist, stimulates megakaryopoiesis through the MK-committed progenitor and MEP bifurcation pathways simultaneously. These findings support an innovative combination of romiplostim and EPO to treat EPO-resistant anemia because the combination therapy further promotes erythropoiesis compared to EPO monotherapy and inhibits platelet production compared to romiplostim monotherapy.

Indomethacin is used commonly in preterm neonates for the prevention of intracranial hemorrhage and closure of an abnormally open cardiac vessel. Due to biomedical advances, the infants who receive this drug in the neonatal intensive care unit setting have become younger, smaller, and less mature (more preterm) at the time of treatment. To develop a pharmacokinetics (PK) model to aid future dosing, we designed a prospective cohort study to characterize indomethacin PK in a dynamically changing patient population. A population PK base model was created using NONMEM, and a covariate model was developed in a primary development cohort and subsequently was tested for accuracy in a validation cohort. Postnatal age was a significant covariate for hepatic clearance (CL

Recombinant human erythropoietin (rHuEPO) is effective in managing chronic kidney disease and chemotherapy-induced anemia. However, hyporesponsiveness to rHuEPO treatment was reported in about 10% of the patients. A decreased response in rats receiving a single or multiple doses of rHuEPO was also observed. In this study, we aimed to develop a quantitative systems pharmacology (QSP) model to examine hyporesponsiveness to rHuEPO in rats. Pharmacokinetic (PK) and pharmacodynamic (PD) data after a single intravenous dose of rHuEPO (100 IU/kg) was obtained from a previous study (Yan et al. in Pharm Res, 30:1026-1036, 2013) including rHuEPO plasma concentrations, erythroid precursors counts in femur bone marrow and spleen, reticulocytes (RETs), red blood cells (RBCs), and hemoglobin (HGB) in circulation. Parameter values were obtained from literature or calibrated with experimental data. Global sensitivity analysis and model-based simulations were performed to assess parameter sensitivity and hyporesponsiveness. The final QSP model adequately characterizes time courses of rHuEPO PK and nine PD endpoints in both control and treatment groups simultaneously. The model indicates that negative feedback regulation, neocytolysis, and depletion of erythroid precursors are major factors leading to hyporesponsiveness to rHuEPO treatment in rats.

Population pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed for extensive data for differing dosage forms and routes for dexamethasone (DEX) and betamethasone (BET) in 48 healthy nonpregnant Indian women in a partial and complex cross-over design. Single doses of 6 mg dexamethasone phosphate (DEX-P), betamethasone phosphate (BET-P), or 1:1 mixture of betamethasone phosphate and acetate (BET-PA) were administered orally (PO) or intramuscularly (IM) where each woman enrolled in a two-period cross-over study. Plasma concentrations collected over 96 h were described with a two-compartment model with differing PO and IM first-order absorption inputs. Overall, BET exhibited slower clearance, similar volume of distribution, faster absorption, and longer persistence than DEX with BET acetate producing extremely slow absorption but full bioavailability of BET. Six biomarkers were assessed over a 24-h baseline period with four showing circadian rhythms with complex baselines. These baselines and the strong responses seen after drug dosing were fitted with various indirect response models using the Laplace estimation methods in NONMEM 7.4. Both the PK and six biomarker responses were well-described with modest variability likely due to the homogeneous ages, weights, and ethnicities of the women. The drugs either inhibited or stimulated the influx processes with some models requiring joint inclusion of drug effects on circadian cortisol suppression. The biomarkers and order of sensitivity (lowest IC50/SC50 to highest) were: cortisol, T-helper cells, basophils, glucose, neutrophils, and T-cytotoxic cells. DEX sensitivities were generally greater than BET with corresponding mean ratios for these biomarkers of 2.86, 1.27, 1.72, 1.27, 2.69, and 1.06. Overall, the longer PK (e.g. half-life) of BET, but lesser PD activity (e.g. higher IC50), produces single-dose response profiles that appear quite similar, except for the extended effects from BET-PA. This comprehensive population modeling effort provides the first detailed comparison of the PK profiles and six biomarker responses of five commonly used dosage forms of DEX and BET in healthy women.

Population analysis of pharmacokinetic data for five differing dosage forms and routes for dexamethasone and betamethasone in 48 healthy nonpregnant Indian women was performed that accounted for a partial and complex cross-over design. Single doses of 6 mg dexamethasone phosphate (DEX-P), betamethasone phosphate (BET-P), or 1:1 mixture of betamethasone phosphate and acetate (BET-PA) were administered orally (PO) or intramuscularly (IM). Plasma concentrations collected for two periods over 96 h were described with a two-compartment model with differing PO and IM first-order absorption inputs. Clearances and volumes were divided by the IM bioavailability $${F}_{IM}$$ F IM . The homogeneous ages, body weights, and ethnicity of the women obviated covariate analysis. Parameter estimates were obtained by the Laplace estimation method implemented in NONMEM 7.4. Typical values for dexamethasone were clearance ($${CL/F}_{IM})$$ C L / F IM ) of 9.29 L/h, steady-state volume ($${{V}_{ss}/F}_{IM})$$ V ss / F IM ) of 56.4 L, IM absorption constant $$\left({k}_{aIM}\right)$$ k aIM of 0.460 1/h and oral absorption constant ($${k}_{aPO})$$ k aPO ) of 0.936 1/h. Betamethasone parameters were CL/FIM of 5.95 L/h, $${Vss/F}_{IM}$$ V s s / F IM of 72.4 L, $${k}_{aIM}$$ k aIM of 0.971 1/h, and $${k}_{aPO}$$ k aPO of 1.21 1/h. The PO to IM F values were close to 1.0 for both drugs. The terminal half-lives averaged about 7.5 h for DEX, 17 h for BET, and 78 h for BET from BET-PA with the latter reflecting very slow release of BET from the acetate ester. Overall, BET exhibited slower clearance, larger volume of distribution, faster absorption, and longer persistence than DEX. These data may be useful in considering exposures when substituting one form of corticosteroid for another.

Serum creatinine (sCr) is a commonly measured biomarker to estimate glomerular filtration rate (GFR) and therefore widely used as a covariate in population pharmacokinetic models of renally excreted drugs. In neonates, sCr dynamically changes during the first few weeks after birth. Missing covariates are a common problem in pharmacokinetic modeling of neonates due to the limited availability of blood sampling in number and volume. The objective of this work is to develop a parsimonious population model describing time courses of sCr in neonates with the intent to be incorporated into pharmacokinetic models of various drugs where sCr values are sparse or missing. The data for model development consisted of sCr measurements in 1080 newborns with a gestational age of 24–42 weeks. The model is based on a pharmacokinetic model of sCr that involves GFR, backflow of creatinine from the renal tubules, and urinary flow. Gestational age is the only covariate explaining between-subject variability of sCr. The model adequately describes distinct features of the sCr time course such as a peak and decline to a plateau. For a neonate with a GA of 35 weeks, the typical value of sCr at birth was 0.584 mg/dL, the peak (0.794 mg/dL) occurred 2.3 days after birth, to reach a plateau of 0.255 mg/dL approximately after 24.7 days. Model simulations reveal that in neonates with a similar postnatal age, sCr decreases with increasing GA. In summary, our model is designed to be a part of full random effects pharmacokinetic models where sCr is a significant covariate.

Serum creatinine (sCr) is a commonly measured biomarker to estimate glomerular filtration rate (GFR) and therefore widely used as a covariate in population pharmacokinetic models of renally excreted drugs. In neonates, sCr dynamically changes during the first few weeks after birth. Missing covariates are a common problem in pharmacokinetic modeling of neonates due to the limited availability of blood sampling in number and volume. The objective of this work is to develop a parsimonious population model describing time courses of sCr in neonates with the intent to be incorporated into pharmacokinetic models of various drugs where sCr values are sparse or missing. The data for model development consisted of sCr measurements in 1080 newborns with a gestational age of 24-42 weeks. The model is based on a pharmacokinetic model of sCr that involves GFR, backflow of creatinine from the renal tubules, and urinary flow. Gestational age is the only covariate explaining between-subject variability of sCr. The model adequately describes distinct features of the sCr time course such as a peak and decline to a plateau. For a neonate with a GA of 35 weeks, the typical value of sCr at birth was 0.584 mg/dL, the peak (0.794 mg/dL) occurred 2.3 days after birth, to reach a plateau of 0.255 mg/dL approximately after 24.7 days. Model simulations reveal that in neonates with a similar postnatal age, sCr decreases with increasing GA. In summary, our model is designed to be a part of full random effects pharmacokinetic models where sCr is a significant covariate.

Delay differential equations (DDEs) are commonly used in pharmacometric models to describe delays present in pharmacokinetic and pharmacodynamic data analysis. Several DDE solvers have been implemented in NONMEM 7.5 for the first time. Two of them are based on algorithms already applied elsewhere, while others are extensions of existing ordinary differential equations (ODEs) solvers. The purpose of this tutorial is to introduce basic concepts underlying DDE based models and to show how they can be developed using NONMEM. The examples include previously published DDE models such as logistic growth, tumor growth inhibition, indirect response with precursor pool, rheumatoid arthritis, and erythropoiesis-stimulating agents. We evaluated the accuracy of NONMEM DDE solvers, their ability to handle stiff problems, and their performance in parameter estimation using both first-order conditional estimation (FOCE) and the expectation–maximization (EM) method. NONMEM control streams and excerpts from datasets are provided for all discussed examples. All DDE solvers provide accurate and precise solutions with the number of significant digits controlled by the error tolerance parameters. For estimation of population parameters, the EM method is more stable than FOCE regardless of the DDE solver. published

In many models of pharmacodynamic systems with delays, a delay of an input is introduced by means of the convolution with the gamma distribution. An approximation of the convolution integral of bound functions based on a system of ordinary differential equations that utilizes properties of the binomial series has been introduced. The approximation converges uniformly on every compact time interval and an estimate of the approximation error has been found $$O\left( {\frac{1}{{N^{\nu } }}} \right)$$ where $$N$$ is the number of differential equations and $$\nu$$ is the shape parameter of the gamma distribution. The accuracy of approximation has been tested on a set of input functions for which the convolution is known explicitly. For tested functions, $$N \ge 20$$ has resulted in an accurate approximation, if $$\nu \ge 1$$ . However, if $$\nu < 1$$ the error of approximation decreases slowly with increasing $$N$$ , and $$N > 100$$ might be necessary to achieve acceptable accuracy. Finally, the approximation was applied to estimate parameters for the distributed delay model of chemotherapy-induced myelosuppression from previously published WBC count data in rats treated with 5-fluorouracil.

The ability to control dosage regimens of erythropoiesis‐stimulating agents (ESAs) to maintain a desired hemoglobin (HGB) target is still elusive. We utilized a Bayesian approach and informative priors to characterize HGB profiles, using simulated drug concentrations, in patients with end‐stage renal disease receiving maintenance doses of epoetin alfa. We also demonstrated an adaptive Bayesian method, applied to individual patients, to improve the accuracy of HGB predictions over time. The results showed that sparse HGB data from daily clinical practice were characterized successfully. The adaptive Bayesian method effectively improved the accuracy of HGB predictions by updating the individual model with new data accounting for within‐subject changes over time. The Bayesian approach presented leverages existing knowledge of the model parameters and has a potential utility in clinical practice to individualize dosage regimens of epoetin alfa and ESAs to achieve target HGB. Further studies are warranted to develop an application for practical use.

Shortened red blood cell (RBC) lifespan is one of the major factors contributing to anemia in end-stage renal disease (ESRD) patients and should be taken into account in anemia management protocols. In this study, we aimed to estimate RBC lifespan and the source of between-subject variability in ESRD patients. The resulting individual parameters (empirical Bayes estimates) were used to predict hemoglobin concentrations 2 weeks in advance. The reticulocyte-based estimation of RBC lifespan (REBEL) and the population modeling of RBC count data were used. A total of 120 blood samples collected biweekly over 10 weeks in 24 patients receiving maintenance doses of recombinant human erythropoietin (rHuEPO) subcutaneously were included in this analysis. Typical RBC lifespan was estimated to be 63.3 days. RBC lifespan was found to increase with erythroferrone, a recently identified hormone participating in iron metabolism. Approximately, a 10% increase in plasma erythroferrone was associated with a 5% increase in RBC lifespan. In addition, RBC lifespan was 18.7% shorter in females compared with males. Out of 24 subjects, 16 had hemoglobin concentrations predicted within 95% prediction intervals. The median absolute prediction error was 15.9% (interquartile range, 9.5 to 24.7%). We demonstrated that REBEL coupled with the population modeling technique can be used effectively to estimate RBC lifespan. Then, individual parameters can be used to predict future hemoglobin concentrations in ESRD patients.

The distributed delay model has been introduced that replaces the transit compartments in the classic model of chemotherapy-induced myelosuppression with a convolution integral. The maturation of granulocyte precursors in the bone marrow is described by the gamma probability density function with the shape parameter (ν). If ν is a positive integer, the distributed delay model coincides with the classic model with ν transit compartments. The purpose of this work was to evaluate performance of the distributed delay model with particular focus on model deterministic identifiability in the presence of the shape parameter. The classic model served as a reference for comparison. Previously published white blood cell (WBC) count data in rats receiving bolus doses of 5-fluorouracil were fitted by both models. The negative two log-likelihood objective function (-2LL) and running times were used as major markers of performance. Local sensitivity analysis was done to evaluate the impact of ν on the pharmacodynamics response WBC. The ν estimate was 1.46 with 16.1% CV% compared to ν = 3 for the classic model. The difference of 6.78 in - 2LL between classic model and the distributed delay model implied that the latter performed significantly better than former according to the log-likelihood ratio test (P = 0.009), although the overall performance was modestly better. The running times were 1 s and 66.2 min, respectively. The long running time of the distributed delay model was attributed to computationally intensive evaluation of the convolution integral. The sensitivity analysis revealed that ν strongly influences the WBC response by controlling cell proliferation and elimination of WBCs from the circulation. In conclusion, the distributed delay model was deterministically identifiable from typical cytotoxic data. Its performance was modestly better than the classic model with significantly longer running time.

Dexmedetomidine (DEX) is a fairly new alfa2-agonist which has been increasingly used in recent years for analgosedation, mostly because it offers a unique ability of providing both moderate level of sedation and analgesia without respiratory depression. Despite of many papers published, there are still only a few concerning the PK of the drug given as long-term infusion in ICU patients. The aim of this work was to characterize the population pharmacokinetics of dexmedetomidine and to investigate the potential benefits of individualization of drug dosing based on patient characteristics in the heterogeneous group of medical and surgical patients staying in intensive care unit. This study was performed in the group of 17 males and 10 females patients with a median age of 59.5 years and median body weight of 75 kg. Blood samples for dexmedetomidine assay were collected from arterial catheter, during and after discontinuation of a standard infusion, that ranged from 24 to 102 h. The following covariates were examined to influence dexmedetomidine PK: age, sex, body weight, patients’ health status described by Sequential Organ Failure Assessment Score (SOFA), inotropes usage, and infusion duration. The dexmedetomidine PK was best described by a two-compartment model. The typical values of PK parameters were estimated as 27 L for the volume of the central compartment, 87.6 L for the volume of the peripheral compartment, 38.5 L/h (9.2 mL/min/kg for a 70 kg patient) for systemic clearance and 46.4 L/h for the distribution clearance. Those values are consistent with literature findings. We were unable to show any significant relationship between collected covariates and dexmedetomidine PK. This study does not provide sufficient evidence to support the individualization of dexmedetomidine dosing based on age, sex, body weight, SOFA, and infusion duration. Electronic supplementary material The online version of this article (10.1007/s10928-017-9564-7) contains supplementary material, which is available to authorized users.

Age-structured cell population model was introduced to describe cell survival. The impact of the environment on the cell population is represented by drug plasma concentration. A key model variable is the hazard of cell removal that is a subject to the environment effect. The model is capable of describing cohort and random labeling cell survival data. In addition, it accounts for cell loss due to labeling of cell sample, but it lacks ability to describe the effect of label elution on the survival data. The model was applied to red blood cell (RBC) survival data in two groups of Wistar rats obtained by two techniques: cohort labeling using 14C-glycine (N = 4) and random labeling using biotin (N = 8). The Weibull probability density function was selected for the RBC lifespan distribution. The data were simultaneously fitted by the mixed effects model implemented in Monolix 4.3.3. The estimated typical values of RBC lifespan and age were 53.7 and 27.8 days, respectively. A noticeable effect of biotinylation on RBC survival was observed that resulted in a significant difference between the means of individual RBC lifespan for two groups. The model provides a mechanistic framework flexible enough to account for various experimental designs to generate the cell survival data. Despite model qualification using animal data, the model has the same potential to be applied to cell survival data analysis in humans.

This study aimed to develop a method for implementing dose correction in a Michaelis-Menten (M-M) approximation of a target-mediated drug disposition (TMDD) model with multiple intravenous (IV) bolus administrations. We derived the formula of a correction factor (Fcorr) for each dose in a multiple IV bolus dosing regimens for M-M model. Fcorr depends on the residual free drug amount prior IV bolus dosing event and dose amount. We conducted a stochastic simulation and estimation (SSE) exercise based on therapeutic antibody PK parameters to evaluate the effect of Fcorr on parameter estimation. Previously published clinical PK data of recombinant human erythropoietin (rHuEPO) from four clinical trials in healthy subjects receiving multiple IV bolus doses were analyzed by both M-M model with and without dose correction (MMC and MMNC) as well as the rapid-binding/quasi-steady-state (RB/QSS) TMDD models. Our results showed that MMNC introduced bias to fixed-effect parameter estimates and overestimated random-effect variables. Compared with MMC, MMNC was not able to adequately characterize the nonlinearity in the PK data of antibody and rHuEPO. The MMC-based simulation demonstrated that thricely weekly 10 IU/kg rHuEPO dosing regimen yielded Fcorr = 0.5. This result suggested that the lower-than-expected exposure for rHuEPO at low dose is due to target binding. An M-M approximation of the TMDD model should include a dose correction to avoid model misfitting and potential bias in the estimated PK parameters.

Body weight is the primary covariate in pharmacokinetics of many drugs and dramatically changes during the first weeks of life of neonates. The objective of this study is to determine if missing body weights in preterm and term neonates affect estimates of model parameters and which methods can be used to improve performance of a population pharmacokinetic model of paracetamol. Data for our analysis were obtained from previously published studies on the pharmacokinetics of intravenous paracetamol in neonates. We adopted a population model of body weight change in neonates to implement three previously introduced methods of handling missing covariates based on data imputation, likelihood function modification, and full random effects modeling. All models were implemented in NONMEM 7.4, and population parameters were estimated using the FOCE method. Our major finding was that missing body weights minimally affect population estimates of pharmacokinetic parameters but do affect the covariate relationship parameters, particularly the one describing dependence of clearance on body weight. None of the tested methods changed estimates of between-subject variability nor impacted the predictive performance of the model. Our analysis shows that a modeling approach towards handling missing covariates allows borrowing information gathered in various studies as long as they target the same population. This approach is particularly useful for handling time-dependent missing covariates. ispartof: AAPS JOURNAL vol:21 issue:4 ispartof: location:United States status: published

Hepcidin (H25) is a hormone peptide synthesized by the liver that binds to ferroportin and blocks iron export. In this study, H25 was inhibited by administration of single and multiple doses of an anti-H25 monoclonal antibody Ab 12B9m in cynomolgus monkeys. The objective of this analysis was to develop a pharmacodynamic model describing the role of H25 in regulating iron homeostasis and the impact of hepcidin inhibition by Ab 12B9m. Total serum H25 and Ab 12B9m were determined in each animal. Corresponding measurements of serum iron and hemoglobin (Hb) were obtained. The PD model consisted of iron pools in serum (FeS), reticuloendothelial macrophages (FeM), hemoglobin (FeHb), and liver (FeL). The iron was assumed to be transported between the FeS, FeHb, and FeM unidirectionally at rates k S, k Hb, and k M. H25 serum concentrations were described by the previously developed PK model with the parameters fixed at their estimates. The serum iron and Hb data were fitted simultaneously. The corresponding estimates of the rate constants were k S/Fe0 = 0.113 h−1, k M = 0.00191 h−1, and k Hb = 0.00817 h−1. The model-based IC50 value for the H25 inhibitory effect on ferroportin activity was 0.398 nM. The PD model predicted a negligible effect of Ab 12B9m on Hb levels for the tested doses. The presented PD model adequately described the serum iron time courses following single and multiple doses of Ab 12B9m. Ab 12B9m-induced inhibition of H25 resulted in a temporal increase in serum and liver iron and a decrease in the iron stored in reticuloendothelial macrophages.

Rhythmicity in baseline responses over a 24-h period for an indirect pharmacological effect R(t) can arise from either a periodic time-dependent input rate $$ k_{in} \left( t \right) $$ or a periodic time-dependent loss constant $$ k_{out} \left( t \right) $$ . If either $$ k_{in} \left( t \right) $$ or $$ k_{out} \left( t \right) $$ follows some nonstationary biological rhythm (e.g., circadian), then the response R(t) also displays a periodic behavior. Indirect response models assuming time-dependent input rates $$ \left[ {k_{in} \left( t \right)} \right] $$ have been utilized to capture drug effects on various physiological responses such as hormone suppression, immune cell trafficking, and gene expression in tissues. This paradigm was extended to consider responses with circadian-controlled loss $$ \left[ {k_{out} \left( t \right)} \right] $$ mechanisms. Theoretical equations describing this model are presented and simulations were performed to examine expected response behaviors. The model was able to capture the chronobiology and pharmacodynamics of applicable drug responses, including the uricosuric effects of lesinurad in humans, suppression of the beta amyloid (Aβ) peptide by a gamma-secretase inhibitor in mouse brain, and the modulation of extracellular dopamine by a dopamine transporter inhibitor in rat brain. This type of model has a mechanistic basis and shows utility for capturing drug responses displaying nonstationary baselines controlled by removal mechanism(s).

Erythropoiesis-stimulating agents (eg, epoetin alfa) are the primary treatment for anemia in patients with end-stage renal disease. Hemoglobin variability in and out of a narrow target range is common and associated with higher morbidity and mortality risk. More robust erythropoiesis-stimulating agent response metrics are needed to define optimal dosing and their association with clinical outcomes. In this cross-sectional, single-center, retrospective study, 49 patients with end-stage renal disease on hemodialysis were followed over 12 months. To quantify hemoglobin deviations outside the target range (10-12 g/dL), the area under the curve of hemoglobin versus time over a 12-month period (AUC-HGB) was calculated using the trapezoidal rule. Patients were categorized into 4 responder groups based on AUC-HGB quartiles. Comparative analyses of demographic and clinical characteristics between responder groups were performed. Correlations between AUC-HGB, erythropoietin resistance index, and time within therapeutic range were calculated. There were no significant differences in laboratory and dialysis parameters between responder groups except hemoglobin concentration and epoetin alfa dose. There was a negative correlation between AUC-HGB and time within therapeutic range (r = -.92; P < .001) and hemoglobin concentration (r = -.85; P < .01), indicating internal validity of the metric. There was a positive correlation between AUC-HGB and erythropoietin resistance index (r = .70; P < .001) indicating external validity. The poor response group received a higher median epoetin alfa dose (160 U/kg/week) compared to the excellent response group (68.8 U/kg/week; P < .001) with a similar number of dose changes between the groups. AUC-HGB is a valid marker of epoetin alfa response and should be considered in future analyses of larger populations.

Prediction of human pharmacokinetics (PK) can be challenging for monoclonal antibodies (mAbs) exhibiting target-mediated drug disposition (TMDD). In this study, we performed a quantitative analysis of a diverse set of six mAbs exhibiting TMDD to explore translational rules that can be utilized to predict human PK. A TMDD model with rapid-binding approximation was utilized to fit PK and PD (i.e., free and/or total target levels) data, and average absolute fold error (AAFE) was calculated for each model parameter. Based on the comparative analysis, translational rules were developed and applied to a test antibody not included in the original analysis. AAFE of less than two-fold was observed between monkey and human for baseline target levels (R 0), body-weight (BW) normalized central elimination rate (K el/BW(-0.25)) and central volume (V c/BW(1.0)). AAFE of less than three-fold was estimated for the binding affinity constant (K D). The other four parameters, i.e., complex turnover rate (K int), target turnover rate (K deg), central to peripheral distribution rate constant (K pt) and peripheral to central rate constant (K tp) were poorly correlated between monkey and human. The projected human PK of test antibody based on the translation rules was in good agreement with the observed nonlinear PK. In conclusion, we recommend a TMDD model-based prediction approach that integrates in vitro human biomeasures and in vivo preclinical data using translation rules developed in this study.

In pharmacokinetics/pharmacodynamics (PKPD) the measured response is often delayed relative to drug administration, individuals in a population have a certain lifespan until they maturate or the change of biomarkers does not immediately affects the primary endpoint. The classical approach in PKPD is to apply transit compartment models (TCM) based on ordinary differential equations to handle such delays. However, an alternative approach to deal with delays are delay differential equations (DDE). DDEs feature additional flexibility and properties, realize more complex dynamics and can complementary be used together with TCMs. We introduce several delay based PKPD models and investigate mathematical properties of general DDE based models, which serve as subunits in order to build larger PKPD models. Finally, we review current PKPD software with respect to the implementation of DDEs for PKPD analysis.

A technique has recently been proposed for obtaining the reticulocyte (RET) age distribution from the flow cytometric reticulocyte count. It allows for a quantitative characterization of reticulocyte dynamics. In this work this technique was applied to characterize the blood, bone marrow and spleen reticulocytes in homeostatic and erythropoietically stimulated rats in order to determine the reticulocyte maturation times in the bone marrow and blood; and to confirm the presence of ineffective erythropoiesis (neocytolysis). The latter was done by comparing the reticulocyte removal rate from blood with bilirubin formation after erythropoiesis stimulation. A single subcutaneous dose (4050 IU/kg) of recombinant human erythropoietin (rHuEPO) was administered to rats, then their reticulocytes were stained with thiazole orange and the distribution of the fluorescent signal measured using flow cytometry. The obtained signal distribution of the reticulocytes was transformed to the age distribution and a set of basic parameters reflecting reticulocyte dynamics was determined. Bilirubin concentrations were measured to directly assess the presence of reticulocyte irreversible removal. The bilirubin formation was found to be considerably modulated by rHuEPO and corresponded well to the determined reticulocyte removal rate. The initial increase and subsequent decrease of the reticulocyte maturation time in blood was quantitated and directly linked with RET mobilization from the bone marrow. A substantial number (60%) of reticulocytes is sequestrated during homeostasis in rats. This number increases and then decreases after rHuEPO administration, as also reflected by bilirubin formation. Flow cytometry seems to be an excellent method for studying RET dynamics and the presence of young RBC neocytolysis. Copyright © 2014 John Wiley & Sons, Ltd.

We introduce a new, minimally invasive laboratory technique called REticulocyte-Based Estimation of Lifespan (REBEL) of erythrocytes in humans. Its major advantage over existing techniques is its applicability to patients with both changing and steady-state erythropoiesis status. The feasibility of REBEL was tested in five hemodialysis dependent End-Stage Renal Disease patients. The RNA degradation half-life was first determined for each subject on day one by flow cytometry measurement of the decay rate of Thiazole Orange stain. Reticulocyte age distribution was then measured from residual RNA content weekly for two months to estimate the RBC production rate time course. Mean RBC lifespan per subject was estimated by fitting the integrated RBC production rate overtime to the measured RBC count and optimizing the integration limits. All calculations were performed in MATLAB. The mean reticulocyte RNA half-life was 0.71±0.11 days. The small coefficient of variation 15.6% indicated that the degradation rate of RNA did not vary substantially between subjects. The mean RBC lifespan TRBC = 76.6±23.8 days was comparable to the reported values for this patient population.

The purpose of this report is to apply multi-scale modeling using the theory of physiologically structured populations (PSP) to develop a mathematical model for antimicrobial resistance based on a heterogeneous distribution of receptors and affinities among bacterial cells. The theory has been tested on data obtained from an in vitro static time-kill infection model analyzing the pharmacodynamics of polymyxin B against Gram-negative bacteria. The drug binding parameter KD (dissociation equilibrium constant) is assumed to vary between the bacterial cells. The PSP model describes the time course of the density distribution of KD upon exposure to cytotoxic drug concentrations. The drug increases the hazard of cell death as a function of receptor occupancy. The initial distribution of KD is described by the Weibull function. Time-kill data were used for model qualification. In vitro static time-kill experiments to evaluate the rate and extent of killing due to polymyxin B against two Klebsiella pneumoniae clinical isolates with differing susceptibilities to polymyxin B were performed over 48 h. The time-kill kinetics data of bacterial load cfu (colony forming units)/mL was used for model qualification. The resistant bacterial population is determined by the balance between growth rate and hazard of cell death controlled by polymyxin B concentrations. There exists a critical KD value below which cells continue to grow. Estimates of shape parameters for distributions of KD yielded unimodal distributions with the modes at 0 nM and the right tails containing approximately 25% of the bacteria. Our findings support a hypothesis that resistance of Klebsiella pneumoniae to polymyxin B can be at least partially attributed to a drug-induced selection of a subpopulation due to heterogeneity of polymyxin B receptor binding in the bacterial population.

We aimed to develop a cell-level pharmacodynamics-mediated drug disposition (PDMDD) model to analyze in vivo systems where the PD response to a drug has an appreciable effect on the pharmacokinetics (PK). An existing cellular level model of PD stimulation was combined with the standard target-mediated drug disposition (TMDD) model and the resulting model structure was parametrically identifiable from typical in vivo PK and PD data. The PD model of the cell population was controlled by the production rate k in and elimination rate k out which could be stimulated or inhibited by the number of bound receptors on a single cell. Simulations were performed to assess the impact of single and repeated dosing on the total drug clearance. The clinical utility of the cell-level PDMDD model was demonstrated by fitting published data on the stimulatory effects of filgrastim on absolute neutrophil counts in healthy subjects. We postulated repeated dosing as a means of detecting and quantifying PDMDD as a single dose might not be sufficient to elicit the cellular response capable of altering the receptor pool to visibly affect drug disposition. In the absence of any PD effect, the model reduces down to the standard TMDD model. The applications of this model can be readily extended to include chemotherapy-induced cytopenias affecting clearance of endogenous hematopoietic growth factors, different monoclonal antibodies and immunogenicity effects on PK.

The target-mediated drug disposition (TMDD) model has been adopted to describe pharmacokinetics for two drugs competing for the same receptor. A rapid binding assumption introduces total receptor and total drug concentrations while free drug concentrations CA and CB are calculated from the equilibrium (Gaddum) equations. The Gaddum equations are polynomials in CA and CB of second degree that have explicit solutions involving complex numbers. The aim of this study was to develop numerical methods to solve the rapid binding TMDD model for two drugs competing for the same receptor that can be implemented in pharmacokinetic software. Algebra, calculus, and computer simulations were used to develop algorithms and investigate properties of solutions to the TMDD model with two drugs competitively binding to the same receptor. A general rapid binding approximation of the TMDD model for two drugs competing for the same receptor has been proposed. The explicit solutions to the equilibrium equations employ complex numbers, which cannot be easily solved by pharmacokinetic software. Numerical bisection algorithm and differential representation were developed to solve the system instead of obtaining an explicit solution. The numerical solutions were validated by MATLAB 7.2 solver for polynomial roots. The applicability of these algorithms was demonstrated by simulating concentration-time profiles resulting from exogenous and endogenous IgG competing for the neonatal Fc receptor (FcRn), and darbepoetin competing with endogenous erythropoietin for the erythropoietin receptor. These models were implemented in Phoenix WinNonlin 6.0 and ADAPT 5, respectively.

The experiments presented in the following paper aimed to investigate the influence of atypical antipsychotic clozapine (CLO, CAS 578-21-0) classified also as normothymic drug on spatial memory in rats in Morris water maze test. The study also investigated the probably occurring side effects (measuring cataleptic activity and motor coordination) following single and chronic administration of CLO compared to haloperidol (HAL, CAS 52-86-8), a conventional antipsychotic. All the tests were carried out on male Wistar rats. CLO 10 mg/kg was administered orally 30 min before the tests and HAL 0.15 mg/kg was administered orally 60 min before the tests. In the Morris test memory improvement only after chronic administration of CLO on the 7th and 14th day of treatment was observed, whereas after 14 days of HAL treatment spatial memory impairment was noted. CLO did not induce catalepsy after single as well as chronic administration, but administrated chronically induced a tendency for its occurrence. HAL evoked a strong cataleptic effect both after acute and chronic treatment. CLO had no impact on motor coordination in the chimney test except on day 14 when statistically significant impairments were observed. HAL disturbed motor coordination in rats after single as well as chronic administration. The wide and unique spectrum of receptor activity of CLO in comparison with HAL as well as some neurotransmit-ters’ interactions may explain its usefulness in long-term therapy of bipolar affective disorder and schizoaffective disorder with co-occurred cognitive deficits.

CNTO 530 and darbepoetin-a are long lived erythropoietin receptor agonists (ERAs). Clinically, anemia of chronic disease (ACD) is associated with increased expression of tumor necrosis factor-a (TNF-a) and mice transgenic for human TNF-a develop ACD. The purpose of this investigation was to compare the effects of these agents in a murine model of ACD. Human TNF-a expressing (Tg 197) mice were administered a single subcutaneous dose of CNTO 530 or darbepoetin-a and the pharmacodynamic response in bone marrow spleen and peripheral blood evaluated. RBC life span and reticulocyte age distribution were also evaluated. CNTO 530 induced a dose responsive increase in reticulocytes, RBCs and Hgb in both wild type and Tg197 mice. Although the reticulocyte response was similar to wild types, the RBC and Hgb response to CNTO 530 in Tg197 mice was blunted. There was no statistically significant difference in RBC life span with either compound. Darbepoetin-α caused a greater peak in % dead Pro/basophilic erythroblasts, greater peak EMH in the spleen and a greater increase in reticulocyte maturation time. In contrast, despite a similar peak increase, CNTO 530 caused a more sustained response of reticulocyte, EMH, RBC and Hgb, consistent with increased exposure. In conclusion, CNTO 530 and darbepoetin-a increased RBC and hemoglobin in a murine model of ACD. Compared to darbepoetin-a, CNTO 530 had a more sustained effect, consistent with increased exposure.

Chemotherapy-induced anemia is a frequent complication in cancer treatment. The aim was to develop a pharmacokinetic (PK) model that describes the time-dependent decline of epoetin alfa (rHuEPO) clearance following thrice (t.i.w.) or once (q.w.) weekly subcutaneous injections in cancer patients using a population PK approach. Serum concentrations of rHuEPO were available retrospectively from a phase I study. A one-compartment model with first-order elimination described rHuEPO PK. Sequential zero- and first-order (ka) processes with duration (tlag) characterized the absorption. Population PK analysis was performed using NONMEM. The influence of several covariates was tested. Model evaluation was performed using visual predictive check. Precision of parameter estimates was assessed by standard errors and confidence intervals determined by bootstrap analysis. Apparent clearance (CL/F) and volume of distribution (Vc/F) were 25.6% lower and 29.2% higher for q.w. than t.i.w. groups. RHuEPO was absorbed for 10% during 24.6 h through the zero-order process. Following which 90% of the dose was absorbed through the first-order process with ka of 0.033 h−1. The most significant covariates were the time-dependent decrease of CL/F with an increase in body weight, a decrease in reticulocyte count, a decrease in hemoglobin baseline, an increase in total number of chemotherapy cycles, and platinum-containing chemotherapy. AGE served as an important covariate on FRAC and ka. Visual predictive check showed no deviation from observed values. The PK model adequately predicted rHuEPO concentration-profiles in all individuals. Relevant covariates were identified and incorporated into the model. Copyright © 2010 John Wiley & Sons, Ltd.

A pharmacokinetics (PK)/pharmacodynamics (PD) model was developed to describe the tolerance and rebound for reticulocyte (RET) and red blood cell (RBC) counts and the hemoglobin (Hb) concentrations in blood after repeated intravenous administrations of 1350 IU/kg of recombinant human erythropoietin (rHuEPO) in rats thrice weekly for 6 weeks. Drug concentrations were described by using a quasi-equilibrium model. The PD model consisted of a lifespan-based indirect response model (LIDR) with progenitor cells [burst colony-forming unit erythroblasts and colony-forming unit erythroblasts (CFUs)], normoblasts (NOR), RETs, and RBCs. Drug-receptor complex stimulatory effects on progenitor cells differentiation and RBC lifespan were expressed by using the E(max) model (S(max-epo) and SC(50-epo), E(max) and EC(50)). The Hb profile was indirectly modeled through a LIDR model for mean corpuscular hemoglobin (with a lifespan T(mch)) including a linear (S(max-mch)) drug stimulatory effect. The negative feedback from RBCs accounted for the time-dependent rHuEPO clearance decline. A simultaneous PK/PD fitting was performed by using MATLAB-based software. PK parameters such as equilibrium dissociation, erythropoietin receptor degradation, production, and internalization rate constants were 0.18 nM (fixed), 0.08 h(-1), 0.03 nM/h, and 2.51 h(-1), respectively. The elimination rate constant and central volume of distribution were 0.57 h(-1) and 40.63 ml/kg, respectively. CFU and NOR, RET, and RBC lifespans were 37.26 h, 17.25 h, and 30.15 days, respectively. S(max-epo) and SC(50-epo) were 7.3 and 0.47 10(-2) nM, respectively. E(max) was fixed to 1. EC(50) and SC(50-epo) were equal. S(max-mch) and T(mch) were 168.1 nM(-1) and 35.15 days, respectively. The proposed PK/PD model effectively described rHuEPO nonstationary PK and allowed physiological estimates of cell lifespans.

Target-mediated drug disposition (TMDD) models have been applied to describe the pharmacokinetics of drugs whose distribution and/or clearance are affected by its target due to high binding affinity and limited capacity. The Michaelis-Menten (M-M) model has also been frequently used to describe the pharmacokinetics of such drugs. The purpose of this study is to investigate conditions for equivalence between M-M and TMDD pharmacokinetic models and provide guidelines for selection between these two approaches. Theoretical derivations were used to determine conditions under which M-M and TMDD pharmacokinetic models are equivalent. Computer simulations and model fitting were conducted to demonstrate these conditions. Typical M-M and TMDD profiles were simulated based on literature data for an anti-CD4 monoclonal antibody (TRX1) and phenytoin administered intravenously. Both models were fitted to data and goodness of fit criteria were evaluated for model selection. A case study of recombinant human erythropoietin was conducted to qualify results. A rapid binding TMDD model is equivalent to the M-M model if total target density R ( tot ) is constant, and R ( tot ) K ( D ) /(K ( D ) + C) ( 2 )1 where K ( D ) represents the dissociation constant and C is the free drug concentration. Under these conditions, M-M parameters are defined as: V ( max ) = k ( int ) R ( tot ) V ( c ) and K ( m ) = K ( D ) where k ( int ) represents an internalization rate constant, and V ( c ) is the volume of the central compartment. R ( tot ) is constant if and only if k ( int ) = k ( deg,) where k ( deg ) is a degradation rate constant. If the TMDD model predictions are not sensitive to k ( int ) or k ( deg ) parameters, the condition of R ( tot ) K ( D ) /(K ( D ) + C) ( 2 )1 alone can preserve the equivalence between rapid binding TMDD and M-M models. The model selection process for drugs that exhibit TMDD should involve a full mechanistic model as well as reduced models. The best model should adequately describe the data and have a minimal set of parameters estimated with acceptable precision.

Background and Objective: Filgrastim is a human granulocyte-colony stimulating factor (G-CSF). The biological activity of filgrastim is identical to that of endogenous G-CSF. It controls neutrophil production within the bone marrow by stimulating the proliferation, differentiation and survival of myeloid progenitor cells and some end-cell function activation. The purpose of this work is to propose a target-mediated drug disposition pharmacokinetic model of filgrastim. Methods: A mechanism-based population pharmacokinetic model was developed to account for receptormediated endocytosis as a mechanism for nonlinear disposition of G-CSF. Time profiles of serum filgrastim concentrations following subcutaneous doses of 2.5, 5 and 10 μg/kg and intravenous infusion of 5 μg/kg over 0.5 hour were studied. The pharmacokinetic model included first-order elimination from the serum, receptor binding, turnover of free receptors and internalization of drug-receptor complexes. The proposed targetmediated drug disposition models served as a tool to study drug absorption and the impact of receptor binding on filgrastim clearance. Results: Filgrastim was found to exhibit parallel absorption with first- and zero-order kinetics and bioavailability of 69.1%. The majority of the drug (58.6%) was absorbed by zero-order processes, presumably through the lymphatic system. The equilibrium dissociation constant (Kd) was estimated as 16.38 pM. Conclusion: The proposed model predicts that clearance is initially mostly governed by the binding of filgrastim to G-CSF receptors. Subsequently, the clearance slows down because of the saturation of binding sites, and occurs mostly via the linear (renal) pathway. Finally, for G-CSF concentrations lower than the Kd, target-mediated clearance dominates. The presented receptor-mediated model adequately describes filgrastim serum concentrations and quantifies the role of receptor binding in G-CSF clearance.

This report generates efficient experimental designs (dose, sampling times) for parameter estimation for four basic physiologic indirect pharmacodynamic response (IDR) models. The principles underlying IDR models and their response patterns have been well described. Each IDR model explicitly contains four parameters, k (in) (production), k (out) (loss), I (max)/S (max) (capacity) and IC (50)/SC (50) (sensitivity). The pharmacokinetics of an IV dose of drug described by a monoexponential function of time with two parameters, V and k (el), is assumed. The random errors in the response variable are assumed to be additive, independent, and normal with zero mean and variance proportional to some power of the mean response. Optimal design theory was used extensively to assess the role of both dose and sampling times. Our designs were generated in Mathematica (ADAPT 5 typically produces identical results). G-optimality was used to verify that the generated designs were indeed D-optimal. Such designs are efficient and robust when good prior knowledge of the estimated parameters is available. The efficiency of unconstrained D-optimal designs (4 dose, sampling time pairs) does not improve much when the drug doses are allowed to differ, compared with constrained single dose designs (4 sampling times) with one maximal feasible dose. Also, explored were efficiencies of alternative study designs and results from parameter misspecification. This analysis substantiates the importance of larger doses yielding greater certainty in parameter estimation in pharmacodynamics.

We have proposed a technique for transforming reticulocyte (RET) flow cytometry count into the age distribution allowing for a quantitative characterization of RETs dynamics. This technique was evaluated in homeostatic and erythropoietically stimulated rats. We administered a single intravenous dose (4,050 IU/kg) of recombinant human erythropoietin to Wistar rats, then stained their RETs with thiazole orange, and measured the distribution of the fluorescent signal using flow cytometry. The RET age distribution was determined assuming an exponential decline of RET RNA. The RET distribution over time was characterized by median age, production rate of mature red blood cell in the blood, bone marrow and blood maturation times, and RET loss from the blood. Erythropoietin was found to cause complex changes not only in RET count but also in age distribution. Generally, an increase in RET count was accompanied by the presence of young RETs and a decrease with the presence of old RETs. The rate at which RETs were removed from the blood was affected considerably by erythropoietin administration, and their death was attributed to it. Flow cytometric analysis of RET age distribution expands our understanding of erythropoiesis and augments the information we can obtain from RET measurements.

Objective The purpose of this work was to develop a flow cytometry method of obtaining RNA-thiazole orange signal distribution of reticulocytes (RET) from bone marrow aspirates and determine the distribution of age for RET in the blood and bone marrow. Methods The thiazole orange (an RNA dye) and LDS-751 (a DNA dye) composition was used to separate RET from other cell populations. LDS 751 was used to separate red blood cells (RBC) from nucleated cells. The differentiation between mature RBC and RET was done by means of the presence of residual RNA. It also served as a marker of age of RET. In vitro changes of the fluorescence signal of the RET population allowed us to obtain the unique relationship between the signal and the age for an individual cell and was used to transform the in vivo signal distribution into the age distribution. Results The determination of age was not possible due to the lack of knowledge of the RET signal distribution at birth. Instead, a relative time (RT) that a RET needs to become a mature RBC was used. The RT distribution of RET in blood and bone marrow was determined. The RT that an average RET spends in the blood is 0.43 days and ranges from 0 to 1.1 days. The time an average RET in bone marrow needs to become a mature RBC, is 1.36 days. The RET born with the highest signal needs about 1.8 days to become a mature RBC. About 37% reticulocytes die in the circulation. Conclusions In summary, the thiazole orange staining can be used for determination of the RET age distribution. However, only the RT distribution can be determined. The proposed method was successfully applied to characterize in vivo aging of blood and bone marrow RET.

Peptidic erythropoiesis receptor agonist is a synthetic, PEGylated peptide that can promote red blood cell production upon binding to the erythropoietin receptor. The objective of this study was to characterize the pharmacokinetics and erythropoietic effects of peptidic erythropoiesis receptor agonist in healthy volunteers. Plasma concentrations of peptidic erythropoiesis receptor agonist and pharmacodynamic responses were obtained after single intravenous injections at doses of 0.025, 0.05, and 0.1 mg/kg. Population pharmacokinetic/pharmacodynamic modeling was performed using NONMEM. Peptidic erythropoiesis receptor agonist exhibited nonlinear pharmacokinetics described by a 1-compartment model with parallel elimination by Michaelis-Menten and linear processes. A catenary, life span-based, indirect response model reflecting bone marrow erythroid and blood cells reflected the pharmacodynamics of peptidic erythropoiesis receptor agonist. A modest tolerance and rebound phenomenon in reticulocytes was modeled with negative feedback regulation related to hemoglobin. This pharmacokinetic/pharmacodynamic model well characterized the prolonged disposition, intrinsic pharmacologic parameters, and typical hematological system properties following single doses of peptidic erythropoiesis receptor agonist in normal subjects.

Based on the paradigm of mechanistic modeling, three types of pharmacodynamic models are introduced: direct effect, indirect response, and signal transduction. The underlying pharmacological and biological assumptions about the model structures and operations are provided along with examples of their applications. A brief historical perspective is introduced for each model class. Mathematical equations defining the model are presented and explored to link model parameters with model characteristics such as the shape of the response curve. The impact of dose on the time courses of pharmacodynamic responses is evaluated for large doses and exemplified with computer simulations. A common theme is the extent of delay between drug pharmacokinetics and response. When relevant, alternative parameterizations and parameter identifiability are discussed. Only the simplest forms of models are provided with some guidelines on how to build more complex models based on a systems pharmacology approach.

A mechanism-based pharmacokinetic–pharmacodynamic (PK/PD) model was developed for recombinant human erythropoietin (rHuEPO) to account for receptor-mediated endocytosis via erythropoietin receptor (EPOR) as a primary mechanism for nonlinear disposition of rHuEPO as well as activation of erythropoietic stimulation. Time profiles of rHuEPO concentrations following a wide range of intravenous (i.v.) doses in rats (10, 100, 450, 1,350, 4,050 IU/kg), monkeys (500, 2,000, 4,000 IU/kg), and man (10, 100, 150, 300, 500 IU/kg) were examined. The mean data of reticulocytes, red blood cells (RBC), and hemoglobin for five different doses in rats were analyzed. The PK model components included receptor binding, subsequent internalization and degradation, EPOR turnover, non-specific tissue distribution, and linear first-order elimination from plasma. The equilibrium dissociation constant (K D ) was similar between rats and monkeys (0.11 nM) and was 10-fold lower in humans (0.012 nM). The PD effects of rHuEPO were described by an indirect response model with lifespan cell loss and driven by the rHuEPO–EPOR complex. A generalized nonlinear PK model for rHuEPO taking into account EPOR binding of the drug in bone marrow was proposed and well described the PK profiles of rHuEPO following i.v. doses in rats, monkeys, and man. The present receptor-mediated PK/PD model for rHuEPO closely reflects underlying mechanisms of disposition and dynamics of rHuEPO.