Pharmacodynamic parameter estimation: Population size versus number of samples

The purpose of this study was to evaluate the effects of population size, number of samples per individual, and level of interindividual variability (IIV) on the accuracy and precision of pharmacodynamic (PD) parameter estimates. Response data were simulated from concentration input data for an inhibitory sigmoid drug efficacy (E(max)) model using Nonlinear Mixed Effect Modeling, version 5 (NONMEM). Seven designs were investigated using different concentration sampling windows ranging from 0 to 3 EC(50) (EC(50) is the drug concentration at 50% of the E(max)) units. The response data were used to estimate the PD and variability parameters in NONMEM. The accuracy and precision of parameter estimates after 100 replications were assessed using the mean and SD of percent prediction error, respectively. Four samples per individual were sufficient to provide accurate and precise estimate of almost all of the PD and variability parameters, with 100 individuals and IIV of 30%. Reduction of sample size resulted in imprecise estimates of the variability parameters; however, the PD parameter estimates were still precise. At 45% IIV, designs with 5 samples per individual behaved better than those designs with 4 samples per individual. For a moderately variable drug with a high Hill coefficient, sampling from the 0.1 to 1, 1 to 2, 2 to 2.5, and 2.5 to 3 EC(50) window is sufficient to estimate the parameters reliably in a PD study.     

Influence of age and sex on pharmacodynamics of propofol in neurosurgical patients： model development

AIM: To determine propofol concentration in the cerebral spinal fluid (CSF) of neurosurgical patients and carry out a preliminary population pharmacodynamic study. METHODS: Twenty-seven elective neurosurgical patients (12 men and 15 women) aged 17-74 years received propofol in a bolus dose of 2 mg/kg for 5 min and an infusion of 10 mg/kg per h for 5 min. Frequent CSF samples were drawn and assayed for propofol concentration. The bispectral index (BIS) was used to measure the drug effect. All data were analyzed first with the Excel software package, then pharmacodynamics modeling was performed using the NONMEM software package. RESULTS: The CSF concentration was related to the drug effect with linear and sigmoid Emax models. The parameters for the linear addictive model were a=1.11 and b=95.4. The parameters for the linear exponential model were a=1.05 and b=92.7. The parameters for the sigmoid E(max) model were E(max)=119, EC(50)=53.6 ng/mL, and N=1.51. When the covariates of age, weight and sex were considered, the parameters of models, objective function, the standard error of the mean and the prediction error were not optimized. CONCLUSION: Linear additive, linear exponential and sigmoid E(max) models can be used to describe the pharmacodynamics of propofol with respect to the concentration in CSF. In this small population, age (17-74 years), weight (47-98 kg) and sex did not influence any of the pharmacodynamic parameters of propofol. To verify these preliminary results, a larger study population is required.     

A novel predictive pharmacokinetic/pharmacodynamic model of repolarization prolongation derived from the effects of terfenadine, cisapride and E-4031 in the conscious chronic av node--ablated, His bundle-paced dog

INTRODUCTION: Terfenadine, cisapride, and E-4031, three drugs that prolong ventricular repolarization, were selected to evaluate the sensitivity of the conscious chronic atrioventricular node--ablated, His bundle-paced Dog for defining drug induced cardiac repolarization prolongation. A novel predictive pharmacokinetic/pharmacodynamic model of repolarization prolongation was generated from these data. METHODS: Three male beagle dogs underwent radiofrequency AV nodal ablation, and placement of a His bundle-pacing lead and programmable pacemaker under anesthesia. Each dog was restrained in a sling for a series of increasing dose infusions of each drug while maintained at a constant heart rate of 80 beats/min. RT interval, a surrogate for QT interval in His bundle-paced dogs, was recorded throughout the experiment. RESULTS: E-4031 induced a statistically significant RT prolongation at the highest three doses. Cisapride resulted in a dose-dependent increase in RT interval, which was statistically significant at the two highest doses. Terfenadine induced a dose-dependent RT interval prolongation with a statistically significant change occurring only at the highest dose. The relationship between drug concentration and RT interval change was described by a sigmoid E(max) model with an effect site. Maximum RT change (E(max)), free drug concentration at half of the maximum effect (EC(50)), and free drug concentration associated with a 10 ms RT prolongation (EC(10 ms)) were estimated. A linear correlation between EC(10 ms) and HERG IC(50) values was identified. DISCUSSION: The conscious dog with His bundle-pacing detects delayed cardiac repolarization related to I(Kr) inhibition, and detects repolarization change induced by drugs with activity at multiple ion channels. A clinically relevant sensitivity and a linear correlation with in vitro HERG data make the conscious His bundle-paced dog a valuable tool for detecting repolarization effect of new chemical entities.     

Drastic effect of cell density on the cytotoxicity of daunorubicin and cytosine arabinoside

White blood cell count (WBC) is generally accepted as a prognostic risk factor in acute myeloid leukemia (AML) outcome and displays a marked interindividual variation. The dose regimen currently used ignores the size of the tumor burden and the standardization of the dose is generally based on body surface area. In this study we have investigated the effect of cell density on the cytotoxic activity of daunorubicin (DNR) and cytosine arabinoside (AraC) towards HL60 cells and leukemic cells isolated from patients with AML. We demonstrate that drug cytotoxicity decreased with cell density and that apoptosis induction by DNR in isolated leukemic cells was greatly reduced at higher cell density. A marked reduction of the uptake of DNR and AraC in HL60 parental and mitoxantrone resistant cells was observed with increasing cell density. Such a drug depleting effect by cells at high density has been previously described for vincristine, doxorubicin and paclitaxel. By extrapolating the in vitro results to the in vivo situation, one could hypothesize that a high WBC can lower the plasma concentration through high uptake in the tumor burden, leading to a shortage of drug in leukemic blasts. Patients with high WBC might therefore benefit from a dose increase of DNR and/or AraC.     

PK-PD modelling of the effect of cefaclor on four different bacterial strains

The effect of cefaclor against relevant bacterial strains was studied by employing a combined in vivo pharmacokinetic (PK)-in vitro pharmacodynamic (PD) approach. For this purpose selected isolates of Escherichia coli, Moraxella catarrhalis, Haemophilus influenzae and Streptococcus pneumoniae were exposed in vitro to the interstitial cefaclor profile obtained in vivo in the interstitial space fluid of human tissue after administration of commonly used doses of cefaclor and the change in the number of colony forming units per millilitre (CFU/ml) versus time was monitored. Fitting of the data using a modified E(max)-model resulted in a set of mean pharmacodynamic parameters (k0, k(max), EC50) for each bacterial strain. The parameters derived from these experiments were used in a computer-simulation of the antibacterial effects for different dosing regimens and formulations of cefaclor, notably an immediate (IR) and a modified (MR) release formulation. Dosage regimens were compared using the ratio between the number of bacteria remaining after 24 h of a given treatment (N24h). The results indicate that the number of bacteria of all investigated strains killed per day is equivalent when the same daily dose is administered twice a day with the MR dosage form than when given three times a day with the IR dosage form, in spite of the fact that the MR dosage form has approximately 20% lower bioavailability. Best results were obtained with the three-times a day regimen of the MR formulation. In conclusion, the present in vivo-PK/in vitro-PD simulations of the antimicrobial effects of cefaclor indicate that a twice-daily treatment with a MR formulation may offer a convenient and safe alternative to the conventional tid treatment.     

Theoretical pharmacokinetic and pharmacodynamic simulations of drug delivery mediated by blood--brain barrier transporters

Pharmacokinetic/pharmacodynamic simulations were performed to assess the feasibility of central nervous system (CNS) drug delivery via endogenous transporters resident at the blood-brain barrier (BBB). Pharmacokinetic models were derived for intravenous bolus dosing of a hypothetical drug in the absence or presence of an endogenous, competing transport inhibitor. These models were linked to CNS pharmacodynamic models where the effect sites were either cell surface receptors or intracellularly localized enzymes. The response of the dependent parameter, the duration of effect (t(dur)), was examined in relationship to changes in the independent parameters, i.e. dose, elimination rate constant (k(e1)), BBB transport parameters (K(m1) and V(max1)) and EC(50) (effective concentration that elicits a 50% response). As expected, t(dur) increased with (a) increases in drug doses, (b) decreases in k(e1) or (c) decreases in EC(50), irrespective of the effect site. Surprisingly, endogenous transport inhibition produced decreases in drug terminal half-life and corresponding decreases in t(dur). Interestingly, t(dur) was independent of assigned transporter K(m) and V(max) when the dose/EC(50) ratio (dose/EC(50)) was >1 (irrespective of endogenous transport inhibition), but highly dependent on K(m1) and V(max1) when dose/EC(50) was (a) <1 (no endogenous transport inhibition) or (b) equal to 1 (with endogenous transport inhibition). Oral input of the endogenous transport inhibitor produced a decrease in t(dur) when the dose/EC(50) range was 0.1-10. These simulations highlight that (a) systemic pharmacokinetic and BBB transport parameters influence t(dur), (b) drug terminal half-life is inversely related to circulating levels of endogenous inhibitors, and (c) oral ingestion of endogenous transport inhibitor(s) reduces t(dur). Overall, these simulations provide insight for the feasibility of rational CNS drug design/delivery via endogenous transporters.     

Imatinib activity in vitro in tumor cells from patients with chronic myeloid leukemia in chronic phase and blast crisis

The aims of this study were to evaluate the feasibility of using the non-clonogenic fluorometric microculture cytotoxicity assay in drug sensitivity testing of tumor cells from patients with chronic myeloid leukemia. In nine samples (six chronic phase, three blast crisis), the drug sensitivities in tumor cells from blood versus from bone marrow and fresh tumor cells versus cryopreserved were compared. In 26 samples obtained in chronic phase (pretreatment), in six samples from patients in blast crisis and in the K 562 cell line, the activity of imatinib alone and in combination with cytarabine, vincristine, daunorubicin, interferon, arsenic trioxide and homoharringtonine was evaluated. All chronic myeloid leukemia chronic phase samples were sensitive to imatinib, with a mean IC50 at 10.3 mumol/l. The chronic myeloid leukemia samples from blast crisis (n=6) were significantly more sensitive to imatinib than the samples from chronic phase (n=26) (P<0.05), with an IC50 mean at 0.4 mumol/l. In blast crisis samples, significant positive interaction effects were observed between imatinib and all other tested drugs except for interferon. In chronic phase samples, interferon, daunorubicin and arsenic trioxide were the drugs with the highest frequency of positive interactions with imatinib (P<0.05). We conclude that the fluorometric microculture cytotoxicity assay may be a useful method for drug sensitivity testing in chronic myeloid leukemia patient samples from both chronic phase and blast crisis, and that testing primary tumor cells may have advantages over cell line studies. Imatinib shows a higher in vitro activity and more positive drug interactions in cells from blast crisis than chronic phase chronic myeloid leukemia patients. Combinations between imatinib and interferon, daunorubicin and arsenic trioxide may be interesting for future clinical trials in patients with chronic myeloid leukemia chronic phase.     

Pharmacokinetic and pharmacodynamic modelling of arterial haemodynamic effects of terazosin in healthy volunteers

OBJECTIVE: This study aimed to investigate, in healthy volunteers, the relationship between the plasma concentrations of the alpha(1)-adrenoceptor antagonist terazosin and its effects on arterial blood pressure after a single oral administration of terazosin 2 mg. M ethods: Twenty-four healthy volunteers participated in this study. Pharmacokinetic and pharmacodynamic modeling were performed subject by subject. First, plasma concentrations were fitted according to a one-compartment model with first-order absorption and monoexponential elimination. Then the maximum drug-induced decrease (E(max)) effect compartment-model was developed to describe the pharmacodynamic relationships between systolic and diastolic blood pressure and plasma concentrations using the pharmacokinetic parameters that were previously estimated. RESULTS: For systolic blood pressure, E(max) was 29.9 +/- 10.6 mmHg. The corresponding value for decrease in diastolic blood pressure was 39.7 +/- 8.6 mmHg. The effects of terazosin on systolic and diastolic blood pressure could be quantified by an inhibitory E(max) effect compartment model. The obtained first-order rate constant values (0.40 +/- 0.006 h(-)(1) for systolic blood pressure and 0.47 +/- 0.012 h(-)(1) for diastolic blood pressure) were consistent with the rapid development of pharmacological effect. EC(50) (concentration of terazosin that induces an effect at 50% of E(max) values) values were similar for systolic (29.9 +/- 4.3 microg/L) and diastolic (28.7 +/- 4.0 microg/L) blood pressure. A decrease in diastolic blood pressure was the most sensitive response after oral administration of a single dose of terazosin. CONCLUSION: The direct haemodynamic effects of terazosin can be characterized by an E(max) effect compartment model.     

Ocular pharmacokinetics of a novel tetrahydroquinoline analog in rabbit: compartmental analysis and PK-PD evaluation

The elimination kinetics of the pharmacologically active compound 1-ethyl-6-fluoro-1,2,3,4-tetrahydroquinoline (MC4) were characterized along with pharmacodynamic (PD) measurements. Four compartmental models based on ocular anatomy, physiology, and possible absorption and disposition pathways were proposed to model the pharmacokinetic (PK) data in WinNonlin and the best model was chosen based on statistical and goodness-of-fit criteria. A three-compartment physiologic-based PK model with a bidirectional transfer between cornea and aqueous humor and a unidirectional transfer between aqueous humor and iris-ciliary body best described the data. The ocular PD parameters, maximum effect attributed to drug (E(max)) and drug concentration which produces 50% of maximum effect (EC(50)), were estimated with change in intraocular pressure (ΔIOP) as the effect (PD response) in the effect compartment model (PK-PD link model) using aqueous humor concentration-time and ΔIOP-time profiles. The link model better described the effect compartment concentrations than a simple E(max) model that used iris-ciliary body concentration-time data, indicating that there is an apparent temporal displacement between aqueous humor concentration (plasma/central compartment equivalent) and pharmacological effect. A physiologically plausible value of 0.0159 min(-1) was obtained for the drug elimination rate constant (k(eo)) from the effect site to account for equilibration time in the biophase. Hysteresis was observed for the iris-ciliary body, aqueous humor drug concentrations, and effect data, further confirming the utility of the link model to describe the PD of MC4.     

Evaluation of in vivo bioequivalence methodology for topical clobetasol 17-propionate based on pharmacodynamic modeling using Chinese skin

The United States Food and Drug Administration recommends pilot dose duration-response and pivotal bioequivalence studies to be conducted using reflectance colorimetry for assessment of the in vivo bioequivalence of topical dermatologic corticosteroids. The major objectives of the present studies were to examine the applicability of the standardized pharmacodynamic modeling-based methodology to super-potent clobetasol 17-propionate (CP) in the Chinese population and to evaluate the bioequivalence of two generic ointments and four generic creams containing 0.05% (w/w) CP with respect to Dermovate formulations using such methodology. In the pilot dose duration-response study, although the E(max) model (where E(max) is the maximum fitted value of AUEC, which is the area under the baseline-corrected, untreated control-site-corrected a* scale data from 0 to 24 h after drug removal) did not provide acceptable model fits, E(max) parameter estimates of -38.97 +/- 3.62 and -41.89 +/- 11.28 a*-scale. h, and ED(50) (dose duration required to achieve 50% of the fitted E(max) value) estimates of 0.40 +/- 0.37 and 0.42 +/- 0.16 h were obtained for Dermovate ointment and cream, respectively, by population analyses. The estimates for the two formulations were not statistically different, so in vivo bioequivalence studies were conducted at an ED(50)dose duration of approximately 0.5 h for both Dermovate formulations. The results demonstrated that one generic ointment was bioequivalent to Dermovate, whereas the other was not. None of the generic creams were shown to be bioequivalent to Dermovate cream. The in vivo bioequivalence data from the vasoconstriction assay were linearly correlated with stratum corneum uptake of the drug at the same dose duration until the maximal vasoconstriction response was achieved. The studies illustrated the applicability of the standardized pharmacodynamic modeling-based methodology in detecting the product differences between a variety of generic 0.05% CP formulations and reference Dermovate formulations in Chinese skin.     

Pharmacokinetics and pharmacodynamics of KR-66223, a novel DPP-4 inhibitor

KR-66223 is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor that is under development for the treatment of type 2 diabetes. We studied the pharmacokinetic and pharmacodynamic characteristics of KR-66223 in rats, monkeys, and dogs to predict PK/PD profiles in humans. KR-66223 exhibited a moderate volume of distribution (0.3-1.8 L/kg), moderate systemic clearance (1-1.76 L/h/kg), long half-life (>3 h), and low oral bioavailability (below 2.5% in all tested species). The EC(50)s for DPP-4 inhibition as calculated by the E(max) model was below 4.25 ng/mL across all species, confirming KR-66223 as a potent DPP-4 inhibitor. In vitro plasma protein binding suggested that it was available (69-89%), correlating with its volume of distribution in animals. Using allometric scaling and the E(max) model, human systemic clearance, volume of the central compartment, volume of the peripheral compartment, and EC?? for DPP-4 inhibition were predicted to be 0.31 L/h/kg, 0.1 L/kg, 2.4 L/kg, and 3 ng/mL, respectively. These results can serve as a valuable foundation for future clinical trials.     

Pharmacokinetic-pharmacodynamic modeling of drug-induced effect on the QT interval in conscious telemetered dogs

INTRODUCTION: To assure drug safety, the investigation of the relationship between plasma concentration and drug-induced prolongation of the QT interval of the ECG is a challenge in drug discovery. For this purpose, dofetilide was utilized to demonstrate the benefits of characterizing the complete time course of concentrations and effect in conscious beagle dogs in the assessment of drug safety. METHOD: On two separate occasions, four male and two female beagle dogs were given vehicle or the test substance, dofetilide (0.25 mumol/kg), over a 3-h intravenous infusion. Cardiovascular parameters, including QT intervals, were recorded for 24-h using radiotelemetry. The QT interval was corrected individually for heart rate, vehicle treatment, and serial correlation (QT(c)). Exposure (plasma concentration) to dofetilide was measured and described by a two-compartment model. The individual concentration-time course of dofetilide was linked to the QT(c) interval via an effect compartment and a pharmacodynamic E(max) model, to account for the observed hysteresis. RESULTS: Dofetilide induced a concentration-dependent increase in the QT(c) interval, with an EC(50) of 9 nM (3-30 nM, 95% C.I.) and an E(max) of 59+/-9 ms. A hysteresis loop was observed by plotting plasma concentrations vs. QT interval in time order, indicating a delay in onset of effect. It was found to have an equilibrium half-life of 11+/-8 min. Based on the parameters potency and E(max), a representation was made of the drug-induced changes to the QT interval. DISCUSSION: An effect compartment model was found to accurately mimic the QT interval prolongation following administration of the test substance, dofetilide. The assessment of the individual concentration-effect relationship and confounding factors such as hysteresis might provide a better prediction of the safety profiles of new drug candidates.     

Anatomical differences in uterine sensitivity to prostaglandin F(2alpha) and serotonin in non-pregnant rats

The ovarian steroids regulate the sensitivity of a population of uterine receptors to prostaglandin F(2alpha), serotonin and oxytocin. However, the uterine sensitivity to prostaglandin F(2alpha) and oxytocin does not coincide with the estrogen-induced increase in the number of receptors. Anatomical differences affect the uterine sensitivity to agonists. We investigated whether anatomical differences between ovarian and cervical uterine regions modulate the hormone-regulated sensitivity to prostaglandin F(2alpha), serotonin and oxytocin. Non-cumulative concentration-response curves for these agonists were recorded for ovarian and cervical uterine segments from adult ovariectomized rats treated with 17beta-estradiol, 17beta-estradiol+progesterone, or vehicle. The ovarian segments displayed a higher maximal response (E(max)) to prostaglandin F(2alpha) and a lower E(max) to serotonin than the cervical segments. Both uterine segments displayed a similar sensitivity to oxytocin. The ovariectomized controls displayed the highest E(max) and the lowest effective concentration 50 (EC(50)) for oxytocin and prostaglandin F(2alpha). Anatomical differences between ovarian and cervical uterine regions modulate the hormonal regulation of uterine sensitivity to serotonin and prostaglandin F(2alpha) in the non-pregnant rat uterus.     

Allosteric modulation of semicarbazide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands

1. Evidence indicates that imidazoline I(2) binding sites (I(2)BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. 2. We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. 3. While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and beta-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. 4. With benzylamine as substrate, clonidine decreased K(M) (EC(50) 8.82 microm, E(max) 75.1% of control) and increased V(max) (EC(50) 164.6 microm, E(max) 154.1% of control). Cirazoline decreased V(max) (EC(50) 2.15 microm, E(max) 91.4% of control), then decreased K(M) (EC(50) 5.63 microm, E(max) 42.6% of control) and increased V(max) (EC(50) 49.0 microm, E(max) 114.4% of decreased V(max) value). 5. Data for clonidine fitted a mathematical model for two-site nonessential activation plus linear intersecting noncompetitive inhibition. Data for cirazoline were consistent with involvement of a fourth site. 6. These results reveal an ability of imidazoline ligands to modulate BPAO kinetics allosterically. The derived mechanism may have functional significance with respect to modulation of MAO by I(2)BS ligands.     

Modulation of the negative inotropic effect of haloperidol by drugs with positive inotropic effects in isolated rabbit heart

Haloperidol is a typical antipsychotic drug with inhibitory effects on dopamine and calcium homeostasis. In this study, the effect of haloperidol on the inotropism of rabbits' isolated heart was investigated by measuring the isovolumetric left ventricular pressure using a balloon in a modified Langendorff perfusion apparatus. Haloperidol at 0.01-0.3 micromol/l induced a negative inotropic effect (E(max) = 77.95 +/- 0.19; EC(50) = 0.043 +/- 0.002 micromol/l). The effect of haloperidol was decreased by Ca(2+) (E(max) = 42.93 +/- 3.22; EC(50) = 0.37 +/- 0.07 micromol/l; pD'(2) = 7.01 +/- 0.16), Bay K 8644 (E(max) = 30.75 +/- 1.33; EC(50) = 10.43 +/- 1.5 micromol/l, pD'(2) = 7.13 +/- 0.12), and digoxin (E(max ) = 42.03 +/- 3.72, EC(50) = 0.32 +/- 0.05 micromol/l, pD'(2) = 6.81 +/- 0.14). The effect of haloperidol was also reduced by norepinephrine (E(max) = 37.16 +/- 1.84; EC(50) = 1.73 +/- 0.24 micromol/l, pD'(2) = 6.97 +/- 0.08) and dopamine (E(max) = 35.68 +/- 2.78; EC(50) = 0.69 +/- 0.01 micromol/l, pD'(2 )7.48 +/- 0.15). However, the effect of haloperidol was nonsignificantly reduced by dobutamine (E(max) = 58.89 +/- 5.18; EC(50) = 0.15 +/- 0.06 micromol/l, pD'(2) = 5.88 +/- 0.47). These results show that the drugs that increase the influx of Ca(2+) into the cardiomyocyte decrease the negative inotropic effect of haloperidol, suggesting that the effect of haloperidol could be mediated via mechanisms involving actions on Ca(2+) entry into the cardiomyocyte. Haloperidol should be used carefully when prescribed for patients with cardiovascular disorders.     

Prognostic factors in adult patients with acute leukemia

An analysis of prognostic factors was performed on a series of 50 adult patients with acute leukemia, treated in our department in the Tokai university Hospital between July, 1975 and June, 1980. The diagnosis was made in all cases on the basis of May-Grünwald-Giemsa-stained smears of peripheral blood and bone marrow. The patients were treated with two successive protocols. One course of induction chemotherapy consisted of 40 units/kg/day of neocarzinostatin, 1.2-1.6 mg/kg/day of cytosine arabinoside, 0.6-0.8 mg/kg/day of daunorubicin and 0.8-1.6 mg/kg/day of prednisolone on days 1-4 for acute nonlymphocytic leukemia, and the other consisted of 0.04 mg/kg/day of vincristine on day 1, 0.6-0.8 mg/kg/day of daunorubicin on days 1-4, 0.8-1.6 mg/kg/day of prednisolone on days 1-4 for acute lymphocytic leukemia. Consolidation and intensification therapies were given every 1-2 months after complete remission, and the protocols were basically the same as those of the induction therapy. Maintenance therapy consisted of 1.2-1.6 mg/kg/day of cytosine arabinoside, twice a week or 2 mg/kg/day of 6 MP orally. Risk factor leading to poor prognosis of acute leukemia were considered to be (1) advanced age (older than 50 years, P less than 0.05), (2) M5 (monocytic leukemia), (3) thrombocytopenia (less than 50,000 cmm-1), and (4) chromosome abnormalities of blast cells.     

兔血清锌浓度及其降低血压作用的药动学－药效学分析

采用药动学－药效学结合模型，分析了锌在兔体内的处置和降低动脉血压的作用。ｉｖＺｎＳＯ１（Ｚｎ２＋，１ｍｇ·ｋｇ－１）后的血清Ｚｎ２＋浓度与时间曲线符合开放型二房室模型。药效－时间曲线符合Ｅｍａｘ模型。降低平均动脉压的作用滞后于血Ｚｎ２＋浓度的变化。其药效学参数为Ｋｅｏ＝０．１６１ｈ－１，Ｔ（ｋｅｏ）＝４．３０１　ｈ，Ｅｍａｘ＝１．６３７ｋＰａ，Ｃｅ（５０）＝９７．４０７μｍｏｌ·Ｌ－１。结果表明Ｚｎ２＋的作用部位可能在效应室。     

Comparison of alamar blue and MTT assays for high through-put screening.

The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 microM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC50 values. Except for daunorubicin, the EC50 values were comparable in both assays. Based on these results and the Z'-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol.     

Sequential topoisomerase targeting and analysis of mechanisms of resistance to topotecan in patients with acute myelogenous leukemia

Resistance to topoisomerase I (TOP1)-targeting drugs such as topotecan often involves upregulation of topoisomerase II (TOP2), with accompanying increased sensitivity to TOP2-targeting drugs such as etoposide. This trial was designed to investigate sequential topoisomerase targeting in the treatment of patients with high-risk acute myelogenous leukemia. An initial cohort of patients received topotecan and cytosine arabinoside daily for 5 days. Serial samples of circulating mononuclear cells were examined to evaluate peak elevations of TOP2-alpha protein expression. In subsequent cohorts, etoposide was administered daily for 3 days, beginning 6 h after initiation of the topotecan infusion. The etoposide dose was escalated to determine a maximum-tolerated dose. Circulating mononuclear cells were analyzed for TOP1 mutations and ABCG2 protein expression. In addition, systemic and intracellular topotecan concentrations were measured. Thirty-one patients were enrolled. On the basis of TOP1-alpha protein levels in three patients with peripheral blast counts greater than 50%, etoposide administration began 6 h after initiation of the topotecan/cytosine arabinoside infusion. Using this schedule of administration, the maximum-tolerated dose of etoposide was 90 mg/m. No TOP1 mutations were identified, but increases in ABCG2 expression during the infusion were observed in mononuclear cells from two of four evaluable patients. Administration of etoposide 6 h after initiation of a topotecan/cytosine arabinoside infusion is feasible and is associated with clinical activity. Analysis of TOP2-alpha protein levels in this small number of patients indicated that peak increases occurred earlier than expected based on earlier publications. Upregulation of ABCG2 was detected in circulating cells and may represent an inducible form of drug resistance that should be investigated further.