Dose-dependent effect and pharmacokinetics of fexinidazole and its metabolites in a mouse model of human African trypanosomiasis.

Human African trypanosomiasis (HAT) is a neglected tropical disease, with a population of 70 million at risk. Current treatment options are limited. In the search for new therapeutics, the repurposing of the broad-spectrum antiprotozoal drug fexinidazole has completed Phase III trials with the anticipation that it will be the first oral treatment for HAT. This study used the recently validated bioluminescence imaging model to assess the dose and rate of kill effect of fexinidazole in infected mice, and the dose-dependent effect of fexinidazole on trypanosome infection. Pharmacokinetics of fexinidazole in plasma and central nervous system (CNS) compartments were similar in both infected and uninfected mice. Drug distribution within the CNS was further examined by microdialysis, showing similar levels in the cortex and hippocampus. However, high variability in drug distribution and exposure was found between mice.

Multiple visceral venous thromboses associated with oral contraceptive use.

Peripheral venous thromboembolism (VTE) is a known complication of oral contraceptive drugs (OCs), yet its association with visceral VTE is rarely reported. We describe a 21-year-old female patient who presented with sudden left loin pain. Plain computed tomography (CT) urography did not show kidney lesion but was suspicious of left renal vein thrombosis. Contrast study confirmed the diagnosis and also disclosed thrombosis of the splenic and left ovarian veins. The patient did not have a family history or laboratory evidence of hypercoagulable disorder. An OC was the only medication she had received in the previous three months. The OC was discontinued, and the patient was anticoagulated with heparin and discharged home on warfarin for a total period of six months. Subsequent CT study with contrast, one month later, showed complete resolution of the thrombosis without any visceral abnormality.

The effect of circadian rhythm on pharmacokinetics and metabolism of the Cdk inhibitor, roscovitine, in tumor mice model.

Roscovitine is a selective Cdk-inhibitor that is under investigation in phase II clinical trials under several conditions, including chemotherapy. Tumor growth inhibition has been previously shown to be affected by the dosing time of roscovitine in a Glasgow osteosarcoma xenograft mouse model. In the current study, we examined the effect of dose timing on the pharmacokinetics, biodistribution and metabolism of this drug in different organs in B6D2F1 mice. The drug was orally administered at resting (ZT3) or activity time of the mice (ZT19) at a dose of 300 mg/kg. Plasma and organs were removed at serial time points (10, 20 and 30 min; 1, 2, 4, 6, 8, 12 and 24 h) after the administration. Roscovitine and its carboxylic metabolite concentrations were analyzed using HPLC-UV, and pharmacokinetic parameters were calculated in different organs. We found that systemic exposure to roscovitine was 38% higher when dosing at ZT3, and elimination half-life was double compared to when dosing at ZT19. Higher organ concentrations expressed as (organ/plasma) ratio were observed when dosing at ZT3 in the kidney (180%), adipose tissue (188%), testis (132%) and lungs (112%), while the liver exposure to roscovitine was 120% higher after dosing at ZT19. The metabolic ratio was approximately 23% higher at ZT19, while the intrinsic clearance (CLint) was approximately 67% higher at ZT19, indicating faster and more efficient metabolism. These differences may be caused by circadian differences in the absorption, distribution, metabolism and excretion processes governing roscovitine disposition in the mice. In this article, we describe for the first time the chronobiodistribution of roscovitine in the mouse and the contribution of the dosing time to the variability of its metabolism. Our results may help in designing better dosing schedules of roscovitine in clinical trials.

Pharmacokinetics and biodistribution of the cyclin-dependent kinase inhibitor -CR8- in mice.

BACKGROUND: CR8 is a second generation inhibitor of cyclin-dependent kinases derived from roscovitine. CR8 was shown to be 50-100 fold more potent than roscovitine in inducing apoptosis in different tumor cell lines. In the present investigation, we have established an analytical method for the quantification of CR8 in biological samples and evaluated its bioavailability, biodistribution and pharmacokinetics in mice. METHODS: A liquid chromatography method utilizing UV-detection was used for the determination of CR8. CR8 was administered either orally (100 mg/kg) or i.v. (50 mg/kg) and the animals were sacrificed at different time points. Blood samples and organs were collected, after which the pharmacokinetic parameters were calculated for plasma and organs. RESULTS: CR8 was eluted at 5 minutes in the high performance liquid chromatography system used. The LLOQ detection was 0.10 µg/ml and linearity was observed within the 0.10-10 µg/ml range (r² > 0.998). The accuracy and precision were >86%, while the recovery from plasma was >95%. CR8 was stable for 2 months at room temperature in both solution and plasma. CR8 pharmacokinetics was fitted to a two-compartment open model after oral administration and to a one compartment model after i.v. injection. The elimination half-life was about 3 hours. Organ exposure to CR8 (expressed as % AUC organ vs. AUC plasma) was highest in liver (205%), adipose tissue (188%) and kidney (150%) and low in bone marrow (30%) and brain (15%) as compared to plasma. The oral bioavailability of CR8 was found to be essentially 100%. CONCLUSIONS: We have developed a rapid and simple method for the analysis of CR8. CR8 pharmacokinetics pattern showed 100% bioavailability, long half-life and limited distribution to brain and bone marrow, which may allow systemic exposure higher than the IC50 reported for cell death in tumor cell lines. CR8 displays favorable pharmacological properties and is therefore a good candidate for future clinical studies.

The Role of Pharmacokinetics and Pharmacodynamics in Early Drug Development with reference to the Cyclin-dependent Kinase (Cdk) Inhibitor - Roscovitine.

Pharmacokinetics, pharmacodynamics and pharmacogenetics play an important role in drug discovery and contribute to treatment success. This is an essential issue in cancer treatment due to its high toxicity. During the last decade, cyclin-dependent kinase inhibitors were recognised as a new class of compounds that was introduced for the treatment of several diseases including cancer. Cyclin-dependent kinases (Cdks) play a key role in the regulation of cell cycle progression and ribonucleic acid transcription. Deregulation of Cdks has been associated with several malignancies, neurodegenerative disorders, viral and protozoa infections, glomerulonephritis and inflammatory diseases. (R)-roscovitine is a synthetic tri-substituted purine that inhibits selectively Cdk1, 2, 5, 7 and 9. Roscovitine has shown promising cytotoxicity in cell lines and tumor xenografts. In this paper, we present several aspects of pharmacokinetics (PK) and pharmacodynamics (PD) of roscovitine. We present also some of our investigations including bioanalysis, haematotoxicity, age dependent kinetics, PK and effects on Cdks in the brain. Unfavourable kinetic parameters in combination with poor distribution to the bone marrow compartment could explain the absence of myelosuppression in vivo despite the efficacy in vitro. Higher plasma and brain exposure and longer elimination half-life found in rat pups compared to adult rats may indicate that roscovitine can be a potential candidate for the treatment of brain tumours in children. Cdk5 inhibition and Erk1/2 activation that was detected in brain of rat pups may suggest the use of roscovitine in neurodegenerative diseases. Early pharmacokinetic/pharmacodynamic studies are important issues in drug discovery and may affect further development of promising drug candidates.

Liver circadian clock, a pharmacologic target of cyclin-dependent kinase inhibitor seliciclib.

Circadian disruption accelerates malignant growth and shortens survival, both in experimental tumor models and cancer patients. In previous experiments, tumor circadian disruption was rescued with seliciclib, an inhibitor of cyclin-dependent kinases (CDKs). This effect occurred at a selective dosing time and was associated with improved antitumor activity. In the current study, seliciclib altered robust circadian mRNA expression of the clock genes Rev-erb alpha, Per2, and Bmal1 in mouse liver following dosing at zeitgeber time (ZT) 3 (i.e., 3 h after the onset of the 12 h light span), when mice start to rest, but not at ZT19, near the middle of the 12 h dark span, when mice are most active. However, liver exposure to seliciclib, as estimated by the liver area under the concentration x time curve (AUC), was approximately 80% higher at ZT19 than at ZT3 (p = 0.049). Circadian clock disruption was associated with increased serum liver enzymes and modified glycogen distribution in hepatocytes, as revealed by biochemical determinations and optic and electronic microscopy. The extent of increase in liver enzymes was most pronounced following dosing at ZT3, as compared to ZT19 (p < 0.04). Seliciclib further up-regulated the transcriptional activity of c-Myc, a cell cycle gene that promotes cell cycle entry and G1-S transition (p < 0.001), and down-regulated that of Wee1, which gates cell cycle transition from G2 to M (p < 0.001). These effects did not depend upon drug dosing time. Overall, the results suggest the circadian time of seliciclib delivery is more critical than the amount of drug exposure in determining its effects on the circadian clock. Seliciclib-induced disruption of the liver molecular clock could account for liver toxicity through the resulting disruption of clock-controlled detoxification pathways. Modifications of cell cycle gene expression in the liver likely involve other mechanisms. Circadian clocks represent relevant targets to consider for optimization of therapeutic schedules of CDK inhibitors.

N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine.

Cyclin-dependent kinases (CDKs) and their regulators show frequent abnormalities in tumors. Ten low molecular weight pharmacologic inhibitors of CDKs are currently in clinical trials against various cancers, including the 2,6,9-trisubstituted purine (R)-roscovitine (CYC202/Seliciclib). We here report the characterization of N-&-N1, a bioisoster of roscovitine displaying improved antitumoral properties. N-&-N1 shows exquisite selectivity for CDKs, with 2- to 3-fold enhanced potency compared with (R)-roscovitine. Inhibition of retinoblastoma protein phosphorylation and RNA polymerase II Ser2 phosphorylation in neuroblastoma SH-SY5Y cells exposed to N-&-N1 indicates that N-&-N1 is able to inhibit CDKs in a cellular context. N-&-N1 also down-regulates the expression of RNA polymerase. Cocrystal structures of N-&-N1 and (R)-roscovitine in complex with CDK2/cyclin A reveal that both inhibitors adopt similar binding modes. A competitive assay shows that, compared with (R)-roscovitine, N-&-N1 has reduced affinity for Erk2 and pyridoxal kinase. N-&-N1 triggers cell death in a panel of diverse cell lines. Cell death is accompanied by events characteristic of apoptosis: cytochrome c release, activation of effector caspases, and poly(ADP-ribose) polymerase cleavage. Induction of p53 and p21CIP1 and down-regulation of the Mcl-1 antiapoptotic factor were also observed. Studies in mice show that N-&-N1 has pharmacokinetics properties similar to those of (R)-roscovitine. Altogether, these results show that analogues of (R)-roscovitine can be designed with improved antitumor potential.

Age-dependent pharmacokinetics and effect of roscovitine on Cdk5 and Erk1/2 in the rat brain.

Roscovitine is a cyclin-dependent kinase (Cdk) and signal-regulated kinase (Erk1/2) inhibitor that has been shown to be effective against several cancer types including brain tumors. We have shown previously that roscovitine crosses the blood brain barrier (BBB) and is rapidly eliminated from both plasma and brain in adult rats. However, age-dependent kinetics and its effects on the brain have not been reported. In the present study, we investigated the pharmacokinetics of roscovitine in adult and in 14 days old rats after the administration of a single dose of 25 mg/kg. Moreover, we studied the effect of the drug on Cdk5 and Erk1/2 activities in three brain regions, hippocampus, frontal cortex and cerebellum. The pharmacokinetics of roscovitine followed a two-compartment model in both plasma and brain in both adult and young rats. The terminal elimination half-life was 7 h in brain as well as in plasma in rat pups compared to < 0.5 h observed in adult rats. Brain exposure expressed as AUC brain/AUC plasma was 100% in rat pups compared to 20% found in adult rats. Roscovitine induced a significant Cdk5 inhibition and significant Erk1/2 activation in all studied pups brain regions at 2 h. This is the first study describing age-dependent pharmacokinetics of roscovitine and showing the high brain exposure of infant rats to the drug. Thus, roscovitine may be a promising candidate for the treatment of brain tumors in children.

Effect of the Cdk-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro.

Myelosuppression is one the most frequent side effects of chemotherapy. New agents that more selectively target cancer cells have been developed in attempt to improve the effects and to decrease the side effects of cancer treatment. Roscovitine is a purine analogue and cyclin-dependent kinase inhibitor. Several studies have shown its cytotoxic effect in cancer cell lines in vitro and in xenograft models in vivo. In this study, we investigated the effect of roscovitine on hematopoietic progenitors in vitro and in vivo in mice. The clonogenic capacity of hematopoietic progenitors was studied using burst-forming unit-erythroid (BFU-E), colony-forming unit granulocyte, macrophage (CFU-GM) and colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). In vitro, bone marrow cells were exposed to roscovitine (25-250 microM) in Iscove's modified Dulbecco's media for 4 h or to roscovitine (1-100 microM) in MethoCult media for 12 days. No effect on colony formation was observed after exposure to roscovitine for 4 h; however, concentration- and cell type-dependent effects were observed after 12 days. Roscovitine in concentration of 100 microM inhibited the growth of all types of colonies, while lower concentrations have shown differential effect on hematopoietic progenitors. The most sensitive were CFU-GEMM, followed by BFU-E and then CFU-GM. In vivo, mice were treated with single dose of roscovitine (50, 100 or 250 mg/kg) and the effect on bone marrow was studied on day 1, 3, 6, 9 or 12 after the treatment. In the second part of experiment, the mice were treated with roscovitine 350 mg/kg/day divided into two daily doses for 4 days. The bone marrow was examined on day 1 and 5 after the last dose of roscovitine. On day 1, BFU-E decreased to less than 50% of the controls (P = 0.019). No decrease in BFU-E formation was observed on day 5. No significant effect was observed on CFU-GM and CFU-GEMM growth after the treatment with multiple doses of roscovitine. Single doses of roscovitine or dimethylsulfoxide did not affect the colony formation. We also studied the distribution of roscovitine to the bone marrow after a dose of 50 mg/kg was administered intraperitoneally. Only 1.5% of the drug was detected in the bone marrow. Thus, the roscovitine effect on hematopoietic progenitors in bone marrow in vivo is only transient. One reason may be that only a small fraction of roscovitine reaches the bone marrow. Another explanation may be the short half-life observed for roscovitine that might not allow enough cell exposure to the drug. However, the toxicity of roscovitine to hematopoietic progenitors in vitro is within the same exposure range as cytotoxicity to cancer cells. Thus, precaution should be taken in clinical trials, especially when combinations with myelosuppressive cytostatics are used.