Screening of NOS activity and selectivity of newly synthesized acetamidines using RP-HPLC.

Nitric Oxide Synthase (NOS) inhibitors could play a powerful role in inflammatory and neurodegenerative diseases. In this work, novel acetamidine derivatives of NOS were synthesized and the inhibitor activity was evalued. To screen the activity and selectivity, the l-citrulline residue, after the enzymatic NOS assay, was derivatized with o-phthaldialdehyde/N-acetyl cysteine (OPA/NAC) and then evaluated by RP-HPLC method with fluorescence detection. All compounds did not affect the activity of endothelial and neuronal isoforms, while nine of them possessed a percentage of iNOS activity at 10µM lower than 50%, and were selected for IC50 evaluation. Among them, a compound emerged as a very potent (IC50 of 53nM) and selective iNOS inhibitor.

Sphingosine kinase type 1 inhibition reveals rapid turnover of circulating sphingosine 1-phosphate.

S1P (sphingosine 1-phosphate) is a signalling molecule involved in a host of cellular and physiological functions, most notably cell survival and migration. S1P, which signals via a set of five G-protein-coupled receptors (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases) from Sph (sphingosine). Interfering RNA strategies and SphK1 (sphingosine kinase type 1)-null (Sphk1-/-) mouse studies implicate SphK1 in multiple signalling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of submicromolar amidine-based SphK1 inhibitors and report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In so doing, we demonstrate that our inhibitors effectively lower S1P levels in cell-based assays, but we have been unable to correlate SphK1 inhibition with changes in cell survival. However, SphK1 inhibition did diminish EGF (epidermal growth factor)-driven increases in S1P levels and Akt (also known as protein kinase B)/ERK (extracellular-signal-regulated kinase) phosphorylation. Finally, administration of the SphK1 inhibitor to wild-type, but not Sphk1-/-, mice resulted in a rapid decrease in blood S1P levels indicating that circulating S1P is rapidly turned over.

Anti-resorptive activity and pharmacokinetic study of N(1),N(1)-diisopropyl-N(2)-(diphenylphosphoryl)-2-(4-nitrophenyl)acetamidine.

In vitro anti-resorptive activity, mechanism of action, pharmacokinetic profile and in vivo anti-resorptive activity of N(1),N(1)-diisopropyl-N(2)-(diphenylphosphoryl)-2-(4-nitrophenyl)acetamidine (1) were evaluated.

Reversible elevations of serum creatinine levels but no effect on glomerular filtration during treatment with the direct thrombin inhibitor AZD0837.

PURPOSE: Reversible mean increases in serum creatinine (approx. 10%) have been observed during clinical investigations of the oral direct thrombin inhibitor AZD0837. The aim of this study was to evaluate whether the increase in s-creatinine is due to a decrease in renal glomerular filtration rate (GFR) or an inhibition of the tubular secretion of creatinine. METHODS: Thirty healthy subjects aged 60-71 years were enrolled in an open-label, randomised, placebo-controlled, two-way crossover study (D1250C00033) in which they received AZD0837 450 mg extended-release formulation once daily for 8 days. Cimetidine was co-administered on Days 6-8 during both treatment periods. Blood and urine samples were collected for assessment of s-creatinine, s-cystatin C, endogenous creatinine clearance (CrCl) and urinary markers of renal damage. GFR was measured by the plasma clearance of iohexol. RESULTS: A 6% increase in mean s-creatinine, but no increase in s-cystatin C, was observed during treatment with AZD0837. Co-administration of cimetidine resulted in a 21% increase in s-creatinine. A significant decrease in CrCl was found during AZD0837 treatment compared with placebo [-5.73 ml/min; 95% confidence interval (CI) -11.3 to -0.12]. No significant difference in GFR (-1.6 ml/min/1.73 m(2); 90% CI -3.7 to 0.5) was seen during treatment with AZD0837 versus placebo. No changes in renal damage markers were found during the treatment periods. CONCLUSIONS: An increase in s-creatinine and a decrease in CrCl, but no decrease in GFR, were found during treatment with AZD0837. These findings suggest that inhibition of the renal tubular secretion of creatinine is the likely cause of the observed increase in s-creatinine.

Metabolism and distribution of two highly potent and selective peptidomimetic inhibitors of matriptase.

Matriptase is a serine protease expressed by several types of cancer cells and it participates in tumour growth and progression through the activation of hepatocyte growth factor (HGF) and urokinase-type plasminogen activator (uPA). The metabolism of two potent and selective peptidomimetic inhibitors of matriptase (CJ-1737 and CJ-672) was examined in vitro with enzyme preparations (9000g supernatants, microsomes, and plasma) from dog, pig, rat, and human. It was found that both compounds displayed interesting species-dependent differences. Though CJ-1737 was not metabolized by microsomes, by 9000g supernatants from all species, or by human or rat plasma, canine and porcine plasma enzymes rapidly hydrolysed this compound. In contrast, CJ-672 was metabolized exclusively by enzymes from human liver (microsomes and 9000g supernatants) via a two-step metabolic pathway. Additionally, the distribution of both compounds was investigated in mice. The highest amounts were measured in the kidney and liver, followed by the spleen, lung, and heart. In contrast to CJ-1737, high concentrations of CJ-672 were detected in the colon, indicating an additional biliary excretion. In summary, this work clarifies both the metabolism and distribution of two new matriptase inhibitors and demonstrates important metabolic differences between human enzymes and those from commonly used laboratory animals.

Functional reinnervation of the rat lower urinary tract after cauda equina injury and repair.

Conus medullaris and/or cauda equina forms of spinal cord injury commonly result in a permanent loss of bladder function. Here, we developed a cauda equina injury and repair rodent model to investigate whether surgical implantation of avulsed lumbosacral ventral roots into the spinal cord can promote functional recovery of the lower urinary tract. Adult female rats underwent sham surgery (n = 6), bilateral L5-S2 ventral root avulsion (VRA) injury (n = 5), or bilateral L5-S2 VRA followed by an acute implantation of the avulsed L6 and S1 ventral roots into the conus medullaris (n = 6). At 12 weeks after operation, the avulsed group demonstrated urinary retention, absence of bladder contractions and external urethral sphincter (EUS) electromyographic (EMG) activation during urodynamic recordings, increased bladder size, and retrograde death of autonomic and motoneurons in the spinal cord. In contrast, the implanted group showed reduced urinary retention, return of reflexive bladder voiding contractions coincident with EUS EMG activation, anatomical reinnervation of the EUS demonstrated by retrograde neuronal labeling, normalization of bladder size, and a significant neuroprotection of both autonomic and motoneurons. In addition, a positive correlation between motoneuronal survival and voiding efficiency was observed in the implanted group. Our results show that implantation of avulsed lumbosacral ventral roots into the spinal cord promotes reinnervation of the urinary tract and return of functional micturition reflexes, suggesting that this surgical repair strategy may also be of clinical interest after conus medullaris and cauda equina injuries.

Synchronization of oxytocin neurons in suckled rats: possible role of bilateral innervation of hypothalamic supraoptic nuclei by single medullary neurons.

We have previously shown that oxytocin neurons located in the four hypothalamic magnocellular nuclei display synchronous bursts of action potentials before each milk ejection. The mechanisms involved in such a synchronization have, however, not yet been elucidated. In this study, we test the hypothesis of an extranuclear synchronization arising from a common extrahypothalamic input innervating bilateral magnocellular nuclei. First, two different retrograde tracers were injected into the right and left supraoptic nuclei of rats that were fixed 5-7 days later. Each tracer labelled numerous neurons in various brain regions ipsilateral or contralateral to the injection site, but colocalization of the two tracers within the same cell body could only be detected bilaterally in neurons in the ventromedial regions of the medulla oblongata. The axonal projections of these medullary neurons were then visualized by the unilateral microinjection of an anterograde tracer (BDA) within the ventromedial medulla oblongata. BDA-labelled axons afferent to the hypothalamus were found to branch towards both supraoptic nuclei through medial portions of the optic chiasma. Finally, in anaesthetized lactating rats, surgical lesions were placed medially through the optic chiasma and the electrical activity of oxytocin neurons in bilateral supraoptic nuclei was pair-recorded during suckling. The incidence of synchronous bursts in oxytocin neurons located within bilateral supraoptic nuclei were dramatically altered only when the medial portions of the optic chiasma were totally lesioned. Taken together, these data suggest that medullary neurons afferent to bilateral supraoptic nuclei are involved in the recruitment and synchronization of bursting in oxytocin neurons during suckling.

Phase I and pharmacokinetic study of the polyamine synthesis inhibitor SAM486A in combination with 5-fluorouracil/leucovorin in metastatic colorectal cancer.

PURPOSE: The purpose of our study was to determine the maximum-tolerated dose, dose-limiting toxicity, safety profile, and pharmacokinetics of the polyamine synthesis inhibitor SAM486A given in combination with 5-fluorouracil/leucovorin (5-FU/LV) in cancer patients. EXPERIMENTAL DESIGN: Patients with advanced colorectal cancer were treated with 5-FU [bolus (400 mg/m(2)) followed by a 22-h infusion (600 mg/m(2))] and LV (200 mg/m(2)) and escalating doses of SAM486A, 1-3-h infusion daily for 3 days. Plasma sampling was performed to characterize the pharmacokinetics and pharmacodynamics of the combination RESULTS: Twenty-seven patients with metastatic colorectal cancer and 1 with pseudomyxoma peritonei were treated. Twenty-six patients received SAM486A in the combination at doses ranging from 25 to 150 mg/m(2)/day. Dose-limiting toxicity consisting of fatigue grade 3 was seen at 150 mg/m(2)/day. Other adverse events included neutropenia, hand and foot syndrome, nausea, vomiting, diarrhea, and constipation. Fifteen of 26 patients evaluable for best response according to the Southwest Oncology Group criteria achieved a partial response [8 (30%) of 26] or stable disease [9 (35%) of 26]. SAM486A did not influence the pharmacokinetics of 5-FU, and SAM486A clearance was similar to that when used as a single agent. CONCLUSIONS: The novel molecular agent SAM486A is tolerable and safe in combination with a standard 5-FU regimen in patients with advanced colorectal cancer. The dose of SAM486A recommended for additional studies with this combination is 125 mg/m(2)/day. A disease-directed evaluation of SAM486A using this regimen is warranted.

Effects of roxifiban on platelet aggregation and major receptor expression in patients with coronary artery disease for the Roxifiban Oral Compound Kinetics Evaluation Trial-I (ROCKET-I Platelet Substudy).

BACKGROUND: It has been expected that therapy with oral glycoprotein (GP) IIb/IIIa blockers including roxifiban will reduce mortality and vascular complications in a long-term. However, platelet-related properties of roxifiban in the clinical setting are not well known. We measured platelet characteristics during chronic treatment in patients with coronary artery disease enrolled in the Roxifiban Oral Compound Kinetics Evaluation Trial (ROCKET-I). METHODS: ROCKET-I was designed as a randomized, double blind, multicenter, dose-ranging study of roxifiban, administered either as monotherapy or concomitantly with aspirin, compared with aspirin alone. Thirty-one patients were assigned for 24 weeks of therapy with aspirin (n = 7), roxifiban (n = 9), or roxifiban plus aspirin (n = 15). Platelets were assessed 5 times in each patient at baseline, and at weeks 2, 4, 12, 18, and 24 thereafter with aggregometry and flow cytometry. RESULTS: Baseline platelet characteristics were similar in all 3 groups. There was a consistent significant decrease of adenosine diphosphate- (P =.0001) and collagen-induced (P =.002) platelet aggregation in the patients treated with roxifiban when compared with patients treated with aspirin alone. Flow cytometry revealed paradoxical late activation of GP IIb/IIIa expression (P =.007) when roxifiban was used without aspirin, which was significant compared with the aspirin and aspirin-roxifiban groups. There were no differences among groups in GP Ib expression, although its rise was more profound in the patients treated with roxifiban. There were substantial differences in the P-selectin expression. Although aspirin time dependently decreased the percent of P-selectin positive platelets (P =.02), treatment with roxifiban resulted in the phasic changes with the early inhibition (P =.01) and then 2-fold activation (P =.0001) starting at week 12 of the therapy. There was an early transient activation of platelet endothelial cell adhesion molecule-1 expression (P =.008) at week 2, followed by the later inhibition of this receptor (P =.003) in patients treated with roxifiban. CONCLUSION: Despite achieving sustained inhibition of platelet aggregation, therapy with roxifiban has been associated with over expression or phasic changes of major platelet receptors. These data may explain clinical concerns about the use of oral GP IIb/IIIa inhibitors linking higher mortality rates and incidence of thrombotic episodes with paradoxical switching to alternative passways of platelet activation.

The use of roxifiban (DMP754), a novel oral platelet glycoprotein IIb/IIIa receptor inhibitor, in patients with stable coronary artery disease.

INTRODUCTION: Intravenous platelet glycoprotein (GP) IIb/IIIa receptor inhibitors have a significant beneficial impact on the outcomes of patients undergoing high-risk coronary interventions and in the stabilization of patients with unstable angina pectoris refractory to conventional medical treatment. The role of long-term treatment with oral platelet GP IIb/IIIa receptor inhibitors in patients with coronary artery disease is unproven. This study examined the dose-response effect on inhibition of platelet aggregation by roxifiban (DMP754), a novel oral platelet GP IIb/IIIa receptor inhibitor, and its safety and tolerability in patients with a history of chronic stable angina pectoris. METHODS: Ninety-eight patients were randomized to receive either a placebo or 1 of 8 oral dosages of roxifiban. Twenty-two patients were enrolled in multiple-dose regimens, bringing the total study population to 120. The oral dosages were 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, or 2.5 mg/day for up to 30 days. RESULTS: Pharmacodynamic response of roxifiban was clearly dose-dependent. Platelet aggregation inhibition in response to 10 micromol/L slope adenosine diphosphate was sustained throughout the study period (up to 1 month). No serious adverse events, including significant major bleeding events, were associated with roxifiban treatment. Minor bleeding was reported in 5% of participants in the placebo group (1 of 21 cases) versus 26% in the study group (26 of 99 cases). Incidence of minor bleeding associated with roxifiban 2 and 2.5 mg/day was significantly (p < or = 0.05) greater than that with placebo. Adverse events, including gastrointestinal disorders, platelet and clotting disorders, and urinary tract disorders, were observed in 1 of 21 cases (5%) in the placebo group and in 12 of 99 cases (12%) in the study group. Reversible thrombocytopenia without other complications developed in two patients. CONCLUSIONS: Roxifiban-induced inhibition of platelet aggregation was dose-dependent and sustained throughout the study period: higher drug dosages correlated with higher levels of platelet inhibition and higher incidence of minor bleeding events. No serious adverse events were observed at any dosage. Thus, roxifiban appears to be a potent oral platelet GP IIb/IIIa receptor inhibitor that is clinically well-tolerated and deserves further study as a new treatment strategy in patients with chronic stable angina pectoris.

A phase I and pharmacokinetic study of SAM486A, a novel polyamine biosynthesis inhibitor, administered on a daily-times-five every-three-week schedule in patients with Advanced solid malignancies.

PURPOSE: SAM486A is a novel inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC). This study was performed to characterize the toxicity profile and the pharmacological behavior and to determine the maximum tolerated dose (MTD) of SAM486A administered by a 1-h i.v. infusion daily for 5 days every 3 weeks in patients with advanced cancer. EXPERIMENTAL DESIGN: Twenty-three patients received 46 cycles of SAM486A at dose levels ranging from 3.6 to 202.8 mg/m(2)/day. SAM486A plasma concentrations were measured during the first cycle for pharmacokinetic and pharmacodynamic evaluations. Paired tumor biopsy specimens pre- and posttreatment were obtained in 1 patient to assess the impact of SAM486A on intratumoral enzymes and metabolites involved in the polyamine biosynthetic pathway. RESULTS: The dose-limiting toxicity of SAM486A on this schedule was myelosuppression. Nonhematological toxicities, including nausea, vomiting, anorexia, and fatigue, were mild to moderate in severity. The MTD of SAM486A was 102.4 mg/m(2)/day. Pharmacokinetic analyses demonstrated a rapid initial decrease in plasma drug concentrations at the end of infusion, followed by a long terminal elimination phase with a mean (+/- SD) terminal elimination half-life of 65.4 +/- 55.6 h. Dose and area under the concentration-time curve correlated with the appearance of grade 4 neutropenia with correlation coefficients of 0.70 and 0.69, respectively. Analysis of paired tumor biopsy specimens taken before and after SAM486A treatment in 1 patient with metastatic melanoma revealed decreased SAMDC activity, increased ornithine decarboxylase activity, increased levels of putrescine, and depleted levels of decarboxylated S-adenosylmethionine and spermine, all of which are consistent with the proposed mode of action of SAM486A. CONCLUSIONS: SAM486A was well tolerated on this schedule of administration with the MTD established at 102.4 mg/m(2)/day. Neutropenia was dose-limiting and correlated with dose and area under the concentration-time curve. Pharmacodynamic assessment of tumoral tissues in 1 study patient demonstrated changes in the levels of polyamines and their biosynthetic enzymes consistent with SAMDC inhibition.

Safety, tolerability, pharmacokinetics, and time course of pharmacologic response of the active metabolite of roxifiban, XV459, a glycoprotein IIb/IIIa antagonist, following oral administration in healthy volunteers.

Roxifiban is an esterprodrug that is hydrolyzed, after oral administration, to the active glycoprotein (GP) IIb/IIIa antagonist, XV459. The objectives of the study were to investigate the safety, tolerability, pharmacokinetics, and the time course of the pharmacologic response of XV459 in escalating doses of roxifiban and to assess the effect of age, loading dose of roxifiban, and aspirin pretreatment on XV459 pharmacokinetics, pharmacologic response, and safety profile in a five-part double-blind, placebo-controlled study. Healthy male volunteers (ages 18-46 years) received 7 (0.75-1.5 mg; n = 20) and 10 (0.75-1.0 mg; n = 8) multiple, oral, qd doses of roxifiban or placebo (n = 5). Healthy older male and female volunteers (ages 47-75 years) received roxifiban qd doses (0.5-0.75 mg; n = 8) or placebo (n = 3) for 7 days. Healthy male subjects (ages 18-46 years; n = 16) received a 1.5 or 1.0 mg loading dose either with or without pretreatment of 325 mg aspirin once daily for 3 days followed by single daily doses of 1.0 mg roxifiban for 6 days. Measurable plasma concentrations of XV459 appeared rapidly and were sustained throughout the dosing interval of 24 hours. The pharmacokinetics of XV459 were nonlinear. Systemic exposure of XV459 plateaued at the 1-mg dose level; plasma concentrations approached steady state in 4 to 6 days for doses greater than 1.0 mg. The time course of pharmacologic response as measured by the inhibition of platelet aggregation in response to an ex vivo 10 microM adenosine 5'-diphosphate (ADP) agonist correlated closely to the plasma concentration of XV459. Potent inhibition of ADP-induced platelet aggregation (IPA) persisted over the entire dosing interval. A clear dose response was achieved with roxifiban doses of 0.5 and 1.0 mg. For doses greater than 1.0 mg, a dose-proportional increase in IPA was not observed. Both the pharmacokinetics and pharmacologic response of XV459 exhibited low intraindividual variability (coefficient of variation [CV] < 15%) and higher interindividual variability (CV < 30%). Pretreatment with aspirin and/or a loading dose of 1.5 mg roxifiban had no significant effect on the pharmacokinetics and pharmacologic response of XV459. A dose-related increase in template bleeding time was observed at 1.25- and 1.5-mg doses of roxifiban, as compared to placebo. However, these bleeding time increases in the 1.25- and 1.5-mg dose groups were not significantly different from those at the lower dose groups. Overall, once-daily oral administration of roxifiban was fairly well tolerated and provided sustained systemic drug exposure and pharmacologic response over the entire administration interval.

In vivo visualization of the cochlear nerve and nuclei with fluorescent axonal tracers.

In recent years multichannel neuroprostheses have been developed which directly stimulate the central auditory pathway. Substantially these have been used in cases of total hearing loss caused by neurofibromatosis type 2 where bilateral damage to the auditory nerve prevents more peripheral stimulation. The electrode carrier of the auditory brainstem implant (ABI) is designed to be placed on the cochlear nucleus complex residing at the lateral brainstem surface. Despite altered anatomy due to tumor growth or preceding surgery, correct electrode placement is essential to maximize the variety of pitch percept elicited during electrical stimulation with the ABI without producing side-effects. In order to assist intraoperative identification of the proximal auditory nerve and cochlear nuclei, the non-toxic fluorescent axonal tracers Fast Blue or Fluorogold were injected into the cochlea of rats and Java monkeys. Four to seven days after tracer application, labeling of the eighth cranial nerve, its entrance into the brainstem and the primary radiation of auditory fibers into the cochlear nucleus could be demonstrated as colored fluorescence on the living brain under appropriate ultraviolet illumination. Additional histological processing revealed groups of retrogradely labeled neuronal cell bodies in both species. Our results suggest that this method could also be used in humans in order to aid surgeons with the proper positioning of the electrode array.

Oxidative stress promotes the development of transformation: involvement of a potent mutagenic lipid peroxidation product, acrolein.

The effect of intracellular oxidative stress on the development of cell transformation was studied. Mouse embryo C3H/10T1/2 fibroblasts pre-treated with benzo[a] pyrene, developed transformed foci on exposure to free radical generators, such as 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1). These compounds generate peroxyl radicals and peroxynitrite, respectively. Neither AAPH nor SIN-1 alone induced transformation. The level of intracellular antioxidants, such as alpha-tocopherol and glutathione (GSH), decreased with time of exposure to the free radical generators, whereas the addition of exogenous alpha-tocopherol, GSH and ebselen showed a reduction in the frequency of transformation. An early event during exposure to AAPH and SIN-1 was the generation of acrolein, a highly mutagenic lipid peroxidation product, which was suppressed by the addition of alpha-tocopherol. Furthermore, it was confirmed that acrolein induced the transformation of cells which were pre-treated with benzo[a]pyrene but not of the untreated cells. These results suggest that acrolein may act as an important mediator of cell transformation under oxidative stress.

Effects of pituitary adenylate cyclase activating polypeptide on lumbosacral preganglionic neurons in the neonatal rat spinal cord.

The effects of PACAP-38 on phasic and tonic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats (5--11 days old) were studied using the whole-cell patch clamp technique. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue, 5 microl of 4% solution) injected into the intraperitoneal space 3--7 days prior to the study. Bath application of pituitary adenylate cyclase activating polypeptide (PACAP) (20 nM) increased the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and spontaneous firing in both types of PGN. PACAP markedly increased the number (200--800%) and frequency of action potentials elicited by depolarizing current pulses in phasic PGN, but had a smaller effect on tonic PGN. PACAP decreased the threshold for action potential generation by approximately 25% in both types of neurons (e.g. -34.0+/-1.5 to -38.4+/-1.7 mV from a holding potential of -50 mV in phasic PGN, P<0.005). PACAP did not affect the duration of the action potential. The amplitude of the spike after hyperpolarization was not changed but the duration was significantly reduced by PACAP from 204.4+/-12.2 to 106.2+/-8.1 ms in tonic but not in phasic PGN. PACAP suppressed a transient outward current that was also suppressed by 4-aminopyridine (0.5 mM). These results coupled with the immunohistochemical identification of a dense collection of PACAP fibers in the region of the PGN, raises the possibility that PACAP may function as an excitatory transmitter in lumbosacral parasympathetic reflex pathways in the neonatal rat.

A phase I study of a new polyamine biosynthesis inhibitor, SAM486A, in cancer patients with solid tumours.

Because tumour cell proliferation is highly dependent upon up-regulation of de-novo polyamine synthesis, inhibition of the polyamine synthesis pathway represents a potential target for anticancer therapy. SAM486A (CGP 48664) is a new inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), more potent and specific than the first-generation SAMDC inhibitor methylglyoxal (bis) guanylhydrazone (MGBG). Preclinical testing confirmed promising antiproliferative activity. In this phase I study, SAM486A was given 4-weekly as a 120 h infusion. 39 adult cancer patients were enrolled with advanced/refractory disease not amenable to established treatments, PS

Effects of ZK-807834, a novel inhibitor of factor Xa, on arterial and venous thrombosis in rabbits.

Inhibition of factor Xa (FXa) may interrupt thrombus progression. This study compared the antithrombotic activity of a novel FXa inhibitor, ZK-807834 [MW, 527 D; Ki (human FXa), 0.11 nM], with recombinant tick anticoagulant peptide [rTAP; MW, 6,685 D; Ki, (human FXa) = 0.28 nM], and DX-9065a [MW 445 D, Ki (human FXa), 40 nM] in rabbits with arterial thrombosis induced by electrical vascular injury. ZK-807834 also was compared with low molecular weight heparin (LMWH; MW, 5,500 D) during venous thrombosis induced by placing a copper wire and threads in the vena cava. Inhibitors were administered as an i.v. bolus and 2-h infusion. Total dosages of ZK-807834, > or =0.7 micromol/kg (n = 18); rTAP, > or =1 micromol/kg (n = 18); or DX-9065a, > or =11 micromol/kg (n = 18) decreased the incidence of arterial thrombotic occlusion compared with control animals (p < 0.05). However, five of six animals given the lowest effective dosage of rTAP and four of six animals given DX-9065a bled from a surgical incision >5 min, but only two of six animals given ZK-807834 bled >5 min. Venous clot weights were reduced compared with controls for dosages of ZK-807834 > or =0.007 micromol/kg (n = 36) or LMWH > or =0.2 micromol/kg (n = 18). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were unchanged from baseline at the minimally effective dose of ZK-807834, whereas aPTT was increased twofold at the effective dose of LMWH. Thus ZK-807834 may be useful to attenuate thrombosis at lower dosages and with less perturbation of systemic hemostasis compared with available agents.

Phase I and pharmacological study of weekly administration of the polyamine synthesis inhibitor SAM 486A (CGP 48 664) in patients with solid tumors. European Organization for Research and Treatment of Cancer Early Clinical Studies Group.

A single-agent dose-escalating Phase I and pharmacological study of the polyamine synthesis inhibitor SAM 486A was performed. A dosing regimen of four weekly infusions followed by 2 weeks off therapy was studied. Fifty patients were entered into the study. Dose levels studied were 1.25, 2.5, 5, 8, 16, 32, 48, 70, 110, 170, 270, and 325 mg/m2/week. Pharmacokinetic sampling was done on day 1, and trough samples were taken weekly during the first treatment cycle. Pharmacodynamic sampling was done on days 1 and 22. At 325 mg/m2/week, dose-limiting toxicity was seen (one patient each with grade 4 febrile neutropenia, grade 3 neurotoxicity, and grade 3 hypotension with syncope and T-wave inversions on electrocardiogram). The recommended dose for further testing was set at 270 mg/m2/week. Infusion time was increased from 10 to 180 min due to facial paresthesias and flushing and somnolence. Drug exposure increased linearly with dose. Mean +/- SD t1,2 at 70-325 mg/m2 doses was 61.4+/-26.2 h, with a large volume of distribution at steady state. In peripheral blood leukocytes, a clear relationship between dose and inhibitory effect on S-adenosylmethionine decarboxylase or changes in intracellular polyamine pools was not recorded. SAM 486A can be administered safely using a dosing regimen of four weekly infusions followed by 2 weeks off therapy. The recommended dose for Phase II studies using this regimen is 270 mg/m2/week.

Population pharmacokinetics/toxicodynamics (PK/TD) relationship of SAM486A in phase I studies in patients with advanced cancers.

SAM486A (previously termed CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, is under clinical development for the treatment of advanced refractory malignancies. Hematological toxicity manifested by dose-dependent neutropenia has been observed in phase I studies. Population methods were used to investigate pharmacokinetics (PK) as a prognostic factor for safety end point (hematological toxicity) in patients with advanced cancers. SAM486A plasma concentrations and neutrophil counts were collected from three ascending-dose tolerability and PK studies (study 1: single 5-day continuous intravenous (IV) infusion with doses ranging from 24-700 mg/m2/cycle; study 2: 10-minute to 3-hour IV infusion once weekly with doses ranging from 16-325 mg/m2/week; study 3: 1-hour IV infusion once daily for 5 days with doses ranging from 3.6-202.8 mg/m2/day). The PK of SAM486A were best estimated by a population linear three-compartment model with NONMEM (version 5) using data from 9 patients in studies 1 through 3. The population pharmacokinetic parameters (SD) were CL = 6.2 (0.4) l/h/m2, Q2 = 15.4 (1.5) l/h/m2, Q3 = 33.6 (5.3) l/h/m2, V1 = 9.5 (1.6) l/m2, V2 = 672 (52) l/m2, and V3 = 39.9 (8.3) l/m2, and the corresponding intersubject variability was 45.4%, 74.0%, 85.3%, 80.1%, 37.0%, and 103%, respectively, where CL is total body clearance, Q2 and Q3 are intercompartmental clearances, and V1, V2, and V3 are the volumes of distribution in central and peripheral compartments, respectively. The intrasubject variability was 24.0%. The cumulative AUC before the onset of neutrophil nadir count (AUC) and the duration of exposure over threshold SAM486A concentrations in the range of 0.05 to 0.1 microM based on Bayesian PK parameter estimates significantly correlated with absolute neutrophil count nadir (< 5 x 10(9)/l). AUC showed the best correlation (R2 = 0.72) with absolute neutrophil count nadir by an inhibitory sigmoid Emax model and also correlated with percent decrease in neutrophil count from baseline to nadir by a simple Emax model (R2 = 0.53). Logistic regression analysis indicated that AUC and the duration of exposure over 0.05 to 0.1 microM, but not Cmax, were strong predictors of grade 4 neutropenia (< 0.5 x 10(9)/l). Drug exposure parameters such as AUC derived from population analysis may be used clinically as a useful predictor of drug-induced neutropenia.

Pharmacokinetics and pharmacodynamics of sibrafiban (Ro 48-3657), an orally active IIb/IIIa antagonist, administered alone or in combination with heparin, aspirin, and recombinant tissue-type plasminogen activator in beagles.

This study characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of sibrafiban (Ro 48-3657) in the presence of aspirin, heparin, and recombinant tissue-type plasminogen activator (rt-PA) in beagles. Sibrafiban is a double prodrug that undergoes bioconversion to the inactive prodrug Ro 48-3656 and to the active IIb/IIIa antagonist, Ro 44-3888, after oral administration. After oral sibrafiban, peak Ro 48-3656 plasma concentrations were observed earlier than Ro 44-3888 and were five- to sixfold higher than Ro 44-3888 peak concentrations. Administration of sibrafiban with heparin and aspirin or heparin and rt-PA did not alter sibrafiban PK. Ro 48-3656 and Ro 44-3888 PK and inhibition of platelet-aggregation profiles in groups treated with sibrafiban and heparin/aspirin or sibrafiban and heparin/rt-PA were similar to those of the group receiving sibrafiban alone. Sibrafiban resulted in >80% inhibition of adenosine diphosphate (ADP)-mediated platelet aggregation and an approximate sixfold increase in bleeding time (BT) compared with baseline measurements. The BT increase was greater in the sibrafiban, heparin, and rt-PA-treated group, during rt-PA administration, compared with the group treated with sibrafiban alone. The recovery of platelet aggregation may be slower after administration of sibrafiban with heparin and rt-PA. Sibrafiban had no effect on rt-PA PK or heparin PD.