The selective phosphodiesterase 9 (PDE9) inhibitor PF-04447943 (6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) enhances synaptic plasticity and cognitive function in rodents.

Inhibition of phosphodiesterase 9 (PDE9) has been reported to enhance rodent cognitive function and may represent a potential novel approach to improving cognitive dysfunction in Alzheimer's disease. PF-04447943, (6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one), a recently described PDE9 inhibitor, was found to have high affinity (Ki of 2.8, 4.5 and 18 nM) for human, rhesus and rat recombinant PDE9 respectively and high selectivity for PDE9 versus PDEs1-8 and 10-11. PF-04447943 significantly increased neurite outgrowth and synapse formation (as indicated by increased synapsin 1 expression) in cultured hippocampal neurons at low (30-100 nM) but not high (300-1000 nM) concentrations. PF-04447943 significantly facilitated hippocampal slice LTP evoked by a weak tetanic stimulus at a concentration of 100 nM but failed to affect response to the weak tetanus at either 30 or 300 nM, or the LTP produced by a theta burst stimulus. Systemic administration of PF-04447943 (1-30 mg/kg p.o.) dose-dependently increased cGMP in the cerebrospinal fluid 30 min after administration indicating target engagement in the CNS of rats. PF-04447943 (1-3 mg/kg p.o.) significantly improved cognitive performance in three rodent cognition assays (mouse Y maze spatial recognition memory model of natural forgetting, mouse social recognition memory model of natural forgetting and rat novel object recognition with a scopolamine deficit). When administered at a dose of 3 mg/kg p.o., which improved performance in novel object recognition, PF-04447943 significantly increased phosphorylated but not total GluR1 expression in rat hippocampal membranes. Collectively these data indicate that PF-04447943 is a potent, selective brain penetrant PDE9 inhibitor that increased indicators of hippocampal synaptic plasticity and improved cognitive function in a variety of cognition models in both rats and mice. Results with PF-04447943 are consistent with previously published findings using a structurally diverse PDE9 inhibitor, BAY73-6199, and further support the suggestion that PDE9 inhibition may represent a novel approach to the palliative remediation of cognitive dysfunction.

Small, noncovalent serine protease inhibitors.

Thrombin and factor Xa (fXa) are the only serine proteases for which small, potent, selective, noncovalent inhibitors have been developed, which are ultimately intended as drug development candidates (in this case as anticoagulants). Noncovalent inhibitors may be more selective and chemically and metabolically less reactive than covalent inhibitors. In addition, noncovalent inhibitors are more likely to have fast-binding kinetics which is particularly important in the development of thrombin inhibitors. TAME derived noncovalent thrombin inhibitors argatroban, napsagatran, and UK 156,406 have entered clinical trials as anticoagulants, the latter as an orally active agent. Serine trap deletion from substrate-like peptides led to the development of inogatran and melagatran, both of which have entered clinical trials as intravenous agents. The use of 3-aminopyridinone and pyrazinone acetamide peptidomimetic templates has resulted in the development of L-375,378 which has been chosen for clinical development as an orally active anticoagulant. Recently, compounds which do not have the conventional hydrogen bonding capabilities of peptides have begun to appear in the thrombin literature. Publications on noncovalent fXa inhibitors cover this type of peptidomimetic almost exclusively.

Potent, orally bioavailable somatostatin agonists: good absorption achieved by urea backbone cyclization.

Backbone cyclization of urea-based somatostatin agonists resulted in novel, orally bioavailable agonists. Binding assays confirmed that the resulting conformationally constrained cyclic ureas retained the potency of their acyclic counterparts. SAR studies subsequently led to highly potent analogs, selective for receptor subtype 2, and having good oral bioavailability.

Antithrombotic efficacy of thrombin inhibitor L-374,087: intravenous activity in a primate model of venous thrombus extension and oral activity in a canine model of primary venous and coronary artery thrombosis.

The small molecule direct thrombin inhibitor L-374,087 was characterized across species in an in vitro activated partial thromboplastin clotting time (aPTT) assay and in vivo in rhesus monkey and dog thrombosis models. In vitro in rhesus, dog, and human plasma, L-374,087 concentrations eliciting 2-fold increases in aPTT were 0.25, 1.9, and 0.28 microM, respectively. In anesthetized rhesus monkeys, 300 microgram/kg bolus plus 12 microgram/kg/min and 300 microgram/kg bolus plus 30 microgram/kg/min L-374,087 i.v. infusions significantly reduced jugular vein thrombus extension, with both regimens limiting venous thrombus extension to 2-fold that of baseline thrombus mass compared with a 5-fold extension observed in the vehicle control group. Antithrombotic efficacy in the rhesus with the lower-dose regimen was achieved with 2.3- to 2.4-fold increases in aPTT and prothrombin time. In a conscious instrumented dog model of electrolytic vessel injury, the oral administration of two 10 mg/kg L-374,087 doses 12 h apart significantly reduced jugular vein thrombus mass, reduced the incidence of and delayed time to occlusive coronary artery thrombosis, and significantly reduced coronary artery thrombus mass and ensuing posterolateral myocardial infarct size. Antithrombotic efficacy in the dog was achieved with 1.6- to 2.0-fold increases in aPTT at 1 to 6 h after oral dosing with L-374,087. These results indicate significant antithrombotic efficacy against both venous and coronary arterial thrombosis with L-374,087 with only moderate elevations in aPTT or prothrombin time. The oral efficacy of L-374,087 characterizes this compound as a prototype for the further development of orally active direct thrombin inhibitors.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Glutathione based approaches to improving cancer treatment.

The use of cytotoxic chemotherapy for cancer therapy has been very successful in the treatment and often cure of patients with particular neoplasms, such as testicular carcinomas and some lymphomas. In addition, the use of adjuvant chemotherapy in patients whose primary tumor has been surgically removed contributes significantly to cure rates in some of the more common malignancies such as breast carcinoma and colon cancer. Nonetheless, for most patients with metastatic malignancies, current antineoplastic drugs provide only brief remissions with few or no long term cures. In addition, the side effects of therapy lead to substantial morbidity in nearly all patients. Insights derived from model system studies on two glutathione based lead compounds, TER286 and TER199, suggest new clinical strategies and raise interesting basic research questions regarding the cell biology foundations of cancer chemotherapy.

Thrombin inhibitor design.

Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.

Tumor efficacy and bone marrow-sparing properties of TER286, a cytotoxin activated by glutathione S-transferase.

TER286 is a latent drug activated by human glutathione S-transferase (GST) isoforms P1-1 and A1-1 to produce a nitrogen mustard alkylating agent. M7609 human colon carcinoma, selected for resistance to doxorubicin, and MCF-7 human breast carcinoma, selected for resistance to cyclophosphamide, both showed increased sensitivity to TER286 over their parental lines in parallel with increased expression of GST P1-1. In primary human tumor clonogenic assays, the spectrum of cytotoxic activity observed for TER286 was both broad and unusual when compared to a variety of current drugs. In murine xenografts of M7609 engineered to have high, medium, or low GST P1-1, responses to TER286 were positively correlated with the level of P1-1. Cytotoxicity was also observed in several other cell culture and xenograft models. In xenografts of the MX-1 human breast carcinoma, tumor growth inhibition or regression was observed in nearly all of the animals treated with an aggressive regimen of five daily doses. This schedule resulted in a 24-h posttreatment decline in bone marrow progenitors to 60% of control and was no worse than for a single dose of TER286. These studies have motivated election of TER286 as a clinical candidate.

Characterization of the two-step pathway for inhibition of thrombin by alpha-ketoamide transition state analogs.

The interaction of thrombin with several potent and selective alpha-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its alpha-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its alpha-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the alpha-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the alpha-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.

C6 modification of the pyridinone core of thrombin inhibitor L-374,087 as a means of enhancing its oral absorption.

1 (L-374,087) is a potent, selective, efficacious, and orally bioavailable thrombin inhibitor that contains a core 3-amino-2-pyridinone moiety. Replacement of the C6 pyridinone methyl group of 1 by a propyl group gave 5 (L-375,052), which retained all the excellent properties of 1, and also yielded higher plasma levels after oral dosing in dogs and rats.

L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor.

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.

Activity of TER286 against human tumor colony-forming units.

Glutathione S-transferase (GST) isozymes have been shown to be elevated in many human cancer types as compared to normal tissues. TER286, one in a class of glutathione-based GST-activated cytotoxins, was tested in a soft agar cloning assay to determine its in vitro activity against primary human tumor colony-forming units. Breast and lung specimens from patients who had received prior therapy and those who were previously untreated were exposed to TER286 at concentrations of 1, 10 and 50 microM using both 1 h and continuous exposures. Overall in vitro responses (50% or less survival compared to untreated controls) were observed in 0% (0/14), 14% (2/14) and 29% (4/14), respectively, in specimens exposed to TER286 for 1 h, and in 5% (2/41), 10% (4/41) and 61% (25/41), respectively, in specimens exposed to TER286 continuously. TER286 has cytotoxic activity against both breast and lung cancer colony-forming units, and demonstrates a concentration-response effect. At 50 microM, there is a significant difference between 1 h and continuous exposures in head-to-head comparisons. These data suggest that TER286 can be activated in human tumor colony-forming units and should be pursued as a treatment candidate for patients whose tumors are resistant to drug treatment based on up-regulation of GST.

Randomized double-blind comparison of three dose levels of intravenous ondansetron in the prevention of cisplatin-induced emesis.

The selective 5-hydroxytryptamine3 (5HT3) antagonist ondansetron has been shown to be an effective antiemetic in patients receiving cisplatin chemotherapy. This double-blind study compared the efficacy and safety of three doses of intravenous ondansetron in the prevention of nausea and vomiting associated with high-dose (> or = 100 mg/m2) cisplatin chemotherapy. A total of 125 patients were randomized (1:1:1) to receive 0.015, 0.15, or 0.30 mg/kg every 4 h for a total of 3 doses. All patients were monitored for emetic episodes, adverse events, and laboratory safety parameters for 24 h following cisplatin administration. The 0.15-mg/kg dose was superior to the 0.015-mg/kg dose with respect to the median number of emetic episodes (P = 0.033) and complete response (no emetic episodes, P = 0.005). No statistically significant difference was found between the 0.15 and the 0.30-mg/kg groups. The most common adverse event was headache. Three 0.15-mg/kg doses of intravenous ondansetron are safe, effective, and adequate for the control of cisplatin-induced emesis.

Ondansetron does not affect alfentanil-induced ventilatory depression or sedation.

Ondansetron is a selective 5-hydroxytryptamine type 3 receptor antagonist effective as an antiemetic in patients experiencing post-operative or cancer chemotherapy-induced nausea and vomiting. Currently, no information is available regarding the interaction of ondansetron with opioids, although a serotonin antagonist might be expected to modify some opioid actions. This study was designed to measure the effects of ondansetron on alfentanil-induced ventilatory depression and sedation in healthy male volunteers. Ventilatory drive (measured as the end-tidal CO2 necessary to produce a minute ventilation of 15 l/min) was determined in 29 subjects using a modification of the Read rebreathing technique. Sedation was measured by asking the subjects to complete visual analog scales. Alfentanil was administered as a bolus (5 micrograms/kg) followed by a continuous infusion (0.25-0.75 micrograms.kg-1.min-1) for at least 90 min. Study medication (ondansetron 8 or 16 mg or vehicle placebo) was then administered in a randomized, double-blind manner, and the alfentanil was infused for an additional 15 min. Measurements of ventilatory drive and sedation were made at baseline, during alfentanil infusion, after study medication, and at 30-min intervals after alfentanil was discontinued. Alfentanil produced significant ventilatory depression (P less than 0.001) and sedation (P less than 0.001) in all three groups. Neither placebo nor ondansetron produced further change in the intensity of either alfentanil effect. After discontinuation of the opioid, both ventilatory depression and sedation decreased, and the rate of recovery was not significantly different between groups. The data indicate that alfentanil-induced sedation and ventilatory depression are not significantly affected by the subsequent administration of ondansetron.

Ondansetron is effective in decreasing postoperative nausea and vomiting.

The efficacy of ondansetron, a selective 5-HT3 receptor antagonist, in preventing postoperative nausea and vomiting in surgical patients was studied. Fifty women were randomized in a double-blind manner to receive either two 8 mg doses of intravenous ondansetron or two doses of placebo vehicle: the first given just before general anesthesia induction and the second 8 hours later. During the first 24 postoperative hours, the number of emetic episodes was recorded and the subjects rated their nausea on a scale from 0 to 10. Ondansetron-treated subjects had fewer emetic episodes (p less than 0.001) and lower subjective nausea scores (p less than 0.001). The number of complete responders (no emetic episodes and no rescue therapy) was 1 of 24 (4%) and 15 of 26 (58%) in the placebo and ondansetron groups, respectively (p less than 0.001). Ondansetron is clearly more effective than placebo in the prophylaxis of postoperative nausea and vomiting. The adverse event profile for ondansetron was similar to that of placebo.

Correlation of anti-emetic efficacy and plasma levels of ondansetron.

Intravenous ondansetron was administered at doses from 0.01 to 0.48 mg/kg every 4 h for three doses to patients receiving cisplatin 60-120 mg/m2 for the first time. Plasma samples were collected from 28 patients at baseline and at suitable times post-dose for pharmacokinetic analysis, and were assayed for ondansetron by high-pressure liquid chromatography. Plasma trough level was defined as the level before the third dose and 4 h area-under-the-curve (AUC4) was calculated with the linear trapezoidal method. Despite wide inter-patient variation, a correlation was seen between both trough level (r = 0.737, P less than 0.0001) and AUC4 (r = 0.903, P less than 0.001) related to dose. Trough level was also predictive of AUC4 (r = 0.824, P less than 0.0001). Frequency of complete protection (no emetic episodes) was equivalent throughout the AUC4 range, suggesting anti-emetic activity even at low AUC4. However, a trend toward better protection against failure (5 or more episodes) was seen when higher values of AUC4 were achieved, suggesting more consistent anti-emetic activity at moderate to high AUC4.