Phase I trial of the thymidylate synthase inhibitor AG331 as a 5-day continuous infusion.

AG331 (N6-[4-(morpholinosulfonyl)benzyl]-N6-methyl-2, 6-diaminobenz-[c,d]-indole glucuronate) is a lipophilic thymidylate synthase inhibitor with activity in solid tumor models. On the basis of preclinical data supporting regimens of frequent drug administration, we performed a Phase I trial of AG331 as a 5-day continuous infusion repeated every 3 weeks. Twenty-nine patients were entered at doses ranging from 25 to 1000 mg/m2/day. The major side effects were mild to moderate fatigue, nausea, vomiting, diarrhea, and fever. At doses >/=400 mg/m2, acute reversible elevation of bilirubin, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltranspeptidase was observed. All patients who received >/=600 mg/m2/day experienced elevated alanine aminotransferase. Elevated liver function tests were evident by day 3 of the infusion and had resolved by day 8 in the majority. This toxicity was dose limiting at 1000 mg/m2/day, at which dose two of two patients developed grade 4 reversible hyperbilirubinemia in addition to the enzyme elevations. Serum and urine samples were analyzed by a novel high-pressure liquid chromatography method for the determination of the pharmacokinetics of AG331. Over the 50-1000 mg/m2/day dose range, mean total clearance ranged from 11.6 to 30.0 liters/h/m2, and volume of distribution at steady state ranged from 279.5 to 758.7 liters/m2. These parameters were dose independent over the dose range tested. The harmonic mean terminal half-life of AG331 was 20.2 h. Less than 5% of an AG331 dose is eliminated unchanged in the urine. Both the administered dose and exposure to the drug were related to the changes in bilirubin and aminotransferase blood levels. Evidence for inhibition of thymidylate synthase was obtained at doses ranging from 100 to 1000 mg/m2 in seven patients; plasma deoxyuridine concentrations at end-infusion were 1.8-3.8-fold higher than pretreatment values. Because of the nature of toxicity on this schedule, more extensive Phase II evaluation is not recommended, although an AG331 dose of 800 mg/m2/day for 5 days is tolerable. Exploration of less frequent dose administration is under way.

AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies.

3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. The aim of the design program was to identify TS inhibitors with different pharmacological characteristics from classical folate analogs and, most notably, to develop non-glutamate-containing molecules which would not require facilitated transport for uptake and would not undergo intracellular polyglutamylation. One molecule which resulted from this program, AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention. In vivo, AG337 was highly active against the thymidine kinase-deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress.

Combined ultrasound and fluorescence spectroscopy for physico-chemical imaging of atherosclerosis.

This paper describes a combined ultrasonic and spectroscopic system for remotely obtaining physico-chemical images of normal arterial tissue and atherosclerotic plaque. Despite variations in detector-tissue separation, R, fluorescence powers corresponding to pixels in the image are converted to the same set of calibrated units using distance estimations from A-mode ultrasound reflection times. An empirical model, validated by Monte Carlo simulations of light propagation in tissue, is used to describe changes in fluorescence power as a function of R. Fluorescence spectra of normal and atherosclerotic human aorta obtained with this system are presented as a function of R. To compensate for changes in fluorescence power with R, the empirical model was used in each case to calculate the fluorescence power at a constant reference value of R(Rref = 1.67 mm). Prior to compensation, tissue fluorescence power decreased more than a factor of two as R was increased from 2.5 to 5 mm. Following compensation, the fluorescence power varied less than +/- 10% of the average compensated peak. The chemical composition of each sample was determined by fitting its fluorescence spectrum (in calibrated units) to a model of tissue fluorescence incorporating structural protein and ceroid fluorescence, as well as structural protein and hemoglobin attenuation. Parameters of the fit were used to classify tissue type. Without compensation for distance variation, classification of tissue type was frequently incorrect; however, with compensation, predictive value was high. A 1-D chemical image of a section of human aorta containing both normal and atherosclerotic regions obtained with this system is also presented. After compensation for detector-sample separation, tissue classifications along the cross-section closely resemble those obtained from histology. Regions of elevated ceroid concentration and intimal thickening are clearly observable in the resultant chemical image. The potential value of this type of system in the diagnosis and treatment of coronary artery disease is discussed.

Types of benzodiazepines abused by chemically dependent inpatients.

Records of inpatients (N = 1,483) over a three-year period at an addiction treatment center were evaluated for the presence of benzodiazepine (BZ) dependence (N = 136). The preferred BZ for 43% of the subjects was diazepam, and alprazolam for 14% of subjects. Chlordiazepoxide, lorazepam, and clorazepate were each the preferred BZ for 4% of patients. Cocaine and opioid abusers were six times more likely to abuse diazepam than any other BZ. Alprazolam patients required a significantly longer period of detoxification than diazepam patients. Four percent of BZ-dependent patients (N = 6) abused BZs only and had no other substance abuse history.

Increased severity of alcohol withdrawal in in-patient alcoholics with a co-existing anxiety diagnosis.

The purpose of this study was to determine whether alcoholics with a co-existing anxiety disorder (dual-diagnosed group) experienced different withdrawal symptomatology from alcoholics without an anxiety disorder (alcohol-only group). Symptoms of alcohol withdrawal were measured on admission to an in-patient treatment program and throughout treatment (days 0, 2, 7, 14 and 21) using the revised Clinical Institute Withdrawal Assessment for Alcohol (CIWA-Ar) scale. The dual-diagnosed group exhibited more severe alcohol withdrawal, as indicated by higher total CIWA-Ar scores, at all time points than the alcohol-only group. The possibility that anxiety disorders and alcohol withdrawal share a common neurochemical basis and that the CIWA-Ar scale may be useful as a screening instrument for anxiety disorders in alcoholics is discussed.

Symptoms of anxiety in inpatient alcoholics with and without DSM-III-R anxiety diagnoses.

Self- and observer-rating scales were administered to alcohol-dependent inpatients during acute withdrawal and regularly for 3 weeks. Following a structured diagnostic interview (SCID) at the end of the 3rd week of hospitalization, subjects were divided into two groups: a dual-diagnosed group (alcohol dependence and anxiety disorder) and an alcohol-only group (no other current Axis I diagnosis). The results demonstrated that the dual-diagnosed subjects experienced higher anxiety levels during and after acute alcohol withdrawal. All rating scales (i.e., Sheehan Patient Rated Anxiety Scale, Spielberger State Anxiety Inventory, Zung Rating Scale for Anxiety, and Hamilton Rating Scale for Anxiety) were analyzed to obtain the best combination of sensitivity and specificity. Taken together, the results indicate that it may be possible to identify alcoholics who require additional psychiatric evaluation early in treatment. This would allow a treatment plan which could be used to address both psychiatric and substance abuse problems.

Sex differences in animal tests of anxiety.

To explore further the meaning of sexually dimorphic behavior in the open-field test, male and female hooded Lister rats were tested in three tests of anxiety. In the social interaction test, the social interaction scores of the female rats were lower and did not increase as readily following familiarization to the apparatus as those of the male rats. In the elevated plus-maze test, female rats showed a reduced aversion to the open arms compared to male rats; and in a modified Vogel conflict test, the punished licking rates of the female rats were lower than those of the male rats. It is concluded that the behavior of male and female rats differs in these tests, but that firm conclusions concerning sex differences in anxiety levels cannot be made because all three tests did not lead to predictions which were in the same direction. It is also suggested that cautious interpretation is necessary because these tests may measure different variables in male and female rats and they may not be valid tests of anxiety for female rats.

Indomethacin does not antagonize the anxiolytic action of ethanol in the elevated plus-maze.

The present study was designed to examine whether the prostaglandin (PG) synthesis inhibitor indomethacin (INDO) could antagonize the anxiolytic effects of ethanol (EtOH) in the elevated plus-maze test of anxiety. EtOH (1.6 g/kg) significantly increased the percentage of open arm entries and time spent on the open arms in both inbred C57BL/6J and outbred CD-1 mouse strains. However, this anxiolytic effect of EtOH was not significantly antagonized by pretreatment with INDO (5 and 10 mg/kg) in either strain. EtOH also significantly increased total arm entries in CD-1 mice, but not in the C57BL/6J strain. These data from C57BL/6J mice indicate that the low-dose stimulant properties of EtOH can be dissociated from the anxiolytic action of the drug in the plus-maze task. Finally, although INDO did not antagonize the stimulant effect of EtOH in the plus-maze task (in CD-1 mice), it did attenuate EtOH-induced stimulation of locomotor activity in an open-field arena. Taken together, these results suggest some specificity with regard to the role of PGs in mediating (or modulating) the neurobehavioral actions of EtOH, and further support the notion that the anxiolytic and stimulant effects of EtOH may be mediated by different mechanisms.

Diazepam reverses the effects of pentylenetetrazole in rat pups by acting at type 2 benzodiazepine receptors.

Pentylenetetrazole (75 mg/kg) induced a characteristic coarse body tremor (accompanied by limb extension) and hyperactivity in 4-day-old rat pups. These effects were reversed by diazepam (0.5 and 2 mg/kg) but not by CL 218,872 (10 and 20 mg/kg) which is selective for type 1 benzodiazepine receptors. Diazepam did not affect the brain concentrations of pentylenetrazole, indicating that the reversal was not based on a pharmacokinetic interaction. Neither diazepam nor CL 218,872 had significant effects on the behavior of the rat pups, although diazepam (2 mg/kg) tended to increase locomotor activity. The results suggest that diazepam displays an anticonvulsant effect in the neonatal rat which is mediated by type 2 receptors.

Antagonistic effects of caffeine and yohimbine in animal tests of anxiety.

The effect of a combination of caffeine and yohimbine was investigated in the social interaction, elevated plus-maze and punished-drinking tests of anxiety. Caffeine (40 mg/kg i.p.) had anxiogenic-like effects in the social interaction and plus-maze tests. Yohimbine (2.5 mg/kg i.p.) was anxiogenic-like in the plus-maze and displayed anticonflict activity. Unexpectedly, caffeine and yohimbine antagonized each others' effects in the social interaction and elevated plus-maze tests.

Yohimbine's anxiogenic action: evidence for noradrenergic and dopaminergic sites.

Yohimbine (2.5 or 4 mg/kg) reduced the percentage of open arm entries and the percentage of time spent on the open arms displayed by rats on an elevated plus-maze indicating anxiogenic activity. These effects were reversed by the alpha 2-adrenoceptor agonist clonidine (0.01 mg/kg) and by the dopamine receptor agonist apomorphine (0.57 mg/kg). The following failed to reverse the effects of yohimbine: the selective alpha 2-adrenoceptor agonists, guanfacine (0.25 and 1 mg/kg), B-HT920 (0.025 and 0.1 mg/kg), B-HT933 (1 and 10 mg/kg); the beta-blocker propranolol (2.5 and 10 mg/kg); the alpha 1-adrenoceptor agonist phenylephrine; the D1 agonist SK&F 38393 (5 and 10 mg/kg) and the D2 agonist LY 171555 (0.5 and 1 mg/kg). Therefore, it is unlikely that activity at only the alpha 1, alpha 2, beta, D1 or D2 sites can entirely account for the anxiogenic actions of yohimbine in the elevated plus-maze. Evidence that clonidine affects the dopaminergic system and that apomorphine affects the noradrenergic system suggest that yohimbine may produce its anxiogenic response by activity on both the noradrenergic and dopaminergic systems.

Lack of effects of 5HT3 receptor antagonists in the social interaction and elevated plus-maze tests of anxiety in the rat.

The effects of three 5HT3 receptor antagonists: BRL 43964 (0.1 and 1 mg/kg, oral), GR 38032F (0.1 and 1 mg/kg, oral), and zacopride (0.01, 0.1 and 1 mg/kg, IP) were examined in low light test conditions of the social interaction test. None of the three 5HT3 receptor antagonists had a significant effect on social interaction. In contrast, in two experiments chlordiazepoxide (7.5 mg/kg) significantly increased social interaction and this effect was greatest in the unfamiliar test condition. In a third experiment, the effects of GR 38032F (0.1 and 1 mg/kg, oral) and zacopride (0.01, 0.1 and 1 mg/kg, oral) were investigated in the high light test conditions of the social interaction test; neither compound had a significant effect. In the elevated plus-maze, chlordiazepoxide (7.5 mg/kg oral or IP) significantly increased both the per cent number of entries made onto open arms and the per cent of time spent on the open arms, indicating an anxiolytic action. Zacopride (0.01, 0.1 and 1 mg/kg, oral or IP) had no significant effect in this test. The effect of the baseline rate of responding in the social interaction test on the effects of 5-HT3 antagonists is discussed. The results from the present experiment and those from other animal tests of anxiety caution against the conclusion that 5HT3 receptor antagonists are anxiolytic.

Sodium phenobarbitone reverses the anxiogenic effects of compounds acting at three different central sites.

Sodium phenobarbitone was tested for its ability to antagonise the anxiogenic effects of compounds acting at three different central sites. These compounds were: FG 7142, a beta-carboline which acts at the benzodiazepine binding site on the GABA-benzodiazepine receptor complex; pentylenetetrazole, which acts at the picrotoxinin site on the GABA-benzodiazepine receptor complex; and yohimbine which is an antagonist at the alpha 2-adrenoceptor. The experiments were carried out in two tests of anxiety using rats. In the social interaction test (the test arena was familiar and dimly lit), FG 7142 (5 mg/kg) and pentylenetetrazole (15 mg/kg) reduced the time spent in social interaction (indicating anxiogenic activity); these effects were reversed by sodium phenobarbitone (35 mg/kg). Sodium phenobarbitone (35 mg/kg) alone decreased locomotor activity as measured in the social interaction test, which was reversed by pentylenetetrazole (15 mg/kg). In the elevated plus-maze, FG 7142 (6.7 mg/kg) pentylenetetrazole (20 mg/kg) and yohimbine (4 mg/kg) reduced the percentage of open-arm entries and the percentage of time spent on the open arms (indicating anxiogenic activity); these effects were reversed by sodium phenobarbitone (35 mg/kg). Sodium phenobarbitone (35 mg/kg) alone significantly increased the percentage of open-arm entries and the percentage of time spent on the open arms (indicating anxiolytic activity). This study, together with previous studies using other clinically-effective anxiolytic drugs, suggests that the ability of a compound to antagonise the effects of anxiogenic agents may be a useful indirect means of predicting anxiolytic activity.

Behavioral effects of acute and chronic administration of caffeine in the rat.

This study investigated the effects of acute and chronic caffeine treatment on behavior in the social interaction, holeboard and home-cage aggression tests and on proconvulsant actions with pentylenetetrazol. Acutely-treated rats received an IP injection of caffeine (20 or 40 mg/kg). Chronically-treated rats received caffeine in their drinking water for 21 days (50 or 100 mg/kg/day) followed by an injection of caffeine on the test day (20 or 40 mg/kg respectively). Acutely, the higher dose of caffeine (40 mg/kg) decreased levels of social interaction. In the holeboard test, 20 mg/kg of acute caffeine increased motor activity whilst 40 mg/kg reduced head-dipping behavior. In the home-cage aggression test, acute caffeine (40 mg/kg) reduced offensive aggressive behaviors. After chronic treatment with caffeine none of these behaviors differed significantly from controls. After both acute and chronic treatment, caffeine (20 and 40 mg/kg) was proconvulsant with pentylenetetrazol.

Profiles of the antipanic compounds, triazolobenzodiazepines and phenelzine, in two animal tests of anxiety.

Two animal tests of anxiety (the elevated plus-maze and the social interaction test) were used to investigate the effects of several antipanic agents. In the elevated plus-maze, the triazolobenzodiazepines adinazolam (2 and 5 mg/kg) and alprazolam (1 mg/kg), tested after 5 days of pretreatment, demonstrated significant anxiolytic effects, while phenelzine (9 mg/kg), after 21 days of pretreatment, demonstrated nonsignificant anxiolytic effects. In the social interaction test, the triazolobenzodiazepines generally did not produce an anxiolytic profile, and phenelzine even revealed significant anxiogenic activity. The antipanic agents therefore distinguish between the two tests of anxiety. The lack of a strong anxiolytic profile with these agents in both tests lends support to the distinction between generalized anxiety and panic disorder.

Kindling with the beta-carboline FG7142 suggests separation between changes in seizure threshold and anxiety-related behaviour.

The aim of this paper was to determine whether the prolonged decrease in seizure threshold produced by chemical kindling was accompanied by behavioural changes in tests of anxiety and aggression. The responses of rats in five tests were examined after chronic treatment with the benzodiazepine inverse agonist FG7142. This treatment caused chemical kindling, so that the originally proconvulsant drug caused full seizures. This effect is very long-lasting, and our previous work with mice had suggested that it might be accompanied by an increase in anxiety-related behavior. In the present work no significant differences were found between the behaviour of FG7142-kindled rats and vehicle-treated controls in social interaction test, elevated plus maze, or the Vogel conflict test of anxiety or in tests of home cage aggression or startle responses. The results therefore show that prolonged changes in seizure threshold can occur without alterations in the apparent level of anxiety.

Measures of anxiety and stress in the rat following chronic treatment with yohimbine.

Yohimbine (2.5 and 5 mg/kg) was investigated in two animal tests of anxiety and on baseline corticosterone plasma concentrations, following both acute and chronic administration. Acute treatment with yohimbine produced the following effects: a reduction in the percentage of total arm entries made onto the open arms and in the percentage of time spent on the open arms of an elevated plus-maze (indicating anxiogenic properties), an increase in baseline plasma corticosterone concentrations, and a reduction in locomotor activity (recorded in the social interaction test). No significant effects were observed on anxiety levels as measured by the social interaction test. Following chronic treatment, we saw no evidence for sensitization to the effects of yohimbine.

Selective agonists and antagonists for 5-hydroxytryptamine receptor subtypes, and interactions with yohimbine and FG 7142 using the elevated plus-maze test in the rat.

The effects of some 5-HT receptor ligands were investigated on measures of anxiety in an elevated plus-maze test in the rat. Quipazine (2 and 4 mg kg-1), a non-specific 5-HT agonist and ritanserin (0.25-10 mg kg-1), a 5-HT2 receptor antagonist displayed anxiogenic profiles by reducing both of the measures of anxiety used in this test. Two 5-HT1A receptor ligands, buspirone (4 and 8 mg kg-1) and ipsapirone (2.5-10 mg kg-1) and the 5-HT1 agonist, RU 24969 (0.1875-1.5 mg kg-1) significantly reduced only the percentage of time spent on the open arms. (-)-Propranolol (5 and 10 mg kg-1), a 5-HT1 receptor antagonist significantly reduced only the percentage of entries made onto the open arms. Metergoline (4 mg kg-1), a non-specific 5-HT antagonist displayed anxiolytic effects in this test by increasing both measures of anxiety. The 5-HT1A receptor agonist, 8-OH-DPAT (0.0625-0.25 mg kg-1) had no effect on either of the measures of anxiety. The results from the non-specific ligands (quipazine and metergoline) are consistent with the theory that a reduction in 5-HT function reduces anxiety. However, in spite of their more selective effects on 5-HT receptors the results in this test from the more specific ligands are not consistent with a strong involvement of any single receptor subtype. The interaction studies with yohimbine and FG 7142 (beta-carboline-3-carboxylate methylamide) provided no clear evidence for a major role of 5-HT pathways in the mediation of their anxiogenic effects.

Chronic treatment with imipramine does not reverse the effects of 3 anxiogenic compounds in a test of anxiety in the rat.

The ability of chronic treatment with imipramine (an antidepressant with anti-panic activity) to antagonise the anxiogenic effects of 3 different compounds was investigated in the elevated plus-maze. The compounds chosen are likely to produce anxiety by activity at different sites in the central nervous system: yohimbine, by blocking the alpha 2-adrenoceptor; FG 7142, by action at the beta-carboline site on the GABA-benzodiazepine receptor complex and pentylenetetrazole, by acting at the picrotoxinin site on this complex. Administration of imipramine following 21 days pre-treatment did not produce a significant consistent anxiolytic effect alone and was unable to reverse the anxiety produced by any of the 3 anxiogenic compounds. Our results are discussed in terms of the nature of the anxiety produced by the anxiogenic drugs and the sensitivity of tests of anxiety to anti-panic agents.