Acute behavioral and EEG effects of NW-1015 on electrically-induced afterdischarge in conscious monkeys.

NW-1015 is a novel Na+ and Ca2+ channel blocker with broad spectrum anticonvulsant activity and an excellent safety margin. As the compound also shows sigma-1 receptor ligand properties it was deemed important to determine whether it possesses anticonvulsant properties in primates without causing behavioral and EEG abnormalities. Thus, the effects of NW-1015 on limbic electrically-induced afterdischarge (AD) were evaluated in four cynomolgus monkeys, and its activity compared to a single effective dose of phenytoin (PHT). The four male cynomolgus monkeys were chronically implanted for EEG recordings, from cortex and limbic structures. AD was induced in limbic areas by electrical stimulation. The effects of NW-1015 on the duration and the behavioral component of the AD were randomly tested at doses from 25 to 75 mg/kg and compared with the effects of PHT 50 mg/kg. Similarly to PHT, 50 mg/kg of NW-1015 significantly shortened the EEG AD and almost abolished AD elicited behavioral seizure. Only the behavioral effects of AD were reduced after administration of 25 mg/kg p.o. NW-1015 did not cause EEG or interictal behavioral alterations at doses up to 75 mg/kg p.o. These data further confirm the broad-spectrum anticonvulsant activity and a good safety profile of NW-1015 even in a primate model of complex partial seizures and suggest that its affinity for sigma-1 receptors is behaviorally irrelevant.

Preclinical evaluation of PNU-151774E as a novel anticonvulsant.

PNU-151774E [(S)-(+)-2-(4-(3-fluorobenzyloxy) benzylamino) propanamide, methanesulfonate] is a structurally novel anticonvulsant having Na+ channel-blocking and glutamate release-inhibiting properties, as well as being a MAOB inhibitor. Its anticonvulsant activity was evaluated in the maximal electroshock (MES) test and in chemically induced seizures (bicuculline, BIC; picrotoxin, PIC; 3-mercaptopropionic acid, 3-MPA; pentylenetetrazole, PTZ; strychnine, STRYC). Behavioral toxicity was evaluated in the rotorod test with measurements of spontaneous locomotor activity and passive avoidance responding. The anti-MES activity of PNU-151774E in both mice and rats, respectively, produced ED50 values of 4.1 mg/kg and 6.9 mg/kg after i.p. administration or 8.0 mg/kg and 11.8 mg/kg after p.o. administration. Oral anti-MES activity in rats peaked between 1 and 2 h after administration and was evident up to 4 h. This activity was related to brain levels of unchanged drug which peaked at 37 mM within 1 h. Oral ED50 values (mg/kg) effective in blocking tonic extension seizures by chemical convulsants in mice were: BIC (26.9), PIC (60.6), 3-MPA (21.5), STRYC (104.1) and PTZ (26.8). This potency was associated with high therapeutic indices relative to: MES (78.2), BIC (23.3), PIC (10.3), 3-MPA (29.1) and STRYC (6.0). No evidence of tolerance to anti-MES activity after repeated dosing was observed. PNU-151774E did not show anti-absence seizure activity as assessed by i.v. infusion of PTZ. PNU-151774E impaired spontaneous activity in rats only at the oral rotorod ED50 dose of 700 mg/kg p.o. PNU-151774E did not impair passive avoidance responding at doses up to 40 times the oral MES ED50 dose in rats. These results indicate that PNU-151774E is an anticonvulsant effective in various seizure models with a wide therapeutic window, and with a low potential to induce tolerance and locomotor or cognitive side effects.

Determination of ethambutol in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 micrograms/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.

Determination of tallimustine in human plasma by high-performance liquid chromatography.

A sensitive and reproducible HPLC method for the determination of tallimustine (I) in human plasma has been developed and validated. Compound I was extracted from plasma by solid-phase extraction using a C18 cartridge from which the test compound was eluted with a methanol-formic acid mixture. The methanol solution was evaporated to dryness and the residue dissolved in a 0.2 M formic acid in methanol-water (1:1, v/v) mixture, then injected onto the HPLC column. The chromatographic separation was performed isocratically by a reversed-phase column filled with ODS, using a 50 mM KH2PO4-acetonitrile mixture as the mobile phase. The flow-rate was 1 ml/min. The eluate was monitored at 314 nm. No peak interfering with that of I was observed when blank human plasma was assayed. Linearity was established in the concentration range 0.5-85.5 nanograms of I per millilitre of plasma. Four calibration curves in plasma, prepared and run on four different days, showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V. = 4.5%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed as C.V. ranged from 0.9 to 14.4%. The inter-day precision evaluated at the same concentrations was better than 10.2%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/added amount of I, ranged from 86.2 to 108.5%. The limit of quantitation was 0.5 ng/ml plasma. The HPLC method described here was successfully employed for the determination of I in some plasma samples obtained during a phase I clinical trial with the test compound.

Phase I study of the novel distamycin derivative tallimustine (FCE 24517).

BACKGROUND: Tallimustine, a benzoyl nitrogen mustard derivative of the antiviral agent distamycin A, is a new alkylating agent which binds to A-T rich regions of DNA in the minor groove producing highly sequence-specific alkylations. Its main preclinical features are a significant antitumor activity in animal models and a lack of cross-resistance in vitro and in vivo with L-PAM. Myelotoxicity was dose-limiting in animals, with a more than 100-fold difference in bone marrow sensitivity between mice and dogs. PATIENTS AND METHODS: Forty adult patients (pts) with solid malignancies were entered in the study. The drug was administered as an IV bolus every 4 weeks. CBC was repeated twice a week and serial assessments of renal function were performed in the week following the first cycle. From the starting dose of 50 micrograms/m2, corresponding to 1/3 of the highest non-toxic dose (HNTD) in dogs, there were increases through 10 dose levels, with reliance only on the features of the myelotoxicity observed. RESULTS: The main toxic effect was neutropenia which was dose-limiting, selective and short-lasting. Only previously-untreated pts received doses of 750 micrograms/m2 or more, with grade 4 neutropenia occurring in > or = 75% of the cycles. The maximally tolerable dose (MTD) was defined as 1250 micrograms/m2, with 3 of 3 pts developing febrile neutropenia requiring IV antibiotics. A platelet count of < 100 x 10(3)/microliters was observed in only one pt. Bone marrow aspiration performed in selected pts on days 8 and 15 confirmed a highly selective impairment by tallimustine of the myeloid lineage, with rapid recovery of the proliferative compartment. Pharmacokinetic studies performed at 1000 micrograms/m2 and 1250 micrograms/m2 showed a rapid fall of the plasma levels within the first 2 hours with drug concentrations between 100 ng/ml and 400 ng/ml within the first hour. A partial response of 4 months' duration was reported in one previously-untreated pt with cutaneous recurrences of malignant mesothelioma. CONCLUSIONS: The report of some antitumour efficacy, the high selectivity of neutropenia, the lack of significant non-hematological toxic effects and the occurrence of detectable but still low plasma drug concentrations suggest that further clinical evaluation of higher doses of tallimustine in combination with colony-stimulating factors would be justified.

The anticonvulsant FCE 26743 is a selective and short-acting MAO-B inhibitor devoid of inducing properties towards cytochrome P450-dependent testosterone hydroxylation in mice and rats.

The effects of the potent anticonvulsant FCE 26743 ((S)-2-(4-3-fluorobenzyloxy)benzylamino)propionamide) on monoamine oxidase (MAO) activity were measured in-vitro and ex-vivo using rat tissue homogenates. In-vitro, FCE 26743 showed potent and selective inhibitory properties towards liver MAO-B, with IC50 values about 10(-7) M for MAO-B and higher than 10(-5) M for MAO-A. When determined ex-vivo in brain, the ED50 value for the inhibition of MAO-B was 1.1 mg kg-1 (p.o.) 1 h post-dosing, whereas MAO-A remained virtually unaffected after administration of 60 mg kg-1. Similar effects were seen in liver. Following oral administration of 5 mg kg-1 FCE 26743 to rats, brain MAO-B inhibition was 79% after 1 h and 13% after 24 h, indicating that FCE 26743 behaves as a short-acting MAO-B inhibitor. The ability of FCE 26743 to act as a MAO substrate was assessed in mice by measuring the urinary excretion of alaninamide, a potential metabolite of FCE 26743 which would result from the action of MAO. No alaninamide was detectable in the 0-8 h urines after administration of a 119 mg kg-1 dose, suggesting that FCE 26743 is not, or only to a small degree, a substrate of MAO. The effects of FCE 26743 on cytochrome P450 enzymes involved in testosterone hydroxylation were determined in rats after repeated administration. No induction of the cytochrome P450 system was noted.

Determination of FCE 23884, a new ergolene derivative, and its possible metabolite, the 6-nor-derivative in plasma by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of FCE 23884 and its 6-nor-derivative (FCE 26506) in plasma has been developed. After buffering the plasma samples, the compounds and the internal standard were extracted with ethyl ether-n-octanol (9:1, v/v), back-extracted into 0.01 M phosphoric acid and then analysed by reversed-phase liquid chromatography. Quantification was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrix was observed. The assay was adequate for the quantification of plasma levels of the two compounds after a single oral dose of 1 mg of FCE 23884 in humans.

Effect of ageing on tissue levels of amino acids involved in the nitric oxide pathway in rat brain.

Nitric oxide (NO) and citrulline are produced from L-arginine by the action of NO synthase after activation of excitatory amino acid receptors. In addition to its role in neurodegeneration, there is convincing evidence that NO is also involved in long-term potentiation, a cellular analog of learning and memory in the mammalian nervous system. In the present study, concentrations of L-arginine, citrulline, aspartic acid and glutamic acid were determined in various brain regions of young and old rats. The aim was to examine whether changes in brain concentrations of these amino acids might be indicative of a possible decrease in NO production with ageing, in relation with the well-established decline of cognitive function. Brain aspartic acid, citrulline and L-arginine concentrations were found to be lower in old rats compared to young animals, although the decrease did not always reach statistical significance. In contrast, no change in glutamic acid levels was found. In all brain structures of young and old rats, concentrations of L-arginine were higher than the concentration for NO synthase to function at maximum velocity in the rat brain. Therefore, the decrease in citrulline concentrations found in some brain regions of old rats might be seen, at least partly, as a reflection of a lower production of NO with ageing, although further work is clearly needed to ascertain a decrease in rat brain NO synthase activity with age.

Pharmacokinetics of iododoxorubicin in the rat, dog, and monkey.

The pharmacokinetics of iododoxorubicin (I-DOX) have been studied after single dose administration in the rat (iv and po), dog (iv and po), and monkey (iv). Plasma levels and amounts in urine were monitored by HPLC for both I-DOX and its biologically active metabolite, iododoxorubicinol (I-DOXOL). Plasma levels of I-DOX after iv administration could be described by a three-exponential curve with extremely fast initial phase. Terminal elimination half-lives of I-DOX were similar, 6-7 hr, in all three species. Body weight-normalized clearance (CL) and distribution volumes (Vd) of I-DOX were lower in the dog, but were similar in rat and monkey. The pharmacokinetic parameters also implied metabolic differences between species. Mean I-DOXOL/I-DOX AUC ratios were 0.02, 0.47, and 0.58, respectively, in rat, dog, and monkey, values considerably lower than reported in human studies. I-DOXOL remained slightly longer in the body than I-DOX, as seen both from terminal half-lives (9-11 hr) and mean residence times. In all species, renal excretion was virtually negligible: the amount of I-DOX + I-DOXOL in urine was less than 2% of dose. Mean bioavailabilities of I-DOX were 0.23 and 0.46 in rat and dog, respectively, and, in the latter, about half of I-DOXOL formation occurred during or before the first pass.

Is the oxidation of milacemide by monoamine oxidase a major factor in its anticonvulsant actions?

The anticonvulsant drug milacemide (2-n-pentylaminoacetamide) is known to be oxidized by monoamine oxidase-B to yield glycinamide which then breaks-down to give glycine. It has been postulated that it is this liberation of glycine in the brain that accounts for the anticonvulsant effects. In order to test this hypothesis, and since amines bearing a methyl-group in the alpha-position have been shown to be resistant to oxidation by monoamine oxidase, the effects of milacemide were compared with those of alpha-methyl-milacemide. Although the latter compound was found to be toxic at higher concentrations, it was found to antagonize bicuculline-induced convulsions in mice. When milacemide was administered to mice (0.5 mmol/kg, p.o.) there was a substantial increase in urinary glycinamide excretion. No such increase was observed after the administration of the same dose of alpha-methyl-milacemide. Furthermore, alpha-methyl-milacemide was not oxidized by either monoamine oxidase-A or -B in vitro to any detectable extent, although it was a competitive inhibitor of both forms of the enzyme. The findings that alpha-methyl-milacemide has anticonvulsant properties in the bicuculline test but is not a substrate for monoamine oxidase or a source of urinary glycinamide cast doubt on the importance of the oxidation or milacemide to form glycinamide as a major factor in its anticonvulsant action.

Effects of ageing on the content in sulfur-containing amino acids in rat brain.

Concentrations of the sulfur-containing amino acids methionine, homocysteic acid, cysteic acid and taurine were measured in brain structures of young and old Wistar rats in an attempt to establish a possible link between the increase in oxidative stress with ageing and changes in tissue levels of these amino acids. Contrary to data reported by others, in all brain structures of young and old rats homocysteic acid levels could not be quantified. Compared with young rats, in old animals taurine and methionine concentrations significantly decreased in striatum and cortex; decreased taurine levels were also found in nucleus accumbens and cerebellum and lower concentrations of methionine were found in midbrain, hippocampus and pons-medulla. Cysteic acid levels either did not change or significantly increased in cortex and hippocampus. These results are discussed taking into account the biosynthesis of sulfur-containing amino acids in rat brain and the decrease in glutathione in relation to oxidative stress with ageing. Changes in aspartic acid, glutamic acid, serine, glutamine, glycine and GABA concentrations with ageing were also determined in the same brain structures and were in good agreement with those previously reported (Strolin Benedetti et al., 1990 a, b).

The effects of aging on MAO activity and amino acid levels in rat brain.

The possibility that age-related changes in amino acid levels in various rat brain areas might correlate with modifications of monoamine oxidase (MAO) activity, already found with aging, has been examined. Taurine, aspartic acid and glutamic acid levels were found to be unchanged or decreased with age, whereas GABA and glutamine concentrations increased or remained unchanged. Serine and glycine (except in pons-medulla) levels were found to be unaffected by age. The increase in total MAO activity with aging in some brain areas might contribute to the changes in amino acid levels. Likewise, the possible influence of age-induced changes in activity of various enzymes involved in H2O2 and NH3 detoxication and in amino acid biosynthesis on rat brain amino acid levels are considered. Oral administration of clorgyline or 1-deprenyl to young rats did not significantly modify the concentrations of most brain amino acids.

The effects of lifelong treatment with MAO inhibitors on amino acid levels in rat brain.

In a previous paper a possible relationship had been suggested to exist between age-induced changes in total MAO activity and amino acid levels in some rat brain areas. To further investigate the possible involvement of MAO activity in changes of brain amino acid levels with aging, moclobemide and Ro 19-6327, short acting MAO-A and MAO-B inhibitors, respectively, were administered to female Wistar rats for their whole life-span. Brain amino acid levels in animals treated with MAO inhibitors were compared to those of young and old nontreated rats. The age-induced changes in brain amino acid concentrations found in the present study were in good agreement with those previously reported. Treatment with both moclobemide and Ro 19-6327 was found to restore taurine and serine concentrations in cortex and glutamine concentrations in cerebellum, to the same values as in young rats, to decrease cerebellum concentrations of serine and to increase taurine concentrations in hypothalamus. Administration of moclobemide brought aspartate concentrations in accumbens and cortex back to the same values as in young rats. A similar effect was observed on hypothalamus glutamate concentrations in rats treated with Ro 19-6327. Some possible causes and consequences of the correction of age-induced brain amino acid levels by chronic administration of MAO inhibitors are discussed.

Pharmacokinetics and bioavailability of metergoline in healthy volunteers after single i.v. and oral administration.

Concentrations of unchanged metergoline and its main metabolite, 1-demethylmetergoline, were measured by HPLC and fluorescence detector in the plasma of 13 healthy male volunteers. The subjects received on various occasions the following single-dose metergoline treatments: 4 mg by i.v. infusion (n = 7), 8 mg orally as aqueous solution (n = 7) and 8 mg orally as two different formulations of film-coated tablets (Formulation A, n = 12; Formulation B, n = 12). The mean plasma t 1/2 of metergoline and of 1-demethylmetergoline were about 50 min and 100 min, respectively, independent of the route of administration. A considerable first-pass effect was evident from the data, with about 75% of metergoline being metabolized by the liver before reaching the systemic circulation. However, the availability of the drug in terms of 1-demethylmetergoline was similar for the i.v. and oral routes of administration indicating a complete absorption of the solution from the gastrointestinal tract. Very low plasma levels of another metabolite (12-hydroxymetergoline) were detected in some patients. The bioavailability of film-coated tablets in Formulation B was slightly better than for Formulation A with regard to both relative absorption (A vs B = 82%) and lower interpatient variation. Compared with oral solution, the absorption of Formulation B was slightly slower but practically complete.