Ivano Rondelli

Simultaneous determination of levodopa methyl ester, levodopa, 3-O-methyldopa and dopamine in plasma by high-performance liquid chromatography with electrochemical detection.

A new procedure is described for the simultaneous determination of levodopa methyl ester (LDME) and its biotransformation products levodopa (L-DOPA), 3-O-methyldopa (3-OMD) and dopamine (DA) in stabilized plasma samples, using reversed-phase high-performance liquid chromatography. A coulometric detector equipped with a dual-electrode system operating in the redox mode was used to simultaneously quantitate all compounds. This system generated a double signal monitored by a dual-channel acquisition data system and allowed quantitation of compounds at the nanogram level. The intra- and inter-assay precision varied in the 2.4-6.9% and 3.2-9.1% ranges respectively, whereas the recoveries were close to 85% for L-DOPA and 3-OMD and 70% for DA and LDME. Samples may be stored at -80 degrees C for 15 days before analysis. The method was applied to plasma samples after oral administration of LDME to rats, but it may also be suitable for human pharmacokinetic studies.

Comparative efficacy of a DA2/α2 agonist and a β-blocker in reducing adrenergic drive and cardiac fibrosis in an experimental model of left ventricular dysfunction after coronary artery occlusion

Attenuation of neuroendocrine activation may be beneficial in congestive heart failure. Sympathetic nervous system overactivity can be reduced by receptors blockade or by reducing norepinephrine (NE) spillover. This study evaluated and compared the effects of a DA2-dopaminergic receptor/alpha2-adrenoceptor agonist (CHF-1024) and a beta1-adrenoreceptor antagonist in terms of hemodynamics, ventricular remodeling, beta-adrenergic drive, and cardiac fibrosis after myocardial infarction (MI) in rats. MI was induced by left coronary artery ligation in 213 rats, whereas 12 were left unoperated on. After 2 months, the operated-on animals were treated for 1 more month with CHF-1024 at either 0.33 mg/kg/day (low dose) or 1 mg/kg/day (high dose) or with metoprolol (10 mg/kg/day), delivered through implanted osmotic minipumps. Plasma concentration and urinary excretion of NE were measured before the rats were killed. Hemodynamic variables were measured and morphometric analysis was done on the diastole-arrested hearts to quantify left ventricular remodeling and interstitial collagen density. Metoprolol treatment tended to normalize LV end-diastolic pressure (LVEDP). CHF-1024 at either dose, and metoprolol, significantly reduced collagen deposition in LV of infarcted animals (from 8.8 +/- 0.5% LV area in vehicle-treated rats to 6.6 +/- 0.2% or 6.4 +/- 0.2% after the low or high dose of CHF-1024, respectively; p < 0.05). Similarly, CHF-1024 at either dose reduced the plasma concentration of NE (from 224 +/- 53 pg/ml to 60 +/- 7 pg/ml or 87 +/- 13 pg/ml; p < 0.05) and urinary excretion of NE in rats with MI, whereas beta-blockade did not affect these variables. In conclusion, CHF-1024 infused for 1 month to rats with LV dysfunction reduced heart rate, NE spillover, and collagen deposition, without unwanted effects, only appearing at the higher dose. Effective beta-blockade with metoprotol reduced LVEDP with no effects on heart function. Neither DA2/alpha2 stimulation nor beta-blockade altered LV remodeling after coronary artery ligation.

Preclinical evaluation of CHF3381 as a novel antiepileptic agent

CHF3381 [n-(2-indanyl)-glycinamide hydrochloride] has been selected on the basis of a screening program as the compound displaying the highest anticonvulsant activity in the maximal electroshock seizure (MES) test and the best therapeutic index with reference to the rotarod test in mice and rats. In this study, the antiepileptic activity and the behavioural toxicity of CHF3381 were characterised in multiple model systems. CHF3381 effectively prevented MES-induced convulsions when administered i.p. (ED50, 24 mg/kg and 7.5 mg/kg) or p.o. (ED50, 21 mg/kg and 21 mg/kg) in both mice and rats, respectively. The time course of oral anti-MES activity in the rat was related to the brain concentration profile of unchanged CHF3381. Interestingly, the brain drug levels were about 4-5 times higher than in plasma. CHF3381 was very effective in mice against picrotoxin-, and i.c.v. N-methyl-D-aspartate (NMDA)-induced hind limb tonic extension (ED50 Approximately/=10 mg/kg), but was a weaker antagonist of 4-amynopyridine- and bicuculline-induced tonic seizures (ED50 approximately/=100 mg/kg), and ineffective against pentylentetrazole- and picrotoxin-induced clonic seizures. CHF3381 antagonised the behavioural effects and lethality of i.p. administered NMDA (ED50 = 57 mg/kg p.o.), indicating that the compound may act as a functional NMDA antagonist. In keeping with this idea, CHF3381 weakly displaced [(3)H]-TCP from binding to NMDA receptor channels (Ki, 8.8 microM). In the rat amygdala kindling model, CHF3381 was more efficient against kindling development than against kindled seizures (minimally active dose = 80 vs. 120 mg/kg i.p). Furthermore, it significantly increased the seizure threshold in kindled rats at relatively low doses (40 mg/kg i.p.). In contrast with MK-801-induced hyperactivity, CHF3381 moderately reduced the spontaneous locomotor activity in mice at anticonvulsant doses. Toxic effects on motor performance (rotarod test) were found at high doses only (TD50 approximately/= 300 mg/kg p.o., congruent with 100 mg/kg i.p. in both mice and rats). Furthermore, CHF3381 did not impair passive avoidance and Morris water maze responding in the therapeutic range of doses. Finally, the development of tolerance after repeated doses was negligible. These data indicate that CHF3381 exerts anticonvulsant and antiepileptogenic effects in various seizure models and possesses good therapeutic window, with scarce propensity to cause neurological side-effects.

Rapid Oral Absorption Profile of Piroxicam from its β-Cyclodextrin Complex

SummaryPlasma concentration-time curves indicate that tablet and sachet formulations of piroxicam-β-cyclodextrin are more rapidly absorbed than capsules of piroxicam (reference formulation). The mean plasma concentrations of piroxicam at 0.25, 0.5 and 1 hour for the tablet, and at 0.25 and 0.5 hours for the sachet, are significantly higher than those for the reference formulation, and the median time to peak plasma concentrations is significantly reduced for the 2 formulations in comparison with the reference formulation.Wagner-Nelson cumulative plots confirm the significant increase in the absorption rate of piroxicam provided by piroxicam-β-cyclodextrin formulations. Absorption of piroxicam was complete within 0.5 hours in 9 of 12 subjects receiving tablets, and within 0.25 hours in 9 of 12 subjects receiving sachets, compared with none of the subjects receiving the reference formulation. As peak plasma concentrations of piroxicam in the sachet study differed from those in the reference study by less than 2% and those in the tablet study were only 5 to 29% higher than reference values, the risk of attaining toxic concentrations was negligible for both formulations.For the other pharmacokinetic variables evaluated, which included area under the plasma concentration-time curve (AUC), mean body residence time (MRT), elimination plasma half-life, clearance, and the apparent volume of distribution, there was little difference between the tablet and reference, and sachet and reference formulations. Enhancement of the absorption rate of piroxicam had no effect on the distribution of the drug.The increase in the rate of absorption of piroxicam obtained with formulations of the complex with β-cyclodextrin may account for the rapid onset of pain relief observed after administration of piroxicam-β-cyclodextrin.

Safety, pharmacokinetics, and pharmacodynamics of CHF 3381, a novel N-methyl-D-aspartate antagonist, after single oral doses in healthy subjects.

A double‐blind, randomized, placebo‐controlled study was performed to assess the safety, tolerability, and pharmacokinetics of single oral doses of CHF 3381 in 56 young healthy male volunteers. The central nervous system effects of CHF 3381 were also evaluated, as well as the effect of food on the rate and extent of CHF 3381 absorption. Seven doses of CHF 3381 (25, 50, 100, 200, 300, 450, and 600 mg) were evaluated in an escalating order. At each dose level, 6 subjects were given CHF 3381, and 2 subjects were given placebo. Safety and tolerability evaluation included adverse events, physical examination, vital functions, electrocardiogram, laboratory tests, and 24‐hour Holter (100‐mg and 450‐mg dose panels). Plasma and urinary concentrations of CHF 3381 and its two main metabolites (CHF 3567 and 2‐aminoindane) were measured with a validated high‐performance liquid chromatography method. Central nervous system effects were evaluated with the simple reaction time (SRT); learning memory task (LMT); Bond & Lader Visual Analog Scale for alertness, contentedness, and calmness; Addiction Research Center Inventory (ARCI); and electroencephalogram. There were no serious adverse events; the most frequent adverse events were dizziness, abnormal thinking, and asthenia. The number of adverse events with moderate intensity increased sharply with the dose, with no or few events up to 450 mg and 17 events with 600 mg. Therefore, 600 mg was defined as the maximum tolerated dose. There were no significant treatment effects on cardiovascular function and electrocardiogram parameters at any CHF 3381 dose or on oral temperature or laboratory tests. There were no clinically significant changes in laboratory variables. CHF 3381 was absorbed rapidly (tmax = 0.5–2 h) and cleared from plasma with a half‐life of 3 to 4 hours. Plasma levels of CHF 3381 and its two major metabolites were found to be proportional to the dose. 2‐Aminoindane formed slowly and reached much lower concentrations compared to the parent compound and the other metabolite (CHF 3567). Within 48 hours after dosing, 2% to 6% of the administered dose was found in the urine as unchanged drug, about 50% to 55% as the acid derivative (CHF 3567), and 2% to 3% as 2‐aminoindane. Ingestion of food did not affect the extent of absorption of the drug, while the rate of absorption was considerably reduced (tmax = 4 h). No significant effects of CHF 3381 were observed on attention (SRT) or memory (LMT). Visual analog scales revealed a decreasing effect of CHF 3381 on alertness at 1 hour that reached statistical significance at 300 and 600 mg. EEG spectral analysis revealed minor decreasing effects of the 200‐mg dose on total electric power measured at 2 hours. A stimulant effect was detected by the ARCI scale 24 hours after the 300‐mg dose and might be related to the slow formation of the 2‐aminoindane metabolite. In conclusion, this study has shown that the maximum tolerated dose of CHF 3381 after single oral administration in young healthy male volunteers is 600 mg. CHF 3381 displays linear pharmacokinetics in the dose range of 25 to 600 mg. The compound is rapidly absorbed and cleared from plasma with a half‐life of 3 to 4 hours. The ingestion of food seems to not affect the extent of absorption of the drug. Minor effects on the central nervous system were detected at doses equal to or greater than 300 mg.

New method for the resolution of the enantiomers of 5,6-Dihydroxy-2-Methyl-Aminotetralin by selective derivatization and HPLC analysis: Application to biological fluids

A new chiral derivatization procedure for the HPLC resolution of chiral catecholamines and structurally related compounds is described. The homochiral reagent, (+)-(R)-1-phenylethyl isocyanate (RPEIC), was added to separate and quantitate the enantiomers of rac-5,6-dihydroxy-2-methyl-aminotetralin, the main metabolite of rac-5, 6-diisobutyryl-2-methyl-aminotetralin, a potent dopamine agonist, by reversed-phase HPLC analysis. To avoid catecholamine degradation in the basic reaction medium and to obtain the selective and quantitative derivatization of the amino group of the compound, the reversible complex formation between diphenylborinic acid (DPBA) and the catechol group, in alkaline medium, was performed before homochiral isocyanate addition. The RPEIC derivatization was completed in 30 min and then the DPBA complex was dissociated by adding dilute acid. The structure of intermediates and urea derivatives was confirmed by mass spectometry. The use of an electrochemical detector, operating in redox mode, allowed HPLC quantitation of enantiomers at the nanogram level in plasma and urine. The derivatization procedure is also suitable for other catecholamine-related compounds.