Analytical developments of p-hydroxy prenylamine reference material for dope control research: Characterization and purity assessment.

Prenylamine was initially used for the treatment of angina pectoris and later on withdrawn from the market in 1988 due to cardiac arrhythmias concern. The major phase I metabolite of prenylamine is p-hydroxy prenylamine that has a chiral center in the structure. Even though p-hydroxy prenylamine was synthesized earlier, it lacked complete analytical developments for chiral high-performance liquid chromatography (HPLC) separation. However, p-hydroxy prenylamine reference material is not commercially available. The innovation of this manuscript is the development and validation of a chiral HPLC separation method and more extensive characterization of the reference material than previously reported method. Therefore, it was hypothesized to develop and validate normal phase HPLC method for p-hydroxy prenylamine reference material. p-Hydroxy prenylamine was synthesized in two batches and characterized successfully using 13 C NMR, 1 H NMR, high-resolution mass spectrometry (HRMS), Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA). A normal phase chiral HPLC method was developed to analyze the p-hydroxy prenylamine purity. Separation of the p-hydroxy prenylamine enantiomers were achieved using ultra-high-performance liquid chromatography (UHPLC) on a ChiralCel ODH column at wavelength of 220 nm. The developed method was validated in terms of its linearity, accuracy, precision, and robustness for purification, purity assessment, and stability studies. Proton and carbon peaks were confirmed by nuclear magnetic resonance (NMR) analysis. Functional groups were confirmed by FT-IR. Loss on drying was 0.3% and 0.6% for Batches 1 and 2, respectively. The purity of the developed reference material for Batches 1 and 2 was found to be 99.59% and 100%, respectively. Therefore, the synthesized batches of p-hydroxy prenylamine can be used in dope testing as reference material.

Interventions for preventing cardiomyopathy due to anthracyclines: a Bayesian network meta-analysis.

Background: The relative efficacy of interventions for primary prevention of anthracycline-associated cardiotoxicity is unknown. Methods: We conducted a systematic review of randomized controlled trials for primary prevention of anthracycline-associated cardiotoxicity in adult cancer patients. We used hierarchal outcome definitions in the following order of priority: (1) composite of heart failure or decline in left ventricular ejection fraction, (2) decline in ejection fraction, or (3) heart failure. Data were analyzed using a Bayesian network meta-analysis with random effects. Results: A total of 16 trials reported cardiotoxicity as a dichotomous outcome among 1918 patients, evaluating dexrazoxane, angiotensin antagonists, beta-blockers, combination angiotensin antagonists and beta-blockers, statins, Co-enzyme Q-10, prenylamine, and N-acetylcysteine. Compared with control, dexrazoxane reduced cardiotoxicity with a pooled odds ratio (OR) of 0.26 (95% credible interval [CrI] 0.11-0.74) and had the highest probability (33%) of being most effective. No other agent was demonstrably better than placebo. Angiotensin antagonists had an 84% probability of being most effective in a sensitivity analysis excluding one outlying study (OR 0.06 [95% CrI 0.01- 0.24]). When the outcome was restricted to heart failure, dexrazoxane was associated with an OR of 0.12 (95% CrI 0.06-0.23) relative to control and had 58% probability of being most effective, while angiotensin antagonists had an OR of 0.18 (95% CrI 0.05-0.55). Available data suggested that dexrazoxane and angiotensin antagonists did not affect malignancy response rate or risk of death. Conclusion: Moderate quality data suggest that dexrazoxane, and low quality data suggest angiotensin antagonists, are likely to be effective for cardiotoxicity prevention.

Identification and characterization of urinary prenylamine metabolites by means of liquid chromatography-tandem mass spectrometry.

Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin(®) 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%.

Deficiency in phylloquinone (vitamin K1) methylation affects prenyl quinone distribution, photosystem I abundance, and anthocyanin accumulation in the Arabidopsis AtmenG mutant.

Phylloquinone (vitamin K(1)) is synthesized in cyanobacteria and in chloroplasts of plants, where it serves as electron carrier of photosystem I. The last step of phylloquinone synthesis in cyanobacteria is the methylation of 2-phytyl-1,4-naphthoquinone by the menG gene product. Here, we report that the uncharacterized Arabidopsis gene At1g23360, which shows sequence similarity to menG, functionally complements the Synechocystis menG mutant. An Arabidopsis mutant, AtmenG, carrying a T-DNA insertion in the gene At1g23360 is devoid of phylloquinone, but contains an increased amount of 2-phytyl-1,4-naphthoquinone. Phylloquinone and 2-phytyl-1,4-naphthoquinone in thylakoid membranes of wild type and AtmenG, respectively, predominantly localize to photosystem I, whereas excess amounts of prenyl quinones are stored in plastoglobules. Photosystem I reaction centers are decreased in AtmenG plants under high light, as revealed by immunoblot and spectroscopic measurements. Anthocyanin accumulation and chalcone synthase (CHS1) transcription are affected during high light exposure, indicating that alterations in photosynthesis in AtmenG affect gene expression in the nucleus. Photosystem II quantum yield is decreased under high light. Therefore, the loss of phylloquinone methylation affects photosystem I stability or turnover, and the limitation in functional photosystem I complexes results in overreduction of photosystem II under high light.

New cytotoxic-antineoplastic prenyl-1,2-naphthohydroquinone derivatives.

Several new prenylnaphthohydroquinone derivatives have been prepared through the Diels-Alder condensation between alpha-myrcene and 1,2-benzoquinone and evaluated for their cytotoxic activity against A-549, HT-29 and MB-231 cultured cell lines. All of them have shown GI50 values in the microM level.

Amphetamine concentrations in human urine following single-dose administration of the calcium antagonist prenylamine-studies using fluorescence polarization immunoassay (FPIA) and GC-MS.

Prenylamine (R,S-N-(3,3-diphenylpropyl-methyl-2-phenethylamine), a World Health Organization class V calcium antagonist, is known to be metabolized to amphetamine. In this study, amphetamine concentrations after a single-dose administration of prenylamine were determined to check if they reached values that could be of analytical and/or pharmacological importance in clinical and forensic toxicology. Enantiomeric composition of amphetamine was also studied. Five volunteers received a single 120-mg oral dose of prenylamine. Urine samples were analyzed using the Abbott TDx immunoassay Amphetamine/Methamphetamine II and using our routine systematic toxicological analysis (STA) gas chromatography-mass spectrometry (GC-MS) procedure. For quantitation purposes, GC-MS was used in the selected-ion monitoring (SIM) mode (ions m/z 118, 122, 240, 244) after solid-phase extraction (Isolute Confirm HCX) and derivatization (heptafluorobutyric anhydride). Amphetamine-d5 was used as internal standard (IS). Chiral separation of the heptafluorobutyrated amphetamine enantiomers was achieved using an Astec Chiraldex G-PN column. The TDx results showed a great variability for the different volunteers. A urine sample of one volunteer showed results as high as 3200 ng/mL, whereas the urine samples of another volunteer never gave results greater than the TDx detection limit (100 ng/mL). Using the STA procedure, the presence of amphetamine could be confirmed in all urine samples with TDx results greater than the cutoff value (300 ng/mL). Using the GC-MS SIM method, amphetamine concentrations up to 1280 ng/mL were determined. Chiral analysis revealed that both enantiomers of amphetamine were present in the samples with a surplus of the S(+)-enantiomer in the early phase of excretion. Forensic implications are discussed.

Arylalkylamines are a novel class of positive allosteric modulators at GABA(B) receptors in rat neocortex.

Using grease-gap recording from rat neocortical slices, the gamma-aminobutyric acid(B) (GABA(B)) receptor agonists baclofen (3-100 microM) and SKF 97541 (3-aminopropyl-methylphosphinic acid) (1-30 microM) elicited reversible and concentration-dependent hyperpolarizing responses, with EC(50) values of 10 and 3 microM, respectively. The hyperpolarizations were antagonised by the GABA(B) receptor antagonist Sch 50911 ((+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid) (1, 5 and 10 microM). Fendiline (N-[3,3-diphenylpropyl)-alpha-methylbenzylamine) (5-50 microM) and its congeners, prenylamine (N-[3,3-diphenylpropyl)-alpha-methylphenylethylamine) (10-100 microM) and F551 (N-[3,3-diphenylpropyl)-alpha-methyl-3-methoxybenzylamine) (1-30 microM) reversibly enhanced hyperpolarizing responses to the agonists; such effects were reduced by Sch 50911. These arylalkylamines produced leftward shifts of the concentration-response curves, with a marked increase in the maximal hyperpolarization obtained, compared with the agonists alone, F551 being the most potent. These findings suggest that these arylalkylamines represent a new class of positive modulators of GABA(B) receptor-mediated function.

Precursor medications as a source of methamphetamine and/or amphetamine positive drug testing results.

Medical Review Officer interpretation of laboratory results is an important component of drug testing programs. The clinical evaluation of laboratory results to assess the possibility of appropriate medical use of a drug is a task with many different facets, depending on the drug class considered. This intercession prevents the reporting of positive results unless it is apparent that drugs were used illicitly. In addition to the commonly encountered prescribed drugs that yield positive drug testing results, other sources of positive results must be considered. This review describes a series of compounds referred to as "precursor" drugs that are metabolized by the body to amphetamine and/or methamphetamine. These compounds lead to positive results for amphetamines even though neither amphetamine nor methamphetamine were used, a possibility that must be considered in the review of laboratory results. Description of the drugs, their clinical indications, and results seen following administration are provided. This information allows for the informed evaluation of results with regard to the potential involvement of these drugs.

Local anesthetic properties of prenylamine.

BACKGROUND: Local anesthetics that produce analgesia of long duration with minimal impairment of autonomic functions are highly desirable for pain management in the clinic. Prenylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels have not been studied thus far. METHODS: The authors characterized the tonic and use-dependent prenylamine block of native Na(+) channels in cultured rat neuronal GH3 cells during whole cell voltage clamp conditions and the local anesthetic effect of prenylamine by neurologic evaluation of sensory and motor functions of sciatic nerve during neural block in rats. RESULTS: Prenylamine elicits both use-dependent block of Na(+) channels during repetitive pulses (3 microm prenylamine produced 50% block at 5 Hz) and tonic block for both resting and inactivated Na(+) channels. The 50% inhibitory concentration for prenylamine was 27.6 +/- 1.3 microm for resting channels and 0.75 +/- 0.02 microm for inactivated channels. Furthermore, in vivo data show that 10 mm prenylamine produced a complete sciatic nerve block of motor function, proprioceptive responses, and nociceptive responses that lasted approximately 27, 34, and 24 h, respectively. Rats injected with 15.4 mm bupivacaine, a known local anesthetic currently used for pain management, had a significantly shorter duration of blockade (< 2 h) compared with rats injected with prenylamine. CONCLUSIONS: The data presented here demonstrate that prenylamine possesses local anesthetic properties in vitro and elicits prolonged local anesthesia in vivo.

Electrophysiologic effects of lercanidipine on repolarizing potassium currents.

Blockade of cardiac repolarizing potassium channels by drugs may result in QT-interval prolongation, eventually degenerating into "torsades de pointes," a life-threatening arrhythmia. Lercanidipine (LER) is a recently introduced lipophilic calcium antagonist with no cardiodepressant activity and long-lasting antihypertensive action. Its chemical structure is characterized by the presence of a diphenylpropylaminoalkyl group, which is present in some of the drugs that have been reported to cause QT-interval prolongation. Our previous data demonstrated that LER blocks L-type calcium channels without affecting sodium current; however, no data are available concerning its effects on cardiac potassium channels. Transient outward (I(to)), delayed rectifier (I(K)), background currents, and action potential (AP) profile were measured from patch-clamped ventricular myocytes isolated from rat, guinea pig, or human hearts using enzymatic dissociation procedures. LER did not affect I(K) (and I(Kr)) density and activation curve in guinea pig myocytes; the reversal potential of the background current (I(K1)) and its slope were not changed by the drug. Maximal diastolic potential (MDP) and duration of the AP measured at -60 mV (APD(-60)) were not significantly changed. I(to) density and activation curves measured in rat myocytes were similar in the absence and presence of 1 or 10 microM LER. Finally, the effect of LER was tested in human ventricular myocytes: superfusion with 1 microM LER did not affect MDP and APD(-60). I(to) density and the midpoint of activation and inactivation curves were similar in the absence and presence of LER. In conclusion, our data demonstrate that LER does not affect repolarizing potassium currents and action potential profile recorded from guinea pig, rat, and human ventricular myocytes. It is unlikely that LER could cause QT prolongation in vivo.

Characteristics of the inhibitory effect of calmodulin on specific [125i]omega-conotoxin GVIA binding to crude membranes from chick brain.

The characteristics of the inhibitory effect of calcium ion (Ca2+)/calmodulin (CaM) on specific [125I]-omega-conotoxin GVIA (125I-omega-CTX) binding and on the labeling of 125I-omega-CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca2+/CaM depended on the concentrations of free Ca2+ and CaM. The IC50 values for free Ca2+ and CaM were about 2.0 x 10(-8) M and 3.0 microg protein/ml, respectively. The inhibitory effect of Ca2+/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290-309), but not by the calcineurin inhibitor FK506. Ca2+/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca2+ channel alpha1 subunits) with 125I-omega-CTX using a cross-linker. These results suggest that Ca2+/CaM affects specific 125I-omega-CTX binding sites, probably N-type Ca2+ channel alpha1 subunits, in crude membranes from chick whole brain.

Effect of a prenylamine analog (MG8926) on spontaneous action potentials in isolated rabbit sinoatrial node.

Effects of verapamil, prenylamine and a prenylamine analog, MG8926 on the intracellular spontaneous action potentials recorded from the isolated rabbit sinoatrial (SA) node were studied. Verapamil (1 microM), a selective inhibitor for slow Ca2+ channels, prolonged the cycle length, decreased the rate of diastolic depolarization, the rate of rise of action potential, the amplitude of action potential and the maximal diastolic potential, and usually arrested showing subthreshold fluctuation of the membrane potential within several ten min. Prenylamine (10 microM), a nonselective inhibitor for slow Ca2+ channels, tended to prolong the cycle length to decrease the diastolic depolarization, the rate of rise of action potential, the amplitude of action potential. However, these changes were statistically insignificant. Prenylamine at the concentration of 10 microM had no effect on the maximal diastolic potential. MG8926 (10 microM) prolonged the cycle length, decreased the rate of diastolic depolarization, the rate of rise of action potential and tended to decrease the amplitude of action potential. MG8926 at the concentration of 10 microM had almost no effect on the maximal diastolic potential. The present findings may indicate that replacement of phenyl residue of prenylamine by cyclohexyl residue increases the inhibitory action on the slow Ca2+ channels in rabbit SA node.

The importance of calcium-related effects on energetics at hypothermia: effects of membrane-channel antagonists on energy metabolism of rat liver.

During normothermic metabolism, the active pumping of Ca2+ across the cell membrane, mitochondria, and specialized sequestration organelles accounts for a large proportion of total energy expenditure in the cell. This study was designed to determine the effects of Ca2+ channel antagonists (chlorpromazine, verapamil, nifedipine, prenylamine, and nisoldipine) on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) during cold ischemia in rat livers. We hypothesized that if the passive channels were blocked during cold ischemia, then the ATP requirement of active ion pumping would be reduced and ATP levels and energy charge ratios would remain higher throughout the ischemic period; thus, viability of the liver would also be increased after prolonged ischemia. The most positive effect on energy metabolism was observed in the chlorpromazine-treated livers, followed by verapamil treatment. In the chlorpromazine treatment, total adenylate (TA) contents were 0.5-1.0 mumol/g (P < 0.05) higher than the sham group for most of the 24-h time course. Energy charge (EC) ratios were 0.05-0.07 higher than the sham values up to 4-10 h ischemia. Verapamil treatment was less effective, but still exhibited positive effects on TA levels at several time points (20 min, 10 h, and 24 h) throughout the entire 24-h period. In both of these groups, TA values by 24 h ischemia were similar to levels at 10 h in the sham group (3.1 mumol/g), thus showing a considerable effect in maintaining adenylate levels. Despite similar pharmacological antagonist activities, ATP levels in the nifedipine, prenylamine, and nisoldipine treatment groups were 1.0-1.5 mumol/g (P < 0.05) less than the corresponding sham group (without Ca2+ antagonists) over the first 1 h ischemia. The decreases in high energy adenylate levels were reflected in lower EC ratios in these three groups; values were 0.06-0.17 (P < 0.05) lower than corresponding sham values. Finally, it was an unexpected finding that the sham injection (0.5 mg/kg ethanol+PEG400) resulted in the sustained elevation of ATP, total adenylates, and EC values over the first h; EC ratios remained at initial (t = 0) values (EC = 0.71 +/- 0.01) up to 1 h.

[Effects of calcium channel blockers on electrical and mechanical responses of rat phrenic nerve-diaphragm preparations]. / Kalcium-csatorna blokkolók hatása patkány nervus phrenicus-diaphragma preparátum elektromos és mechanikus válaszára.

The effects of diltiazem, prenylamine and verapamil on the electrical and mechanical responses of a rat phrenic nerve-diaphragm preparation were investigated as a function of the indirect stimulating frequency. The amplitude of the muscle compound action potential was depressed by all three drugs with stimulation between 3 and 50 Hz. The contractile force of the diaphragm was increased by these drugs in concentrations of 3 x 10(-8)-3 x 10(-5) mol/l at low frequency (3-10 Hz), but the contractility was decreased at higher concentrations (above 3 x 10(-5) mol/l). In contrast, a facilitating effect was not observed at a higher stimulating frequency (above 20 Hz), but a concentration depressant effect did develop above 1 x 10(-6) mol/l. The literature data and our results suggest that low concentrations of calcium channel blocking agents can not influence the trigger calcium current at low frequency (under 15 Hz), but calcium liberation and the contractile force are increased by these drugs. The intracellular calcium liberation triggered by the calcium current or the muscle force was inhibited by these drugs at higher concentrations. Above 15 Hz, the liberated intracellular calcium is insufficient for the contraction, so an adequate quantity of calcium is obtained from the extracellular space. It is suggested that an adequate calcium uptake is inhibited at low concentrations of these drugs at higher frequency, and the contractility is thereby inhibited.

Female gender as a risk factor for torsades de pointes associated with cardiovascular drugs.

OBJECTIVE: To test the hypothesis that female prevalence is greater than expected among reported cases of torsades de pointes associated with cardiovascular drugs that prolong cardiac repolarization. DATA SOURCES: A MEDLINE search of the English-language literature for the period of 1980 through 1992, using the terms torsade de pointes, polymorphic ventricular tachycardia, atypical ventricular tachycardia, proarrhythmia, and drug-induced ventricular tachycardia, supplemented by pertinent references (dating back to 1964) from the reviewed articles and by personal communications with researchers involved in this field. STUDY SELECTION: Ninety-three articles were identified describing at least one case of polymorphic ventricular tachycardia (with gender specified) associated with quinidine, procainamide hydrochloride, disopyramide, amiodarone, sotalol hydrochloride, bepridil hydrochloride, or prenylamine. A total of 332 patients were included in the analysis following application of prospectively defined criteria (eg, corrected QT [QTc] interval of 0.45 second or greater while receiving drug). DATA EXTRACTION: Clinical and electrocardiographic descriptors were extracted for analysis. Expected female prevalence for torsades de pointes associated with quinidine, procainamide, disopyramide, and aminodarone was conservatively estimated from gender-specific data reported for antiarrhythmic drug prescriptions in 1986, as derived from the National Disease and Therapeutic Index, a large pharmaceutical database; expected female prevalence for torsades de pointes associated with sotalol, bepridil, and prenylamine was assumed to be 50% or less since these agents are prescribed for male-predominant cardiovascular conditions. RESULTS: Women made up 70% (95% confidence interval, 64% to 75%) of the 332 reported cases of cardiovascular-drug-related torsades de pointes, and a female prevalence exceeding 50% was observed in 20 (83%) of 24 studies having at least four included cases. When analyzed according to various descriptors, women still constituted the majority (range, 51% to 94% of torsades de pointes cases), irrespective of the presence or absence of underlying coronary artery or rheumatic heart disease, left ventricular dysfunction, type of underlying arrhythmia, hypokalemia, hypomagnesemia, bradycardia, concomitant digoxin treatment, or level of QTc at baseline or while receiving drug. When cases of torsades de pointes were analyzed by individual drug, observed female prevalence was always greater than expected, representing a statistically significant difference (P < .05) for all agents except procainamide. CONCLUSIONS: These findings strongly suggest that women are more prone than men to develop torsades de pointes during administration of cardiovascular drugs that prolong cardiac repolarization. The pathophysiological basis for, and therapeutic implications of, this gender disparity should be further investigated.

Effects of prenylamine and AQ-A 39 on reentrant ventricular arrhythmias induced during the late myocardial infarction period in conscious dogs.

The effects of prenylamine (PNL) and AQ-A 39 on sustained ventricular tachycardia (SVT) were studied by programmed stimulation in conscious dogs 4-10 days after ligation of the left anterior descending (LAD) coronary artery. In 8 of 16 dogs developing SVT in the control, PNL (3 mg/kg intravenously, i.v.) suppressed inducibility of SVT and slowed the rate of tachycardia in 6 other animals. In a separate group of 10 dogs with inducible SVT, AQ-A 39 (4 mg/kg i.v.) abolished elicitation of tachycardia in 3 dogs and decreased its rate in 6 other dogs. Neither drug affected normal conduction significantly, but PNL impaired slow conduction in the infarct zone, as indicated by prolongation of late potential. Both agents increased the effective refractory period (ERP) of infarcted and normal ventricular myocardium and prolonged the corrected QT interval. PNL and AQ-A 39 exert notable efficacy in preventing infarcted heart from severe ventricular arrhythmias. Prolongation of ventricular refractoriness and repolarization, as well as decreased slow conduction in ischemically damaged myocardium, are major mechanisms accounting for the effectiveness of these drugs against ventricular arrhythmias.

Release of intracellular calcium by prenylamine in human ovarian tumour cells.

The effects of the calcium antagonist prenylamine on intracellular calcium concentration were studied in a human ovarian carcinoma cell line, OVCAR-3. Exposure of cells to 100 microM prenylamine resulted in nearly a 10-fold increase in cytosolic free calcium concentration ([Ca2+]i) as measured by Fura-2 fluorescence. In calcium-free medium, although the increase in [Ca2+]i caused by prenylamine was smaller, it was still substantial compared with the basal level. Efflux experiments with 45Ca showed that 100 microM prenylamine increased calcium efflux by 70% compared with control, indicating active extrusion of the elevated [Ca2+]i. The sluggish nature of calcium release and its independence from the pool activated by ionomycin suggest that the calcium was probably not released from endoplasmic reticulum. These results, although paradoxical, provide a new insight into the possible mechanism of action of prenylamine in causing cancer cell death.

Calcium mobilization and cell proliferation activated by extracellular ATP in human ovarian tumour cells.

The effects of ATP on cell proliferation and intracellular calcium concentration ([Ca2+]i) were examined in a human ovarian cancer cell line (OVCAR-3). Micromolar concentrations of ATP promoted a biphasic rise in [Ca2+]i representing a phase with a rapid peak followed by a phase in which the rise was slower and sustained. When the influx of extracellular calcium was blocked by calcium chelation to EGTA, the ATP stimulated rise in [Ca2+]i was rapid and monophasic. Voltage-sensitive calcium channel blockers like nifedipine and verapamil had no effect on the action of ATP while prenylamine totally blocked calcium influx. ATP inclusion in the medium significantly stimulated growth of OVCAR-3 cells. Fetal calf serum (FCS) increased [Ca2+]i with similar biphasic kinetics representing both the entry of extracellular calcium and release of calcium from intracellular stores. FCS also caused a substantial increase in cell growth. From these experiments it was concluded that an increase in [Ca2+]i is obligatory for stimulation of cell growth in OVCAR-3 cells and that this increase probably requires a contribution from the entry of extracellular calcium. The involvement of both pertussis toxin sensitive G-protein and protein kinase C in ATP induced responses was indicated by the data showing interference of the response by pertussis toxin and phorbol-myristate acetate.

A gas chromatographic/mass spectrometric assay for prenylamine suitable for pharmacokinetic studies of the racemate and the enantiomers.

A sensitive assay for prenylamine and dideuteroprenylamine (racemic or pseudo-racemate) has been developed and used in human pharmacokinetic studies. Plasma levels of prenylamine could be measured up to 50 h after a single oral therapeutic dose. The extracted drug was derivatized with pentafluoropropionic anhydride in acetonitrile. The dried samples were reconstituted in decane; an aliquot was injected into a fused-silica capillary in a cooled on-column injector. The base peaks in the electron impact mass spectra of the compounds--derived by loss of a benzyl radical--at m/z 384, 386 and 390 were measured for prenylamine, (D2)-prenylamine and the internal standard hexahydroprenylamine, respectively. The sensitivity of this assay--limit of detection 0.2 ng ml-1 plasma with a signal-to-noise ratio of 5:1--allowed measurement of the kinetics of the racemate and of both stereoisomers for the first time. In man, the (+)-isomer was eliminated considerably faster than the (-)-prenylamine; the area under the plasma concentration time curve (AUC) of the (+)-isomer was only about 1/4 of the AUC of (-)-prenylamine.

The role of calcium in neutrophil migration: the effect of calcium and calcium-antagonists in electroporated neutrophils.

Chemotaxis by electroporated rabbit peritoneal neutrophils in the absence of Ca2+ is only slightly different from that in the presence of Ca2+. Pretreatment of neutrophils with quin2-AM causes inhibition of chemotaxis. Calcium antagonists as nitrendipine and verapamil are inhibitory in nanomolar concentrations, while 10(5) times higher concentrations are required for inhibition of chemotaxis by neutrophils which were not electroporated. The results support the hypothesis that Ca2+ from Ca(2+)-storing organelles is of importance for chemotaxis, but that chemotaxis is not dependent on changes in cytoplasmic Ca2+ concentrations.