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PubMed1995

Antipeptide antibodies against overlapping sequences differentially inhibit human CYP2D6.

Two peptides that correspond to sequences within the major 33-amino acid sequence recognized by human liver-kidney microsomal-1 autoantibodies were used to elicit antibodies in rabbits (four per peptide) against CYP2D6. Peptide 1(DPAQPPRDLTEAFLA) corresponded to amino acids 263-277, and peptide 2 (LLTEHRMTWDPAQPPRDLTE) corresponded to amino acids 254-273 of CYP2D6. The peptide-keyhole limpet hemocyanin conjugates elicited good immune responses against their respective peptides as judged by enzyme-linked immunosorbent assay (titers of 1/10,000 to 1/30,000). The antisera recognized CYP2D6 on Western blots and, to varying extents, inhibited recombinant CYP2D6 and liver microsomal CYP2D6 activity. Immunization with peptide 2 produced antisera with the greatest inhibitory potency. Antiserum from a rabbit (#236) immunized with peptide 2 inhibited up to 95% of dextromethorphan O-demethylase activity in human liver microsomes at the highest concentration tested (40% v/v) but did not significantly inhibit CYP1A2, CYP2C9, CYP2E1, or CYP3A4 marker activities. On Western blot, only a single immunoreactive protein comigrating with recombinant CYP2D6 was recognized. In liver microsomes from a CYP2D6-deficient individual, no proteins were recognized, and the antisera did not cross-react with recombinant CYP1A2, CYP2C9, CYP2E1, or CYP3A4. There was a significant correlation between the quantity of immunoreactive CYP2D6 as determined by immunoblotting with anti-peptide 2 antiserum and dextromethorphan O-demethylation in a panel of 10 human liver microsomes (r = 0.95). These data identify a peptide sequence (peptide 2) that can be used to raise antisera that specifically recognize and inhibit CYP2D6.

Amino Acid Sequence↗
PubMed1994

Characterization of specific (+)-[3H]N-allylnormetazocine and [3H]1,3-di(2-tolyl)guanidine binding sites in porcine gastric fundic mucosa.

We have identified and characterized sigma receptor sites in porcine gastric fundic mucosa by receptor binding assay techniques using two highly selective radioligands of sigma receptor, (+)-[3H]N-allylnormetazocine (SKF 10,047) and [3H]1,3-di(2-tolyl)guanidine (DTG). Specific binding of (+)-[3H]SKF 10,047 and [3H]DTG in porcine gastric fundic mucosa were saturable, reversible and of high affinity and capacity with Kd: 90.5 nM, Bmax: 1058 fmol/mg of protein and Kd: 53.6 nM, Bmax: 3573.3 fmol/mg of protein, respectively. The inhibitory effects of sigma receptor ligands on specific (+)-[3H]SKF 10,047 binding decreased in the following order: haloperidol > DTG > or = (+)-3-(3-hydroxyphenol)-N- (1-propyl)piperidine (3-PPP) > (+)-SKF 10,047 > (-)-3-PPP > or = dextromethorphan > rimcazole > (-)-SKF 10,047. Specific (+)-[3H]SKF 10,047 binding sites showed stereoselectivity for stereoisomers of SKF 10,047 and 3-PPP and were highly correlated with the profile of sigma-1 sites. On the other hand, the inhibitory effects on specific [3H]DTG binding decreased in the following order: DTG > haloperidol > rimcazole > (+)-3-PPP > or = (-)-3-PPP > dextromethorphan > (+)-SKF 10,047 = (-)-SKF 10,047. Specific [3H]DTG binding sites did not show stereoselectivity and were highly correlated with the profile of sigma-2 sites. These findings indicate that porcine gastric fundic mucosa contains sigma receptor sites with the characteristic of sigma-1 sites and sigma-like sites showing several of the characteristics of sigma-2 sites (putative sigma-2 sites).

Animals↗
PubMed1993

Identification of a 27-kDa high affinity phenylalkylamine-binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand-directed antibodies.

The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047]. The apparent affinities of these and other drugs closely corresponded to those for 1,3-[3H]ditolylguanidine-labeled sigma binding sites. Based on its high affinity for the (+)-enantiomer [but not the (-)-enantiomer] of SKF10,047 (Ki = 51 nM), pentazocine (Ki = 3 nM), and dextromethorphan (Ki = 30 nM), the (-)-[3H]azidopamil-labeled site on the 27-kDa polypeptide was classified as being of the sigma 1 subtype. Using antiphenylalkylamine antibodies, we developed a novel immunological detection method that allows the rapid and sensitive staining of the photolabeled 27-kDa polypeptide after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the phenylalkylamines emopamil and azidopamil represent a novel class of sigma ligands, highly suitable for the further structural characterization of polypeptides carrying sigma 1 binding sites.

Affinity Labels↗
PubMed1995

Bioequivalence and absolute bioavailability of oblong and coated levomepromazine tablets in CYP2D6 phenotyped subjects.

The bioequivalence and absolute bioavailability of oblong and coated levomepromazine tablets were studied in 12 healthy volunteers. A 1-hour intravenous infusion served as the reference. Serum concentrations of levomepromazine were quantified with a specific high-performance liquid chromatographic method and electrochemical detection. The 2 oral formulations were bioequivalent. After oral administration of oblong and coated levomepromazine tablets the mean serum concentration versus time profiles were similar and the pharmacokinetic parameters showed wide interindividual variations. There was a 21% absolute bioavailability of levomepromazine, indicating a pronounced presystemic metabolism. The total serum clearance and the apparent volume of distribution at steady state were 48 +/- 14 l/min and 980 +/- 213 l, respectively. These pharmacokinetic parameters were also investigated with respect to the CYP2D6 polymorphism, i.e. via dextromethorphan phenotyping of 9 subjects, 3 subjects were poor metabolizers, and 6 extensive metabolizers. The Spearman's rank-ordered correlation analysis did not reveal a significant correlation between the pharmacokinetic parameters AUC, Cmax and t1/2 after oral administration of oblong and coated levomepromazine tablets and the metabolic ratios of dextromethorphan, suggesting that levomepromazine is not metabolized to any major extent by the isoenzyme CYP2D6.

Administration, Oral↗
PubMed1998

Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities.

There is a need for methodology to predict clinically significant drug-drug interactions so that clinical studies can be directed toward interactions which are likely to be clinically relevant. To this end, we evaluated selective assays for the seven drug-metabolizing cytochrome P450 (P450) isozymes 1A2 (caffeine N3-demethylation), 2A6 (coumarin 7-hydroxylation), 2C9 (tolbutamide hydroxylation), 2C19 (S-mephenytoin 4-hydroxylation), 2D6 (dextromethorphan O-demethylation), 2E1 (chlorzoxazone 6-hydroxylation), and 3A4/5 (dextromethorphan N-demethylation). Using initial rate conditions, we determined the Km and Vmax values of each reaction in human liver microsomes from three individuals. Because organic solvents (usually methanol) are frequently used as solubilization aids for drugs/inhibitors, we also screened several solvents for inhibitory activity. Methanol was the least inhibitory toward P450s 2A6, 2D6, and 3A4, dimethylformamide was the least inhibitory toward P450s 1A2 and 2C9, and acetonitrile was the least inhibitory toward P450s 2C19 and 2E1. Using substrate concentrations close to the determined Km and an appropriate solvent (where necessary), we used the selective inhibitors furafylline (1A2), 8-methoxypsoralen (2A6), sulfaphenazole (2C9), S-mephenytoin (2C19), quinidine (2D6), diethyldithiocarbamate (2E1), and troleandomycin (3A4) to assess the limitations of each probe assay as an indicator of the P450 isoform in question. Our results were consistent with these inhibitors and probes, being selective tools for studying P450 drug metabolism.

Acetonitriles↗
PubMed1976

Antitussive properties of butorphanol.

Butorphanol (levo-N-cyclobutylmethyl-3, 14-dihydroxy morphinan), a potent analgetic agent of the narcotic antagonist type with a low abuse potential in laboratory animals, was evaluated for antitussive activity in unanesthetized guinea-pigs and dogs. Subcutaneously, it was over 100 times more active than codeine, dextromethorphan and dl-pentazocine and about 20 times more active than morphine in the guinea-pig, while in the dog it was 100, 10 and 4 times more active than codeine, dl-pentazocine and morphine, respectively. Orally, butorphanol was 15-20 times more active than either codeine or dextromethrophan in both species. Naloxone reversed the antitussive effects of butorphanol, codeine, morphine and dl-pentazocine while those of dextromethorphan were not antagonized. The antitussive effect of butorphanol and morphine lasted about 4 hr and both compounds were longer acting than codeine. Butorphanol was also shown to be as effective against cough of pathological origin as against experimentally induced cough in the dog.

Animals↗
PubMed1998

Effects of gestational and overt diabetes on human placental cytochromes P450 and glutathione S-transferase.

The placenta possesses the ability to metabolize a number of xenobiotics and endogenous compounds by processes similar to those seen in the liver. Animal and in vivo studies have observed that the presence of diabetes alters the expression of hepatic metabolizing enzymes (cytochrome P450 and glutathione S-transferase); however, it is unknown whether similar alterations occur in the human placenta. To evaluate whether diabetes has any effect of placental xenobiotic metabolizing activity, the catalytic activities of 7-ethoxyresorufin O-deethylation (EROD, CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2, 4-dinitrobenzene (CDNB) conjugation with glutathione (glutathione S-transferase, GST) from placentas of diet (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared with matched controls. EROD activity (CYP1A1) ranged from 0.29 to 2.67 pmol/min/mg protein. However, no differences were observed among overt or gestational diabetics and their respective matched controls. CDNB conjugation (GST) ranged from 0.275 to 1.65 units/min/mg protein. In contrast to that observed with CYP1A1, a small but statistically significant reduction in GST activity was noted in overt diabetics as compared with their matched controls and gestational diabetics. CYP2E1, 2D6, and 3A4 enzymatic activities were not detected in human placental tissue. GST protein was detectable in all tissues studied, but no CYP protein could be detected in any of the tissues. Thus, it seems that pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in the exposure of the fetus to harmful electrophiles. However, the full clinical significance of this finding remains to be elucidated.

Adolescent↗
PubMed1998

Interaction of terfenadine and its primary metabolites with cytochrome P450 2D6.

The substrate structure-activity relationships described for the major human drug-metabolizing cytochrome P450 (P450 or CYP) enzymes suggest that the H1 receptor antagonist terfenadine could interact with CYP2D6 either as a substrate or as an inhibitor, in addition to its known ability to act as a substrate for CYP3A4. Based on this substrate structure-activity relationship, computer modeling studies were undertaken to explore the likely interactions of terfenadine with CYP2D6. An overlay of terfenadine and dextromethorphan, a known substrate of CYP2D6, showed that it was possible to superimpose the site of hydroxylation (t-butyl group) and the nitrogen atom of terfenadine with similar regions in dextromethorphan. These observations were substantiated by the ease of docking of terfenadine into a protein model of CYP2D6. Experimentally, terfenadine inhibited CYP2D6 activity in human liver microsomes with an IC50 of 14-27 microM, depending on the CYP2D6 substrate used. The inhibition of CYP2D6 was further defined by determining the Ki for terfenadine against bufuralol 1'-hydroxylase activity in four human livers. Terfenadine inhibited bufuralol 1'-hydroxylase activity with a Ki of approximately 3.6 microM. The formation of the hydroxylated metabolite (hydroxyterfenadine) in microsomes prepared from human liver and specific P450 cDNA-transfected B lymphoblastoid cells indicated that only CYP2D6 and CYP3A4 were involved in this transformation. As expected, the rate of formation was greatest with CYP3A4 (Vmax = 1257 pmol/min/nmol of P450), with CYP2D6 forming the metabolite at a 6-fold lower rate (Vmax = 206 pmol/min/nmol of P450). The two enzymes had similar KM values (9 and 13 microM, respectively). These data indicate that, as predicted from modeling studies, terfenadine has the structural features necessary for interaction with CYP2D6.

Chromatography, High Pressure Liquid↗
PubMed2006

Randomized, controlled trial of dextromethorphan/quinidine for pseudobulbar affect in multiple sclerosis.

OBJECTIVE: To evaluate the efficacy and safety of DM/Q (capsules containing dextromethorphan [DM] and quinidine [Q]) compared with placebo, taken twice daily, for the treatment of pseudobulbar affect over a 12-week period in multiple sclerosis patients. METHODS: A total of 150 patients were randomized in a double-blind, placebo-controlled study to assess pseudobulbar affect with the validated Center for Neurologic Study-Lability Scale. Each patient also recorded the number of episodes experienced between visits, estimated quality of life and quality of relationships on visual analog scales, and completed a pain rating scale. RESULTS: Patients receiving DM/Q had greater reductions in Center for Neurologic Study-Lability Scale scores than those receiving placebo (p < 0.0001) at all clinic visits (days 15, 29, 57, and 85). All secondary end points also favored DM/Q, including the number of crying or laughing episodes (p <or= 0.0077), quality of life (p < 0.0001), quality of relationships (p = 0.0001), and pain intensity score (p = 0.0271). DM/Q was well tolerated; only dizziness occurred with greater frequency than with placebo. INTERPRETATION: Results in multiple sclerosis patients were similar to those of a previous study in amyotrophic lateral sclerosis, demonstrating that DM/Q may be beneficial in treating potentially disabling pseudobulbar affect in a variety of neurological disorders.

Affect↗
PubMed2007

Drug release properties of polymer coated ion-exchange resin complexes: experimental and theoretical evaluation.

Although ion-exchange resins have been used widely as drug delivery systems, their exact release kinetics has not been reported yet. Usually only the rate-limiting step has been taken into account and the rest of the steps have been ignored as instantaneous processes. To investigate the exact release kinetics of polymer-coated drug/ion-exchange resin complexes for sustained drug delivery, the results of new mathematical modeling were compared with experimental results. Drug/resin complexes with a model drug, dextromethorphan, were prepared and used as cores for fluid-bed coating. An aqueous colloidal dispersion of poly(vinyl acetate) was applied for the coating. A comprehensive mathematical model was developed using a mechanistic approach by considering diffusion, swelling, and ion-exchange processes solved by numerical techniques. The rate-limiting factor of the uncoated resin particles was diffusion through the core matrix. Similarly, in the coated particles the rate-limiting factor was diffusion through the coating membrane. The mathematical model has captured the phenomena observed during experimental evaluations and the release dynamics from uncoated and coated (at different coat levels) particles were predicted accurately (maximum RMSE 2.4%). The mathematical model is a useful tool to theoretically evaluate the drug release properties from coated ion-exchange complexes thus can be used for design purposes.

Anion Exchange Resins↗
PubMed2003

Supraspinal N-methyl-D-aspartate receptor inhibition influences the micturition reflex and function of the upper urinary tract of anesthetized and conscious rats.

AIMS: To examine the effects of N-methyl-D-aspartate (NMDA) receptor antagonists on the frequency/volume (F/V) characteristics of micturition of conscious and anesthetized rats in relation to the mechanisms of renal urine output and pyelo-ureteral transport function of the upper urinary tract. METHODS: Micturition F/V characteristics of 24 conscious female Sprague-Dawley rats, each weighting 160-180 g, were evaluated in a metabolic chamber. Control values of (F/V) were first obtained after administration of a 5-mL loading dose of saline solution and compared with intraperitoneal (i.p.) injection of 0.3-6.0 mg/kg of LY274614 (LY) and 0.3-40 mg/kg of dextromethorphan (DEX). Upper urinary tract studies were performed on two groups of urethane anesthetized rats (1.2 g/kg, s.c.). A group of 17 rats was used for intravenous (i.v.) injection of 1-30 mg/kg of LY and 1-10 mg/kg of DEX. In a second group of 12 rats, 0.2 mg/kg of LY and DEX were administered intracerebroventricularly (i.c.v.), by placing a catheter into the third ventricle. In anesthetized rats, continuous cystometrograms (CMG) were done while perfusing the renal pelvis with indigo carmine, and measuring pelvic and arterial pressure and as well as visualizing the transport of urine within the upper urinary tract by using videomicroscopic imaging. RESULTS: In conscious rats, the i.p. injection of LY and DEX produced a significant and dose-dependent increase in functional bladder capacity and a considerable increase in diuresis. In anesthetized rats, i.v. and i.c.v. administration of LY and DEX affected the micturition reflex, reflected as a significant increase in bladder capacity and preleakage pressure. Videomicroscopic imaging shows dilation of the ureter and a dilution of the dyed urine after the i.v. and i.c.v. injection of LY and DEX. The frequency of ureteral peristalsis and renal pelvic contractions were significantly decreased after i.v. and i.c.v. injection of LY as well as DEX. CONCLUSIONS: The present study shows that the NMDA receptor antagonists LY and DEX given systemically (i.v. and i.p.) or intracerebroventricularly influence the micturition reflex and modulate pyelo-ureteral motility as well as the rate of urine production. From the present study, it is suggested that NMDA receptors are directly involved in regulating pyelo-ureteral peristalsis as well as in the coordination of urine transport between upper and lower urinary tract.

Anesthesia↗
PubMed2005

Functional analysis of CYP2D6.31 variant: homology modeling suggests possible disruption of redox partner interaction by Arg440His substitution.

Cytochrome P450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (microM) and kcat (min(-1))] of debrisoquine 4-hydroxylation (wild-type, 25; 0.92; variants, 43-68; 0.05-0.11) and dextromethorphan O-demethylation (wild-type, 1; 4.72; variants, 12-23; 0.64-1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild-type enzyme. The rates of oxidation of rac-metoprolol at single substrate concentrations of 40 and 400 microM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding.

Amino Acid Substitution↗
PubMed2005

High-throughput biological sample analysis using on-line turbulent flow extraction combined with monolithic column liquid chromatography/tandem mass spectrometry.

A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, which combines on-line sample extraction through turbulent flow chromatography with a monolithic column separation, has been developed for direct injection analysis of drugs and metabolites in human plasma samples. By coupling a monolithic column into the system as the analytical column, the method enables running 'dual-column' extraction and chromatography at higher flow rates, thus significantly reducing the time required for the transfer and mixing of extracted fraction onto the separation column as well as the time for gradient separation. A strategy of assessing and reducing the matrix suppression effect on the on-line extraction LC/MS/MS has also been discussed. Experiments for evaluating the resolution, peak shape, sensitivity, speed, and matrix effect were conducted with dextromethorphan and its metabolite dextrorphan as model compounds in human plasma matrix. It was demonstrated that the total run time for this assay with a baseline separation of two analytes is less than 1.5 min.

Analgesics, Opioid↗
PubMed2001

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1.

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.

Aryl Hydrocarbon Hydroxylases↗
PubMed2005

Phenytoin differentially modulates the affinity of agonist and antagonist ligands for sigma 1 receptors of guinea pig brain.

We evaluated the effects of phenytoin (DPH) on the affinity for sigma-1 (sigma(1)) receptors of agonist or antagonist sigma(1) ligands in guinea pig brain. Heterologous competition experiments showed that DPH (250 microM and 1 mM) concentration-dependently increased the affinity of the sigma(1) agonists dextromethorphan, (+)-SKF-10,047, (+)-3-PPP, and PRE-084. However, neither DPH 250 microM nor 1 mM increased (in fact, they slightly decreased) the affinity of the sigma(1) receptor antagonists haloperidol, BD 1063, NE-100, progesterone, and BD 1047. These findings suggest that allosteric modulation by DPH of the affinity of sigma(1) receptor ligands depends on the agonist or antagonist characteristics of the ligand. Therefore, determining in vitro the differential modulation by DPH of sigma(1) ligand affinity appears to constitute a procedure that can predict the pharmacological profile of different sigma(1) ligands.

Allosteric Regulation↗
PubMed1999

N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture.

Whereas excessive activation of the NMDA receptor may contribute to ischemic neuronal injury, physiologic activation may promote neuronal survival under certain conditions. Consistently, it has recently been shown that NMDA antagonists induce apoptosis of central neurons in immature rats. In the present study, we have examined whether NMDA antagonists induce neuronal apoptosis also in a culture condition. Exposure of cortical cultures (DIV 10-13) to MK-801 (1-10 microM) for 48 h resulted in death of about 30-40% of neurons. Similar neuronal death was induced by exposure to other NMDA antagonists, D-AP5 and dextromethorphan. The neuronal death was dependent on the culture age; MK-801 induced much less neuronal death in younger (DIV 7) and older (DIV 16-19) cultures. The NMDA antagonist-induced neuronal death was accompanied by cell body shrinkage, nuclear fragmentation, and cleavage/activation of caspase-3. Furthermore, it was attenuated by cycloheximide and zVAD-fmk, indicating that the death occurred mainly by the apoptosis mechanism. As in several other apoptosis models, high-potassium medium blocked the NMDA antagonist-induced apoptosis, which was reversed by voltage-gated calcium channel blockers. The present results demonstrate that NMDA antagonists induce neuronal apoptosis in cortical culture, consistent with the findings obtained in immature rats. Since the activation of the voltage-gated calcium channels attenuated the NMDA antagonist-induced apoptosis, it may be another example of the "calcium set point hypothesis."

2-Amino-5-phosphonovalerate↗
PubMedDate not supplied

Evaluation of antitussive agents in man.

Methodology to evaluate the efficacy of antitussive drugs rely largely on subjective methods and cough counts. There are few studies in cough due to natural disease especially using objective techniques. This paper presents data from a series of randomized, double blind, placebo controlled clinical trials in cough due to both chronic bronchopulmonary disease and acute upper respiratory tract infections. In these studies, cough was quantified using a standardized and validated computerized system for the acquisition and multidimensional analysis of the cough sound. Key objective parameters like cough counts, intensity, latency and total effort expended were studied. Guaiphenesin and bromhexine showed significant expectorant effects in patients with productive cough due to chronic bronchopulmonary disease. Differences were observed in speed of action, and objective and subjective measures, that probably indicate differences in drug action. More recently, three studies evaluated the antitussive drug dextromethorphan in non-productive cough due to uncomplicated upper respiratory tract infections. Reproducible cough suppressant effects were demonstrated after a single 30 mg dose using objective measures of cough counts, latency and total effort. These results establish the sensitivity and robustness of the cough quantitation methodology in the objective evaluation of cough treatments.

Acute Disease↗
PubMed2001

The placebo response to citric acid-induced cough: pharmacodynamics and gender differences.

Characteristics of the response to placebo in a citric acid-induced cough challenge were investigated as part of a randomized, double-blind crossover trial to assess the antitussive effect of dextromethorphan. Baseline cough responses were established on two occasions in 22 healthy subjects. They received 60 ml placebo antitussive syrup and cough frequency following five inhalations of 10% citric acid over 5 min was measured at regular intervals up to 12 h. Response-time models of varying complexity were used to describe the placebo cough suppression data. The cough response to placebo was also compared to that of the untreated state. The placebo cough response was best characterized by a non-linear increase in cough suppression up to a maximum reduction of 1.6 coughs from baseline at 4-4.5 h, followed by a non-linear return to baseline. The cough response in the untreated state was not different from that of placebo (P=0.99). Females coughed more frequently than males (median number of coughs=10.5 vs. 9.0, respectively P<0.001; Mann-Whitney U test), and adaptation to the cough stimulus was significantly more rapid in females (P<0.025). Accordingly, in trials that use citric acid-induced cough, gender should be considered in study design, particularly in relation to the timing of measurements.

Administration, Inhalation↗