Enhancing the uptake of dextromethorphan in the CNS of rats by concomitant administration of the P-gp inhibitor verapamil

Clinical trials evaluating high doses of dextromethorphan hydrobromide (DM) for the treatment of neurological disorders have resulted in numerous adverse events due to the presence of its active metabolite dextrorphan (DX). Since the uptake of drugs in the CNS can be modulated by P-glycoprotein (P-gp) inhibition at the blood-brain barrier (BBB), we propose to determine whether the P-gp inhibitor verapamil can enhance the uptake of DM in the CNS. Rats (n=42) received an oral dose of DM (20 mg/kg) alone or 15 min after an intravenous dose of verapamil (1 mg/kg). Rats were euthanized at different time points over 12 h, and concentrations of DM and DX (conjugated and unconjugated) were assessed in plasma, brain and spinal cord using a LC-ESI/MS/MS method. Pharmacokinetic parameters were calculated using noncompartmental methods. Verapamil treatments did not affect the biodisposition of DM in plasma. On the other hand, verapamil treatments increased the area under curve of DM in the brain (from 1221 to 2393 ng h/g) and spinal cord (from 1753 to 3221 ng h/g) by approximately 2-fold. The uptake of DX in brain and spinal cord were markedly lower than those of DM and increased by only 15% and 22% following verapamil treatments, respectively. These results suggest that the P-gp inhibitor verapamil can enhance the uptake of DM in the CNS without affecting that of DX. This change is most likely related to an inhibition of P-gp or other transporters located in the BBB since the biodisposition of DM in plasma remained unaffected by verapamil treatments.     

Changes in Histamine Metabolism in the Mouse Hypothalamus Induced by Acute Administration of Ethanol

The effect of acute ethanol administration on histamine (HA) dynamics was examined in the mouse hypothalamus. The steady-state level of HA did not change after intraperitoneal administration of ethanol (0.5-5 g/kg), whereas the level of tele-methylhistamine (t-MH), a predominant metabolite of brain HA, increased when 3 and 5 g/kg of ethanol was given. Pargyline hydrochloride (80 mg/kg, i.p.) increased the level of t-MH by 72.2% 90 min after the treatment. Ethanol at any dose given did not significantly affect the t-MH level in the pargyline-pretreated mice. Decrease in the t-MH level induced by metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, was suppressed by ethanol (5 g/kg), thereby suggesting inhibition of the elimination of brain t-MH. Ethanol (5 g/kg) significantly delayed the depletion of HA induced by (S)-alpha-fluoromethylhistidine (50 mg/kg, i.v.), a specific inhibitor of histidine decarboxylase. Therefore, a large dose of ethanol apparently decreases HA turnover in the mouse hypothalamus.     

The effect of ozagrel sodium on photochemical thrombosis in rat: therapeutic window and combined therapy with heparin sodium

The therapeutic efficacy of ozagrel sodium (ozagrel), alone and in combination with heparin, and its therapeutic time window were studied in a photochemically induced thrombotic cerebral infarction rat model. Cerebral artery thrombosis was induced by irradiating the brain with green light through intact skull using rose bengal as the photosensitizing dye. One set of animals was treated immediately after thrombosis with (1) vehicle, (2) 10 mg/kg ozagrel in saline, intravenously (i.v.), (3) 150 U/kg unfractioned heparin, subcutaneously (s.c.), or (4) ozagrel, i.v. plus heparin, s.c. Infarct volume was significantly smaller and edema was reduced in the ozagrel-treated groups compared to the vehicle-treated group; heparin did not convey additional benefit. In another set of animals, rats were given either vehicle or 10 mg/kg ozagrel in saline, i.v., 60 min or 120 min after induction of thrombosis. Ozagrel reduced infarct volume, but its effect diminished with delayed administration. The therapeutic window was determined to be less than 60 minutes after induction of thrombosis.     

Effect of verapamil on ventilation and chemical control of breathing in anesthetized rats

The calcium channel blocker, verapamil (0.1-1.0 mg/kg, i.v.) was administered to anesthetized rats to determine its effects on ventilation and on ventilatory responses to hypoxia and CO2. Verapamil produced a dose-dependent increase in tidal volume (VT) and a decrease in respiration rate (f). The bradypnea due to verapamil was characterized by an increase in expiratory duration (TE) and no change of inspiratory duration (TI). Verapamil produced similar changes in VT and f in vagotomized rats. The increase in respiration rate and minute volume due to hypoxia were inhibited by verapamil (0.5 and 1.0 mg/kg) but the increase in tidal volume due to hypoxia was depressed only with the 1.0 mg/kg dose. On the other hand, the increase in VT due to breathing CO2 was not changed by verapamil (0.1-1.0 mg/kg), but depression of the respiratory frequency response to CO2 occurred with 1.0 mg/kg of verapamil. These results indicate that verapamil produced slow, deep breathing and these responses were not mediated by vagal mechanisms. Ventilatory responses to hypoxia were depressed by verapamil. However, since the calcium blocker demonstrated no effect on the VT-CO2 relationship, verapamil did not change ventilatory chemosensitivity to CO2. The data also suggest that mechanisms governing the control of respiratory frequency are more sensitive to verapamil than tidal volume responses.     

Strain and sex differences in repeated ethanol treatment-induced motor activity in quasi-congenic mice

The B6.C quasi-congenic Recombinant QTL Introgression (RQI) strains of the b4i5 series have similar genetic background, but differ in about 5% of their genome from the C57BL/6ByJ (B6) background strain because they carry short chromosome segments introgressed from the BALB/cJ (C) donor strain. These RQI strains were derived from mouse lines selectively bred for high activity of mesencephalic tyrosine hydroxylase (TH/MES), therefore genetic variation in dopamine system-related behaviours, such as ethanol-induced motor activity, can be expected. Males and females of 17 RQI and two progenitor strains were tested for initial motor activity for 15 min after a habituating injection of saline, which was followed by an i.p. injection of saline or ethanol (2 g/kg) and an additional test of motor activity for 30 min. This procedure was repeated during 4 subsequent days. In all strains, the first-day ethanol treatment showed an inhibitory effect. With repetition of the treatment the inhibitory effect decreased, and a stimulatory effect could be observed with significant strain- and sex-dependent variation. Females exhibited higher activity in the saline group than males, and reached an equilibrium of inhibition and stimulation sooner than males with repetition of the ethanol treatment. The highest (> 25-fold) difference in activity after repeated ethanol treatment was detected between females of the two strains B6.Cb4i5-Alpha4/Vad and B6.Cb4i5-Beta13/Vad. These results firstly suggest that females are more sensitive to repeated ethanol exposure than males, secondly they support the observations that ethanol has both inhibitory and stimulatory effects on motor activity, which are affected by sex, genotype, and repetition of treatment, and thirdly offer new quasi-congenic animal models with highly different responses to ethanol allowing one to more quickly move to gene detection.     

Development of tolerance to ethanol-induced suppression of breathing movements and brain activity in the near-term fetal sheep during short-term maternal administration of ethanol

The effect of short-term maternal ethanol administration on the ethanol-induced suppression of fetal breathing movements, electrocortical (ECoG) activity, and electroocular (EOG) activity was determined in the near-term fetal sheep. Twelve conscious instrumented pregnant ewes (between 125 and 139 days of gestation; term, 147 days) received 1-h intravenous infusion of 1 g ethanol/kg total body weight daily for six days (n = 6) or an equivalent volume of normal saline daily for six days (n = 6). On the seventh day, the ethanol- and saline-pretreated animals were administered 1 g ethanol/kg total body weight. A further six ewes received 1-h intravenous infusion of 1 g ethanol/kg total body weight (n = 3) or an equivalent volume of normal saline (n = 3) daily for thirteen days with both groups receiving 1 g ethanol/kg total body weight on day fourteen. Fetal ECoG and EOG activities, and fetal breathing movements were monitored continuously over the post- operative and experimental periods. Saline infusion had no significant effect on the parameters studied. Fetal breathing movements were suppressed for 8 h after the first ethanol dose, and were not significantly suppressed after fourteen days of once-daily, maternal ethanol administration. Low-voltage ECoG and EOG activities were suppressed for 3 h after the first ethanol dose, and were not significantly suppressed after seven days of repeated ethanol administration. Maternal and fetal blood gases and acid-base balance were not significantly affected by maternal ethanol administration. These data demonstrate that short-term maternal administration of ethanol results in the development of tolerance to ethanol in the mature fetus.     

Ethanol-induced hypothermia and ethanol consumption in the rat are affected by low-level microwave irradiation

Microwave irradiation of rats by circularly polarized, 2,450-MHz, pulsed waves (2-μs pulses; 500 pps) was performed in waveguides to determine effects on ethanol-induced hypothermia and on ethanol consumption. Rats injected intraperitoneally with ethanol (3 g/kg in a 25% v/v water solution) immediately after 45 min of microwave irradiation exhibited attenuation of the initial rate of fall in body temperature, which was elicited by the ethanol, but exhibited no significant difference in maximal hypothermia as compared with that of sham-irradiated rats. Microwave irradiation did not affect the consumption of a 10% sucrose (w/v) solution by water-deprived rats. However, it enhanced the consumption of a solution of 10% sucrose (w/v) + 15% ethanol (v/v) by water-deprived animals. These results were obtained at a specific absorption rate (SAR) of 0.6 W/kg, which rate of energy dosing would require a power density of 3–6 mW/cm² if exposure of the animals had occurred to a 12-cm plane wave.     

Effects of hypotension and fluid depletion on central angiotensin-induced thirst and salt appetite

We examined the effects of hypotension and fluid depletion on water and sodium ingestion in rats in response to intracerebroventricular infusions of ANG II. Hypotension was produced by intravenous infusion of the vasodilator drug minoxidil (25 microg x kg(-1) x min(-1)) concurrently with the angiotensin-converting enzyme inhibitor captopril (0.33 mg/min) to prevent endogenous ANG II formation. Hypotension increased water intake in response to intracerebroventricular ANG II (30 ng/h) but not intake of 0.3 M NaCl solution and caused significant urinary retention of water and sodium. Acute fluid depletion was produced by subcutaneous injections of furosemide (10 mg/kg body wt) either alone or with captopril (100 mg/kg body wt sc) before intracerebroventricular ANG II (15 or 30 ng/h) administration. Fluid depletion increased water intake in response to the highest dose of intracerebroventricular ANG II but did not affect saline intake. In the presence of captopril, fluid depletion increased intakes of both water and saline in response to both doses of intracerebroventricular ANG II. Because captopril administration causes hypotension in fluid-depleted animals, the results of the two experiments suggest that hypotension in fluid-replete animals preferentially increases water intake in response to intracerebroventricular ANG II and in fluid-depleted animals increases both salt and water intake in response to intracerebroventricular ANG II.     

Nimodipine's interactions with other drugs: I. Ethanol

Adult mice (Binghamton Heterogeneous stock) received different doses of ethanol (0.5, 1.0, or 2.0 g/kg) administered alone or in combination with the voltage-sensitive calcium channel antagonist, nimodipine (Bay e 9736). Both 20 and 60 minutes later, sensitivity to ethanol was assessed in terms of rotorod activity and changes in rectal temperatures. Nimodipine (5 mg/kg) alone did not alter rectal temperature or motor coordination, but at both observation periods nimodipine potentiated the hypothermia induced by the highest dose of alcohol (2.0 g/kg) and exaggerated alcohol-induced motor incoordination at all doses. The present set of results indicates that the inhibition of voltage-dependent calcium channels can exaggerate ethanol-induced effects.     

Microwave attenuation of ethanol-induced hypothermia: ethanol tolerance, time course, exposure duration, and dose response studies

Four experiments were conducted to quantify the reported attenuation by microwave (MW) irradiation of ethanol-induced hypothermia. In one experiment rats were irradiated (continuous wave 2.45 GHz, specific absorption rate = 0.3 W/kg) or sham irradiated for 45 min, injected with 3.6 g/kg, 20% (v/v) ethanol (EtOH) or saline (NaCl) i.p.. Colonic temperature was monitored at 20-min intervals for 2 h. This procedure was repeated for 8 days to determine the rate of tolerance development to the hypothermic effect of ethanol. While MW irradiation did significantly attenuate EtOH-induced hypothermia, it did not enhance or retard the rate of tolerance development. To determine the duration of irradiation necessary to attenuate EtOH-induced hypothermia, groups of rats were irradiated or sham irradiated for 5, 15, 30, or 60 min prior to EtOH injection and subsequent temperature measurements. The attenuation was apparent only after 60 min of irradiation. To determine the duration of the attenuation effect after irradiation, rats were injected with EtOH or NaCl at 0, 30, 60, 120, or 480 min after 45 min of irradiation or sham irradiation. The attenuation effect was apparent among rats injected 0 to 30 min after irradiation and for the first 40 min for groups injected at 120 min. Additional rats were injected with NaCl or 0.9, 1.8, or 2.7 g/kg of EtOH i.p. following 45 min of irradiation or sham irradiation to determine if the attenuation effect depends on the dose of EtOH administered. Attenuation of EtOH-induced hypothermia was more apparent at lower doses of EtOH than at higher doses. These results indicate that the effect is an acute response to irradiation, and rule out several other potential explanations.     

Effects of ethanol on central dopamine functions

Apomorphine-elicited stereotypic behavior and motor activity were studied in rats given one-dose (3g/kg,p.o.) or 14 days (6g/kg/day, p.o.) of ethanol. Apomorphine-elicited stereotypic behavior was enhanced while motor activitu was not significantly affected in both the acute- and chronic ethanol-treated animals. Acute ethanol treatment did not affect but chronic treatment increased the concentration of ³H-spiroperidol binding sites in the striatum. No significant change in binding affinity was observed.     

Effects of verapamil on sympathetic tone in normal and chronic sinoaortic-denervated conscious dogs

Previous studies have shown that calcium channel blockers from the dihydropyridine group (such as nicardipine) induce an increase in sympathetic tone from a central origin in chronic sinoaortic-denervated (SAD) dogs. In the present study, we investigated to see if verapamil possesses such properties. The effects of acute injection of verapamil (0.2 mg/kg i.v.) were compared in normal and SAD conscious dogs. Verapamil induced a decrease in blood pressure in the two groups of animals, and an increase in heart rate and plasma catecholamines (noradrenaline and adrenaline) in normal but not in SAD dogs. Contrary to the dihydropyridine studies (nicardipine), we did not find any evidence for a centrally mediated sympathoexcitatory effect of verapamil in conscious SAD dogs.     

Neuronal nitric oxide synthase is resistant to ethanol.

To test for a possible role of nitric oxide (NO) in the neurotoxicity of ethanol, we studied the effects of ethanol on the neuronal NO synthase (nNOS) both in vitro and in vivo. Ethanol, up to 200 mM, did not change the NOS activity in the cerebellar homogenate or the production of NO by the cultured cerebellar granule cells. The number of NADPH diaphorase-positive cells in the culture did not change after the exposure to 200 mM ethanol in vitro. The NOS activity in the various brain regions of mice remained similar to the controls after the acute (3 g/kg) and the chronic (33 g/kg/day, 3.5 days) administration of ethanol. N(omega)-nitro-L-arginine, a NOS inhibitor, did not affect the ethanol-withdrawal behavior. These results indicate that nNOS is resistant to ethanol at clinically relevant concentrations and that ethanol affects the NO-operated system in the brain through a pathway other than that of nNOS.     

Effect in vivo of a sub-hypnotic dose of ethanol on nerve conduction velocity in mice

Relative nerve conduction time (reciprocal of velocity), before and after administration of ethanol, was studied in the caudal nerves of 58 mice. Doses of 1.4, 2.0, 2.5, 3.0, and 4.24 g ethanol/kg body wt., given i.p., were used. The tail was maintained at 37.0° and a control (saline) injection preceeded the ethanol injection. Doses of 1.4 and 2.0 g/kg did not produce significant effects on conduction time but doses of 2.5, 3.0, and 4.24 g/kg did, the relative conduction times twelve minutes after ethanol increasing by 1.2, 1.8, and 3.1 percent, respectively. This appears to be the first controlled demonstration of an acute $\text{in}$$\text{vivo}$ effect of ethanol on conduction velocity.     

Effect of Withania somnifera Dunal in ethanol-induced anxiolysis and withdrawal anxiety in rats

Withania somnifera (WS) or its psychotropic preparation is known to play a critical role in morphine, alcohol and benzodiazepines addiction. This study investigates the role of WS in acute ethanol and withdrawal from chronic ethanol consumption using elevated plus maze paradigm in rats. Acute administration of ethanol (1.5-2 g/kg, ip) triggered anxiolytic effect and withdrawal from prolonged ethanol (9% v/v ethanol, 15 days) consumption elicited enhanced behavioral despair (anxiety). Acute administration of WS (50 mg/kg, oral) potentiated the anxiolytic action of subeffective dose of ethanol (0.5 or 1 g/kg, ip). Moreover, the ethanol withdrawal anxiety was markedly antagonized in dose dependent manner by WS at 200 and 500 mg/kg or higher dose of ethanol (2.5 g/kg). However, co-administration of subeffective doses of WS (50 mg/kg, oral) and ethanol also attenuated withdrawal-induced anxiety due to chronic ethanol (9% v/v ethanol, 15 days) consumption. The results suggest the protective effect of WS in the management of ethanol withdrawal reactions.     

Increased Levels of Monoamine-Derived Potential Neurotoxins in Fetal Rat Brain Exposed to Ethanol

Pregnant SD rats were exposed to ethanol (25 % (v/v) ethanol at 1.0, 2.0 or 4.0 g/kg body weight from GD8 to GD20) to assess whether ethanol-derived acetaldehyde could interact with endogenous monoamine to generate tetrahydroisoquinoline or tetrahydro-beta-carboline in the fetuses. The fetal brain concentration of acetaldehyde increased remarkably after ethanol administration (2.6 times, 5.3 times and 7.8 times as compared to saline control in 1.0, 2.0 and 4.0 g/kg ethanol-treated groups, respectively) detected by HPLC with 2,4-dinitrophenylhydrazine derivatization. Compared to control, ethanol exposure induced the formation of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol, Sal), N-methyl-salsolinol (NMSal) and 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline (6-OH-MTHBC) in fetal rat brains. Determined by HPLC with electrochemical detector, the levels of dopamine and 5-hydroxytryptamine in whole fetal brain were not remarkably altered by ethanol treatment, while the levels of homovanillic acid and 5-hydroxyindole acetic acid in high dose (4.0 g/kg) of ethanol-treated rats were significantly decreased compared to that in the control animals. 4.0 g/kg ethanol administration inhibited the activity of mitochondrial monoamine oxidase (51.3 % as compared to control) and reduced the activity of respiratory chain complex I (61.2 % as compared to control). These results suggested that ethanol-induced alteration of monoamine metabolism and the accumulation of dopamine-derived catechol isoquinolines and 5-hydroxytryptamine-derived tetrahydro-beta-carbolines may play roles in the developmental dysfuction of monoaminergic neuronal systems.     

Mechanism of tachycardia caused by intracarotid PGE2 in conscious ewes

Conscious adult ewes prepared with nonocclusive indwelling vascular catheters were used to determine the mechanism by which heart rate increases during central administration of prostaglandin E2 (PGE2). Heart rate increased 14 bpm during steady-state intracarotid infusion of PGE2, 10 ng/kg/min (P less than 0.05). Intravenous atropine methyl bromide, 1 mg/kg, increased heart rate 26 bpm (P less than 0.05) 5 min after injection. Heart rate remained elevated 30 min after injection. The heart rate response to PGE2 plus atropine was greater than the heart rate response to either atropine or PGE2 alone (P less than 0.05). Propranolol, 1 mg/kg bolus plus intravenous infusion, 0.025 mg/kg/min, did not change resting heart rate. Propranolol attenuated but did not abolish the increase in heart rate caused by intracarotid PGE2. Although heart rate increased in response to PGE2 after administration of either propranolol or atropine alone, the combination of propranolol and atropine prevented any further increase in heart rate during subsequent PGE2 infusion. The increase in heart rate when all three drugs were given together was not different from the increase observed during atropine alone. Thus, both beta-adrenergic activation and muscarinic deactivation contribute to the PGE2-induced tachycardia.     

Effects of verapamil on the development of pain syndrome in rats after section of the sciatic nerve]

The effect of daily verapamil administration (5 mg/kg) on pain sensitivity, bioelectric activity of cerebral cortex and microcirculation was studied in rats with cut sciatic nerve. Verapamil untreated rats showed hyperalgesia, autotomies and hypersynchronic discharges in contralateral hemisphere, increased range of evoked potentials and disorders in microcirculatory system which evidence developing pain syndrome. Early introduction of verapamil after the nerve cut prevented the emergence of pain syndrome.     

Influence of verapamil on central and peripheral effects of prostacyclin on circulatory system in rats

The influence of verapamil on cardiovascular effects of prostacyclin (PGI2) in rats was examined. PGI2 administered into the lateral brain ventricle (i.c.v.) or intravenously (i.v.) in a dose of 2.7 x 10(-8)mol evoked hypotension and tachycardia. Pretreatment with verapamil in a dose of 2.0 x 10(-5)mol/kg given intraperitoneally (i.p.) diminished hypotensive effect of PGI2 i.c.v. as well as inhibiting the influence of PGI2 i.c.v. and i.v. upon the heart rate. Bolus injection of PGI2 in a dose of 2.7 x 10(-10), 2.7 x 10(-9) or 2.7 x 10(-8)mol evoked biphasic inotropic and chronotropic effects on isolated rat heart. Short-term increase of the contractile force together with bradycardia and afterwards long-lasting decrease of contractility with sustained, slight tachycardia were observed. Verapamil in a concentration of 1.0 x 10(-6)M blocked biphasic inotropic effect and bradycardia after PGI2 administration. Because some central and peripheral cardiovascular effects of PGI2 were inhibited by verapamil, it is concluded that PGI2 may participate in transmembrane calcium ions movements.     

The ethanol-induced open-field activity in rodents treated with isethionic acid, a central metabolite of taurine.

The effect of isethionic acid, a central metabolite of taurine, on ethanol-induced locomotor activity was investigated in rodents. Ten minutes following an (i.p.) simultaneous administration of ethanol (0.0, 1.5, 2.0, 2.5, 3.0, 3.5 g/kg) and isethionic acid (0.0, 22.5, 45.0, 90.0, 180.0 mg/kg), mice were placed in the open-field chambers and locomotor activity was measured during a ten-minute testing period. A significant interaction was found between isethionic acid and ethanol. Isethionic acid pre-treated mice (45.0, 90.0 and 180.0 mg/kg) showed a higher locomotor activity than the saline group at 2.5 and 3 g/kg of ethanol. In a second study, isethionic acid (45 mg/kg) and ethanol (1 g/kg) were simultaneously injected to rats. Ten minutes after the two treatments, rats were placed in the open-field chamber for a 30-minute period. The depressant effects that ethanol produced on rat locomotion were amplified by the same dose of isethionic acid as it affected ethanol-induced locomotion in mice (45 mg/kg). However, isethionic acid did not change the spontaneous locomotion at any of the doses tested in mice or rats. Since no differences in blood ethanol levels were detected in both mice and rats, the interaction between isethionic acid's action and ethanol-related locomotion does not seem to be due to different rates of absorption of ethanol or any other pharmacokinetic process related to ethanol levels. The current study displayed that isethionic acid, administered intraperitoneally, behaves in a similar way to its immediate precursor, taurine, by amplifying ethanol-induction of the locomotor activity.