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Several studies indicate greater sensitivity to morphine (MOR) analgesia in male compared to female rats under the acute dosing condition. The present study investigated whether the same sex difference in sensitivity persists in MOR-tolerant rats. MOR was administered chronically (7 mg/kg twice daily) until tolerance developed in each rat. Tolerant rats were treated randomly with higher graded doses of MOR (10-25 mg/kg). Analgesia (tail-flick test) and spontaneous motor activity (total locomotion) were measured. The present data confirmed previous studies showing a greater sensitivity to acute MOR in male than in female rats. However, the sex differences seen in MOR sensitivity were abolished in tolerant rats. The rate of acquisition of tolerance was similar in male and female rats. The analgesic response was not affected by motor depression.

Sex and age influence morphine analgesia in humans and animals. Mature rats show greater morphine analgesia in males than in females. Ultra-low doses of naltrexone enhance morphine analgesia. In mature rats (18-22 weeks), naltrexone (0.002-2.0 mg/kg)-morphine (2 mg/kg) cotreatment enhanced morphine analgesia in females, an effect inversely related to naltrexone dose. Conversely, in mature male rats, naltrexone tended to decrease morphine analgesia with increasing dose. In young rats (8-10 weeks), morphine analgesia was unrelated to sex and in both sexes the naltrexone-morphine interaction was negligible. These data show that dose, age, and sex alter the naltrexone-morphine interaction in rats.

This study assessed the effect of the central benzodiazepine receptor antagonist, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (flumazenil), on morphine-induced analgesia, locomotor effects, and development of tolerance in rats. The thermally evoked pain (tail flick) response was determined after acute and chronic intraperitoneal (i.p.) administration of morphine and flumazenil, alone and in combination. In acute studies, flumazenil induced weak analgesia unrelated to dose and sex, whereas morphine-induced analgesia was dependent on both dose and sex (male>female). Flumazenil dose-dependently enhanced the analgesic effect of morphine in female but not in male rats. Isobolographic analysis suggested synergism between flumazenil and morphine in female rats, but antagonism in male rats. Flumazenil-induced locomotor changes (alone and with morphine) were related to sex but not dose. Chronic coadministration of flumazenil with morphine enhanced analgesia and attenuated tolerance development in female rats. The findings suggest a possible role for flumazenil as an adjunct with opioids in acute and chronic pain therapy.

The 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Flumazenil)-morphine interaction on analgesia (acute pain model, tail-flick test) was tested after intraperitoneal (IP) and intrathecal (IT) routes of administration in female rats. Analgesia was enhanced by the concurrent administration of Flumazenil with morphine (IP), in a dose-related way. Flumazenil alone (IP) did not produce analgesia. In contrast, morphine analgesia was not enhanced by Flumazenil by the IT route. These data demonstrate that Flumazenil enhances morphine-mediated antinociception by mechanisms that are likely to involve benzodiazepine receptors at sites other than the spinal cord.

Male and female rats were exposed for 3 weeks to diazepam (DZ)-filled or empty capsules (CTR) prior to the daily administration of morphine (MOR, 5 mg/kg, IP) for 5 days. Thereafter, capsules were removed and 48 h later MOR was injected for the next 5 days. The tail-flick latency (TFL) was measured prior to and 15, 30, and 60 min after MOR assessed analgesia. Locomotion (LOC) was determined before and 15 min after injection. Prior to MOR injection (baseline), male rats were more sensitive to the thermal stimulus and were less active than female rats. Daily MOR injections neither affected the baseline TFL nor LOC. Regardless of gender, MOR produced greater analgesia in DZ-dependent and withdrawn rats than in CTR. MOR analgesia was greater in DZ-dependent male than in female rats. Gender differences in MOR analgesia were not of statistical significance in DZ-withdrawn rats. The first dose of MOR produced more depression of LOC in DZ-dependent female than in male rats. Across the time of MOR injections, female DZ-dependent and withdrawn rats were less active than CTR. LOC increased with repeated administration of MOR in all groups of rats. In summary, DZ dependence and withdrawal enhanced MOR analgesia in rats of both sexes. Regardless of chronic treatment, MOR produced more analgesia and less depression of LOC in male than in female rats. It is suggested that a decrease in the function of the GABAergic system plays a role in alteration of MOR analgesia.

Control of malignant pain and related symptoms is paramount to clinical success in caring for cancer patients. To achieve the best quality of life for patients and families, oncologists and palliative care clinicians must work together to understand problems related to psychologic, social, and spiritual pain. Pain is the primary problem targeted for control using the World Health Organization's (WHO) analgesic ladder. This article focuses on increased knowledge of analgesic action that may enable expansion of the WHO analgesic ladder to fulfill the broader objectives of palliative medicine. We discuss clinical experience with several classes of drugs that are currently used to treat cancer pain: 1) nonsteroidal anti-inflammatory drugs, with emphasis on cyclooxygenase-2 inhibitors; 2) opioid analgesics, with specific emphasis on methadone and its newly recognized value in cancer pain; 3) ketamine, an antagonist at N-methyl-d-aspartate receptors; and 4) bisphosphonates, used for pain resulting from bone metastases. New concepts that compare molecular actions of morphine at excitatory opioid receptors, and methadone at nonopioid receptor systems, are presented to underscore the importance of balancing central nervous system excitatory (anti-analgesic) versus inhibitory (analgesic) influences.

This study was undertaken to evaluate the effect of acute and repeated (daily and weekly) intravenous (IV) administration of PK 11 195 (PK; 10 mg kg(-1)) on body weight (BW) in Sprague-Dawley male and female rats exposed for 4-8 weeks to diazepam (DZ) slowly released from Silastic capsules (120 and 90 mg/capsule/week for males and females, respectively). Rats implanted with empty capsules served as controls. Both acute and repeated (daily and weekly) administration of PK inhibited BW gain to a greater extent in male than in female rats that received identical treatment. There was no difference in PK effect between DZ-treated and control rats. Furthermore, regardless of treatment or gender, PK-induced inhibition of BW gain lessened during repeated administration of PK. The present data indicate that PK-induced inhibition of BW gain is related to gender rather than to chronic DZ treatment., (Copyright 2000 Academic Press.)

These studies were undertaken to 1) determine whether repeated dosing with the peripheral benzodiazepine antagonist PK 11195 alters its ability to precipitate withdrawal abstinence in diazepam-dependent rats; 2) whether the administration of PK 11195 and the central benzodiazepine antagonist, flumazenil, 3 days apart to the same rat produces an ordering effect in the intensity of withdrawal abstinence; 3) whether there are gender differences in these effects. Age-matched male and female Sprague Dawley rats had capsules implanted weekly that contained approximately equal (mg/kg) doses of diazepam (120 and 90 mg, respectively) or empty capsules (controls). After 5 implants, the maximum precipitated withdrawal score (PAS(MAX)) induced by PK 11195 and/or flumazenil (10 mg/kg/IV, respectively) was measured. Repeated administration of PK 11195 (1x/day for 5 days) induced tolerance with regard to the intensity of the PAS(MAX) and with gender-related differences. When PK 11195 was administered weekly (5 weeks), rather than daily, tolerance did not develop in either sex. The PK 11195- and flumazenil-induced PAS(MAX) was not changed by the order in which they were administered. There were gender differences in that females had a higher PAS(MAX) after flumazenil than after PK 11195 and vocalized more after all treatments than males.

Control of malignant pain and related symptoms is paramount to clinical success in caring for cancer patients. To achieve the best quality of life for patients and families, oncologists and palliative care clinicians must work together to understand problems related to psychologic, social, and spiritual pain. Pain is the primary problem targeted for control using the World Health Organization's (WHO) analgesic ladder. This article focuses on increased knowledge of analgesic action that may enable expansion of the WHO analgesic ladder to fulfill the broader objectives of palliative medicine. We discuss clinical experience with several classes of drugs that are currently used to treat cancer pain: 1) nonsteroidal anti-inflammatory drugs (NSAIDs), with emphasis on cyclooxygenase-2 (COX-2) inhibitors; 2) opioid analgesics, with specific emphasis on methadone and its newly recognized value in cancer pain; 3) ketamine, an antagonist at N-methyl d-aspartate (NMDA) receptors; and 4) bisphosphonates, used for pain resulting from bone metastases. New concepts that compare molecular actions of morphine at excitatory opioid receptors, and methadone at non-opioid receptor systems, are presented to underscore the importance of balancing central nervous system excitatory (anti-analgesic) versus inhibitory (analgesic) influences.

In spite of the extensive use of the peripheral benzodiazepine (BZ) receptor antagonist, PK 11195 (PK), in pharmacological studies, there is a lack of information of its pharmacokinetics in the rat. In this study the pharmacokinetics of PK were determined after bolus intravenous (i.v.) administration in rat. The effects of dose and gender were evaluated in Sprague-Dawley age-matched male and female rats after the injection of PK (5, 10, 20 mg kg(-1)). Plasma was collected at 5-300 min. Levels of PK in plasma and brain were determined by a novel HPLC method. The stability of PK in blood in vitro was determined. PK is stable in rat blood in vitro. The pharmacokinetics of PK are described by a two-compartment model. The half-lives for distribution ( approximately 0.14 h) and elimination ( approximately 5.4 h) are not related to dose. The large volume of distribution (9-24 l kg(-1)) indicates an extensive distribution outside plasma. Total plasma clearance increases with increasing dose (23-42 ml min(-1)kg(-1)). The brain/plasma ratio ( approximately 3) is not related to dose. These finding suggest that the pharmacokinetics of PK are related to dose (5-20 mg kg(-1)) and gender in rat., (Copyright 2000 Academic Press.)

Female rats were exposed to diazepam (DZ) implants (90 mg/week) or to empty capsules (controls) for 5 weeks. Rats were focally injected (1 microl) into the substantia nigra (SNR) with the central (CBR) and peripheral (PBR) benzodiazepine receptor antagonists, flumazenil [(FLU) 6.25, 12.5, or 25 microg], and PK 11195 [(PK) 3.125, 6.25, 12.5, or 25 microg], respectively. Rats were observed for behavioral and EEG manifestation of withdrawal syndrome. In female rats, both FLU and PK induced a dose-related precipitated abstinence score (PAS), tachypnea, and head bobbing. Twitches and jerks tended to increase with increasing dose of both FLU and PK. Furthermore, FLU evoked dose-related turning and head and body tremors. The FLU- and the PK-induced PAS were accompanied by an increase in total power of the EEG in the SNR. The involvement of the CBR and PBR in physical dependence on DZ in the SNR is suggested. The present data in female rats are discussed with regard to similarities and differences with previous studies in male rats.

Male rats chronically exposed to diazepam (DZ) slowly released from subcutaneously implanted silastic capsules along with empty capsule control rats were focally injected (1 microl) into the substantia nigra (SNR) with the central (CBR) and peripheral (PBR) benzodiazepine receptor antagonists, flumazenil [(FLU) 6.25, 12.5, 25 microg] and PK 11195 [(PK) 3.125, 6.25, 12.5, 25 microg], respectively (weekly intervals; Latin square design). Rats were observed for signs of withdrawal and the EEG was recorded simultaneously from the site of injection (SNR), caudate putamen, thalamus, hippocampus, and frontal cortex. In DZ-dependent rats the Precipitated Abstinence Score (PAS) was significantly related to dose of FLU. The PAS increased with increasing doses of PK (3.125-12.5 microg); however, the highest dose of PK (25 microg) showed less effect. The rapid onset of the PAS was accompanied by a rise in the total power (1-32 Hz) of the EEG (TP(EEG)) in the SNR and other brain areas. The PAS and TP(EEG) had similar time courses. Intranigrally injected FLU and PK did not evoke clonic and tonic-clonic convulsions; however, both antagonists induced dose-related twitches and jerks. Additionally, FLU precipitated a dose-related tachypnea and increases in turning and backing. Chronic DZ treatment altered the spectral content of the EEG, as indicated by a decrease and an increase of the slow and fast frequency bands, respectively. FLU and PK rapidly but transiently reversed the EEG. Data suggest that in the SNR the CBR mediate autonomic and motor signs of DZ withdrawal, while both the CBR and PBR are responsible for twitches and jerks and alteration of the EEG. It is possible that PK also acts on the site linked to a GABA(A)/CBR/ionophore.

Six female rats had a loading dose of 180 mg of diazepam (DZ) contained in two Silastic capsules implanted in their backs. Thereafter, a single 90-mg capsule was implanted weekly for 4 weeks prior to weekly microinjections of 1 microl of flumazenil (6.25, 12.5, or 25 microg) and PK 11195 (3.125, 6.25, or 12.5 microg) or vehicle into the CA1. Three control rats had empty capsules implanted but received only the high dose of flumazenil after 5 weeks. The time of DZ exposure spanned 8 weeks. Mean steady-state plasma levels of DZ were 1.06 +/- 0.11, and the mean total (DZ + metabolites) was 2.46 microg/ml +/- 0.37. Flumazenil elicited a dose-related precipitated withdrawal score (PAS) in DZ-treated rats (but not in controls) characterized by dose-related increases in convulsive (twitches and jerks), motor and autonomic signs, dose-related increases in the percent of total power in the low frequency (1-4 Hz), and decreases in the high-frequency (18-26 Hz) bands of the EEG recorded from the dentate and the amygdala. PK 11195 produced a dose-related increase in the 4-12 Hz band of the EEG recorded from the CA1, whereas the PAS was mild and not dose-related. However, the 6.25 and 12.5-microg doses elicited a significant PAS that tended to increase with dose. These data indicate that chronic DZ produces dependence, and that in the CA1 it involves the participation of central and possibly peripheral benzodiazepine (BZ) receptors located within this structure.

Changes in body weight (BW) in female rats treated for 5 weeks (wk) with weekly subcutaneous implantation of silastic capsules containing different benzodiazepines (BZs): diazepam (DZ) 90, 180, 360 and 540 mg wk-1; nordiazepam (ND) 600 mg wk-1; oxazepam (OX) 600 mg wk-1 and flunitrazepam (FN) 540 mg wk-1 and in male rats exposed to DZ (540 mg wk-1) were evaluated herein. Rats (female and male) implanted with empty capsules served as controls. The BW gain was significantly higher in male than in female rats (both DZ-treated and controls). The BW gain increased with increasing doses of DZ but slowed with time of exposure. In comparison to control rats, the BW gain was significantly higher in DZ-(540 mg wk-1) and OX- but not in ND- and FN-treated female rats. However, the differences between BZs were not of statistical significance. In rats exposed to empty capsules (male, female); DZ (male); ND and OX (female) the BW gain increased with time (1-4 wk) while in rats exposed to DZ and FN (female) the BW stabilised within 2 wk. Acute injection of the central BZ receptor antagonist, flumazenil (40 mg kg-1, i.v., 5th wk of chronic exposure), tended to inhibit the time-related BW gain in rats exposed to empty capsules (male, female), DZ (male), ND and OX (female) but did not affect the BW in DZ- (540 mg wk-1) and FN-exposed rats (female) where BW stabilised prior to FLU injection. Repeated administration of flumazenil (30 mg kg-1 wk-1, i.p.) did not affect the BW gain in DZ- and ND-treated female rats. The present data indicate that different BZs have different effects on BW gain in the rat suggesting that different subtypes of BZ receptors are involved.

The abilities of the central (CBR) and the peripheral (PBR) benzodiazepine receptor antagonists, flumazenil (FLU) and PK 11195 (PK), to precipitate an abstinence syndrome in diazepam (DZ)-dependent rats have been evaluated. Female rats were exposed for 5 weeks to DZ slowly released from SC implanted silastic capsules (90 mg/capsule per week) and thereafter they were challenged in weekly intervals with IV injections of FLU (10, 20, 40 mg/kg) or PK (5, 10, 20 mg/kg), respectively. The maximum abstinence scores tended to increase with the dose of FLU but not with the dose of PK. Although FLU and PK precipitated some common abstinence signs, there were marked differences between these antagonists. FLU evoked dose-related tonic-clonic and clonic convulsions (five out of six rats), whereas PK (10 mg/kg) induced convulsions in only one rat (out of five); tachypnea tended to increase with the dose of both FLU and PK; twitches and jerks, backing and writhing had a significant regression on the dose of FLU; rearing tended to decrease with the dose of PK whereas FLU-evoked head bobbing and PK-evoked twitches and jerks had inverse U-shaped dose-response curves. In comparison to FLU, similar doses of PK (10 and 20 mg/kg) induced a lower precipitated abstinence score (P < 0.05) and a less intense tachypnea (P < 0.05). The data indicate that the chronic continuous exposure to DZ (and/or its active metabolites) affects both CBR and PBR in the rat; however, the abstinence syndromes produced by the CBR and PBR antagonists, FLU and PK, differ in overall intensities and in the diversity of evoked abstinence signs.

Flumazenil (FLU; 25 micrograms) and DMSO-vehicle were focally injected (1 microliter) into the substantia nigra (SN) and the dorsal raphe nucleus (DR) in rats chronically implanted with silastic capsules containing diazepam (DZ; 540 mg/week). FLU precipitated an abstinence syndrome in the SN as indicated by a significant abstinence score, several abstinence signs and reduced total power of the fast frequency bands of the electroencephalogram (EEG) in the injections sites frontal cortex, (FC) and hippocampus (H). In contrast, FLU did not produce an abstinence syndrome in the DR, and its effect on the power of the EEG in DR, FC and H was not significantly different from that of the DMSO-vehicle. The data show regional heterogeneity in the response of the SN and the DR to chronic DZ treatment in terms of a focally precipitated abstinence syndrome.

The central and peripheral benzodiazepine (BZ) receptor antagonists, flumazenil (FLU) and PK 11195 (PK), administered intrathecally (IT) to diazepam (DZ)-dependent rats produced a precipitated abstinence syndrome. The scores for abstinence increased with increasing dose of FLU but not with increasing dose of PK. Twitches and jerks increased with increased doses of both. Head and body tremors were produced by FLU, but not by PK. Neither FLU nor PK precipitated abstinence in controls. In DZ-dependent rats IT administered FLU and PK did not significantly change the spectral content and the total power of the EEG. The data indicate that an abstinence syndrome is precipitated at the spinal level in DZ-dependent rats and that both central and peripheral BZ receptors are involved.

Steady-state levels of oxazepam (OX), nordiazepam (ND), and diazepam (DZ) in plasma, brain tissue, cerebrospinal fluid (CSF), and intracranial microdialysis perfusate were determined in dogs dependent on 0.56, 4.5, 9, and 36 mg/kg per day of DZ. There was a linear relationship between the total plasma and brain levels of DZ, ND, and OX and the chronic dose of DZ. Levels of free benzodiazepines in plasma and CSF and levels in microdialysis perfusates from plasma and brain were significantly correlated. With increasing dependence on DZ there was progressively more free ND and OX and less free DZ in plasma, CSF, and brain. There was a correlation between several signs of precipitated abstinence and free ND in the brain interstitial fluid, whereas convulsions emerged only when free metabolites exceeded free DZ. The changes in contribution of free DZ, ND, and OX to the overall levels of benzodiazepines present in the CNS may explain differences in signs of abstinence for different levels of dependence on DZ.

The effect of flumazenil (FLU) on plasma protein binding of diazepam (DZ), nordiazepam (ND) and oxazepam (OX) was determined in plasma from drug-naive dogs to which graded concentrations of tested drugs alone and in combination were added. The results revealed that as the concentration of FLU added to plasma alone was increased its binding with plasma proteins decreased and that there were no significant binding interactions between FLU and OX, ND and DZ.

The rates of in vitro release of flunitrazepam (FN), nordiazepam (ND) and diazepam (DZ) from silastic capsules were compared and found to be in the following order: DZ > FN > ND. Rats that were implanted subcutaneously with capsules filled with FN or ND for 5 to 7 weeks before administering flumazenil (FLU) (40 mg/kg, i.v.) showed precipitated abstinence as measured by the Precipitated Abstinence Score (PAS) which included a rapid onset of clonic and tonic-clonic convulsions. Rats implanted with DZ also demonstrated significant PAS and seizures. Implantation of similar doses of DZ, FN and ND resulted in different plasma levels of parent benzodiazepines and their metabolites that corresponded with their in vitro release: DZ > FN > ND. These data indicate that, as for DZ, the capsule implantation is an effective method of producing physical dependence on FN and ND in the rat.

Physical dependence was produced in the rat by exposure to continuous release of diazepam from silastic capsule implants (recrystallized diazepam) or by dosing through a gastric fistula. The precipitated abstinence syndrome induced by the IV infusion of flumazenil was characterized by clonic and tonic-clonic seizures, retropulsion, digging, rearing, head, limb and body tremors, twitches and jerks of the body, and ear twitches. This abstinence syndrome differed both qualitatively and quantitatively from the milder syndrome induced in previous experiments by the intragastric administration of flumazenil in the diazepam-dependent gastric fistula rat. Capsule-implanted rats had free plasma and extraneuronal brain levels of diazepam, oxazepam, and nordiazepam in the 10(-3) and 10(-4) mg/ml range, and their brain: plasma ratios were not significantly different from 1. The diazepam capsules had a sustained release of over 28 days. These studies show that the capsule implantation technique is an efficacious way of maintaining plasma levels of diazepam and its metabolites, and producing a high level of physical dependence in the rat.

The ability of different chronic doses of diazepam to produce dependence was studied in groups of six dogs who received diazepam p.o. (0.05625, 0.225, 0.5625, 4.5, 9 or 36 mg/kg/day) every 8 hr. After 5 to 6 weeks of stabilization, the intensity of precipitated abstinence was measured by benzodiazepine-precipitated abstinence scores (BPAS) after the p.o. administration of graded doses of the benzodiazepine antagonist, flumazenil (0.66, 2, 6, 18, 36 and 72 mg/kg or a placebo). A modified Latin square design was used. Dogs receiving the two lowest stabilization doses of diazepam showed only liminal signs of precipitated abstinence even with 72 mg/kg of flumazenil. The intensity of the precipitated abstinence syndrome increased with the stabilization dose of diazepam. There was also a dose-related increase in BPAS for increasing doses of flumazenil for all doses of diazepam except the 9-mg/kg/day dose. Not only were quantitative differences observed in precipitated abstinence signs for different levels of diazepam dependence, but the pattern of abstinence signs differed also. Dogs dependent on high doses of diazepam were more sensitive to flumazenil than those dependent on lower doses. Furthermore, seizure activity was seen only in dogs dependent on 9 and 36 mg/kg/day of diazepam. BPAS increased linearly with plasma and brain total and free levels of the sum of diazepam and its metabolites (oxazepam and nordiazepam), but not with free plasma and brain levels of diazepam.

Previous studies suggested that the extensive accumulation of benzodiazepines is an important factor in the induction of physical dependence. The mechanistic basis for accumulation of nordiazepam (ND) and its metabolite, oxazepam (OX), have been examined in crossover studies in drug-naive and in ND-dependent dogs that exhibited a flumazenil-precipitated abstinence syndrome. ND and parent OX have similar pharmacokinetic profiles. Steady-state plasma levels of ND and OX cannot be predicted from single-dose pharmacokinetics. Reduced plasma clearance of ND and altered plasma protein binding were observed in dogs physically dependent upon ND. The benzodiazepine antagonist, flumazenil, significantly reduces steady-state plasma levels of total and free ND.

Halazepam administered chronically to dogs in oral doses of 180 and 450 mg/kg/day produced physical dependence which was revealed by a flumazenil precipitated abstinence syndrome and measured by the Nordiazepam Precipitated Abstinence Scale score (NPAS) (McNicholas et al., 1988; Sloan et al., 1990). This abstinence as measured by the NPAS score was more severe in diazepam- and halazepam-dependent than in nordiazepam-dependent dogs whereas the incidence of precipitated clonic seizures was greater in the diazepam- and nordiazepam-dependent than in the halazepam-dependent dogs. Pharmacokinetic studies showed that in the dog the major conversion of halazepam, like diazepam, was to nordiazepam and an oxazepam conjugate. Appreciable quantities of oxazepam, 3-OH halazepam and its conjugated metabolite were also identified in plasma. The NPAS score obtained in the halazepam-dependent dogs, however, was greater than the NPAS score obtained in nordiazepam-dependent dogs who had nordiazepam plasma levels over three times higher than those obtained in the halazepam-dependent dogs. Further, the precipitated abstinence observed in the halazepam-, diazepam- and nordiazepam-dependent dogs differed in qualitative as well as in quantitative aspects including marked differences in the time course of abstinence signs. These data argue that the different dependencies produced by halazepam, diazepam and nordiazepam are not due solely to either the parent compound or to a single metabolite but most likely to their combined effects.

Dogs dosed chronically (4-7 weeks) with oral flunitrazepam (7.6 mg/kg/day) or diazepam (24-36 mg/kg/day) administered in 4 equally divided doses had dose-related flumazenil precipitated benzodiazepine abstinence scale scores (BPAS) of comparable intensities despite the fact that plasma levels of flunitrazepam and its metabolites were much lower than nordiazepam levels in the diazepam-dependent dog. Both groups of dependent dogs had clonic and tonic-clonic seizures after oral and IV flumazenil. Precipitated abstinence signs persisted longer in the diazepam than in the flunitrazepam-dependent dogs. Differences in the pharmacokinetics of the drugs of dependence, their metabolites, and their interactions at receptor sites offer a partial explanation for the high level of dependence seen in the flunitrazepam dog. The finding that the estimated plasma free concentration of flunitrazepam and its metabolites is equal to or greater than that of diazepam and its metabolites together with the fact that flunitrazepam has a higher affinity for the benzodiazepine receptor than either diazepam, nordiazepam or oxazepam can explain why the intensity of the precipitated abstinence syndrome is comparable in flunitrazepam- and diazepam-dependent dogs. Although the flumazenil-induced precipitated abstinence syndromes observed in flunitrazepam- and diazepam-dependent dogs differed qualitatively they did not differ quantitatively. It is therefore concluded from these data that the doses of flunitrazepam and diazepam, chosen for producing comparable degrees of weight loss during dose escalation, did not differ in the degree of physical dependence that they produced in the dog.

The pharmacokinetic profiles of halazepam (HL) and its metabolites, desmethyldiazepam (DMDZ), oxazepam (OX), 3-hydroxyhalazepam (OH-HL), and conjugates of oxazepam (OX-CONJ) and 3-hydroxyhalazepam (OH-HL-CONJ) were studied in 4 naive dogs following single intravenous (2 mg/kg) and oral (112.5 mg/kg) administrations of HL and in 5 dependent dogs chronically dosed with HL (450 mg/kg/day q.i.d.). HL is rapidly metabolized to DMDZ as the principal metabolite but appreciable levels of HL, OX and OH-HL were measured in plasma and the brain tissue. High levels of conjugated metabolites were measured in plasma. The steady-state plasma concentrations of HL and its unconjugated metabolites can be predicted from the single dose study. Halazepam does not serve as a simple prodrug for DMDZ in producing physical dependence in dogs.

The compartmental distribution of diazepam (DZ) and nordiazepam (ND) and their metabolites was studied in DZ and ND dependent dogs. The levels of DZ, and ND and their metabolites were determined during the last week of stabilization in the extraneuronal brain space, in brain tissue, in plasma and in CSF. In these studies dependent dogs were anesthetized with pentobarbital and microdialysis probes were inserted bilaterally into the parietal cortex and perfused with artificial cerebrospinal fluid. Microdialysis probes were also used to determine the unbound parent drugs and their metabolites in plasma. The brain-plasma distribution of total ND and oxazepam (OX) is about equal in ND dependent dogs but in DZ dependent dogs total ND and OX are about 2-fold higher in brain than in plasma. The levels of DZ, ND, and OX in the extraneuronal brain space are similar to their unbound levels in plasma. These data suggest that the concentration of free benzodiazepines in plasma is a good approximation of the concentration in the vicinity of the membrane receptors in the dependent dogs.

The ability of graded doses of flumazenil (2, 6, 18, 36 and 72 mg/kg) and a lactose placebo to precipitate abstinence was studied in dogs treated chronically with diazepam, nordiazepam, flunitrazepam, alprazolam, oxazepam, halazepam and lorazepam by oral dosing. A scale comprised of 10 precipitated abstinence signs, the Benzodiazepine Precipitated Abstinence Scale, was developed, which yielded linear flumazenil log-dose response lines with significant slopes in dogs dependent on diazepam, nordiazepam and flunitrazepam. The effects of 18, 36 and 72 mg/kg of flumazenil in otherwise drug naive dogs were studied. In naive dogs, the most prominent effect of flumazenil was to reduce activity. All benzodiazepines studied produced dependence that was characterized by signs of precipitated abstinence; however, the intensity and quality of abstinence varied from one benzodiazepine to another. Precipitated abstinence in dogs treated chronically with diazepam and flunitrazepam was characterized by a dose-related increase in clonic convulsions and Benzodiazepine Precipitated Abstinence Scale scores. This pattern differed from that seen in nordiazepam- and alprazolam-dependent dogs, which showed a comparable flumazenil dose-related increase in clonic convulsion but only a modest increase in Benzodiazepine Precipitated Abstinence Scale scores. Oxazepam and lorazepam produced dependence that was less intense than that seen with the other benzodiazepines. Plasma levels of the benzodiazepines and their metabolites were repeatedly determined after single doses and during addiction cycles. Nordiazepam accumulated in diazepam- and nordiazepam-dependent dogs and alpha-OH alprazolam accumulated in alprazolam-dependent dogs. Other drugs and metabolites did not. These observations suggest that: 1) different benzodiazepines or their metabolites produce different types of physical dependence, suggesting that they or their metabolites have different mechanisms and sites of action; 2) plasma cumulation of the benzodiazepines or their active metabolites is an important factor in the genesis of physical dependence; and 3) metabolism of benzodiazepines plays an important role in their dependence-producing capacity, and because of differences in the way species metabolize benzodiazepines, the type of dependence produced in different species may differ.

Alprazolam (48 mg/kg/day) administered orally to dogs 4 times a day in equally divided doses produced physical dependence. This dependence was revealed by a precipitated abstinence syndrome which occurred after either oral administration of flumazenil (6, 18 and 36 mg/kg) or intravenous administration of a liposomal suspension of flumazenil. Flumazenil alone (18, 36 and 72 mg/kg) produced no significant signs of precipitated abstinence in naive dogs. This precipitated abstinence syndrome in alprazolam-dependent dogs was characterized by both clonic and tonic-clonic seizures. Other signs of precipitated abstinence which comprise the NPAS score were less intense in the alprazolam-dependent than in diazepam-dependent dogs. Alprazolam is extensively metabolized in the dog and does not accumulate whereas its predominant metabolite, alpha hydroxyalprazolam, does accumulate. The data suggest that alpha hydroxyalprazolam plays a role in the dependence-producing properties of alprazolam in the dog as revealed by the precipitated abstinence syndrome.

The pharmacokinetics of oxazepam was studied in naive dogs and in oxazepam-dependent dogs without and with administered flumazenil (6 mg/kg). Oxazepam is eliminated with a relatively short elimination half life (ca. 150 min) in both acutely and chronically treated dogs. It exhibits only a modest first pass metabolism (ca. 10%) and its bioavailability following oral administration is about 22%. The steady state concentration of oxazepam in chronically treated dogs was lower than was predicted from single dose studies. Flumazenil did not change the rate of absorption or elimination of oxazepam-dependent dogs. The total steady state plasma concentration of oxazepam was significantly reduced by flumazenil administration suggesting a displacement interaction between flumazenil and oxazepam.

Previous studies have shown that (+/-)-[3H]nicotine binds to multiple sites in the rat brain P2 preparation. Using a series of pyridine, piperidine and pyrrolidine analogues, the present studies identified drugs with specificity for a separate up-regulatory site that increases the density of nicotine binding at another site. Of these compounds, (+/-)-2-methylpiperidine was the most specific. Some compounds inhibited without enhancing (+/-)-[3H]nicotine binding, but none bound with the very high affinity exhibited by nicotine and none could be classified as specific in inhibiting binding at a specific site. Structural changes in the 1- and 2-positions of pyridine and piperidine appear to be important for conferring specificity for the up-regulatory site whereas 3-position changes may be important for binding specificity.

Nicotine produced a distinct reproducible syndrome in the conscious dog when injected intravenously or intracerebroventricularly. Intravenously administered nicotine (40 micrograms/kg/min for 20 minutes) increased cardiac and respiratory rates and produced analgesia, miosis, hypothermia, behavioral restlessness and emesis. When microinjected into the third cerebral ventricle, nicotine (100-200 micrograms) similarly increased cardiac and respiratory rates and pupillary diameter; and produced behavioral restlessness, emesis, erratic analgesia and maintained wakefulness and a desynchronized EEG. Microinjection of nicotine (5-25 micrograms) into the periaqueductal gray failed to alter any of the parameters studied. Intravenous pretreatment with the opioid antagonist naltrexone (2 mg/kg) influenced the action of intravenous nicotine on certain physiological systems. While naltrexone alone produced a significant degree of tachycardia, miosis, and analgesia, it potentiated the tachypnea and antagonized the miotic response evoked by nicotine. Methionine-enkephalin was detected in perfusates obtained from the lateral cerebral ventricles of conscious dogs. Nicotine produced a non-significant decrease in enkephalin levels. These observations suggest that there are interactions between endogenous opioid and nicotinic processes. However, they are complex and may differ from one functional system to another.

In the urethane-pentobarbital anesthetized rat, the respiratory depressant and lethal effects of intravenously infused (-)-nicotine (120 micrograms/kg/min) or (+)-nicotine (600 micrograms/kg/min) were effectively prevented by pretreatment with the opioid antagonist, naltrexone, whereas the lethal effect of (-)-nicotine (120 micrograms/kg/min) was not altered by bilateral adrenalectomy. Further, pretreatment with either the nicotinic ganglion-blocker, mecamylamine, a secondary amine, or the quarternary nicotinic ganglion-blocker, hexamethonium, completely prevented the lethal effects of (-)-nicotine (120 micrograms/kg/min). These data suggest that central opioidergic and nicotinic processes are involved in nicotine's respiratory depressant and lethal effects.

The effects of ethylketazocine (EKC) administered intraperitoneally and the nicotinic ligands (-)- and (+)-nicotine, (-)-cytisine, (-)-lobeline, and (+)-2-methylpiperidine administered into the 4th ventricle on the latency of the thermally evoked withdrawal reflex of the decerebrate rat were investigated. EKC administered intraperitoneally produced both hyperalgesia and analgesia. (-)-Nicotine administered into the 4th ventricle produced a biphasic dose related effect on the latency of the withdrawal reflex; low doses produced a dose related analgesia while higher doses produced hyperalgesia. (-)-Cytisine and (-)-lobeline administered into the 4th ventricle produced biphasic effects. (+)-2-Methylpiperidine administered into the 4th ventricle produced a significant degree of hyperalgesia. Both the analgesic and hyperalgesic effects of (-)-nicotine were antagonized by mecamylamine (1 mg/kg) and naltrexone (5 mg/kg). The hyperalgesic action of (+)-2-methylpiperidine was antagonized by naltrexone but not by mecamylamine. These observations suggest that there are both medullary opioidergic and nicotinic cholinergic mechanisms for modulating both analgesic and hyperalgesic processes and that nicotinic ligands have multiple mechanisms of action in the brain.

Five drugs [(-)- and (+)-nicotine, (-)-lobeline, (-)-anabasine and (-)-cytisine] were infused IV into the urethane-pentobarbital anesthetized rat. Changes in heart rate, blood pressure, respiratory rate, minute and tidal volume, which appeared to be largely centrally mediated, were studied. Each of these compounds produced different pharmacologic profiles. The nature of these dissimilarities is not readily explained on the basis of pharmacokinetic considerations suggesting that the drugs have different mechanisms of action. Binding data obtained with these compounds using the rat brain P2 preparation also show differences. (-)-Lobeline and (-)-anabasine, like the nicotinic antagonists mecamylamine and hexamethonium, bind predominantly to low affinity sites with KDs in the micromolar range whereas (-)-cytisine binds only to a single high affinity site with a KD in the nanomolar range. Further, the binding patterns of these drugs are different from (-)- and (+)-nicotine which bind to both high and low affinity sites but differ from each other in binding characteristics. Thus the binding data are consistent with the pharmacologic data in suggesting that the drugs have different modes of action and support the concept that the low affinity site has an important role in the central nervous system action of these compounds.

Tryptamine produces pharmacologic effects in man and the chronic spinal dog which are similar to those produced by LSD, mescaline, psilocin, DMT, DOM and DOB. These effects include tachycardia, tachypnea, mydriasis, hyperreflexia, behavioral changes and in man, hallucinations. Chronic spinal dogs treated chronically with LSD became tolerant to its ability to produce mydriasis, tachycardia, tachypnea and hyperreflexia, and were cross tolerant to the ability of tryptamine, psilocin, mescaline, DMT, DOM and DOB to produce these same effects. Further, it was found that the brain and spinal cord contained tryptamine and could release it. Further tryptamine levels were higher in the brainstem and spinal cord above the level of transection in the chronic spinal dog that in intact dogs, and the same in the spinal cord below the level of transection. These observations suggested that there were both ascending and descending tryptaminergic pathways. Supporting this hypothesis were the observations that L-tryptophan also produced hyperreflexia in the acute, but not the chronic, spinal dog and cat, and that L-tryptophan hyperreflexia was antagonized by alpha-methyldopa but not pCPA. These observations and others argue that the spinal cord and brain have tryptaminergic mechanisms which are distinct from serotoninergic mechanisms, and that LSD-like hallucinogens act in part through a tryptaminergic mechanism.

(-)-Nicotine may bind to as many as 5 sites in the rat brain P2 preparation: A very high affinity site (KD approximately 2.2 X 10(-11) M); a positive cooperativity site; a high affinity site (KD approximately 5.2 X 10(-9) M); a low affinity site (KD approximately 4.5 X 10(-5) M) and a very low affinity site. The curvilinear nature of both Scatchard plots and kinetic curves indicates the presence of multiple binding sites. Evidence for a positive cooperativity site includes: (1) The configuration of Scatchard plots (at low concentrations) of saturation as well as inhibition curves for (-)- and (+)-nicotine. (2) The Hill number of 1.37 for the binding of low concentrations of (+/-)-[3H]nicotine. (3) Selectivity among cholinergic drugs for producing positive cooperativity. (4) Markedly different specificities of drugs for the positive cooperativity site. Thus while only (+)- and (-)-nicotine interacted with the very high affinity site, acetylcholine, atropine, mecamylamine, lobeline, carbachol, (+)-nicotine and (-)-nicotine enhanced the binding of (+/-)-[3H]nicotine and cytisine, anabasine, cotinine and choline selectively inhibited binding at the high affinity site. Several lines of evidence indicate that there is stereospecificity. (+)-Nicotine was more potent than (-)-nicotine in inducing positive cooperativity whereas (-)-nicotine was 80 times more potent than (+)-nicotine in inhibiting binding at the high affinity site. Further, the specificity of the binding sites can be altered by changing the concentration of the buffer which gives additional evidence for the lability of the nicotine binding site. Although the pharmacologic significance of the different binding sites has not been determined, these data taken together indicate that (+/-)-[3H]nicotine binds with specificity to multiple sites in the rat brain P2 preparation with a complexity not addressed heretofore.

The brain and spinal cord of the chronic spinal dog contained higher levels of tryptamine than comparable regions of the intact dog. The most significant brain elevations were found in the cerebellum and mesencephalon. Further, tryptamine in the white matter of the spinal cord above the level of transection was higher than below. These findings have been interpreted as indicating that there are tryptaminergic pathways descending in the white matter of the spinal cord from the mesencephalon, cerebellum and rostral spinal cord. The level of tryptamine below the transection was not different from that found in the intact dog, suggesting that there are not only descending but ascending tryptaminergic pathways and that when the axons are transected. tryptamine accumulates proximal to the level of transection.

Saturation studies employing (-)- and (+)-[3H]nicotine indicate that the isomers bind to different very high and high affinity sites since the binding density for (-)-[3H]nicotine is 10 times that for (+)-[3H]nicotine. Both isomers also bind to a low affinity site (KDS = approximately 10(-5) to 10(-4) M). Competition studies employing unlabelled (-)- and (+)-nicotine reveal greater complexities. The isomers also appear to bind to a separate site which enhances binding at the (-)- and (+)-nicotine high affinity sites. (+)-Nicotine is more effective in increasing the binding of (-)-[3H]nicotine at its high affinity site than (-)-nicotine. Further, (+)-nicotine has a greater specificity for enhancing binding than (-)-nicotine in that it enhances (-)-[3H]nicotine binding at lower concentrations and inhibits binding at higher concentrations than (-)-nicotine.

Four distinguishable nicotinic binding sites have been identified as well as four nicotinic ligands with different specificities: (+/-)-2-methylpiperidine which binds to a very high affinity site (Site 1) and produces up-regulation of the high affinity site (Site 2); (-)-nicotine which binds to Site 1 and Site 2 as well as to a low affinity site (Site 4); (+)-nicotine which binds to Site 1, Site 4 and Site 3 which is also a high affinity site; and (-)-cytisine which binds to Sites 1 and 2. These drugs were injected into the 4th ventricle of 5 dogs in graded concentrations (12.5 to 400 micrograms) and their effects on the EEG, skin twitch reflex latency, heart rate, rectal temperature, pupillary diameter, blood pressure and the amplitude of the flexor reflex were measured. Drugs which act predominantly on Site 1 [(+/-)-2-methylpiperidine and (+)-nicotine] produced EEG synchronization and hyperalgesia while drugs which interact with Sites 2 and 4 produce EEG desynchronization, analgesia and tachycardia. These data indicate that nicotinic ligands which have different binding specificities have different actions in medullary function and support the hypothesis that the different binding sites have different pharmacologic significance.

Flumazenil is rapidly absorbed after oral or gastric fistula administration to the dog reaching peak plasma concentrations in about an hour. Plasma level decrease rapidly thereafter reaching barely detectable levels by four hours. The onset of signs of flumazenil precipitated abstinence in diazepam-dependent dogs is well correlated with the rise of flumazenil plasma levels, however, precipitated abstinence seizures occur when plasma levels have markedly decreased. Oral dosing is a more efficient way of administering flumazenil than gastric fistula dosing.

Dogs were made dependent on p.o. administered diazepam (24 or 36 mg/kg/day) or nordiazepam (18 mg/kg/day). Flumazenil (2, 6 or 18 mg/kg) administered p.o. once a week according to a Latin Square design precipitated abstinence in both groups of dogs. Abstinence was evaluated using a Nordiazepam Precipitated Abstinence Scale (NPAS) of various signs of abstinence and by counting seizure episodes. Flumazenil caused dose-related increases in the NPAS scores of both diazepam- and nordiazepam-dependent dogs; the slopes of the two dose-response lines were not different. Both groups of dogs also had both clonic and tonic-clonic seizures after flumazenil administration. CGS-8216 (2, 6 or 18 mg/kg) administered p.o. did not cause a dose-related elevation in NPAS scores for either group of dogs but clonic and tonic-clonic seizures were seen. Thus, flumazenil precipitates the benzodiazepine abstinence syndrome, as evidenced by tremors, tachypnea and other signs, including seizures, whereas CGS-8216 may have some selectivity in precipitating seizures without other signs of abstinence.

(+/-)-2-Methylpiperidine has a high degree of specificity in enhancing the binding of (-)-[3H]nicotine in the rat brain P2 preparation. (-)- and (+)-2-Methylpiperidine have been resolved. The (+) but not the (-) isomer increased the binding of (-)-[3H]nicotine. The two isomers were equally effective in inhibiting the binding of (-)-[3H]nicotine in high concentrations. These data provide additional support for a stereospecific nicotinic up-regulatory site.