Development and Characterization of Lyophilized Diazepam-Loaded Polymeric Micelles

Polymeric micelles were studied as delivery carriers of diazepam, a practically insoluble drug in water, for rectal administration. The diazepam-loaded polymeric micelles were developed by using poloxamer 407 (P407), poloxamer 188, and d-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS). Among the used polymers, TPGS resulted in polymeric micelles with good characteristics for encapsulation of diazepam which had the small particle size of 8–12 nm and narrow size distribution (PI 0.053–0.275). Additionally, 7.5% w/v of TPGS could entirely entrap the desired concentration of diazepam (5 mg/mL). To improve the physical stability upon lyophilization, an addition of P407 of 1% w/v prevented aggregation, increased physical stability, and maintained chemical stability of the lyophilized powders of diazepam-loaded polymeric micelles for 3 months storage at 4°C. The rate and amount of diazepam release from TPGS polymeric micelles mainly depended on the concentration of TPGS. The release data were fitted to Higuchi''s model suggesting that the drug release mechanism was controlled by Fickian diffusion. In conclusion, 10% w/v TPGS and 1% w/v P407 were the optimum formulation of lyophilized diazepam-loaded polymeric micelles.Key words: diazepam, lyophilization, poloxamer 407, polymeric micelles, d-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS)     

Diazepam and Tests of Thyroid Function

The effect of diazepam on thyroid function tests was examined in 12 euthyroid patients requiring the drug for psychiatric reasons and in six patients with thyrotoxicosis. Assessment was made before and after four weeks'' therapy.There was no significant difference in results from tests of thyroid iodide trapping and binding (thyroid radioiodine uptake, thyroid clearance, and absolute iodine uptake) except in the one-hour thyroid uptake in the euthyroid group, which was increased after diazepam. This increase occurred without alteration in serum thyroid stimulating hormone levels. No change occurred in either group in tests of thyroid hormone release (protein-bound iodine, T-3 resin uptake, or Thyopac-3 and free thyroxine index).Patients with suspected thyroid disease who are taking diazepam do not need to stop therapy while their thyroid status is being determined.     

Diazepam sensitive mice: differential sensitivity to the depressant and anticonvulsant effects of diazepam

A mouse line was developed by selecting for increased sensitivity to the hypnotic effect of diazepam. These "diazepam sensitive" mice showed a mean duration of loss of righting reflex (LORR) of approximately 150 min at a dose of 20 mg/kg diazepam, this dose failed to induce LORR in the control outbred mice. Rotarod treading times of the diazepam sensitive mice were significantly shorter than that of the control mice over the same dose range indicating that these mice are also more sensitive to the sedative/muscle relaxant effects of diazepam. On the contrary, the ability of diazepam to protect against pentylenetetrazole-induced convulsion was found to be the same in the sensitive and control mice. These observations strongly suggest that the heightened sensitivity to the sedative-hypnotic effects of diazepam in the sensitive mice is unlikely to be due entirely to changes in drug disposition.     

Influence of age and previous use of diazepam dosage required for endoscopy.

In 19 patients (10 men and 9 women) a 22-fold variation was observed in the intravenous dose of diazepam necessary as preparation for endoscopy (median dose, 20 mg; range, 5 to 110 mg). Analysis of plasma samples for diazepam and N-desmethyldiazepam revealed that the clinical response did not relate to the rate or character of initial drug distribution. There was a high correlation (r = 0.96) between the dose and the plasma concentration 10 minutes after administration. Users of diazepam displayed tolerance to its pharmacologic effects, requiring a significantly larger (P less than 0.05) dose than nonusers (median doses, 35.0 mg and 14.5 mg respectively). Older patients required less than younger patients (r = -0.54, P less than 0.05). The variation between individuals in the dose of diazepam required as preparation for endoscopy cannot be explained by variation in drug disposition but instead reflects previous diazepam use, age and probably differences in sensitivity at the site or sites of drug action.     

An ethological analysis of the influence of perinatally-administered diazepam on murine behaviour

1. A number of studies have found that the perinatal exposure of rodents to various tranquillizing agents alters their adult behaviour.2. Given the known anti-aggressive influence of acute doses of diazepam it was hypothesized that, when administered during pregnancy, this drug would change the adult social behaviour.3. The social interactions of adult male mice whose mothers were treated with diazepam or appropriate controls during pregnancy were video-taped and subjected to an ethological analysis that involved counting the incidences of 43 distinct postures.4. Prenatal but not postnatal diazepam treatment was associated with a large increase in the incidence of the sideways offensive posture. No significant differences resulted, however, in the case of other postures; in general exposure to prenatal diazepam produced few changes in adult social behaviour.     

Failure to confirm consistent stimulation of growth hormone by diazepam

Diazepam has been reported to influence pituitary hormone secretion, with several studies claiming that diazepam provokes growth hormone release. Normal volunteers were therefore examined for anterior pituitary responsiveness to 10 mg diazepam i.v. and p.o. The drug had no significant effect on the secretion of prolactin or thyrotropin when compared to control saline injections in all subjects. Growth hormone response was variable; serum growth hormone increased significantly in only 4 of 10 patients after i.v. diazepam and in only 1 of 7 subjects tested with oral diazepam. Serum cortisol rose in only 1 subject, precluding a stress-related explanation for the increase in growth hormone. We conclude that diazepam inconsistently stimulates growth hormone secretion and should not be relied upon as a test of growth hormone reserve.     

The effect of diazepam on potassium contractures, contraction threshold, and resting tension in rat skeletal muscles

The effects of diazepam on potassium contractures, contraction threshold, and resting tension have been examined in rat soleus muscle fibres. Two actions of the drug were defined that could not be attributed to changes in the resting membrane potential or depolarization in high potassium solutions. The major effect was an increase in the amplitude of submaximal tension during either twitches or potassium contractures and an increase in resting tension. At 400 microM diazepam, there was (a) a fourfold increase in 40 mM potassium contracture tension, (b) a negative shift of 8 mV in the membrane potential for half maximum tension estimated from the best fit of a Boltzmann-type equation to average potassium contracture data, (c) a negative shift of 8 mV in the threshold for contraction measured under voltage clamp conditions, and (d) a contracture of variable amplitude to a level that was occasionally equivalent to maximum tetanic tension. These potentiating actions of diazepam depended on drug concentration within the range of 100-800 microM. In contrast, the second effect of diazepam, depression of maximum tension by 10-15%, was independent of drug concentration between 100 and 400 microM. The results support the idea that diazepam produces an increase in resting myoplasmic calcium concentrations.     

Characteristics of the abstinence syndrome induced by the administration of the benzodiazepine antagonist CGS 8216 to rats following chronic treatment with diazepam

Male rats were treated with 5 and 10 mg/kg diazepam once daily for 5-30 days. After the drug discontinuation a benzodiazepine receptor antagonist CGS 8216 (2.5-10 mg/kg) induced a behavioural syndrome that might be characterized as an abstinence syndrome. The most typical signs of abstinence were head twitches, myoclonic seizures of forepaws, emotional hyperirritability, increased muscle tone of the tail, sniffing and chewing. These behavioural changes could be observed within 1-1.5 hours after CGS 8216 injection. The abstinence syndrome was induced by repeated CGS 8216 injections for 10-15 days after diazepam discontinuation. Further analysis has shown that that the intensity of abstinence was dependent on the dose and duration of chronic diazepam, as well as on CGS 8216 dose. It is suggested that CGS 8216-induced abstinence syndrome in rats chronically treated with diazepam might be used as a tool for studying the addictive potential of benzodiazepines.     

Improving benzodiazepine prescribing in family practice through review and education.

Indications for and dosages of four commonly prescribed benzodiazepines were recorded at a family medicine centre with the aid of a computerized data collection system. Four guidelines were then developed for appropriate prescribing of these drugs: (a) benzodiazepines should be used less frequently with increasing age; (b) short-acting drugs are preferable to long-acting drugs; (c) patients 65 years of age and over should receive half the daily dose prescribed for younger patients; and (d) use of these drugs for more than 1 month should be discouraged. After a year''s observation it was evident that none of the guidelines were being followed. The 30 physicians in the practice were then informed of the findings by an educational program. Another 6 months of observation showed a reduction in the prescribing of benzodiazepines to patients 65 years of age and over, a significant shift to the use of short-acting benzodiazepines, and some reduction in the daily dose and duration of administration of diazepam. Thus, such a review of drug prescribing in family practice can be a practical and effective method of improving prescribing patterns.     

Effects of diazepam on photosynthesis,respiration, rubidium uptake,and finestructure of Scenedesmus obliquus in synchronous cultures

Effects of diazepam (Valium) on photosynthesis, chlorophyll/photosynthesis ratios, respiration, uptake of rubidium ions, and ultrastructure of Scenedesmus obliquus synchronized by a light-dark regimen of \(14:\overline {10}\) hrs were determined. 80 and 160 μM diazepam, added to the nutrient medium at the start of the light-dark change (i.e., start of the cell cycle) gradually reduced rates of photosynthesis below the initial rates from the beginning of the experiment. Contents of chlorophyll, however, remained nearly unaffected. Consequently, the diazepam-treated cells had a higher chlorophyll/photosynthesis ratio—also with regard to respiration in order to calculate the gross photosynthesis. The occurrence of photorespiration cannot be assumed. The net influx or rubidium was slightly reduced by 100 μM diazepam 0.5 and 2.0 hrs after the start of the cell cycle and was strongly inhibited after 5 to 14 hrs. 80 and 160 μM diazepam caused separation of thylakoids, formation of giant mitochondria and enlargement of vacuoles. The results are discussed and it is finally suggested that diazepam acts on different membrane systems. Furthermore an ATP deficiency cannot be excluded.     

Diazepam withdrawal syndrome: its prolonged and changing nature.

The diazepam withdrawal syndrome was studied in 10 patients who had abused the drug for 3 to 14 years. In the previous 6 months their consumption of diazepam had ranged from 60 to 120 mg daily; none had used other drugs during this period. The withdrawal period lasted about 6 weeks. The intensity of the symptoms and signs was high initially, fell during the first 2 weeks, then rose again in the third week, before finally declining. Three groups of symptoms and signs were identified. Group A symptoms occurred throughout withdrawal and included tremor, anorexia, insomnia and myoclonus. Group B symptoms and signs were largely confined to the first 10 days and were those of a toxic psychosis. Group C symptoms reached a peak in the third and fourth weeks of withdrawal and were characterized by sense perceptions that were either heightened or lowered. The symptom groups, the presence of tremor and myoclonus, and the relief of symptoms by a test dose permit diazepam withdrawal to be distinguished from anxiety. The biphasic course of the symptoms is probably related to the pharmacokinetics of diazepam.     

Effects of denzimol on benzodiazepine receptors in the CNS: Relationship between the enhancement of diazepam activity and benzodiazepine binding sites induced by denzimol in the rat

Denzimol, a new anticonvulsant drug with a pharmacological profile similar to that of phenytoin, enhances the ataxic and antimetrazol activity of diazepam in rats without affecting its activity against picrotoxin-induced seizures. $\text{In vivo}$ and $\text{ex vivo}$ denzimol enhances the binding of ³H-flunitrazepam in cortex and in hippocampus but not in cerebellum.The possibility of this increase in the number of benzodiazepine binding sites contributing in some way to enhancement of the depressive and anticonvulsant activity of diazepam is discussed.     

Diazepam induces relaxation of chick embryo muscle fibers in vitro and inhibits myosin synthesis

Diazepam (Valium/Roche) causes an immediate cessation of spontaneous contraction in chick embryo skeletal muscle fibers growing in vitro. Between 24–48 h later in the presence of 100 μM diazepam the relaxed muscle fibers no longer accumulate myosin as measured by the total amount of myosin heavy-chain peptide extracted from the cell cultures and identified by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The myosin heavy chain assay procedure was standardized by quantitative precipitation of myosin with antibody to column purified chicken skeletal muscle myosin. Failure to accumulate myosin is related to a progressive inhibition of myosin synthesis. Diazepam-treated cultures showed an 80% inhibition of myosin heavy-chain synthesis over a period of 4 days. At the same time the rate of myosin heavy-chain degradation increases in diazepam-treated cultures relative to matched control cultures. Total protein synthesis was only marginally affected suggesting that diazepam may differentially inhibit myofibrillar protein synthesis. All of the observed effects of diazepam were reversible if drug exposure was limited to 48 h. The apparent specificity and reversibility of diazepam suggests that the drug will be useful in probing the mechanisms of terminal skeletal muscle cell differentiation and the hypotrophic relationship between chronic relaxation and inhibition of accumulation of myosin and perhaps other myofibrillar proteins.     

Use of the overturn end point in goldfish to measure the interaction of diazepam and ethanol and the effects of the binding of diazepam to bovine serum albumin

Over the ranges 2.8 X 10(-5) to 8.78 X 10(-5) M diazepam and 4.85 X 10(-2) to 1.22 X 10(-1) M ethanol, addition of the effects of these agents on the overturn end point in goldfish was observed. The addition of bovine serum albumin (1.56 X 10(-5) M) to aqueous solutions of diazepam modifies the diazepam effect by reducing the "free" drug concentrations.     

Effects of diazepam at the neuromuscular junction

Diazepam (Valieum, Roche) is a centrally-acting drug belonging to the benzodiazepine group of tranquillisers with anxiolytic, hypnotic, anti-convulsant and myorelaxant properties (1). It has been reported that in addition to its central effects (1), diazepam also produces relaxation of the skeletal muscle (2, 3). The myorelaxation produced by diazepam is thought to be of central origin (2), although at least some of the effects is due to a peripheral effect of diazepam, i.e. at the neuromuscular junction.Although the effects and interactions of diazepam with neuromuscular blocking agents have been studied by many workers (2–12), the results reported are somehow are controversial (4–8). In sum, diazepam can either enhance or depress neuromuscular transmission, the effect being dependent on the concentration and the type of the preparation used. A multi-site of action of diazepam may provide an explanation for some of the anomalies reported in the literature.     

Prevention of alcohol withdrawal seizures with oral diazepam loading

Twenty patients withdrawing from alcohol who had reliable histories of previous alcohol-withdrawal seizures and thus were at high risk for a subsequent seizure were treated in hospital with oral diazepam loading: 20 mg of the drug was given every hour to a minimum total of 60 mg. None of the patients had a seizure during the stay in hospital. We believe that phenytoin prophylaxis is not necessary in these circumstances. However, if the patient is already taking phenytoin, this drug should not be abruptly discontinued in the withdrawal period in favour of diazepam loading.     

An isobolographic analysis of the hypnotic effects of combinations of dexmedetomidine with fentanyl or diazepam in rats.

The effects of the combinations of dexmedetomidine-fentanyl and dexmedetomidine-diazepam on the righting reflex were studied in rats. The doses that block the righting reflex for the agents given alone and for their combinations were determined with a probit procedure and compared with an isobolographic analysis. The interactions between dexmedetomidine and fentanyl or diazepam were found to be synergistic. In the dexmedetomidine-diazepam combination studies, less than one-fourth of the single drug dose (for each of two agents) was needed to produce the required effect. These data confirm synergistic interactions between dexmedetomidine and fentanyl or diazepam in producing hypnotic-anesthetic action.     

Pentazocine and codeine: effects on human performance and mood and interactions with diazepam

Effects of two opioid analgesics on performance and their interactions with diazepam were studied double-blind and cross over in ten healthy students. At two-week intervals the subjects received first a single oral dose of placebo, codeine (100 mg) or pentazocine (75 mg). Then, 1 h 30 min later they were all given diazepam (0.25 mg/kg) orally. Lastly, naloxone (0.4 mg) was injected intravenously at 4 h. In addition to this, the subjects on pentazocine received a second 75 mg dose at 3 h. Codeine and pentazocine alone failed to affect performance in objective tests (body sway, digit symbol substitution, flicker fusion, Maddox wing, nystagmus) recorded at 1 h 30 min. Visual analogue scales showed subjective drug effects: pentazocine made the volunteers talkative, contented, interested and energetic, whilst codeine rendered them mentally slow. 75 mg of pentazocine and 100 mg of codeine produced comparable plasma opiate activity (determined in morphine equivalents) according to radioreceptor bioassay with [3H]-dihydromorphine as a ligand. Impaired performance was clear at the tests done 1.5 and 2.5 h after diazepam. No major interactions were found between opiates and diazepam in objective tests with the exception that nystagmus was stronger after the combined treatment than after diazepam alone. Codeine reduced the absorption of diazepam. Subjectively codeine and pentazocine counteracted the effects of diazepam. The subjects overestimated their performance after opiate + diazepam when compared to diazepam alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of diazepam in the mitochondria of Tetrahymena pyriformis as detected by quantitative high resolution autoradiography

Tritiated diazepam accumulates mainly in the mitochondria of the unicellular Tetrahymena. This is the case in both a single (the first encounter) and a repeated (one day or a week after the first) administration of the drug. When imprinting of Tetrahymena by diazepam (the first encounter) is followed a week later by the administration of the labelled drug, the membranes of the vesicles, too, show the appearance of label. Regarding the studies presented here, the unicellular Tetrahymena also contain diazepam receptors in the mitochondria as suggested for cells of higher rank animals.     

Benzodiazepine Binding to Cultured Human Pituitary Cells

Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.