Alfred A. Smith

Inhibition of growth in young mice treated with d,l-methadone☆☆☆

Neonatal mice were treated with d,1-methadone, 1-alpha-acetylmethadol (LAAM) or the narcotic antagonists, naloxone, nalorphine or levallorphan. Litter mates were injected with normal saline solution and handled in the same way. Treatment began on the second postpartum day and continued daily or on alternate days for up to 6 weeks. Injection of methadone in dosages of 2 mg/kg to mg/kg inhibited weight gain in a log dose-related fashion. LAAM, 1 mg/kg or 2 mg/kg also retarded weight gain. Mice gained weight normally when naloxone, 10 mg/kg was injected with methadone, 2 mg/kg. Furthermore the daily injection of d-methadone, 4 mg/kg, did not inhibit weight gain nor did any of the narcotic antagonists. There findings indicate that growth inhibition induced by methadone is a stereospecific, opioid effect.

Inhibition by propranolol of ethanol‐induced narcosis

Large doses of propranolol increase the sleeping time of mice or rats injected with barbiturates (Leszkovsky & Tardos, 1965) or chloral hydrate (Laverty & Taylor, 1968). This action is attributed to the central depressant property, rather than the ,&blocking effect of propranolol. Ethanol also depresses the central nervous system. When biogenic amines are given with ethanol the sleeping time of mice is prolonged (Rosenfeld, 1960) and the lethal effect of large doses of amine is potentiated. We now report on the influence of propranolol on the depressant effect of ethanol. Female, Swiss-Webster mice, 20-30 g in groups of 5, were tested for each dose; controls numbered 10 or more. Only 5 mice were observed at each time and observation was continuous. The drugs propranolol, 1 mg/kg, given 15 min before ethanol or ethanol as a 25% solution, w/v in normal saline, or sodium pentobarbitone, 60 mg/kg, were injected intraperitoneally. As shown in Fig. 1, the time for the return of righting reflex after pentobarbitone injection was increased by propranolol. However the return of the righting reflex after ethanol was shortened by low doses of propranolol but increasing the dose caused the righting reflex time to rise, roughly paralleling the slope for the barbiturate curve.

Interaction of Biogenic Amines with Ethanol

Ethanol through its primary catabolite, acetaldehyde, competitively inhibits oxidation of aldehyde dehydrogenase substrates. As a consequence biogenic amines form increased quantities of alcohols rather than the corresponding acids. During this biotransformation, condensation reactions between deaminated and intact amines may occur which can yield tetrahydropapaverolines. These compounds are closely related to precursors of opioids which is cause to link ethanol abuse to morphine addiction. There is, however, no pharmacological or clinical evidence suggesting similarities between ethanol dependence or opiod addiction. Acetaldehyde plays an additional role in alkaloidal formation in vitro. Biogenic amines may react with acetaldehyde to form isoquinoline or carboline compounds. Some of these substances have significant pharmacological activity. Furthermore, they may enter neural stores and displace the natural neurotransmitter. Thus, they can act as false neurotransmitters. Some investigators believe that chronic ethanol ingestion leads to significant formation of such aberrant compounds which may then upset autonomic nervous system balance. This disturbance may explain the abnormal sympathetic activity seen in withdrawal. While these ideas about the etiology of alcohol abuse have a definite appeal, they are naturally based on in vitro preliminary work. Much study of the quantitative pharmacology of these compounds in animals is required before judgement can be made as to the merits of the proposed hypotheses. In the meantime, pharmacological studies on the ability of ethanol to depress respiration in the mouse has revealed that unlike opioids or barbituates, respiratory depression induced by ethanol requires the presence in brain of serotonin. This neurotransmitter also mediates the respiratory effects of several other alcohols but curiously, not chloral hydrate, yet this compound is purported to alter biogenic amine metabolism much like ethanol. Thus, the response to ethanol can be pharmacologically separated from other major narcotic classes such as opioids and barbiturates by respiratory depression effects. The specific requirement for serotonin mediation exhibited by ethanol and several other alcohols opens the door for a rational therapeutic approach to the treatment of alcohol abuse. At the same time, this finding tends to lessen the probability that alcoholism is in some way connected with the formation of addictive alkaloids.

Inhibitory by d,1-methadone of RNA and protein synthesis in neonatal mice: Antagonism by naloxone or naltrexone

Neonatal mice were injected once daily with d,l-methadone in a dosage of 2 mg/kg. The remaining half of the litter was injected with saline. After one week the incorporation of labeled uridine and labeled leucine was significantly (P less than 0.05) reduced in skeletal muscle. Longer treatment with methadone impaired RNA and protein synthesis in liver, heart, skeletal muscle and brain. The percentage reduction RNA was log-dose related. When methadone was discontinued for 1 week following 4 weeks of treatment, incorporation of precursors into RNA and protein was found to be normal. A specific opioid effect is suggested by the finding that naltrexone or in part, naloxone, given concomitantly with with the methadone prevents development of the biochemical lesion.

Inhibition of neurotrophic activity in salamanders treated with opioids

Abstract Regeneration of the amputated hind limb of the salamander was prevented by the injection of methadone or levorphanol. Blastema failed to develop despite formation of a thick epidermal cap at the wound site. Injection of the narcotic antagonist, levallorphan, blocked blastemal development for 4 wk but normal growth then began. Phalanges appeared 4 wk later than in controls. Naloxone, a potent narcotic antagonist without agonist activity, neither altered blastemal development nor prevented normal regeneration and differentiation. Treatment with the opioids methadone or levorphanol produced some atrophy of the lingual epithelium and taste buds of the salamanders. Neither of the antagonists caused these changes.

Reversal by sotalol of the respiratory depression induced in mice by ethanol

Smith & Hayashida (1 970) reported that treatment with a P-adrenoceptive antagonist inhibited respiratory depression in mice subsequently given graded doses of ethanol, and correlated with this effect was a shortening of the sleeping time (Smith, Hayashida & Kim, 1970). While prevention of ethanol intoxication is of interest, reversal of the central depression would imply a therapeutic potential. Swiss-Webster female mice, 18-22 g, had food and water available up to the time of the experiment. Groups of at least 10 mice for each dose were then injected with ethanol as a 25 % solution (w/v) in normal saline, in doses ranging from 1 to 5 g/kg, The groups receiving only ethanol solution were tested 45 min after iiijection. An incision was then made in the ventral surface of the proximal third of the tail and the capillary blood collected into 125 p1 heparinized tubes without significant air exposure. The contents of the tube were then transferred to a Radiometer microelectrode system for determination of the pH and pC0,. A dose-related fall in pH with a concomitant rise in pC0, was found (Fig. 1). The horizontal bars indicate the standard error. The curves are plotted in a linear fashion to allow comparison with normal values and the influence of the drug on cmtrol pH or pC0,. Sotalol, (MJ 1999) 10mg/kg, was given in aqueous solution 15 min after ethanol, at a time when many mice injected with ethanol were already asleep. Thirty min later capillary blood was obtained from the tails. Mice develop maximal respiratory depression about 30 min after parenteral injection of a moderate dose of ethanol. The pH and pC0, changes begin within minutes after ethanol injection. In the mice treated with sotalol, capillary blood pH did not fall except at the highest doses of ethanol, while pC0, rose only slightly. The fact that mice treated with large doses of ethanol were already narcotized at the time of sotalol injection suggests that the drug acts not by preventing entry of ethanol into the brain but by antagonizing some action of ethanol or possibly acetaldehyde. Since the respiratory depressive effect of ethanol is closely related to dose, whereas acetaldehyde concentration is probably constant, it would seem that ethanol alone depresses respiration. The sleeping time was slightly attenuated in mice treated with ethanol (4 g/kg) and subsequently with sotalol (40 f 3.9 rnin versus 30 f 3.6 min). There was however, no shortening of sleeping time for mice given the larger dose of 5 g/kg despite relief of the respiratory depression and acidosis. Evidently respiratory depression and narcosis are not equivalents.

Modulation of the respiratory depressant effect of ethanol by 5-hydroxytryptamine

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Inhibitors of Tolerance Development

Tolerance to opioids is a complex phenomenon that depends in large measure on dose and duration of treatment. Recently the use of pharmacological agents to prevent tolerance has permitted a sharper view of some of the mechanisms responsible for this phenomenon. In order to examine more easily some of the experiments involving inhibition of tolerance to opioids, a brief review of several of the proposed mechanisms seems appropriate. This subject will be more thoroughly reviewed elsewhere in this volume.

Inhibition of nucleic acid synthesis in the regenerating limb of salamanders treated with a dl-methadone or narcotic antagonists

Intraperitonal injection of single dose of methadone, 2 mg/kg/day, cyclazocine, 1 mg/kg/day, or naltrexone, 10 mg/kg/2 days to Ambystoma mexicanum significantly retarded limb regeneration rates. [H+]) uridine incorporation into RNA of the regenerating limb in Ambystoma tigrinum was profoundly inhibited (P < 0.01) by each of these drugs. Significant, but a lesser inhibition (P < 0.05), was observed in DNA synthesis. Individual variation was eliminated by comparison of the specific activities in the regenerated limb with a similar portion of the contralateral nonregenerating limb. These findings parallel the inhibition of RNA and DNA synthesis caused by acute denervation of regenerated amphibian limbs.

Unmyelinated nerves in familial dysautonomia.

Familial dysautonomia is a rare neurologic disorder characterized by alacrima, areflexia, faulty blood pressure regulation, multiple sensory deficits, and subnormal growth.’ The protean manifestations of this inherited disorder suggest a central cause, but no consistent brain abnormality has been reported.’ A peripheral etiology is suggested by the finding of relatively few unmyelinated nerve fibers in the tongue of one child with dysautonomia3 and in the sural nerve of a n ~ t h e r . ~ If generalized, these findings would account for many of the sensory and autonomic disturbances affecting patients with dysautonomia. A study was then made of a lacrimal gland, the muscle spindle from an extraocular muscle, and sections of tongue t o detcrminc whether unmyelinated fibers were lacking in these organs, which were clearly involved in the dysautonomic process.