Evaluation of non-clinical toxicity studies of COVID-19 vaccines.

In this study we evaluated the outcomes of non-clinical toxicity studies of various SARS-CoV-2 vaccines produced with different manufacturing technologies, with focus on Repeated Dose Toxicity (RDT) and Developmental and Reproductive Toxicity (DART) studies. We found that RDT and DART studies at doses relevant for human treatment showed no adverse effects while remaining observations were expected findings including local reactogenicity, immune response and macroscopic findings at the injection site. We have also reviewed the European Medicines Agency (EMA) nonclinical assessment reports for market authorization. Regardless of utilized vaccine manufacturing technology EMA assessment of the non-clinical studies consisted most frequently of comments related to study design, species selection and missing data. Sponsors have often submitted platform studies (vaccine studies with the same technology/construct but using other antigens) as supplementary data. Animal model-based toxicity testing has shown rather small effects, which have been never serious adverse effects. The translational value to support clinical development is mainly to inflammatory effects, indicative of the primary action of the vaccines. From a 3R perspective supportive platform technology data consisting of previously executed RDT and DART studies from the same platform technology are encouraged to be implemented in the vaccine assessment process.

The pre- and post-authorisation data published by the European medicines agency on the use of biologics during pregnancy and lactation.

AIMS: The effects of biologics on reproduction/lactation are mostly unknown although many patients that receive biologics are women of reproductive age. The first objective of this study was to investigate the publicly available data on pregnancy/lactation before and after marketing authorization in Europe of biologics for the indications of rheumatologic inflammatory autoimmune diseases and inflammatory bowel disease. Secondary objectives included the assessment of the clinical relevance of the provided data and comparison of initial and post-authorization data. METHODS: Initial and post-authorization data were extracted from the European Public Assessment Reports and the latest versions of Summary of Product Characteristics using publicly available documents on the European Medicines Agency's website. Four sections were categorized regarding pregnancy outcomes: pre-clinical/animal studies, human female fertility, pregnancy-related outcomes and congenital malformations in the human fetus. Three sections were categorized regarding lactation outcomes: pre-clinical/animal studies, excretion in human breast milk and absorption in children through breastfeeding. The clinical applicability of each category was scored by specified criteria, based on scientific literature, and further as defined by the authors. RESULTS: For the 16 included biologics, post-authorization data were delivered only for adalimumab, certolizumab pegol, etanercept and infliximab. For the 12 remaining biologics limited data on pregnancy and lactation during the post-marketing period of 2-21 years were available. CONCLUSIONS: In this article several suggestions are provided for improving a multidisciplinary approach to these issues. The initiation of suitable registries by marketing authorization holders and data transparency for clinicians and academics are highly endorsed.

Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

Classifying the adverse mitogenic mode of action of insulin analogues using a novel mechanism-based genetically engineered human breast cancer cell panel.

Insulin analogues are widely used in clinical practice. Modifications on the insulin molecular structure can affect the affinity and activation towards two closely related receptor tyrosine kinases: the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R). A switch towards higher IGF1R affinity is likely to emphasize mitogenesis rather than glucose metabolism. Relevant well-validated experimental tools to address the insulin analogue activation of either INSR or IGF1R are missing. We have established a panel of human MCF-7 breast cancer cell lines either ectopically expressing the INSR (A or B isoform) in conjunction with a stable knockdown of the IGF1R or ectopically expressing the IGF1R in conjunction with a stable knockdown of the INSR. In these cell lines, we systematically evaluated the INSR and IGF1R receptor activation and downstream mitogenic signalling of all major clinical relevant insulin analogues in comparison with insulin and IGF1R. While most insulin analogues primarily activated the INSR, the mitogenic activation pattern of glargine was highly similar to IGF1 and insulin AspB10, known to bind IGF1R and induce carcinogenesis. Yet, in a long-term proliferation assay, the proliferative effect of glargine was not much different from regular insulin or other insulin analogues. This was caused by the rapid enzymatic conversion into its two metabolic active metabolites M1 and M2, with reduced mitogenic signalling through the IGF1R. In summary, based on our new cell models, we identified a similar mitogenic potency of insulin glargine and AspB10. However, rapid enzymatic conversion of glargine precludes a sustained activation of the IGF1R signalling pathway.

Use of statins is associated with an increased risk of rheumatoid arthritis.

OBJECTIVES: Statins offer significant cardiovascular benefits. Their use, however, influences immune regulation, which may potentially facilitate autoimmunity, eventually resulting in autoimmune diseases such as rheumatoid arthritis (RA).The authors studied whether statin use was associated with an increased risk of developing RA by conducting a case-control study using the Netherlands Information Network of General Practice database. METHODS: The authors identified 508 patients aged 40 years or older with a first-time diagnosis of RA in the period 2001-2006. Each RA case was matched to five controls for age, sex and index date, which was selected 1 year before the first diagnosis of RA. Odds ratios for the first-time diagnosis of RA were verified by a referral to a rheumatologist and/or at least one prescription of disease-modifying anti-rheumatic drugs and/or two prescriptions of corticosteroids after the date of first diagnosis. RESULTS: Cases were more often users of statins (15.9%) compared to controls (8.6%). After adjustment for cardiovascular risk factors and use of comedication, statin use was associated with an increased risk of incident RA (adjusted OR, 1.71 (95% CI 1.16 to 2.53); p=0.007). A consistent trend of increasing risk with increased cumulative duration, cumulative defined daily doses and number of prescriptions was not observed. However, a small trend between the potency of statin treatment and the risk of RA was found. CONCLUSIONS: Statin use seems to be associated with an increased risk of developing RA. Our findings should be replicated by additional studies.

Release of outer membrane fragments from normally growing Escherichia coli.

A complex containing lipopolysaccharides, phospholipids and proteine separated from the medium by gelfiltration on Sephadex G-200 or by centrifugation. Electron microscopy revealed that this material is released as vesicles and membrane fragements. To determine the origin of these fragments, they were compared to outer and cytoplasmic membranes with respect to keto-deoxyoctulosonic acid, phospholipid, and protein content, phospholipid composition, fatty acid composition, protein distribution on sodium dodecyl sulfate-polyacrylamide gels, buoyant density, and content of several membrane marker enzymes. The results of this comparison indicate that the membrane fragments found in the culture supernatant of normally growing Escherichia coli consist of practically unmodified outer membrane. Possible mechanisms as to the cause of the release of outer membrane fragments, and its relationship to cell-division, are discussed.

Effects of inhibitors of GABA-transaminase on hole-board exploration and on temperature. Relation with effects on quasi-morphine abstinence behaviour induced by sodium dipropylacetate.

Four inhibitors of gamma-aminobutyric acid transaminase (GABA-T) were investigated together with respect to their effects on hole-board exploration and temperature and the relation with effects on quasi-morphine-abstinence behaviour induced by dipropylacetate (DPA) in rats. Amino-oxyacetic acid (AOAA), gamma-acetylenic-GABA (GAG), gamma-vinyl-GABA (GVC) and ethanolamine-O-sulfate (EOS) were found to reduce hole-board exploration especially in the higher doses used, although the time-course of the effect was different for the compounds. For EOS and GVG the decrease in hole-board exploration paralleled a strong hypothermic effect. The compounds AOAA and GAG exerted a less and more transient hypothermic effect. However, the decrease in hole-board exploration did not fall in with this decrease in temperature. AOAA and GAG were found to decrease DPA-induced body shakes and locomotor activity, while GVG and EOS had no effect on body shakes and transient effects but opposite to each other, on locomotor activity. The efficacy of the GABA-T-inhibitors was measured biochemically, and the influence on the activity of glutamate decarboxylase (GAD) was also determined. AOAA and GAG were found to be strong inhibitors of GABA-T whereas the other two compounds were less efficient in the used doses. In addition AOAA and GAG influenced the activity of GAD strongly, while using GVG only a small decrease was found. The results suggest that the anti-quasi-withdrawal, the sedative and the hypothermic effects are not related to each other nor related to an effect on GABA-T. The suppressive effects on quasi-withdrawal body shakes, however, could be related to the inhibition of GAD and a hypothesis involving a compartmentalized action of DPA on GABA-metabolism has been proposed.

A dual role for GABA in quasi-morphine abstinence behavior induced by di-n-propylacetate involving both initiation and termination.

Di-n-propylacetate (DPA) induces a behavioral syndrome in rats resembling morphine abstinence behavior. The inhibitory action of DPA on GABA degradation, resulting in an enhanced release of GABA, is probably responsible for this behavioral effect, since GABA antagonists, like bicuculline and picrotoxin, have been shown to suppress this behavior. However, the time-course of the DPA-induced behavior is much shorter than that of the DPA-induced increase of GABA concentrations. Therefore, we have studied the influence of enhanced GABA levels caused by a first injection of DPA and the behavior evoked by a second injection of DPA at different time intervals after the first injection. The results indicate that GABA fulfills a role in both the initiation and termination of DPA-induced behavior. The mechanism responsible for this dual action of GABA is ascribed to a differential sensitivity to DPA of the nerve terminal and glial metabolic compartments of GABA in the brain. The increase of GABA in the nerve terminal caused by DPA is probably responsible for the initiation of the quasi-abstinence behavior, whereas the overflow of GABA into the synaptic cleft may be responsible for the suppression of this behavior via stimulation of presynaptic autoreceptors. Another mechanism responsible for the rapid termination of the DPA-evoked behavior could be the formation of DPA metabolites which antagonize this behavior. From the results of experiments using some primary metabolites of DPA, a role for these metabolites in the termination of the DPA-induced behavior seems unlikely.

Chronic infusion of clonidine does not alleviate spontaneous morphine withdrawal symptoms in rats.

Opiate withdrawal is associated with behavioural symptoms and a sympathetic hyperactivity, the latter being sensitive to clonidine. The central question is whether behavioural symptoms would be also sensitive to clonidine. A rat model was used in which the locomotor activity was measured 24 h a day during the morphine withdrawal phase. Spontaneous withdrawal of morphine reduced strongly the high nocturnal locomotor activity, concomitantly decreasing food intake and body weight. Chronic infusion of clonidine (30-120 micrograms/kg/day) using osmotic minipumps had no effect on the withdrawal symptoms. Higher dosages (250-1000 micrograms/kg/day) potentiated rather than alleviated the withdrawal symptoms, suggesting an alpha 1-adrenergic effect of clonidine rather than an alpha 2-action. Therefore, we studied the action of a more specific alpha 2-agonist UK-14.304. UK-14.304 was less potent than clonidine in naive animals. It slightly alleviates the decrease of nocturnal activity during spontaneous morphine withdrawal. Furthermore, we have tested whether the effects of high dosages of clonidine could be altered by a specific alpha 1-antagonist doxazosine. Doxazosine reduced only slightly the potentiation in the decrease in food intake by clonidine during morphine withdrawal. For the other symptoms no interaction between doxazosine and clonidine was found. The data suggest that the use of clonidine in the detoxification of opiate dependent people is based on the suppression of the sympathetic hyperactivity rather than on symptoms with a more behavioural character.

Dipropylacetate-induced quasi-morphine abstinence behaviour in the rat: suppression by alpha 2-adrenoceptor stimulation.

The anti-withdrawal effect of clonidine was studied using quasi-morphine abstinence behaviour induced by dipropylacetate (DPA) in naive rats. Clonidine potently suppressed body shakes and locomotor activity (ID50 30 and 40 micrograms/kg IP respectively). Phenoxybenzamine and prazosine did not antagonize the anti-withdrawal effect of clonidine, whereas piperoxane and yohimbine were effective with respect to locomotor activity and a total abstinence score. Piperoxane also reversed the suppressive action of clonidine on body shakes. Other alpha 2-agonists (guanfacine, azepexole and BHT 920) also suppressed DPA-induced behaviour, whereas the lipophilic alpha 1-agonist ST-587 had such an effect only at high doses. The relative potencies of the alpha 2-agonists correlated well with their potency to exert other alpha 2-adrenoceptor mediated actions such as blood pressure lowering and sedation.

Dipropylacetate-induced quasi-morphine abstinence behaviour in the rat: involvement of amygdaloid and thalamic structures.

Quasi-morphine abstinence behaviour induced by di-n-propylacetate (DPA) in rats is thought to be caused by an increased GABAergic activity in the CNS. Behavioural responses after intracerebral injections of DPA were studied to gain insight into the centre median-parafascicularis (Cm-Pf) resulted in a large number of body shakes and greater locomotor activity when compared to other brain areas. Injection of DPA into the central amygdala (Ac) resulted in an enhanced number of chewing episodes. Administration of bicuculline methiodide (BMI) into the Cm-Pf, 5 min after intraperitoneal administration of DPA, suppressed by body shakes but had only minor effects on horizontal activity, whereas injection of morphine into the same structure suppressed both behavioural symptoms. It is concluded that GABAergic and opioid mechanisms in the Cm-Pf are involved in the DPA-induced behaviour. Injection of BMI into the central amygdala shortly after i.p. injection of DPA resulted in an increase in the number of body shakes, whereas no effect was observed on activity. Morphine applied to this structure slightly potentiated the locomotor activity, but had no effect on the body shakes induced by DPA. The present results suggest a facilitatory role for a GABAergic system in the Cm-Pf on body shakes, while in the central amygdala a GABAergic system exerts an inhibitory influence on this symptom of abstinence.

Dipropylacetate-induced shaking behaviour in the rat: a role of spinal alpha 2-adrenoceptors.

The role of spinal adrenoceptors in di-n-propylacetate (DPA)-induced shaking behaviour was studied. The alpha 2-agonist p-aminoclonidine in a dose of 5 micrograms was found to suppress the DPA-induced body shakes when injected intrathecally but not when given intraventricularly. There was an enhancement of DPA-induced body shakes after the intrathecal injection of idazoxan while only a slight decrease was found after the intrathecal injection of prazosin. Intrathecal injection of idazoxan but not of prazosin proved to be effective to reverse the DPA-induced body shakes suppressed by p-aminoclonidine. Although the difference in effectiveness after intrathecal and intravenous injection was less than has been described for opiates there are several arguments that indicate an effect confined to the spinal cord. The present results further evidence the notion that the DPA-induced shaking behaviour shares common mechanisms with some of the morphine abstinence symptoms. The data indicate that spinal alpha 2-adrenoceptors are at least partly involved in the suppressive effect of p-aminoclonidine on DPA-induced shaking behaviour.

The effects of centrally acting adrenergic agonists on temperature and on explorative and motor behaviour. Relation with effects on quasi-morphine withdrawal behaviour.

Seven structurally unrelated adrenergic agonists were compared in three models to measure sedation viz. hole-board exploration, open-field activity and rotarod performance. It appeared that all compounds exerted the same order of potency in these models except for BHT-920 which was more active in the open-field. The other compounds were clonidine, azepexole, guanfacine, lofexidine, UK-14.304 and ST-587. The sedative effect appeared to be not causally related with the hypothermic activity since much higher doses were necessary for the latter effect. There was a relation between the sedative effects and the suppression of body shakes and enhanced locomotor activity induced by dipropylacetate, a potent model for morphine-withdrawal behaviour. The body shakes appeared to be more sensitive than locomotor activity suggesting that the antiwithdrawal action of alpha 2-agonists might be intrinsically higher than their sedative action. The data support earlier findings indicating that the antiwithdrawal action of clonidine is mediated by alpha 2-adrenoceptors.

Effect of chronic prenatal morphine treatment of mu-opioid receptor-regulated adenylate cyclase activity and neurotransmitter release in rat brain slices.

Timed-pregnant rats received a semisynthetic diet with or without morphine (0.5-1 mg/g) for 2 weeks. After 21 days of gestation the morphine-dependent dams were decapitated and the foetal brains were dissected. Chronic morphine administration caused a profound increase of adenylate cyclase activity stimulated by postsynaptic D1 dopamine receptors in striatal slices. The relative inhibitory effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) on D1-stimulated cyclic AMP (cAMP) production was unaffected. In contrast, cAMP production induced via direct activation of the catalytic unit of adenylate cyclase with forskolin was not changed upon long-term morphine treatment, although DAGO strongly inhibited the effect of forskolin. The electrically evoked release of [3H]noradrenaline (NA) from superfused neocortical slices was strongly enhanced upon morphine treatment, whereas release induced by the calcium ionophore A23187, bypassing voltage-sensitive calcium channels, was unchanged. Again, the inhibitory effect of the mu receptor agonist DAGO was unaffected in neocortical slices from morphine-treated rats. It is suggested that tolerance to morphine may be caused by the fact that the opiate is acting against up-regulated signal transduction mechanisms, rather than by desensitization of central mu-opioid receptors. The pre- and postsynaptic changes may include an enhanced expression and/or biochemical modification of D1 receptors, Gs proteins and calcium channels in central neurons on which mu-opioid receptors are present. At the same time, these adaptive changes may underlie morphine withdrawal phenomena.

Tolerance and withdrawal after chronic lorazepam treatment in rats.

The purpose of the study was to develop an animal model for benzodiazepine tolerance and dependence on the basis of oral administration, using lorazepam as the test drug. We have used the continuous measurement of locomotor activity in home cages to obtain a narrow estimation of the time course of withdrawal related hyperactivity as an observer-independent symptom. Acute administration of lorazepam (9.5-37.5 mg/kg body weight/day) resulted in the first week in a dose-dependent muscle relaxation on the accelerod and sedation in the open field. The most striking manifestation of sedation, however, was the decrease of nocturnal locomotor activity in home cages. After 5 weeks of administration tolerance to the sedative effect had developed. In a second study, using a lower dose range (2.5-9.5 mg/kg body weight/day), a decrease of nocturnal locomotor activity was also observed as was the development of tolerance. The latter can be partly explained by dispositional tolerance, i.e., decreased serum concentrations after administration of lorazepam for more than 2 weeks. Withdrawal of lorazepam in the experiment using high doses led to three symptoms, i.e., a decrease in food intake, loss of body weight and an increase in daytime locomotor activity. The decrease in food intake and the loss of body weight were maximal on the first day of withdrawal. The increase in the daytime locomotor activity was present in the high dose experiment only, with a maximum on days 2-3 and a duration of at least 1 week. The increase however, was not dependent on the dose previously administered. The symptoms 'loss of body weight' and 'decrease in food intake' appeared to be more sensitive in benzodiazepine withdrawal: they were dose dependently present over the whole dosage range (2.5-37.5 mg/kg body weight/day). It is concluded that the model represents a sensitive model to measure lorazepam tolerance and dependence in animals. Comparative studies with other benzodiazepines are in progress.

Benzodiazepines preferentially affect mesolimbic dopamine turnover in rats.

Withdrawal of benzodiazepines in man may induce hallucinatory symptoms and can evoke delusional depressions, which can be treated with dopamine-antagonistic drugs. Withdrawal of benzodiazepines in rats induces a strong hyperactivity during daytime, leaving the nighttime activity relatively undisturbed. This hyperactivity may be related to an enhanced dopaminergic activity in the mesolimbic area, especially in the nucleus accumbens. Mesolimbic dopaminergic activity may be specifically involved in the development of benzodiazepine withdrawal. Acute administration of benzodiazepines in otherwise non-treated rats, has been described not to affect the dopamine-turnover in the nucleus accumbens, measured by synthesis inhibition. However, activation by administration of haloperidol (feedback activation) can be suppressed by benzodiazepines effectively. Five different benzodiazepines viz. desmethyldiazepam (DMD), lorazepam (LRZ), brotizolam (BTZ), triazolam (TRZ) and flunitrazepam (FNZ) have been compared with respect to their acute effects. Using a 3-fold increase in dopamine turnover (determined by measuring the DOPAC-concentration), benzodiazepines were capable to reduce this increase maximally for 70-80% in the nucleus accumbens. The results point to a selective effect of benzodiazepines in the nucleus accumbens. The increase induced by haloperidol in the corpus striatum was found to be much less sensitive to benzodiazepines. In contrast to the other compounds lorazepam appeared to have no effect on haloperidol-induced increase in DOPAC concentration. Flunitrazepam and brotizolam did affect not only the haloperidol-induced DOPAC increase but also the basal DOPAC concentrations. Linear dose-response curves could not be obtained for the compounds, but minimal effective doses could be assessed. Flunitrazepam and triazolam appeared to be the most active compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in locomotor-activity patterns as a measure of spontaneous morphine withdrawal: no effect of clonidine.

The anti-withdrawal action of clonidine was studied in a model of spontaneous morphine withdrawal in rats. After withdrawal the behaviour of the animals was registered continuously for several days. In the initial phase of induction of dependence the locomotor activity was enhanced during daytime. Partial tolerance to this increase developed in the course of 3 weeks. In morphine withdrawn animals the activity decreased strongly at night, and this effect was maximal on the second night after removal of morphine. After four nights the nightly activity was restored. Treatment with clonidine (100 micrograms/kg s.c. twice daily) changed neither the observed decrease in nightly locomotor activity nor other withdrawal symptoms such as a decrease in food intake and loss of body weight. In non-dependent animals clonidine induced a biphasic effect in locomotor activity, i.e. a decrease in the first few hours of the night and an increase in the second part of the night. The latter can be interpreted as a rebound phenomenon occurring after only three injections. It was concluded that clonidine was not effective as an anti-withdrawal compound in a model for spontaneous morphine abstinence. The low incidence of symptoms relating to a hyperactive sympathetic system during spontaneous withdrawal may be an explanation for the absence of an effect of clonidine.

Effects of alpha 2-agonists on morphine withdrawal behaviour: potentiation of jumping mediated by alpha 2-receptors.

Clonidine has a dual action on naloxone-precipitated morphine withdrawal symptoms in rats: a suppressive action on body shakes and body weight loss and a potentiating action on jumping and aggression. It has been suggested that this potentiating, excitatory action is mediated by alpha 1-receptors. More specific alpha 2-agonists therefore should have a less excitatory effect on the latter symptoms. This hypothesis has been studied in rats dependent on morphine. Withdrawal was precipitated using naloxone. Prior to naloxone the alpha 2-agonists clonidine, guanfacine, azepexole, BHT-920, UK 14304 or the centrally acting alpha 1-agonist ST 587 were administered. All alpha 2-agonists but not the alpha 1-agonist potentiated the jumping and decreased body shakes and body weight loss. The effects of clonidine and azepexole were characterized pharmacologically using the alpha-antagonists yohimbine and prazosin. Jumping potentiated by clonidine was antagonized by yohimbine whereas prazosin had no effect. Azepexole induced jumping was decreased by yohimbine both with respect to incidence and frequency, whereas prazosin only lowered the frequency. The suppressive actions of clonidine and azepexole on body shakes were reversed by yohimbine and not by prazosin. The data indicate that the potentiation of jumping by alpha 2-agonists as well as the suppression of body shakes in morphine withdrawal behaviour is mediated by alpha 2-receptors.

Effects of prolonged exposure to morphine and methadone on in vivo parameters of immune function in rats.

In rats, two 6-week repeated dose oral toxicity studies were performed with morphine (250 and 500 mg/kg food) and methadone (200 and 400 mg/kg food), respectively. Alterations in immune function were studied by assessing primary and secondary immune responses to sheep red blood cells. In addition, the ability to resist challenge with infectious agents was measured in host resistance models employing the parasite Trichinella spiralis and the bacterium Listeria monocytogenes. The primary and secondary antibody responses to sheep red blood cells were not affected by treatment with either morphine or methadone. The clearance of L. monocytogenes bacteria in the spleen was not affected either. Prolonged treatment with morphine, however, resulted in a decrease in host resistance to T. spiralis infection, as indicated by a 1.5-fold increase in numbers of muscle larvae counted in the carcass, but did not affect the T. spiralis-specific IgM, IgG and IgE antibody responses. In contrast to morphine, the methadone-treated animals did not show a significant change in host resistance to T. spiralis. Total serum IgG levels, however, were increased in high-dose methadone-treated animals. Apparently, prolonged administration of morphine to rats resulted in immune suppression, mediating a slight, though biologically relevant, exacerbation of the T. spiralis infection, whereas methadone did not.

Quasi-morphine withdrawal behaviour: indication for a specific involvement of the GABA-ergic nerve terminal compartment.

Wet-dog-shaking resembling morphine withdrawal behaviour can be evoked in rats by administration of dipropylacetate (DPA). It has been postulated that DPA elicits the withdrawal like behaviour through specifically inhibiting the GABA-degradation in nerve terminals. Inhibition of GABA-transaminase (GABA-T) by aminooxyacetic acid (AOAA) in other compartments of the brain would result in an inhibition of the stimulated GABA-release via feedback on autoreceptors and therefore suppress the DPA-evoked behaviour. This hypothesis has been tested using the GABA-T inhibitors gamma-acetylenic-GABA (GAG), gamma-vinyl-GABA (GVG) and ethanolamine-O-sulphate (EOS). Although GAG and AOAA were found to suppress the body shakes, both GVG and EOS had no effect. Both GAG and AOAA have possibly also effect on glutamate decarboxylase (GAD) whereas GVG and EOS did not affect this enzyme, suggesting a nerve terminal-specific effect on DPA-induced behaviour. GVG stimulated DPA-evoked body shakes after 36 and 60 h, when a specific GABA-increase in nerve terminals will be present.