Karen L. Houseknecht

Acute Effects of Atypical Antipsychotics on Whole-Body Insulin Resistance in Rats: Implications for Adverse Metabolic Effects

Although it is generally accepted that atypical antipsychotics differ in their risk for diabetic side effects, the underlying pharmacological mechanisms are unknown. Studies on the mechanisms of antipsychotic-induced hyperglycemia or insulin resistance are often confounded by the concomitant weight gain and dyslipidemia, known diabetic risk factors. To investigate whether antipsychotics can acutely cause metabolic effects before any change in body composition, we studied the effects of four atypical antipsychotics on whole-body insulin resistance. Using the hyperinsulinemic, euglycemic clamp technique in conscious rats, insulin and somatostatin were infused at a constant rate to provide constant hyperinsulinemia and to suppress pancreatic insulin secretion. Glucose was infused at a variable rate, adjusted to maintain euglycemia. At steady state, animals were administered vehicle (V) or antipsychotic and the glucose infusion rate was monitored as an index of insulin sensitivity. Clamp experiments using radiotracers and studies on glucose uptake into isolated skeletal muscle were conducted to differentiate between effects on hepatic glucose production (HGP) and on peripheral glucose uptake. Olanzapine (OLAN) and clozapine (CLOZ) acutely impaired whole-body insulin sensitivity in a dose-dependent manner (P<0.001 vs V), whereas ziprasidone and risperidone had no effect. CLOZ also induced profound insulin resistance after dosing 10u2009mg/kg/day for 5 days (P<0.05 vs V). Tracer studies indicated that acute changes mainly reflect increased HGP, consistent with the lack of effect on glucose uptake. OLAN and CLOZ can thus rapidly induce marked insulin resistance, which could contribute to the hyperglycemia and ketoacidosis reported for patients receiving those therapies.

Mephedrone (4-methylmethcathinone) supports intravenous self-administration in Sprague-Dawley and Wistar rats.

Recreational use of the drug 4‐methylmethcathinone (mephedrone; 4‐MMC) became increasingly popular in the United Kingdom in recent years, spurred in part by the fact that it was not criminalized until April 2010. Although several fatalities have been associated with consumption of 4‐MMC and cautions for recreational users about its addictive potential have appeared on Internet forums, very little information about abuse liability for this drug is available. This study was conducted to determine if 4‐MMC serves as a reinforcer in a traditional intravenous self‐administration model. Groups of male Wistar and Sprague‐Dawley rats were prepared with intravenous catheters and trained to self‐administer 4‐MMC in 1‐hour sessions. Per‐infusion doses of 0.5 and 1.0u2009mg/kg were consistently self‐administered, resulting in greater than 80% discrimination for the drug‐paired lever and mean intakes of about 2–3u2009mg/kg/hour. Dose‐substitution studies after acquisition demonstrated that the number of responses and/or the total amount of drug self‐administered varied as a function of dose. In addition, radiotelemetry devices were used to show that self‐administered 4‐MMC was capable of increasing locomotor activity (Wistar) and decreasing body temperature (Sprague‐Dawley). Pharmacokinetic studies found that the T1/2 of 4‐MMC was about 1 hour in vivo in rat plasma and 90 minutes using in vitro liver microsomal assays. This study provides evidence of stimulant‐typical abuse liability for 4‐MMC in the traditional pre‐clinical self‐administration model.

Effect of Ambient Temperature on the Thermoregulatory and Locomotor Stimulant Effects of 4-Methylmethcathinone in Wistar and Sprague-Dawley Rats

The drug 4-methylmethcathinone (4-MMC; aka, mephedrone, MMCAT, “plant food”, “bath salts”) is a recent addition to the list of popular recreational psychomotor-stimulant compounds. Relatively little information about this drug is available in the scientific literature, but popular media reports have driven recent drug control actions in the UK and several US States. Online user reports of subjective similarity to 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) prompted the current investigation of the thermoregulatory and locomotor effects of 4-MMC. Male Wistar and Sprague-Dawley rats were monitored after subcutaneous administration of 4-MMC (1–10 mg/kg ) using an implantable radiotelemetry system under conditions of low (23°C) and high (27°C) ambient temperature. A reliable reduction of body temperature was produced by 4-MMC in Wistar rats at 23°C or 27°C with only minimal effect in Sprague-Dawley rats. Increased locomotor activity was observed after 4-MMC administration in both strains with significantly more activity produced in the Sprague-Dawley strain. The 10 mg/kg s.c. dose evoked greater increase in extracellular serotonin, compared with dopamine, in the nucleus accumbens. Follow-up studies confirmed that the degree of locomotor stimulation produced by 10 mg/kg 4-MMC was nearly identical to that produced by 1 mg/kg d-methamphetamine in each strain. Furthermore, hypothermia produced by the serotonin 1A/7 receptor agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) was similar in each strain. These results show that the cathinone analog 4-MMC exhibits thermoregulatory and locomotor properties that are distinct from those established for methamphetamine or MDMA in prior work, despite recent evidence of neuropharmacological similarity with MDMA.

Contrasting effects of d-methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxypyrovalerone, and 4-methylmethcathinone on wheel activity in rats.

BACKGROUNDnReports from U.S., U.K. and European drug policy entities, and ongoing media accounts, show increasing recreational use of 4-methylmethcathinone (4-MMC, mephedrone) and 3,4-methylenedioxypyrovalerone (MDPV). Severe sympathomimetic symptoms, hallucinations, psychoses, and even deaths have been reported, yet little scientific information is available on the effects of these compounds in laboratory models. Available studies on the neurochemistry of these drugs show that 4-MMC and MDPV enhance DA neurotransmission, while 4-MMC additionally enhances 5-HT neurotransmission--a pattern much like that reported for methamphetamine versus 3,4-methylenedioxymethamphetamine (MDMA). As is the case for designer amphetamines, these neurochemical distinctions may predict differential potential for repetitive versus episodic abuse and distinct lasting toxicities.nnnMETHODSnThis study determined relative locomotor stimulant effects of 4-MMC (1-10 mg/kg, s.c.) and MDPV (0.5-5.6 mg/kg, s.c.), in comparison with d-methamphetamine (MA; 0.5-5.6 mg/kg, s.c.) and MDMA (1-7.5 mg/kg, s.c.) on a measure of locomotor activity--voluntary wheel running--in male Wistar rats (N=8).nnnRESULTSnCompared to counts of wheel rotations after saline, a biphasic change in the pattern of counts was observed after injections of MA and MDPV, with relatively higher counts following lower doses and lower counts following the highest dose. However, monophasic, dose-dependent reductions in counts were observed in response to injections of MDMA and 4-MMC.nnnCONCLUSIONnThus, voluntary wheel running yielded the same categorical distinctions for these drugs as did prior experiments testing the effects of these drugs on monoaminergic neurotransmission. These data indicate that MDPV produces prototypical locomotor stimulant effects whereas 4-MMC is more similar to the entactogen MDMA.

Changes in ambient temperature differentially alter the thermoregulatory, cardiac and locomotor stimulant effects of 4-methylmethcathinone (mephedrone)

BACKGROUNDnThe substituted cathinone compound known as mephedrone (4-methylmethcathinone; 4-MMC) has become popular with recreational users of psychomotor-stimulant compounds. Only recently have the first preclinical studies provided information about this drug in the scientific literature; nevertheless, media reports have led to drug control actions in the UK and across several US states. Rodent studies indicate that 4-MMC exhibits neuropharmacological similarity to 3,4-methylenedioxymethamphetamine (MDMA) and prompt investigation of the thermoregulatory, cardiac and locomotor effects of 4-MMC. This study focuses on the role of ambient temperature, which has been shown to shift the effects of MDMA from hyperthermic to hypothermic.nnnMETHODSnMale Sprague-Dawley rats were monitored after subcutaneous administration of 4-MMC (1.0-5.6 mg/kg) using an implantable radiotelemetry system under conditions of low (20 °C) and high (30 °C) ambient temperature.nnnRESULTSnA pharmacokinetic study found a T(max) of 0.25 h and a C(max) of 1206 ng/ml after 5.6 mg/kg 4-MMC. A dose-dependent reduction of body temperature was produced by 4-MMC at 20 °C but there was no temperature change at 30 °C. Increased locomotor activity was observed after 4-MMC administration under both ambient temperatures, however, significantly more activity was observed at 30 °C. Heart rate was slowed by 1.0 and 5.6 mg/kg 4-MMC at 20°C, and was slower in the 30 °C vs. 20 °C condition across all treatments.nnnCONCLUSIONnThese results show that the cathinone analog 4-MMC exhibits in vivo thermoregulatory properties that are distinct from those produced by MDMA.

Trabecular bone loss after administration of the second-generation antipsychotic risperidone is independent of weight gain

Second generation antipsychotics (SGAs) have been linked to metabolic and bone disorders in clinical studies, but the mechanisms of these side effects remain unclear. Additionally, no studies have examined whether SGAs cause bone loss in mice. Using in vivo and in vitro modeling we examined the effects of risperidone, the most commonly prescribed SGA, on bone in C57BL6/J (B6) mice. Mice were treated with risperidone orally by food supplementation at a dose of 1.25 mg/kg daily for 5 and 8 weeks, starting at 3.5 weeks of age. Risperidone reduced trabecular BV/TV, trabecular number and percent cortical area. Trabecular histomorphometry demonstrated increased resorption parameters, with no change in osteoblast number or function. Risperidone also altered adipose tissue distribution such that white adipose tissue mass was reduced and liver had significantly higher lipid infiltration. Next, in order to tightly control risperidone exposure, we administered risperidone by chronic subcutaneous infusion with osmotic minipumps (0.5 mg/kg daily for 4 weeks) in 7 week old female B6 mice. Similar trabecular and cortical bone differences were observed compared to the orally treated groups (reduced trabecular BV/TV, and connectivity density, and reduced percent cortical area) with no change in body mass, percent body fat, glucose tolerance or insulin sensitivity. Unlike in orally treated mice, risperidone infusion reduced bone formation parameters (serum P1NP, MAR and BFR/BV). Resorption parameters were elevated, but this increase did not reach statistical significance. To determine if risperidone could directly affect bone cells, primary bone marrow cells were cultured with osteoclast or osteoblast differentiation media. Risperidone was added to culture medium in clinically relevant doses of 0, 2.5 or 25 ng/ml. The number of osteoclasts was significantly increased by addition in vitro of risperidone while osteoblast differentiation was not altered. These studies indicate that risperidone treatment can have negative skeletal consequences by direct activation of osteoclast activity and by indirect non-cell autonomous mechanisms. Our findings further support the tenet that the negative side effects of SGAs on bone mass should be considered when weighing potential risks and benefits, especially in children and adolescents who have not yet reached peak bone mass.

Identification of Clinically Viable Quinolinol Inhibitors of Botulinum Neurotoxin A Light Chain

Botulinum neurotoxins (BoNT) are the most potent toxins known and a significant bioterrorist threat. Few small molecule compounds have been identified that are active in cell-based or animal models, potentially due to toxin enzyme plasticity. Here we screened commercially available quinolinols, as well as synthesized hydroxyquinolines. Seventy-two compounds had IC50 values below 10 μM, with the best compound exhibiting submicromolar inhibition (IC50 = 0.8 μM). Structure–activity relationship trends showed that the enzyme tolerates various substitutions at R1 but has a clear preference for bulky aryl amide groups at R2, while methylation at R3 increased inhibitor potency. Evaluation of the most potent compounds in an ADME panel showed that these compounds possess poor solubility at pH 6.8, but display excellent solubility at low pH, suggesting that oral dosing may be possible. Our data show the potential of quinolinol compounds as BoNT therapeutics due to their good in vitro potencies and favorable ADME properties.

Propranolol Attenuates Risperidone-Induced Trabecular Bone Loss in Female Mice.

Atypical antipsychotic (AA) drugs cause significant metabolic side effects, and clinical data are emerging that demonstrate increased fracture risk and bone loss after treatment with the AA, risperidone (RIS). The pharmacology underlying the adverse effects on bone is unknown. However, RIS action in the central nervous system could be responsible because the sympathetic nervous system (SNS) is known to uncouple bone remodeling. RIS treatment in mice significantly lowered trabecular bone volume fraction (bone volume/total volume), owing to increased osteoclast-mediated erosion and reduced osteoblast-mediated bone formation. Daytime energy expenditure was also increased and was temporally associated with the plasma concentration of RIS. Even a single dose of RIS transiently elevated expression of brown adipose tissue markers of SNS activity and thermogenesis, Pgc1a and Ucp1. Rankl, an osteoclast recruitment factor regulated by the SNS, was also increased 1 hour after a single dose of RIS. Thus, we inferred that bone loss from RIS was regulated, at least in part, by the SNS. To test this, we administered RIS or vehicle to mice that were also receiving the nonselective β-blocker propranolol. Strikingly, RIS did not cause any changes in trabecular bone volume/total volume, erosion, or formation while propranolol was present. Furthermore, β2-adrenergic receptor null (Adrb2(-/-)) mice were also protected from RIS-induced bone loss. This is the first report to demonstrate SNS-mediated bone loss from any AA. Because AA medications are widely prescribed, especially to young adults, clinical studies are needed to assess whether β-blockers will prevent bone loss in this vulnerable population.

A novel role for dopamine signaling in the pathogenesis of bone loss from the atypical antipsychotic drug risperidone in female mice

Atypical antipsychotic (AA) drugs, including risperidone (RIS), are used to treat schizophrenia, bipolar disorder, and autism, and are prescribed off-label for other mental health issues. AA drugs are associated with severe metabolic side effects of obesity and type 2 diabetes. Cross-sectional and longitudinal data also show that risperidone causes bone loss and increases fracture risk in both men and women. There are several potential mechanisms of bone loss from RIS. One is hypogonadism due to hyperprolactinemia from dopamine receptor antagonism. However, many patients have normal prolactin levels; moreover we demonstrated that bone loss from RIS in mice can be blocked by inhibition of β-adrenergic receptor activation with propranolol, suggesting the sympathetic nervous system (SNS) plays a pathological role. Further, when, we treated ovariectomized (OVX) and sham operated mice daily for 8weeks with RIS or vehicle we demonstrated that RIS causes significant trabecular bone loss in both sham operated and OVX mice. RIS directly suppressed osteoblast number in both sham and OVX mice, but increased osteoclast number and surface in OVX mice alone, potentially accounting for the augmented bone loss. Thus, hypogonadism alone cannot explain RIS induced bone loss. In the current study, we show that dopamine and RIS are present in the bone marrow compartment and that RIS can exert its effects directly on bone cells via dopamine receptors. Our findings of both direct and indirect effects of AA drugs on bone are relevant for current and future clinical and translational studies investigating the mechanism of skeletal changes from AA drugs.

Long-term morphine delivery via slow release morphine pellets or osmotic pumps: Plasma concentration, analgesia, and naloxone-precipitated withdrawal

Aims: Slow‐release morphine sulfate pellets and osmotic pumps are common routes of chronic morphine delivery in mouse models, but direct comparisons of these drug delivery systems are lacking. In this study, we assessed the efficacy of slow‐release pellets versus osmotic pumps in delivering morphine to adult mice. Main methods: Male C57BL/6NCr mice (8 weeks old) were implanted subcutaneously with slow‐release pellets (25 mg morphine sulfate) or osmotic pumps (64 mg/mL, 1.0 &mgr;L/h). Plasma morphine concentrations were quantified via LC‐MS/MS, analgesic efficacy was determined by tail flick assay, and dependence was assessed with naloxone‐precipitated withdrawal behaviors (jumping) and physiological effects (excretion, weight loss). Key findings: Morphine pellets delivered significantly higher plasma drug concentrations compared to osmotic pumps, which were limited by the solubility of the morphine sulfate and pump volume/flow rate. Within 96 h post‐implantation, plasma morphine concentrations were indistinguishable in pellet vs. pump‐treated samples. While osmotic pump did not have an antinociceptive effect in the tail flick assay, pumps and pellets induced comparable dependence symptoms (naloxone‐precipitated jumping behavior) from 24–72 h post‐implantation. Significance: In this study, we compared slow‐release morphine pellets to osmotic minipumps for morphine delivery in mice. We found that osmotic pumps and subcutaneous morphine sulfate pellets yielded significantly different pharmacokinetics over a 7‐day period, and as a result significantly different antinociceptive efficacy. Nonetheless, both delivery methods induced dependence as measured by naloxone‐precipitated withdrawal.