Discovery of APL-1030, a Novel, High-Affinity Nanofitin Inhibitor of C3-Mediated Complement Activation.

Uncontrolled complement activation contributes to multiple immune pathologies. Although synthetic compstatin derivatives targeting C3 and C3b are robust inhibitors of complement activation, their physicochemical and molecular properties may limit access to specific organs, development of bifunctional moieties, and therapeutic applications requiring transgenic expression. Complement-targeting therapeutics containing only natural amino acids could enable multifunctional pharmacology, gene therapies, and targeted delivery for underserved diseases. A Nanofitin library of hyperthermophilic protein scaffolds was screened using ribosome display for C3/C3b-targeting clones mimicking compstatin pharmacology. APL-1030, a recombinant 64-residue Nanofitin, emerged as the lead candidate. APL-1030 is thermostable, binds C3 (KD, 1.59 nM) and C3b (KD, 1.11 nM), and inhibits complement activation via classical (IC50 = 110.8 nM) and alternative (IC50 = 291.3 nM) pathways in Wieslab assays. Pharmacologic activity (determined by alternative pathway inhibition) was limited to primate species of tested sera. C3b-binding sites of APL-1030 and compstatin were shown to overlap by X-ray crystallography of C3b-bound APL-1030. APL-1030 is a novel, high-affinity inhibitor of primate C3-mediated complement activation developed from natural amino acids on the hyperthermophilic Nanofitin platform. Its properties may support novel drug candidates, enabling bifunctional moieties, gene therapy, and tissue-targeted C3 pharmacologics for diseases with high unmet need.

Samidorphan, an opioid receptor antagonist, attenuates drug-induced increases in extracellular dopamine concentrations and drug self-administration in male Wistar rats.

Opioid receptors modulate neurochemical and behavioral responses to drugs of abuse in nonclinical models. Samidorphan (SAM) is a new molecular entity that binds with high affinity to human mu- (µ), kappa- (κ), and delta- (δ) opioid receptors and functions as a µ-opioid receptor antagonist with partial agonist activity at κ- and δ-opioid receptors. Based on its in vitro profile, we hypothesized that SAM would block key neurobiological effects of drugs of abuse. Therefore, we assessed the effects of SAM on ethanol-, oxycodone-, cocaine-, and amphetamine-induced increases in extracellular dopamine (DAext) in the nucleus accumbens shell (NAc-sh), and ethanol and cocaine self-administration behavior in rats. In microdialysis studies, administration of SAM alone did not result in measurable changes in NAc-sh DAext when given across a large range of doses. However, SAM markedly decreased average and maximal increases in NAc-sh DAext produced by each of the drugs of abuse tested. In behavioral studies, SAM attenuated fixed-ratio ethanol self-administration and progressive ratio cocaine self-administration. These results highlight the potential of SAM to counteract the neurobiological and behavioral effects of several drugs of abuse with differing mechanisms of action.

Hyporesponsivity to mu-opioid receptor agonism in the Wistar-Kyoto rat model of altered nociceptive responding associated with negative affective state.

ABSTRACT: Chronic pain is often comorbid with anxiety and depression, altering the level of perceived pain, which negatively affects therapeutic outcomes. The role of the endogenous mu-opioid receptor (MOP) system in pain-negative affect interactions and the influence of genetic background thereon are poorly understood. The inbred Wistar-Kyoto (WKY) rat, which mimics aspects of anxiety and depression, displays increased sensitivity (hyperalgesia) to noxious stimuli, compared with Sprague-Dawley (SD) rats. Here, we report that WKY rats are hyporesponsive to the antinociceptive effects of systemically administered MOP agonist morphine in the hot plate and formalin tests, compared with SD counterparts. Equivalent plasma morphine levels in the 2 rat strains suggested that these differences in morphine sensitivity were unlikely to be due to strain-related differences in morphine pharmacokinetics. Although MOP expression in the ventrolateral periaqueductal gray (vlPAG) did not differ between WKY and SD rats, the vlPAG was identified as a key locus for the hyporesponsivity to MOP agonism in WKY rats in the formalin test. Moreover, morphine-induced effects on c-Fos (a marker of neuronal activity) in regions downstream of the vlPAG, namely, the rostral ventromedial medulla and lumbar spinal dorsal horn, were blunted in the WKY rats. Together, these findings suggest that a deficit in the MOP-induced recruitment of the descending inhibitory pain pathway may underlie hyperalgesia to noxious inflammatory pain in the WKY rat strain genetically predisposed to negative affect.

Chronic administration of buprenorphine in combination with samidorphan produces sustained effects in olfactory bulbectomised rats and Wistar-Kyoto rats.

BACKGROUND: The combination of buprenorphine, a partial mu-opioid receptor agonist and a functional kappa-opioid receptor antagonist, with samidorphan, a functional mu-opioid receptor antagonist, is being developed as an adjunct therapy for major depressive disorder, in order to harness the mood-enhancing effects of opioids without unwanted side-effects such as a risk of addiction. Acute and subacute administration of the combination of buprenorphine and samidorphan is effective in reducing forced swim immobility in the Wistar-Kyoto rat, but the chronic effects have not been examined. AIMS AND METHODS: The purpose of this study was to assess if chronic (14-day) administration of buprenorphine (0.1 mg/kg, subcutaneous) alone or in combination with samidorphan (0.3 mg/kg, subcutaneous) maintains antidepressant-like activity in the olfactory bulbectomised rat model and the Wistar-Kyoto rat, two models that exhibit ongoing behavioural deficits in tests commonly used to study effects of antidepressants. RESULTS: Olfactory bulbectomised-induced hyperactivity was attenuated by chronic administration of buprenorphine alone and in combination with samidorphan, to that of sham control activity levels. Neither buprenorphine nor samidorphan altered stress-associated defecation in sham or olfactory bulbectomised rats in the open field. In Wistar-Kyoto rats, buprenorphine alone significantly reduced forced swim immobility and increased locomotor activity three hours post-final dosing. Buprenorphine plus samidorphan significantly reduced forced swim immobility without changing locomotor activity at this time point. Buprenorphine alone also significantly reduced forced swim immobility 24 h post-final dosing. CONCLUSION: Chronic treatment of buprenorphine alone or buprenorphine plus samidorphan is effective in reversing behavioural deficits in distinct non-clinical paradigms. These non-clinical results complement the antidepressant effect of this combination observed in clinical studies.

Samidorphan mitigates olanzapine-induced weight gain and metabolic dysfunction in rats and non-human primates.

BACKGROUND: Olanzapine, regarded as one of the most efficacious antipsychotic medications for the treatment of schizophrenia, is associated with a high risk of weight gain and metabolic dysfunction. ALKS 3831, a clinical candidate for treatment of schizophrenia, is a combination of olanzapine and samidorphan, an opioid receptor antagonist. The addition of samidorphan is intended to mitigate weight gain and the metabolic dysregulation associated with the use of olanzapine. METHODS: Non-clinical studies were conducted to assess the metabolic effects of olanzapine and samidorphan alone and in combination at clinically relevant exposure levels. RESULTS: Chronic olanzapine administration in male and female rats shifted body composition by increasing adipose mass, which was accompanied by an increase in the rate of weight gain in female rats. Co-administration of samidorphan normalized body composition in both sexes and attenuated weight gain in female rats. In hyperinsulinemic euglycemic clamp experiments conducted prior to measurable changes in weight and/or body composition, olanzapine decreased hepatic insulin sensitivity and glucose uptake in muscle while increasing uptake in adipose tissue. Samidorphan appeared to normalize glucose utilization in both tissues, but did not restore hepatic insulin sensitivity. In subsequent studies, samidorphan normalized olanzapine-induced decreases in whole-body glucose clearance following bolus insulin administration. Results from experiments in female monkeys paralleled the effects in rats. CONCLUSIONS: Olanzapine administration increased weight gain and adiposity, both of which were attenuated by samidorphan. Furthermore, the combination of olanzapine and samidorphan prevented olanzapine-induced insulin insensitivity. Collectively, these data indicate that samidorphan mitigates several metabolic abnormalities associated with olanzapine in both the presence and the absence of weight gain.

Opioid system modulators buprenorphine and samidorphan alter behavior and extracellular neurotransmitter concentrations in the Wistar Kyoto rat.

Approximately two-thirds of major depressive disorder (MDD) patients do not respond adequately to current therapies. BUP/SAM (ALKS 5461), a combination of buprenorphine (BUP) and samidorphan (SAM), is a novel opioid system modulator in development as an adjunct treatment for MDD. Using a rat strain (Wistar Kyoto rat) that is predisposed to stress and has an inadequate response to selective serotonin reuptake inhibitors (SSRIs), we investigated the effect of BUP and SAM, individually and in combination, in established nonclinical assays used to study antidepressants (the forced swim test, FST) and anxiolytics (marble burying test). As opioids and their receptors are expressed in mesocorticolimbic regions of the brain, we analyzed extracellular concentrations of dopamine, serotonin, and/or their metabolites in brain areas associated with mood and motivation. BUP alone and in combination with SAM significantly reduced immobility in the FST. Similarly, the BUP/SAM combination significantly reduced immobility in SSRI (escitalopram)-treated rats. BUP/SAM also decreased burying behavior. SAM attenuated BUP-induced changes of extracellular levels of serotonin and dopamine in the medial prefrontal cortex and nucleus accumbens shell. The latter suggests that the addition of SAM to BUP may limit activation of the mesolimbic dopamine reward pathway and thereby reduce BUP's reinforcing properties. SAM alone had no effect on neurochemistry or immobility in the FST. Collectively, these data indicate that opioid system modulation may offer an alternative mechanism that does not rely on enhanced serotonergic neurotransmission in neurocircuits associated with antidepressant and anxiolytic activity in nonclinical models.

Regulation of glutamate release by α7 nicotinic receptors: differential role in methamphetamine-induced damage to dopaminergic and serotonergic terminals.

Regulation of glutamate release is an important underlying mechanism in mediating excitotoxic events such as damage to dopamine (DA) and serotonin (5-HT) neurons observed after exposure to methamphetamine (Meth). One way to regulate glutamate release may be through the modulation of α7 nicotinic acetylcholine (nACh) receptors. Meth administration is known to increase acetylcholine release; however, it is unknown whether Meth increases glutamate release and causes long-term damage to both DA and 5-HT terminals through the activation of α7 nACh receptors. To test this hypothesis, the α7 nACh receptor antagonist, methyllycaconitine (MLA), was administered before the administration of repeated doses of Meth while simultaneously monitoring extracellular striatal glutamate with in vivo microdialysis. In addition, the subsequent long-term decreases in markers of dopaminergic and serotonergic terminals, including DA reuptake transporter (DAT), serotonin reuptake transporter (SERT), vesicular monoamine transporter-2, vesicular DA, and vesicular 5-HT content in the rat striatum, were measured. The results show that MLA pretreatment prevented Meth-induced increases in striatal glutamate and protected against the subsequent long-term decreases in striatal DAT and vesicular DA content without affecting the hyperthermia produced by Meth. In contrast, the Meth-induced decreases in striatal SERT immunoreactivity and vesicular 5-HT content were not affected by MLA. This suggests that the α7 nACh receptor differentially mediates glutamate-dependent damage to DA but not 5-HT terminals in a manner that is independent of hyperthermia. Furthermore, antagonism of α7 nACh receptors may be a possible therapeutic strategy for decreasing extracellular glutamate and preventing the excitotoxic damage observed in other DA-related neurodegenerative disorders.

A rapid oxidation and persistent decrease in the vesicular monoamine transporter 2 after methamphetamine.

Methamphetamine (METH) produces long-term decreases in markers of dopamine (DA) terminals in animals and humans. A decrease in the function of the vesicular monoamine transporter 2 (VMAT2) has been associated with damage to striatal DA terminals caused by METH; however, a possible mechanism for this decrease in VMAT2 function has not been defined. The current study showed that METH caused a rapid decrease to 68% of controls in VMAT2 protein immunoreactivity of the vesicular fraction from striatal synaptosomes within 1 h after a repeated high-dose administration regimen of METH. This decrease was associated with a 75% increase in nitrosylation of VMAT2 protein in the synaptosomal fraction as measured by nitrosocysteine immunoreactivity of VMAT2 protein. The rapid decreases in VMAT2 persisted when evaluated 7 days later and were illustrated by decreases in VMAT2 immunoreactivity and DA content of the vesicular fraction to 34% and 51% of control values, respectively. The decreases were blocked or attenuated by prior injections of the neuronal nitric oxide synthase inhibitor, S-methyl-l-thiocitrulline. These studies demonstrate that METH causes a rapid neuronal nitric oxide synthase-dependent oxidation of VMAT2 and long-term decreases in VMAT2 protein and function. The results also suggest that surviving DA terminals after METH exposure may have a compromised capacity to buffer cytosolic DA concentrations and DA-derived oxidative stress.

Lobeline attenuates methamphetamine-induced changes in vesicular monoamine transporter 2 immunoreactivity and monoamine depletions in the striatum.

L-Lobeline is an alkaloid that inhibits the behavioral effects of methamphetamine (METH) in rats. No studies have examined the effects of lobeline on the acute and long-term neurochemical changes produced by neurotoxic doses of METH. The effects of lobeline on METH-induced dopamine release, alterations in vesicular monoamine transporter 2 (VMAT-2) distribution, and long-term depletions of dopamine and serotonin (5-HT) content in the rat striatum were examined. METH increased body temperature and dopamine release, decreased VMAT-2 immunoreactivity at 1 and 24 h after METH, and decreased dopamine and 5-hydroxytryptamine (5-HT) content in striatum when examined 7 days later. Prevention of METH-induced hyperthermia attenuated the decrease in VMAT-2 as well as dopamine and 5-HT content. Lobeline pretreatment did not affect METH-induced dopamine release but attenuated the decreases in VMAT-2 after METH and the long-term decreases in striatal dopamine and 5-HT content. These effects of lobeline were due partly to the attenuation of METH-induced hyperthermia. The maintenance of hyperthermia during lobeline + METH exposure restored the effects of METH on decreases in VMAT-2 as well as dopamine and 5-HT content. To examine the effects of lobeline independent of its effects on METH-induced hyperthermia, lobeline was administered after METH when body temperature returned to normal. Lobeline treatment at 5 and 7 h after METH attenuated the METH-induced decreases in synaptosomal, membrane-associated, and vesicular VMAT-2 24 h after METH, as well as the METH-induced decreases in dopamine and 5-HT content 7 days later. Therefore, lobeline has both temperature-dependent and -independent neuroprotective effects against METH toxicity.