Pharmacologic Profile of Naloxegol, a Peripherally Acting µ-Opioid Receptor Antagonist, for the Treatment of Opioid-Induced Constipation.

Opioid-induced constipation (OIC) is a common side effect of opioid pharmacotherapy for the management of pain because opioid agonists bind to µ-opioid receptors in the enteric nervous system (ENS). Naloxegol, a polyethylene glycol derivative of naloxol, which is a derivative of naloxone and a peripherally acting µ-opioid receptor antagonist, targets the physiologic mechanisms that cause OIC. Pharmacologic measures of opioid activity and pharmacokinetic measures of central nervous system (CNS) penetration were employed to characterize the mechanism of action of naloxegol. At the human µ-opioid receptor in vitro, naloxegol was a potent inhibitor of binding (Ki = 7.42 nM) and a neutral competitive antagonist (pA2 - 7.95); agonist effects were <10% up to 30 µM and identical to those of naloxone. The oral doses achieving 50% of the maximal effect in the rat for antagonism of morphine-induced inhibition of gastrointestinal transit and morphine-induced antinociception in the hot plate assay were 23.1 and 55.4 mg/kg for naloxegol and 0.69 and 1.14 mg/kg by for naloxone, respectively. In the human colon adenocarcinoma cell transport assay, naloxegol was a substrate for the P-glycoprotein transporter, with low apparent permeability in the apical to basolateral direction, and penetrated the CNS 15-fold slower than naloxone in a rat brain perfusion model. Naloxegol-derived radioactivity was poorly distributed throughout the rat CNS and was eliminated from most tissues within 24 hours. These findings corroborate phase 3 clinical studies demonstrating that naloxegol relieves OIC-associated symptoms in patients with chronic noncancer pain by antagonizing the µ-opioid receptor in the ENS while preserving CNS-mediated analgesia.

Naloxegol, an opioid antagonist with reduced CNS penetration: Mode-of-action and human relevance for rat testicular tumours.

Naloxegol is an opioid antagonist which has been developed for the treatment of patients with opioid induced constipation. In the nonclinical safety program naloxegol was shown to have a very benign toxicity profile. In the rat, but not the mouse, 2-year carcinogenicity study a change in tumour pattern with an increase in testicular Leydig cell tumours (LCT) was observed after dosing at high (supra-pharmacological) concentrations. To establish the basis of the increase in LCT and to assess its potential relevance to humans, studies to exclude and potentially identify mode-of-action (MoA) were performed. A genotoxic mechanism was ruled out following negative results in the Ames, mouse lymphoma, and micronucleus assays. An effect on androgen metabolism was excluded since the treatment of rats with naloxegol for 14days did not result in any induction of CYP protein levels. It was demonstrated that administration of centrally restricted opioid antagonists naloxegol or methylnaltrexone at high doses induced an increase in LH release with no clear increase in testosterone, in contrast to the centrally acting opioid antagonist naloxone, which showed marked increases in both LH and testosterone. LCT due to increased LH stimulation is common in rats but not documented in humans. Collectively, the lack of genotoxicity signal, the lack of androgen effect, the increase in LH secretion in rats, which is no considered to be relevant for LCT formation in humans, and high margins to clinical exposures, the observed increase in LCT in the rat is not expected to be clinically relevant.

Pharmacological inhibition of DGAT1 induces sebaceous gland atrophy in mouse and dog skin while overt alopecia is restricted to the mouse.

Diacylglycerol O-acyltransferase 1 (DGAT1) plays an important role in synthesizing lipids, and inhibitors of DGAT1 have been investigated as potential treatments for diabetes and metabolic diseases. DGAT1 knockout (-/-) mice are resistant to obesity, have increased sensitivity to insulin, and exhibit sebaceous gland atrophy and alopecia. Prolonged pharmacological inhibition of DGAT1 with AZD7687 in mice results in the same skin phenotype, including sebaceous gland atrophy and alopecia, as seen in the skin of DGAT1 (-/-) mice. AZD7687-mediated effects on the skin were dose- and time-dependent and reversible. They occurred only at substantial levels of continuous DGAT1 inhibition. Prolonged treatment of dogs with AZD7687 also resulted in sebaceous gland atrophy but did not result in the more adverse skin changes of hair loss and skin lesions. Our findings highlight a significant risk of generating the same lesions that were seen in mouse skin during clinical development of DGAT1 inhibitors in humans and also reveal a species difference in the effects on the skin, indicating that the mouse may be an especially sensitive species. Therefore, although human therapeutic doses may not have the same influence on skin morphology as seen in mice, monitoring of skin changes will be essential in clinical trials with DGAT1 inhibitors.

Prolonged inhibition of glycogen phosphorylase in livers of Zucker Diabetic Fatty rats models human glycogen storage diseases.

The preclinical efficacy and safety of GPi921, a glycogen phosphorylase inhibitor, was assessed following twenty-eight days of administration to Zucker Diabetic Fatty (ZDF) rats. The ZDF rat is an animal model of type 2 diabetes mellitus (TTDM) which develops severe hyperglycemia. Inhibition of glycogen phosphorylase throughout the duration of the study was demonstrated by reductions in twenty-four-hour glucose profiles and glycated hemoglobin levels. In addition, progression towards hyperglycemia was halted in treated but not control animals, which developed hyperglycemia over the twenty-eight days of the study. Biochemical and histopathological analysis revealed large increases in hepatic glycogen, which closely paralleled the development of hepatomegaly and ultimately resulted in increases in hepatic lipids. Furthermore, prolonged glycogen phosphorylase inhibition resulted in an increased incidence and severity of other adverse pathological findings in the liver, such as inflammation, fibrosis, hemorrhage, and necrosis. The observed biochemical and histopathological phenotype of the liver closely resembled that seen in severe cases of human glycogen storage diseases (GSD) and hepatic glycogenosis in poorly controlled diabetes mellitus. These findings revealed that although glycogen phosphorylase inhibitors are efficacious agents for the control of hyperglycemia, prolonged treatment might have the potential to cause significant clinical hepatic complications that resemble those seen in GSD and hepatic glycogenosis.

Imaging kidney in conscious rats with high-frequency ultrasound and detection of two cases of unilateral congenital hydronephrosis.

A quick noninvasive screen of laboratory animal organ phenotype by high-resolution ultrasound is useful in biomedical research and new drug discovery. During new drug testing, imaging animal at the conscious state avoiding anaesthesia does not only speed up the screening process, but also avoids the potential compounding interaction of anaesthetic agents with the new drugs. The feasibility of imaging kidney in conscious rats with high-frequency ultrasound was explored in this study. Two operators were involved with the procedure, with one operator holding the rat and the other operator doing the imaging process. A VisualSonics ultrasound system was used, with a 30 MHz central frequency probe at the resolution of 115 x 55 microm. It was feasible to hold the conscious rats still, allowing ultrasound imaging of kidneys, without causing stress to the animals. In a group of 12 male Han Wistar rats (Charles River, UK), two cases of unilateral congenital hydronephrosis of the right kidney were identified. The right kidneys with hydronephrosis showed an echolucent dilated pelvis and overall parenchymal hypotrophy.

Clinical Pharmacokinetics and Pharmacodynamics of Naloxegol, a Peripherally Acting µ-Opioid Receptor Antagonist.

Naloxegol is a peripherally acting µ-opioid receptor antagonist approved for use as an orally administered tablet (therapeutic doses of 12.5 and 25 mg) for the treatment of opioid-induced constipation. Over a wide dose range (i.e. single supratherapeutic doses up to 1000 mg in healthy volunteers), the pharmacokinetic properties of naloxegol appear to be time- and dose-independent. Naloxegol is rapidly absorbed, with mean time to maximum plasma concentration of <2 h. Following once-daily administration, steady state is achieved within 2-3 days and minimal accumulation is observed. The primary route of naloxegol elimination is via hepatic metabolism, with renal excretion playing a minimal role. In clinical studies, six metabolites were found in feces, urine or plasma, none of which have been identified as unique or disproportionate human metabolites. The major plasma circulating species is naloxegol. There are small effects of mild and moderate renal impairment, age, race, and body mass index on the systemic exposure of naloxegol; however, gender has no effect on the pharmacokinetics of this agent. Naloxegol is a sensitive substrate of cytochrome P450 (CYP) 3A4 and its exposure can be significantly altered by strong or moderate CYP3A modulators. Food increases the bioavailability of naloxegol, and the relative bioavailability of the tablet formulation was not limited by dissolution. Naloxegol in the dose range of 8-125 mg can antagonize morphine-induced peripheral effects without impacting the effect of morphine on the central nervous system, consistent with a peripheral mode of action.

Latent membrane protein 1 inhibits Epstein-Barr virus lytic cycle induction and progress via different mechanisms.

Epstein-Barr virus (EBV) is a potent growth-transforming agent of human B cells. It has previously been shown that viral latent membrane protein 1 (LMP1) is essential for EBV-induced transformation of normal B cells and contributes to maintenance of latency in vitro. Using the EBV-positive Burkitt's lymphoma line P3HR1-c16, which lacks LMP1 during latency and which can readily be activated into virus-productive lytic cycle, we found that LMP1 inhibits lytic cycle induction via the transcription factor NF-kappa B. In addition, LMP1 inhibits lytic cycle progress via two distinct NF-kappa B-independent mechanisms: one involving the cytosolic C-terminal activating regions and the other involving the transmembrane region of LMP1. These findings indicate that in B cells EBV self-limits its lytic cycle via three distinct LMP1-mediated mechanisms.

Multi-parameter analysis of the kinetics of NF-kappaB signalling and transcription in single living cells.

Proteins of the NF-kappaB transcription factor family normally reside in the cytoplasm of cells in a complex with IkappaB inhibitor proteins. Stimulation with TNFalpha leads to proteosomal degradation of the IkappaB proteins and nuclear translocation of the NF-kappaB proteins. Expression of p65 and IkappaBalpha fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IkappaBalpha-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression, the half-time of IkappaBalpha-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IkappaBalpha and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IkappaBalpha is the true substrate for TNFalpha stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor, leptomycin B (LMB), there was nuclear accumulation of IkappaBalpha-EGFP and p65-dsRed, with IkappaBalpha-EGFP accumulating more rapidly. No NF-kappaB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IkappaBalpha-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFalpha, degradation of IkappaBalpha-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-kappaB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-kappaB-dependent transcription occur even when nuclear export of NF-kappaB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

Hit-to-lead studies: the discovery of potent, orally active, thiophenecarboxamide IKK-2 inhibitors.

A hit-to-lead optimisation programme was carried out on the thiophenecarboxamide high throughput screening hits 1 and 2 resulting in the discovery of the potent and orally bioavailable IKK-2 inhibitor 22.