Pharmacological Characterization of a Novel Neuroactive Steroid Prodrug, NTS-104, and Its Neuroprotective Activity in Experimental Stroke Models.

BACKGROUND: Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke. METHODS: The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly. RESULTS: Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1ß, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1. CONCLUSIONS: NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

rHIgM22 enhances remyelination in the brain of the cuprizone mouse model of demyelination.

Failure of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is thought to be a major cause of the limited ability of the central nervous system to repair plaques of immune-mediated demyelination in multiple sclerosis (MS). Current therapies for MS aim to lessen the immune response in order to reduce the frequency and severity of attacks, but these existing therapies do not target remyelination or stimulate repair of the damaged tissue. Thus, the promotion of OPC differentiation and remyelination is potentially an important therapeutic goal. Previous studies have shown that a recombinant human-derived monoclonal IgM antibody, designated rHIgM22, promotes remyelination, particularly of the spinal cord in rodent models of demyelination. Here, we examined the effects of rHIgM22 in remyelination in the brain using the mouse model of cuprizone-induced demyelination, which is characterized by spontaneous remyelination. The myelination state of the corpus callosum of cuprizone-fed mice treated with rHIgM22 was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated corpus callosum. The remyelination-enhancing effects of rHIgM22 were found across different, anatomically distinct regions of the corpus callosum, and followed a spatiotemporal pattern that was similar to that of the spontaneous remyelination process. These enhancing effects were also accompanied by increased differentiation of OPCs into mature oligodendrocytes. Our data indicate strong remyelination-promoting capabilities of rHIgM22 and further support its therapeutic potential in MS.

Species-specific effects of neuregulin-1ß (cimaglermin alfa) on glucose handling in animal models and humans with heart failure.

Neuregulin-1ß is a member of the neuregulin family of growth factors and is critically important for normal development and functioning of the heart and brain. A recombinant version of neuregulin-1ß, cimaglermin alfa (also known as glial growth factor 2 or GGF2) is being investigated as a possible therapy for heart failure. Previous studies suggest that neuregulin-1ß stimulation of skeletal muscle increases glucose uptake and, specifically, sufficient doses of cimaglermin alfa acutely produce hypoglycemia in pigs. Since acute hypoglycemia could be a safety concern, blood glucose changes in the above pig study were further investigated. In addition, basal glucose and glucose disposal were investigated in mice. Finally, as part of standard clinical chemistry profiling in a single ascending-dose human safety study, blood glucose levels were evaluated in patients with heart failure after cimaglermin alfa treatment. A single intravenous injection of cimaglermin alfa at doses of 0.8mg/kg and 2.6mg/kg in mice resulted in a transient reduction of blood glucose concentrations of approximately 20% and 34%, respectively, at 2h after the treatment compared to pre-treatment levels. Similar results were observed in diabetic mice. Treatment with cimaglermin alfa also increased blood glucose disposal following oral challenge in mice. However, no significant alterations in blood glucose concentrations were found in human heart failure patients at 0.5 and 2h after treatment with cimaglermin alfa over an equivalent human dose range, based on body surface area. Taken together, these data indicate strong species differences in blood glucose handling after cimaglermin alfa treatment, and particularly do not indicate that this phenomenon should affect human subjects.

An optimized dosing regimen of cimaglermin (neuregulin 1ß3, glial growth factor 2) enhances molecular markers of neuroplasticity and functional recovery after permanent ischemic stroke in rats.

Cimaglermin (neuregulin 1ß3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb- and hindlimb-placing scores (proprioceptive behavioral tests), body-swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin-mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose-frequency study used the dose-ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose- and frequency-dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth-associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment.

NTS-105 decreased cell death and preserved long-term potentiation in an in vitro model of moderate traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of hospitalization and death. To mitigate these human costs, the search for effective drugs to treat TBI continues. In the current study, we evaluated the efficacy of the novel neurosteroid, NTS-105, to reduce post-traumatic pathobiology in an in vitro model of moderate TBI that utilizes an organotypic hippocampal slice culture. NTS-105 inhibited activation of the androgen receptor and the mineralocorticoid receptor, partially activated the progesterone B receptor and was not active at the glucocorticoid receptor. Treatment with NTS-105 starting one hour after injury decreased post-traumatic cell death in a dose-dependent manner, with 10 nM NTS-105 being most effective. Post-traumatic administration of 10 nM NTS-105 also prevented deficits in long-term potentiation (LTP) without adversely affecting neuronal activity in naïve cultures. We propose that the high potency pleiotropic action of NTS-105 beneficial effects at multiple receptors (e.g. androgen, mineralocorticoid and progesterone) provides significant mechanistic advantages over native neurosteroids such as progesterone, which lacked clinical success for the treatment of TBI. Our results suggest that this pleiotropic pharmacology may be a promising strategy for the effective treatment of TBI, and future studies should test its efficacy in pre-clinical animal models of TBI.

Dalfampridine improves sensorimotor function in rats with chronic deficits after middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: Stroke survivors often have permanent deficits that are only partially addressed by physical therapy. This study evaluated the effects of dalfampridine, a potassium channel blocker, on persistent sensorimotor deficits in rats with treatment initiated 4 or 8 weeks after stroke. METHODS: Rats underwent permanent middle cerebral artery occlusion. Sensorimotor function was measured using limb-placing and body-swing symmetry tests, which normally show a partial recovery from initial deficits that plateaus ≈4 weeks after permanent middle cerebral artery occlusion. Dalfampridine was administered starting at 4 or 8 weeks after permanent middle cerebral artery occlusion in 2 blinded, vehicle-controlled studies. Plasma samples were collected and brain tissue was processed for histologic assessment. RESULTS: Dalfampridine treatment (0.5-2.0 mg/kg) improved forelimb- and hindlimb-placing responses and body-swing symmetry in a reversible and dose-dependent manner. Plasma dalfampridine concentrations correlated with dose. Brain infarct volumes showed no differences between treatment groups. CONCLUSIONS: Dalfampridine improves sensorimotor function in the rat permanent middle cerebral artery occlusion model. Dalfampridine extended-release tablets (prolonged release fampridine outside the United States) are used to improve walking in patients with multiple sclerosis, and these preclinical data provide a strong rationale for examining the potential of dalfampridine to treat chronic stable deficits in stroke patients. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01605825.

Neurosteroid Receptor Modulators for Treating Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers wide-ranging pathology that impacts multiple biochemical and physiological systems, both inside and outside the brain. Functional recovery in patients is impeded by early onset brain edema, acute and chronic inflammation, delayed cell death, and neurovascular disruption. Drug treatments that target these deficits are under active development, but it seems likely that fully effective therapy may require interruption of the multiplicity of TBI-induced pathological processes either by a cocktail of drug treatments or a single pleiotropic drug. The complex and highly interconnected biochemical network embodied by the neurosteroid system offers multiple options for the research and development of pleiotropic drug treatments that may provide benefit for those who have suffered a TBI. This narrative review examines the neurosteroids and their signaling systems and proposes directions for their utility in the next stage of TBI drug research and development.

Torcetrapib produces endothelial dysfunction independent of cholesteryl ester transfer protein inhibition.

OBJECTIVE: Torcetrapib, a prototype cholesteryl ester transfer protein (CETP) inhibitor with potential for decreasing atherosclerotic disease, increased cardiovascular events in clinical trials. The identified hypertensive and aldosterone-elevating actions of torcetrapib may not fully account for this elevated cardiovascular risk. Therefore, we evaluated the effects of torcetrapib on endothelial mediated vasodilation in vivo. METHODS AND RESULTS: In vivo endothelial mediated vasodilation was assessed using ultrasound imaging of acetylcholine-induced changes in rabbit central ear artery diameter. Torcetrapib, in addition to producing hypertension and baseline vasoconstriction, markedly inhibited acetylcholine-induced vasodilation. A structurally distinct CETP inhibitor, JNJ-28545595, did not affect endothelial function despite producing similar degrees of CETP inhibition and high-density lipoprotein elevation. Nitroprusside normalized torcetrapib's basal vasoconstriction and elicited dose-dependent vasodilation of norepinephrine preconstricted arteries in torcetrapib-treated animals, indicating torcetrapib did not impair smooth muscle function. CONCLUSIONS: Torcetrapib significantly impairs endothelial function in vivo, independent of CETP inhibition and high-density lipoprotein elevation. Given the well-documented association of endothelial dysfunction with cardiovascular disease and risk, this activity of torcetrapib may have contributed to increased cardiovascular risk in clinical trials.

Pharmacological characterization of RWJ-676070, a dual vasopressin V(1A)/V(2) receptor antagonist.

The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V(1A) and V(2) receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V(1A) and V(2) receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V(1A), V(1B) or V(2) receptors. Its inhibition of V(1A) receptor-mediated contraction of vascular rings and platelet aggregation was determined. V(2) receptor-medated aquaresis was determined in rats, dogs and monkeys. V(1A) receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V(1A) and V(2) receptors (Ki=1 and 14 nM, respectively). RWJ-676070 inhibited V(1A) receptor-induced intracellular calcium mobilization and V(2) receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V(1A) receptors. RWJ-676070 inhibited V(1A) receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V(1A) and V(2) receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V(1A) and V(2) receptors.

The antibody rHIgM22 facilitates hippocampal remyelination and ameliorates memory deficits in the cuprizone mouse model of demyelination.

Multiple sclerosis (MS) is a chronic, inflammatory demyelinating disease of the CNS. In addition to motor, sensory and visual deficits, MS is also characterized by hippocampal demyelination and memory impairment. We recently demonstrated that a recombinant human-derived monoclonal IgM antibody, which is designated rHIgM22 and currently in clinical development for people with MS, accelerates remyelination of the corpus callosum in the brains of cuprizone-treated mice. Here, we investigated the effects of rHIgM22 in the hippocampus and on hippocampal-dependent learning and memory in the same mouse model of cuprizone-induced demyelination and spontaneous remyelination. The degree of hippocampal myelination of cuprizone-fed mice treated with a single dose of rHIgM22 (10 mg/kg of body weight) was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. Four or five mice per treatment group were examined for each time point. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated hippocampus. Using two additional cohorts of mice and eight animals per treatment group for each cohort, we also demonstrate that the enhancing effects of rHIgM22 on hippocampal remyelination were accompanied by improved performance in the Morris water maze and amelioration of the memory deficits induced by cuprizone. These results further confirm the remyelination-promoting capabilities of rHIgM22 and support additional investigation of its therapeutic potential in MS.

Transient Changes in Hepatic Physiology That Alter Bilirubin and Bile Acid Transport May Explain Elevations in Liver Chemistries Observed in Clinical Trials of GGF2 (Cimaglermin Alfa).

GGF2 is a recombinant human neuregulin-1ß in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations approximately 0.8-fold average plasma Cmax for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified approximately 50% reductions in mRNAs coding for bilirubin transporters and bile acid conjugating enzymes, as well as changes in expression of additional genes mimicking the interleukin-6-mediated acute phase response. Similar gene expression changes were observed in GGF2-treated HepG2 cells and primary monkey hepatocytes. Additional studies conducted in sandwich-cultured human hepatocytes revealed a transient and GGF2 concentration-dependent decrease in hepatocyte bile acid content and biliary clearance of taurocholate without affecting biliary taurocholate efflux. Taken together, these data suggest that GGF2 does not cause significant hepatocellular death, but transiently modifies hepatic handling of bilirubin and bile acids, effects that may account for the elevations in serum bilirubin observed in the clinical trial subjects.

Phenylpiperidine-benzoxazinones as urotensin-II receptor antagonists: synthesis, SAR, and in vivo assessment.

Various 4-phenylpiperidine-benzoxazin-3-ones were synthesized and biologically evaluated as urotensin-II (U-II) receptor antagonists. Compound 12i was identified from in vitro evaluation as a low nanomolar antagonist against both rat and human U-II receptors. This compound showed in vivo efficacy in reversing the ear-flush response induced by U-II in rats.

Clinically Relevant Levels of 4-Aminopyridine Strengthen Physiological Responses in Intact Motor Circuits in Rats, Especially After Pyramidal Tract Injury.

BACKGROUND: 4-Aminopyridine (4-AP) is a Food and Drug Administration-approved drug to improve motor function in people with multiple sclerosis. Preliminary results suggest the drug may act on intact neural circuits and not just on demyelinated ones. OBJECTIVE: To determine if 4-AP at clinically relevant levels alters the excitability of intact motor circuits. METHODS: In anesthetized rats, electrodes were placed over motor cortex and the dorsal cervical spinal cord for electrical stimulation, and electromyogram electrodes were inserted into biceps muscle to measure responses. The motor responses to brain and spinal cord stimulation were measured before and for 5 hours after 4-AP administration both in uninjured rats and rats with a cut lesion of the pyramidal tract. Blood was collected at the same time as electrophysiology to determine drug plasma concentration with a goal of 20 to 100 ng/mL. RESULTS: We first determined that a bolus infusion of 0.32 mg/kg 4-AP was optimal: it produced on average 61.5 ± 1.8 ng/mL over the 5 hours after infusion. This dose of 4-AP increased responses to spinal cord stimulation by 1.3-fold in uninjured rats and 3-fold in rats with pyramidal tract lesion. Responses to cortical stimulation also increased by 2-fold in uninjured rats and up to 4-fold in the injured. CONCLUSION: Clinically relevant levels of 4-AP strongly augment physiological responses in intact circuits, an effect that was more robust after partial injury, demonstrating its broad potential in treating central nervous system injuries.

Recovery of sensorimotor function following sciatic nerve injury across multiple rat strains.

BACKGROUND: Peripheral nerve injury (PNI) can result in neurodegenerative changes leading to motor, sensory and autonomic dysfunction. Injury to the rat sciatic nerve is used to model pathophysiologic processes following PNI and assess the efficacy of therapeutic interventions. Frequently, temporal changes in the sciatic functional index (SFI), a measure of sensorimotor integration are measured in rats to assess functional recovery following sciatic nerve injury. However, multiple rat strains and behavioral endpoints have been employed to investigate pathophysiology of PNI and impact of therapeutic intervention on recovery, raising the possibility that rat strain may influence the outcome of such studies. NEW METHOD: The temporal course of recovery from sham, sciatic nerve crush or transection injury was assessed using SFI determined by two methods (footprint and DigiGait), and proprioceptive hind limb placement (a measure of proprioceptive integrity) of the sciatic nerve innervation, in male Sprague Dawley, Lewis, Fischer, Wistar and Long Evans rats. RESULTS: The SFI profile, as assessed by both inked footprint analysis and DigiGait, following sciatic nerve injury was remarkably conserved across strains. Dramatic strain-related differences were observed in the latency to place the crush- or transection-injured hind limb following proprioceptive hind limb stimulation. COMPARISON WITH EXISTING METHOD: The novelty of this study is the parallel comparison of multiple strains using existing and novel tests. CONCLUSION: These results suggest that some sensorimotor function tests may be sensitive to the choice of strain, as evidenced by the differences between SFI and proprioceptive function outcomes.

AC105 Increases Extracellular Magnesium Delivery and Reduces Excitotoxic Glutamate Exposure within Injured Spinal Cords in Rats.

Magnesium (Mg2+) homeostasis is impaired following spinal cord injury (SCI) and the loss of extracellular Mg2+ contributes to secondary injury by various mechanisms, including glutamate neurotoxicity. The neuroprotective effects of high dose Mg2+ supplementation have been reported in many animal models. Recent studies found that lower Mg2+ doses also improved neurologic outcomes when Mg2+ was formulated with polyethylene glycol (PEG), suggesting that a PEG/ Mg2+ formulation might increase Mg2+ delivery to the injured spinal cord, compared with that of MgSO4 alone. Here, we assessed spinal extracellular Mg2+ and glutamate levels following SCI in rats using microdialysis. Basal levels of extracellular Mg2+ (∼0.5 mM) were significantly reduced to 0.15 mM in the core and 0.12 mM in the rostral peri-lesion area after SCI. A single intravenous infusion of saline or of MgSO4 at 192 µmoL/kg did not significantly change extracellular Mg2+ concentrations. However, a single infusion of AC105 (a MgCl2 in PEG) at an equimolar Mg2+ dose significantly increased the Mg2+ concentration to 0.3 mM (core area) and 0.25 mM (rostral peri-lesion area). Moreover, multiple AC105 treatments completely restored the depleted extracellular Mg2+ concentrations after SCI to levels in the uninjured spinal cord. Repeated MgSO4 infusions slightly increased the Mg2+ concentrations while saline infusion had no effect. In addition, AC105 treatment significantly reduced extracellular glutamate levels in the lesion center after SCI. These results indicate that intravenous infusion of PEG-formulated Mg2+ normalized the Mg2+ homeostasis following SCI and reduced potentially neurotoxic glutamate levels, consistent with a neuroprotective mechanism of blocking excitotoxicity.

Effects of neuregulin GGF2 (cimaglermin alfa) dose and treatment frequency on left ventricular function in rats following myocardial infarction.

Neuregulins are important growth factors involved in cardiac development and response to stress. Certain isoforms and fragments of neuregulin have been found to be cardioprotective. The effects of a full-length neuregulin-1ß isoform, glial growth factor 2 (GGF2; USAN/INN; also called cimaglermin) were investigated in vitro. Various dosing regimens were then evaluated for their effects on left ventricular (LV) function in rats with surgically-induced myocardial infarction. In vitro, GGF2 bound with high affinity to erythroblastic leukemia viral oncogene (ErbB) 4 receptors, potently promoted Akt phosphorylation, as well as reduced cell death following doxorubicin exposure in HL1 cells. Daily GGF2 treatment beginning 7-14 days after left anterior descending coronary artery ligation produced improvements in LV ejection fraction and other measures of LV function and morphology. The improvements in LV function (e.g. 10% point increase in absolute LV ejection fraction) with GGF2 were dose-dependent. LV performance was substantially improved when GGF2 treatment was delivered infrequently, despite a serum half-life of less than 2h and could be maintained for more than 10 months with treatment once weekly or once every 2 weeks. These studies confirm previous findings that GGF2 may improve contractile performance in the failing rat heart and that infrequent exposure to GGF2 may improve LV function and impact remodeling in the failing myocardium. GGF2 is now being developed for the treatment of heart failure in humans.

Effects of drug combinations on smooth muscle cell proliferation: an isobolographic analysis.

Although sirolimus is a potent inhibitor of vascular smooth muscle cell (VSMC) proliferation and is effective at preventing restenosis in the majority of clinical revascularization procedures employing sirolimus-eluting stents, some VSMC may escape the antiproliferative effects of sirolimus. The present study examines the effects of combining sirolimus with other known cell cycle-specific antiproliferative agents (cladribine, topotecan or etoposide) on cultured coronary artery VSMC proliferation and utilizes a novel isobolographic approach to determine whether sirolimus/antiproliferative agent combinations produce subadditive, additive or supraadditive potentiation of antiproliferative activity. All agents were found to inhibit coronary artery VSMC proliferation in a dose-dependent manner. Cladribine was found to potentiate the antiproliferative activity of sirolimus in either an additive or supraadditive manner, depending upon the cladribine concentration. Topotecan potentiated the sirolimus antiproliferative activity by simple additivity while etoposide yielded subadditive potentiation. The present results demonstrate the utility of isobolographic analysis for identifying and optimizing antiproliferative drug combinations.

Drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and injured arteries.

Sirolimus and paclitaxel eluted from stents inhibit cell proliferation and other cellular processes by dramatically different mechanisms. In this study, the effects of sirolimus and paclitaxel on cultured human coronary artery smooth muscle and endothelial cell function or cell cycle changes in balloon-injured arteries were directly compared. Both sirolimus and paclitaxel inhibited smooth muscle and endothelial cell proliferation. However, only paclitaxel inhibited smooth muscle and endothelial cell migration at low (nM) concentrations. Sirolimus arrested smooth muscle and endothelial cells in the G0/G1 phase of the cell cycle without inducing apoptosis while paclitaxel produced apoptosis in both cell types at low nanomolar concentrations. Although both agents blocked neointimal formation, sirolimus applied locally to injured rat carotid arteries increased the percentage of cycling vascular cells in G0/G1 without inducing apoptosis while paclitaxel increased the percentage of cycling cells in S and G2/M phases while inducing apoptosis. These results suggest that sirolimus reduces neointimal hyperplasia through a cytostatic mechanism while paclitaxel produces apoptotic cell death.

Albutropin: a growth hormone-albumin fusion with improved pharmacokinetics and pharmacodynamics in rats and monkeys.

Growth hormone (GH) replacement therapy is used to treat GH deficiency. Treatment requires daily administration because of the short plasma t(1/2) of GH. Albutropin, human GH fused at its N-terminus with human serum albumin, should be cleared from the circulation more slowly than GH. Pharmacokinetic and pharmacodynamic studies of albutropin were conducted in rats and monkeys. After subcutaneous (s.c.) dosing in rats, a twofold decrease in clearance and a fourfold increase in plasma half-life were seen with albutropin compared to GH. In monkeys, s.c. administered albutropin (0.3 mg/kg) had a sixfold longer terminal half-life and an eightfold slower clearance than GH (0.3 mg/kg). A single subcutaneous administration of albutropin (0.3, 1.5 and 4 mg/kg) increased plasma insulin-like growth factor 1 (IGF-1) levels for up to 7 days. Seven consecutive daily s.c. injections of GH at 0.3 mg/kg resulted in an increase in IGF-1 equivalent to that induced by a single administration of albutropin at 4 mg/kg. Albutropin (1-20 microg/kg) dosed daily, every other day or every 4 days significantly increased cumulative body weight gain and tibial epiphyseal growth plate width in hypophysectomized rats compared to equimolar doses of GH. These results suggest that albutropin could be given less frequently than GH and achieve therapeutic effects in patients.

Pharmacologic and pharmacokinetic profile of repifermin (KGF-2) in monkeys and comparative pharmacokinetics in humans.

Repifermin (truncated, recombinant human keratinocyte growth factor-2, KGF-2) was evaluated in cynomolgus monkeys and healthy humans during a phase 1 trial. Monkeys received vehicle or repifermin at 20, 75, or 200 microg/kg IV or 750 microg/kg subcutaneous (SC) daily for 29 days. Clinical observations were made during the entire dosing period. Gross and microscopic changes were assessed at necropsy. Pharmacokinetic parameters and immunogenicity were evaluated in these monkeys and in humans, following a single or 7 daily IV bolus injections of 1, 5, 25, or 50 microg/kg repifermin. In monkeys, repifermin was well tolerated, and histologic evaluation demonstrated dose-dependent, reversible thickening of the mucosa throughout the alimentary tract, except for the stomach. In the alimentary tract tissues, nonepithelial tissues were not affected, indicating a specificity of repifermin for epithelial cells. Pharmacokinetics in both monkeys and humans were dose proportional, showed lack of drug accumulation with repeated daily dosing, and were characterized by high volumes of distribution and clearance rates, indicating substantial tissue binding and metabolism. Repifermin was not markedly immunogenic following multiple daily IV injections in either species. Serum repifermin concentrations in humans were comparable to those attained in monkeys that produced significant pharmacological effects on epithelial cells in the alimentary tract. These findings provide additional support for the ongoing clinical development of repifermin for diseases involving epithelial injury.