Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.

BACKGROUND: Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease and calcific aortic valve stenosis. No effective therapies to lower plasma Lp(a) concentrations exist. We have assessed the safety, pharmacokinetics, and pharmacodynamics of ISIS-APO(a)Rx, a second-generation antisense drug designed to reduce the synthesis of apolipoprotein(a) (apo[a]) in the liver. METHODS: In this randomised, double-blind, placebo-controlled, phase 1 study at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK), we screened for healthy adults aged 18-65 years, with a body-mass index less than 32·0 kg/m(2), and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. Via a randomisation technique, we randomly assigned participants to receive a single subcutaneous injection of ISIS-APO(a)Rx (50 mg, 100 mg, 200 mg, or 400 mg) or placebo (3:1) in the single-dose part of the study or to receive six subcutaneous injections of ISIS-APO(a)Rx (100 mg, 200 mg, or 300 mg, for a total dose exposure of 600 mg, 1200 mg, or 1800 mg) or placebo (4:1) during a 4 week period in the multi-dose part of the study. Participants, investigators, and study staff were masked to the treatment assignment, except for the pharmacist who prepared the ISIS-APO(a)Rx or placebo. The primary efficacy endpoint was the percentage change from baseline in Lp(a) concentration at 30 days in the single-dose cohorts and at 36 days for the multi-dose cohorts. Safety and tolerability was assessed 1 week after last dose and included determination of the incidence, severity, and dose relation of adverse events and changes in laboratory variables, including lipid panel, routine haematology, blood chemistry, urinalysis, coagulation, and complement variables. Other assessments included vital signs, a physical examination, and 12-lead electrocardiograph. This trial is registered with European Clinical Trials Database, number 2012-004909-27. FINDINGS: Between Feb 27, 2013, and July 15, 2013, 47 (23%) of 206 screened volunteers were randomly assigned to receive ISIS-APO(a)Rx as a single-dose or multi-dose of ascending concentrations or placebo. In the single-dose study, we assigned three participants to receive 50 mg ISIS-APO(a)Rx, three participants to receive 100 mg ISIS-APO(a)Rx, three participants to receive 200 mg ISIS-APO(a)Rx, three participants to receive 400 mg ISIS-APO(a)Rx, and four participants to receive placebo. All 16 participants completed treatment and follow-up and were included in the pharmacodynamics, pharmacokinetics, and safety analyses. For the multi-dose study, we assigned eight participants to receive six doses of 100 mg ISIS-APO(a)Rx, nine participants to receive six doses of 200 mg ISIS-APO(a)Rx, eight participants to receive six doses of 300 mg ISIS-APO(a)Rx, and six participants to receive six doses of placebo. Whereas single doses of ISIS-APO(a)Rx (50-400 mg) did not decrease Lp(a) concentrations at day 30, six doses of ISIS-APO(a)Rx (100-300 mg) resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentration of 39·6% from baseline in the 100 mg group (p=0·005), 59·0% in the 200 mg group (p=0·001), and 77·8% in the 300 mg group (p=0·001). Similar reductions were observed in the amount of oxidized phospholipids associated with apolipoprotein B-100 and apolipoprotein(a). Mild injection site reactions were the most common adverse events. INTERPRETATION: ISIS-APO(a)Rx results in potent, dose-dependent, selective reductions of plasma Lp(a). The safety and tolerability support continued clinical development of ISIS-APO(a)Rx as a potential therapeutic drug to reduce the risk of cardiovascular disease and calcific aortic valve stenosis in patients with elevated Lp(a) concentration. FUNDING: Isis Pharmaceuticals.

Simultaneous quantification of davalintide, a novel amylin-mimetic peptide, and its active metabolite in beagle and rat plasma by online SPE and LC-MS/MS.

BACKGROUND: Davalintide, an investigational therapeutic peptide for the treatment of obesity, is rapidly metabolized by enzymatic cleavage of its N-terminal lysine residue to produce an active des-Lys metabolite in vivo. While a sensitive ELISA assay is available, it is unable to distinguish davalintide from its metabolite. Consequently, we developed an online SPE-LC-MS/MS method for simultaneous quantification of the drug and its active metabolite in beagle and rat plasma samples and compared the resulting pharmacokinetic profiles with those determined by ELISA. RESULTS: The total concentration of active drug measured by ELISA correlated well with the total concentration of davalintide and its metabolite using online SPE-LC-MS/MS. CONCLUSION: The technique is a viable alternative to immunochemistry-based methods for peptide quantitation in terms of sensitivity, reproducibility and specificity, and importantly, does not require developing antibody-based reagents.

Pharmacological and pharmacokinetic properties of a structurally novel, potent, and selective metabotropic glutamate 2/3 receptor agonist: in vitro characterization of agonist (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic acid (LY404039).

Group II metabotropic glutamate (mGlu) receptor agonists, including (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), have demonstrated efficacy in animal models of anxiety and schizophrenia, and LY354740 decreased anxiety in human subjects. Herein, we report the in vitro pharmacological profile and pharmacokinetic properties of another potent, selective, and structurally novel mGlu2/3 receptor agonist, (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) and provide comparisons with LY354740. Similar to LY354740, LY404039 is a nanomolar potent agonist at recombinant human mGlu2 and mGlu3 receptors (K(i) = 149 and 92, respectively) and in rat neurons expressing native mGlu2/3 receptors (Ki = 88). LY404039 is highly selective for mGlu2/3 receptors, showing more than 100-fold selectivity for these receptors, versus ionotropic glutamate receptors, glutamate transporters, and other receptors targeted by known anxiolytic and antipsychotic medications. Functionally, LY404039 potently inhibited forskolin-stimulated cAMP formation in cells expressing human mGlu2 and mGlu3 receptors. Electrophysiological studies indicated that LY404039 suppressed electrically evoked excitatory activity in the striatum, and serotonin-induced l-glutamate release in the prefrontal cortex; effects reversed by LY341495. These characteristics suggest LY404039 modulates glutamatergic activity in limbic and forebrain areas relevant to psychiatric disorders; and that, similar to LY354740, it works through a mechanism that may be devoid of negative side effects associated with current antipsychotics and anxiolytics. Interestingly, despite the slightly lower potency (approximately 2-5-fold) of LY404039 versus LY354740 in binding, functional, and electrophysiological assays, LY404039 demonstrated higher plasma exposure and better oral bioavailability in pharmacokinetic experiments. Collectively, the current data indicate that LY404039 may be valuable in the treatment of neuropsychiatric disorders, including anxiety and psychosis.

Disposition of [14C]ruboxistaurin in humans.

Ruboxistaurin is a potent and specific inhibitor of the beta isoforms of protein kinase C (PKC) that is being developed for the treatment of diabetic microvascular complications. The disposition of [(14)C]ruboxistaurin was determined in six healthy male subjects who received a single oral dose of 64 mg of [(14)C]ruboxistaurin in solution. There were no clinically significant adverse events during the study. Whole blood, urine, and feces were collected at frequent intervals after dosing. Metabolites were profiled by high performance liquid chromatography with radiometric detection. The total mean recovery of the radioactive dose was approximately 87%, with the majority of the radioactivity (82.6 +/- 1.1%) recovered in the feces. Urine was a minor pathway of elimination (4.1 +/- 0.3%). The major route of ruboxistaurin metabolism was to the N-desmethyl ruboxistaurin metabolite (LY338522), which has been shown to be active and equipotent to ruboxistaurin in the inhibition of PKC(beta). In addition, multiple hydroxylated metabolites were identified by liquid chromatography-mass spectrometry in all matrices. Pharmacokinetics were conducted for both ruboxistaurin and LY338522 (N-desmethyl ruboxistaurin, 1). These moieties together accounted for approximately 52% of the radiocarbon measured in the plasma. The excreted radioactivity was profiled using radiochromatography, and approximately 31% was structurally characterized as ruboxistaurin or N-desmethyl ruboxistaurin. These data demonstrate that ruboxistaurin is metabolized primarily to N-desmethyl ruboxistaurin (1) and multiple other oxidation products, and is excreted primarily in the feces.

Pharmacokinetics of ruboxistaurin are significantly altered by rifampicin-mediated CYP3A4 induction.

AIMS: The aim of this study was to evaluate the effect of rifampicin co-administration on the pharmacokinetics of ruboxistaurin and its active metabolite, N-desmethyl ruboxistaurin and, in addition, to compare the changes in pharmacokinetics of ruboxistaurin and N-desmethyl ruboxistaurin with the urinary 6beta-hydroxycortisol : cortisol ratio. Ruboxistaurin is a specific protein-kinase-C beta inhibitor in clinical development for the treatment of diabetic microvascular complications. METHODS: This was a two-period, one-sequence study. Sixteen healthy male subjects completed both study periods. In period one, a single 64 mg oral dose of ruboxistaurin was administered. In period two, 600 mg rifampicin was administered daily for 9 days, during which another single 64 mg ruboxistaurin dose was administered on day 7. Blood samples were collected and assayed for ruboxistaurin and N-desmethyl ruboxistaurin. CYP3A4 induction was assessed by ratios of urinary 6beta-hydroxycortisol : cortisol (6beta-OHC : C) obtained via 24 h and morning-spot sampling techniques. Results Following repeated doses of rifampicin, both the mean C(max) and AUC(0,infinity) of ruboxistaurin were significantly reduced by approximately 95% (P < or = 0.001). For the metabolite, the mean C(max) decreased by 68% (P < or = 0.001), and AUC(0,infinity) decreased by 77% (P < or = 0.001). The t(max) values did not appear affected. The 6beta-OHC : C ratios from both 24 h and morning spot methods increased significantly, consistent with CYP3A4 induction. CONCLUSIONS: The effect of rifampicin co-administration on the exposure of ruboxistaurin is consistent with ruboxistaurin being a substrate of CYP3A4. Therefore, co-administration with known CYP3A4 inducing agents (rifampicin, carbamazepine, phenobarbital, etc.) may decrease the concentrations of ruboxistaurin and N-desmethyl-ruboxistaurin. In this study, 6beta OHC : C ratios substantially underestimated the impact of rifampicin on ruboxistaurin.

In vivo metabolism of [14C]ruboxistaurin in dogs, mice, and rats following oral administration and the structure determination of its metabolites by liquid chromatography/mass spectrometry and NMR spectroscopy.

Ruboxistaurin (LY333531), a potent and isoform-selective protein kinase C beta inhibitor, is currently undergoing clinical trials as a therapeutic agent for the treatment of diabetic microvascular complications. The present study describes the disposition and metabolism of [14C]ruboxistaurin following administration of an oral dose to dogs, mice, and rats. The study revealed that ruboxistaurin was highly metabolized in all species. Furthermore, the results from the bile duct-cannulated study revealed that ruboxistaurin was well absorbed in rats. The primary route of excretion of ruboxistaurin and its metabolites was through feces in all species. The major metabolite detected consistently in all matrices for all species was the N-desmethyl metabolite 1, with the exception of rat bile, in which hydroxy N-desmethyl metabolite 5 was detected as the major metabolite. Other significant metabolites detected in dog plasma were 2, 3, 5, and 6 and in mouse plasma 2, 5, and 19. The structures of the metabolites were proposed by tandem mass spectrometry with the exception of 1, 2, 3, 5, and 6, which were additionally confirmed either by direct comparison with authentic standards or by nuclear magnetic resonance spectroscopy. To assist identification by nuclear magnetic resonance spectroscopy, metabolites 3 and 5 were produced via biotransformation using recombinant human CYP2D6 and, likewise, metabolite 6 and compound 4 (regioisomer of 3 which did not correlate to metabolites found in vivo) were produced using a microbe, Mortierella zonata. The unambiguous identification of metabolites enabled the proposal of clear metabolic pathways of ruboxistaurin in dogs, mice, and rats.