The effect of the REG2 Anticoagulation System on thrombin generation kinetics: a pharmacodynamic and pharmacokinetic first-in-human study.

The REG2 Anticoagulation System consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous active control agent, anivamersen. Its effect on thrombin generation is unknown. A prospectively designed thrombin generation study was conducted within the phase 1 ascending dose study of REG2 to assess the effect of REG2 on thrombin generation kinetics. A total of 32 healthy volunteers were recruited into four cohorts of ascending dose pegnivacogin for the phase 1 study. In this pre-specified substudy, blood samples were drawn in the presence or absence of corn trypsin inhibitor at specified times within each dosing cohort. Thrombin generation was initiated with tissue factor and thrombin generation kinetics were measured using the Calibrated Automated Thrombogram (CAT). REG2 attenuated thrombin generation in a dose-dependent manner. All parameters of the CAT assay, except for lag time, showed a dose and concentration-dependent response to pegnivacogin [time to peak thrombin generation (PTm), endogenous thrombin potential, peak thrombin generation, and velocity index (VIx)]. Reversal of the effect of pegnivacogin with anivamersen demonstrated restoration of thrombin generation without rebound effect. This first-in-human study of the effect of the REG2 Anticoagulation System on thrombin generation demonstrates concentration-dependent suppression of thrombin generation that is reversible without rebound effect, as measured by the CAT assay.

A phase 1 ascending dose study of a subcutaneously administered factor IXa inhibitor and its active control agent.

BACKGROUND: The REG2 anticoagulation system consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous control agent, anivamersen. OBJECTIVES: To assess the safety, tolerability and pharmacokinetic and pharmacodynamic responses of REG2. PATIENTS/METHODS: In this phase 1a study, 36 healthy volunteers were enrolled into five cohorts and given one dose of pegnivacogin. Cohorts 1 (n = 6) and 1A (n = 4) received 0.5 mg kg(-1); cohort 2 (n = 6) received 1.0 mg kg(-1); cohort 3 (n = 6) received 3.0 mg kg(-1); and cohort 4 (n = 8) received 2.0 mg kg(-1) . In cohorts 1-3, two subjects were randomized to placebo. Cohort 4 subjects were subsequently randomized to single-dose (n = 4) or multidose (n = 4) anivamersen. RESULTS: The mean maximum observed concentrations of pegnivacogin in cohorts 1, 1A, 2 and 3 at median time were 5.16 µg mL(-1) at 84 h, 5.19 µg mL(-1) at 72 h, 9.32 µg mL(-1) at 90 h, and 32.5 µg mL(-1) at 84 h, respectively. The maximum relative activated partial thromboplastin time and time needed to achieve this were 1.18 at 2 days, 1.16 at 2 days, 1.27 at 3 days, and 1.85 at 2 days, respectively. The calculated mean half-life and mean residence times of pegnivacogin were 6.12 days and 9.6 days, respectively. There was rapid reversal with intravenous anivamersen, although subsequent reaccumulation of pegnivacogin was observed. CONCLUSIONS: In our first-in-human study, REG2 was well tolerated and provided dose-proportional anticoagulation for several days after a single subcutaneous dose, with complete, although transient, reversal by its control agent. This study demonstrates the first application of a subcutaneously administered aptamer, and represents a potential advance in aptamer therapeutics.

Dose selection for a direct and selective factor IXa inhibitor and its complementary reversal agent: translating pharmacokinetic and pharmacodynamic properties of the REG1 system to clinical trial design.

We performed detailed pharmacokinetic and pharmacodynamic modeling of REG1, an anticoagulation system composed of the direct factor IXa (FIXa) inhibitor pegnivacogin (RB006) and its matched active control agent anivamersen (RB007), with a focus on level of target inhibition to translate phase 1 results to phase 2 dose selection. We modeled early-phase clinical data relating weight-adjusted pegnivacogin dose and plasma concentration to prolongation of the activated partial thromboplastin time (aPTT). Using an in vitro calibration curve, percent FIXa inhibition was determined and related to aPTT prolongation and pegnivacogin dose and concentration. Similar methods were applied to relate anivamersen dose and level of reversal of pegnivacogin anticoagulation. Combined early-phase data suggested that ≥0.75 mg/kg pegnivacogin was associated with >99% inhibition of FIX activity and prolongation of plasma aPTT values ≈2.5 times above baseline, leading to selection of a 1 mg/kg dose for a phase 2a elective percutaneous coronary intervention study to achieve a high intensity of anticoagulation and minimize intersubject variability. Phase 2 validated our predictions, demonstrating 1 mg/kg pegnivacogin yielded plasma concentrations ≈25 µg/ml and >99% inhibition of FIX activity. The relationship between the anivamersen to pegnivacogin dose ratio and degree of pegnivacogin reversal was also validated. Our approach decreased the need for extensive dose-response studies, reducing the duration, complexity and cost of clinical development. The 1 mg/kg pegnivacogin dose and a range of anivamersen dose ratios are being tested in the phase 2b RADAR study (NCT00932100).

A randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor IXa inhibitor.

BACKGROUND: Active and safe reversibility of anticoagulation is an unmet need in clinical care. Factor IXa, required for rapid thrombin generation on platelet surfaces, is a novel target for modulating coagulation. REG1 comprises RB006 (drug) and RB007 (antidote). RB006, a ribonucleic acid aptamer, exerts its anticoagulant effect by selectively binding FIXa. RB007, the complementary oligonucleotide antidote, binds to RB006 by Watson-Crick base pairing, neutralizing its anti-FIXa activity. OBJECTIVE: To test the multiple repeat-dose safety, intraindividual pharmacodynamic reproducibility and graded active reversibility of REG1. METHODS: We randomized 39 healthy volunteers to receive either three consecutive weight-adjusted, drug-antidote treatment cycles, or double placebo. Each treatment cycle included an intravenous bolus of 0.75 mg kg(-1) RB006, followed 60 min later by a descending dose of RB007, ranging from a 2 : 1 to 0.125 : 1 antidote/drug ratio (1.5 mg kg(-1) to 0.094 mg kg(-1) RB007). Serial clinical assessments and coagulation measurements were performed through 14 days postrandomization. RESULTS: Repeat doses of RB006 achieved highly reproducible activated partial thromboplastin time (APTT) levels with low intrasubject variability (coefficient of variation 5.5%, intraclass correlation coefficient 5.8 at 15 min postdose), while repeat doses of RB007 reversed the APTT levels dose-dependently and reproducibly. There was no major bleeding and there were no other serious adverse events. CONCLUSIONS: This is the first human study demonstrating multiple repeat-dose safety, intraindividual pharmacodynamic reproducibility and graded active reversibility of an RNA aptamer-oligonucleotide antidote pair. The results lay the foundation for studying the translation of this novel anticoagulation platform to a wide variety of clinical applications.

Nucleic acid aptamers and their complimentary antidotes. Entering an era of antithrombotic pharmacobiologic therapy.

The translation of fundamental science-based constructs to the preemptive identification and optimal management of individuals with or those at risk for thrombotic disorders of the cardiovascular system has taken a step closer to being realized with the development of molecular technologies that include nucleic acid aptamers and their complimentary oligonucleotide antidotes. Herein, we summarize our experience with factor IX and von Willebrand factor aptamers, and introduce the era of antithrombotic pharmacobiologic therapy.

A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Blocking the initiation of coagulation by RNA aptamers to factor VIIa.

The tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factor-dependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of approximately 15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells.

To determine the presence and identity of isopentenyladenosine-containing transfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs. This method was based on 3' polyadenylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning and DNA sequencing. Using this unique procedure, we report the cloning and sequencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and other small RNAs where the primary sequence is unknown.

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

The import of nuclear-encoded RNAs into mitochondria is required for proper mitochondrial function in most organisms. However, the mechanisms used to achieve RNA import are largely unknown. In particular, the RNA elements that direct import have not been identified in any organism. In Tetrahymena, only one of three nuclear-encoded glutamine accepting tRNAs is imported into mitochondria. We transform Tetrahymena with marked glutamine tRNAs and quantitate their level of accumulation in mitochondria. Of several isostructural nucleotide substitutions tested, alteration of the anticodon sequence uniquely abolishes import. Furthermore, substitution of a single anticodon nucleotide (UUA-->UUG) confers import on a normally nonimported glutamine tRNA. Thus, the anticodon functions as a mitochondrial localization signal and is both necessary and sufficient for tRNA import. Given the prior evidence that neither the cytoplasmic nor the mitochondrial glutaminyl-tRNA synthetase distinguishes between the imported and nonimported glutamine tRNAs with respect to aminoacylation, we propose that some mitochondrial import factor distinct from a synthetase recognizes the anticodon of the imported glutamine tRNA.

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila.

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.