A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia.

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8(-/-)) did not rescue the embryonic death of TFPI null (Tfpi(-/-)) mice. Tfpi(+/-) did not alter the bleeding phenotype of F8(-/-) mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8(-/-) mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi(+/-);F8(-/-) mice with hematopoietic Tfpi(-/-) cells compared with Tfpi(+/-);F8(-/-) mice with Tfpi(+/+) hematopoietic cells. Additionally, following femoral vein injury, Tfpi(+/-);F8(-/-) mice with Tfpi(-/-) hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi(+/+) hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide.

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin.

NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.

Selective agonists at group II metabotropic glutamate receptors: synthesis, stereochemistry, and molecular pharmacology of (S)- and (R)-2-amino-4-(4-hydroxy[1,2,5]thiadiazol-3-yl)butyric acid.

Homologation of analogues of the central excitatory neurotransmitter glutamic acid (Glu), in which the distal carboxy group has been bioisosterically replaced by acidic heterocyclic units, has previously provided subtype selective ligands for metabotropic Glu receptors (mGluRs). The (S)-form of the 1,2,5-thiadiazol-3-ol Glu analogue, 2-amino-3-(4-hydroxy[1,2,5]thiadiazol-3-yl)propionic acid (TDPA, 6), is an 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, which in addition stereospecifically activates group I mGluRs. We have now synthesized the (S)- and (R)-forms of 2-amino-4-(4-hydroxy[1,2,5]thiadiazol-3-yl)butyric acid (homo-TDPA, 7) and shown that whereas neither enantiomer interacts with AMPA receptors, (S)- and (R)-7 appear to be selective and equipotent agonists at group II mGluRs as represented by the mGluR2 subtype. The activities of (S)- and (R)-7 are rationalized by conformational analysis, comparison with the potent and specific group II mGluR agonist (-)-LY379268 [(-)-12], and docking to a homology model of mGluR2.

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.

Ibotenic acid and thioibotenic acid: a remarkable difference in activity at group III metabotropic glutamate receptors.

In this study, we have determined and compared the pharmacological profiles of ibotenic acid and its isothiazole analogue thioibotenic acid at native rat ionotropic glutamate (iGlu) receptors and at recombinant rat metabotropic glutamate (mGlu) receptors expressed in mammalian cell lines. Thioibotenic acid has a distinct pharmacological profile at group III mGlu receptors compared with the closely structurally related ibotenic acid; the former is a potent (low microm) agonist, whereas the latter is inactive. By comparing the conformational energy profiles of ibotenic and thioibotenic acid with the conformations preferred by the ligands upon docking to mGlu1 and models of the other mGlu subtypes, we propose that unlike other subtypes, group III mGlu receptor binding sites require a ligand conformation at an energy level which is prohibitively expensive for ibotenic acid, but not for thioibotenic acid. These studies demonstrate how subtle differences in chemical structures can result in profound differences in pharmacological activity.

Prolonged half-life of glycoPEGylated rFVIIa variants compared to native rFVIIa.

INTRODUCTION: Bleeding episodes in haemophilia patients with inhibitors are primarily treated with by-passing agents such as recombinant activated FVII (rFVIIa). Prophylactic treatment with rFVIIa has been shown to significantly reduce the number of bleeding episodes as compared to conventional on-demand haemostatic therapy, and a reduced dosing frequency could present an improved treatment option in inhibitor patients. MATERIALS AND METHODS: A series of glycoPEGylated rFVIIa derivatives (5-40K PEG) has been produced and their effect and pharmocokinetics have been investigated in several animal species. RESULTS: The glycoPEGylated rFVIIa derivatives exhibit significant prolongation of half-life in mice, dogs and pigs as measured by rFVIIa clot activity. The clearance of rFVIIa, rFVIIa-5K PEG, rFVIIa-10K PEG, rFVIIa-20K PEG and rFVIIa-40K PEG in minipigs were estimated to 59, 27, 22, 8.7 and 3.1 ml/h/kg, respectively. Across species a reduction in clearance as a function of the size of the attached PEG was observed. By allometric scaling, the compiled pharmacokinetics predicts a human half-life for rFVIIa-10K PEG and rFVIIa-40K PEG of approximately 7 and 12h, respectively. The rFVIIa-10K PEG and rFVIIa-40K PEG are efficacious in stopping a bleed in the haemophilia A mouse tail-bleeding model after intravenous administration. CONCLUSIONS: GlycoPEGylation of rFVIIa significantly increases the rFVIIa exposure in three animal models, glycoPEGylated rFVIIa compounds are effective in vivo and thus, represents a potential prophylactic treatment option for patients with inhibitors.

Prolonged effect of GlycoPEGylated rFVIIa (40k-PEG-rFVIIa) in rabbits correlates to activity in plasma.

The pharmacokinetics and pharmacodynamics of 40k-PEG-rFVIIa, a GlycoPEGylated derivative of recombinant wild-type FVIIa, were compared with rFVIIa in rabbits. The procoagulant effect was determined as the weight of the clot formed in a defined segment of a facial vein. A time course study was conducted where ligation was made 10 minutes, 12 or 24 hours after i.v. injection of equimolar doses of 40k-PEG-FVIIa or rFVIIa (2 mg/kg). This dose was selected based on a dose response study and a duration of effect study with rFVIIa. The clot weight increased with increasing doses of rFVIIa, and the duration of effect correlated with the plasma FVIIa clot activity. The plasma half-life of 40k-PEG-rFVIIa measured as FVIIa clot activity was found to be 25 hours, which was 5-6 times longer than rFVIIa. The aPTT and PT were reduced, and the measured increase in thrombin-antithrombin correlated to the effect on clot formation. Thus, the effect was similar at ligation 10 minutes after administration of 40k-PEG-rFVIIa or rFVIIa. At 12 hours, the effect of rFVIIa was absent while significant effect was seen 12 and 24 hours post dosing with 40k-PEG-rFVIIa. No consumption of platelets or fibrinogen was found and no thrombi formation was seen in histological examination of various organs. In conclusion, 40k-PEG-rFVIIa has shown prolonged duration of effect that correlated to various plasma markers and FVIIa clot activity. In perspective, the data support further clinical development of 40k-PEG-rFVIIa to potentially become a long-acting recombinant treatment option for prophylaxis in haemophilia patients with inhibitors.

Mutation-induced quisqualic acid and ibotenic acid affinity at the metabotropic glutamate receptor subtype 4: ligand selectivity results from a synergy of several amino acid residues.

The metabotropic glutamate receptors (mGluRs) are key modulators of excitatory neurotransmission in the central nervous system. The eight mGluR subtypes are seven trans-membrane-spanning proteins that possess a large extracellular amino-terminal domain in which the endogenous ligand binding pocket resides. In this study, we have identified four non-conserved amino acid residues that are essential for differentiating mGluR1 from mGluR4. Our approach has been to increase the affinity of the classic mGluR1 agonists, quisqualic acid and ibotenic acid, at mGluR4 by making various point mutations that mimicked mGluR1 residues. Based on ligand docking to homology models, the non-conserved residues, Lys-74, Glu-287, Ser-313, and Lys-317, were chosen for the mutational studies and all of the mutations proved capable of partially or completely restoring the affinities of the ligands. In particular, the mutations K74Y and K317R induced dramatic triple-order-of-magnitude increases in the affinity of ibotenic acid at mGluR4, making the affinity equivalent to that of mGluR1. Furthermore, the affinity of quisqualic acid at mGluR4 was increased to the same level as mGluR1 by the two double mutations, K74Y/K317R and K74Y/E287G. Advanced analysis of ligand conformation and docking procedures were used for the interpretation of these results. The study shows that mGluR subtype selectivity results from a complex interplay of residues shaping the binding pocket, rather than being attributable to a single specific ligand-receptor interaction.