A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors.

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-D-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K(i)=8µM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.

Palladium-catalyzed alpha-arylation of tetramic acids.

A mild, racemization-free, palladium-catalyzed alpha-arylation of tetramic acids (2,4-pyrrolidinediones) has been developed. Various amino acid-derived tetramic acids were cleanly arylated by treatment with 2 mol % of Pd(OAc)(2), 4 mol % of a sterically demanding biaryl phosphine, 2.3 equiv of K(2)CO(3) or K(3)PO(4), and aryl chlorides, bromides, or triflates in THF. With conventional heating, conversions >95% could be attained after 1 h at 80 degrees C, whereas microwave-induced heating led to much shorter reaction times (5 min at 110 degrees C). The electron density of the aryl electrophile had no effect on their reactivity: both electron-rich and electron-poor aryl chlorides and bromides or triflates led to good yields. Ortho-substituted aryl halides and heteroaryl halides, however, did not undergo the title reaction.

Antagonistic targeting of the histamine H3 receptor decreases caloric intake in higher mammalian species.

The main purpose of this study was to examine the effects of a selective histamine H(3) receptor antagonist, NNC 38-1202, on caloric intake in pigs and in rhesus monkeys. The compound was given intragastrically (5 or 15 mg/kg), to normal pigs (n=7) and subcutaneously (1 or 0.1mg/kg) to obese rhesus monkeys (n=9). The energy intake recorded following administration of vehicle to the same animals served as control for the effect of the compound. In addition, rhesus monkey and pig histamine H(3) receptors were cloned from hypothalamic tissues and expressed in mammalian cell lines. The in vitro antagonist potencies of NNC 38-1202 at the H(3) receptors were determined using a functional GTPgammaS binding assay. Porcine and human H(3) receptors were found to have 93.3% identity at the amino acid level and the close homology between the monkey and human H(3) receptors (98.4% identity) was confirmed. The antagonist potencies of NNC 38-1202 at the porcine, monkey and human histamine H(3) receptors were high as evidenced by K(i)-values being clearly below 20 nM, whereas the K(i)-value on the rat H(3) receptor was significantly higher (56+/-6.0 nM). NNC 38-1202, given to pigs in a dose of 15 mg/kg, produced a significant (p<0.05) reduction (55%) of calorie intake compared with vehicle alone, (132.6+/-10.0 kcal/kgday versus 59.7+/-10.2 kcal/kgday). In rhesus monkeys administration of 0.1 and 1mg/kg decreased (p<0.05) average calorie intakes by 40 and 75%, respectively. In conclusion, the present study demonstrates that antagonistic targeting of the histamine H(3) receptor decreases caloric intake in higher mammalian species.

2-(4-alkylpiperazin-1-yl)quinolines as a new class of imidazole-free histamine H3 receptor antagonists.

With the aim of identifying structurally novel, centrally acting histamine H(3) antagonists, a series of 2-(4-alkylpiperazin-1-yl)quinolines was prepared. Systematic variation of the substituents led to highly potent histamine H(3) antagonists with low polar surface area and appropriate log P for blood-brain barrier penetration.

The influence of conformational restriction in the C-terminus of growth hormone secretagogues on their potency.

In order to obtain more potent growth hormone secretagogues, a comparison of ipamorelin and NN703 suggested the addition of a polar group at the C-terminus of NN703. A study was conducted using constrained amines for this purpose. Here, substituted 4-piperidinylamino- and 4-dimethylaminopiperidino-substituents were found to give the most active compounds. A replacement of the 4-dimethylaminopiperidino-substituent with 4-hydroxypiperidino resulted in a series of compounds, which showed in vitro activity with EC(50) values in the low nanomolar range, and favourable kinetic properties, such as 40% oral bioavailability. The most promising compound was also tested in a swine in vivo model, resulting in a growth hormone level with a C(max) of over 40 ng mL(-1).

Controlled coupling of peptides at their C-termini.

Fusion of two proteins has become an important tool in biotechnology. Whereas biotechnological methods easily can produce C-terminal to N-terminal fused compounds, methods to couple two proteins to each of their C-termini are not easily accessible. Herein, peptides are used as models for larger proteins. A method is described exploiting the possibility to attach different reactive handles to their C-termini using a reaction catalyzed by the enzyme carboxypeptidase Y (CPY). It is possible to attach pairs of reaction handles which can react with each other to each of the peptides to be coupled. In a second step, the two modified peptides can be linked together by a chemical reaction, such as an oxime-forming reaction or a copper(I) catalyzed [2+3]-cycloaddition reaction of an azide with an alkyne.

C-Terminally PEGylated hGH-derivatives.

A two-step strategy was used for the preparation of C-terminally PEGylated hGH-derivatives. In a first step a CPY-catalyzed transpeptidation was performed on hGH-Leu-Ala, introducing reaction handles, which were used in the second step for the ligation of PEG-moieties. Both oxime-ligation and copper(I) catalyzed [2+3]-cycloaddition reactions were used for the attachment of PEG-moieties. The biological data show a dependency of the potency of the hGH-derivatives on both size as well as shape of the PEG-group.

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides are potent antagonists of the human H3-receptor.

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides have been found to be antagonists on the human histamine-3-receptor, showing a Ki value of as low as 4 nM.

Increase of neuronal histamine in obese rats is associated with decreases in body weight and plasma triglycerides.

OBJECTIVE: The purpose of the present study was to examine the metabolic effects of a specific histamine H(3) receptor antagonist, the cinnamic amide NNC 0038-0000-1202 (NNC 38-1202). RESEARCH METHODS AND PROCEDURES: Effects of NNC 38-1202 on paraventricular levels of histamine and acute effects on food intake were followed in normal rats, whereas effects on body weight homeostasis and lipid metabolism were studied in a rat model of diet-induced obesity (DIO). RESULTS: NNC 38-1202, administered as single oral doses of 15 and 30 mg/kg, significantly (p < 0.01) increased paraventricular histamine by 339 +/- 54% and 403 +/- 105%, respectively, compared with basal levels. The same doses produced significant (p < 0.01) reductions in food intake. In DIO rats receiving NNC 38-1202 in a daily dose of 5 mg/kg for 22 days, a decrease in food intake was associated with a significant (p < 0.001) net loss of body weight (-11.0 +/- 4.8 grams), compared with rats receiving vehicle, which gained 13.6 +/- 3.0 grams. Also, NNC 38-1202 significantly (p < 0.05) reduced plasma triglycerides by approximately 42%, in parallel with increases in plasma free fatty acids and beta-hydroxybutyrate levels. Despite reductions in food intake and body weight following administration of NNC 38-1202, no sign of a decrease in energy expenditure was observed, and whole-body lipid oxidation was significantly (p < 0.05) increased in the period after dosing. DISCUSSION: The present study suggests that antagonistic targeting of the histamine H(3) receptor decreases food intake, body weight, and plasma TG levels and, thus, represents an interesting approach to treatment of obesity and associated hyperlipidemia.

Head-to-backbone cyclization of peptides on solid support by nucleophilic aromatic substitution.

A new versatile synthetic route is presented for the cyclization of tripeptides on solid support using nucleophilic aromatic substitution in the cyclization step. Identification of all conformers within a limit of 3 kcal/mol from the identified global minimum conformations by Monte Carlo conformational searching reveals that five out of six synthesized compounds have well-defined peptide backbone conformational properties. This was determined by clustering the identified conformers against a filter of seven to nine torsion angles in the peptide backbone. Thus, the results meet our goal to find synthetic routes to peptides that are conformationally sufficiently locked to serve as convenient leads for further development of pharmacophoric models. The strategy is based on Fmoc-peptide chemistry on a N-aminoethyl-substituted glycine bound to the commercially available Rink amide PS-resin. After deprotection of the N-terminus of the tripeptide, it is acylated with a fluoronitrobenzoic acid. Subsequently, a Boc group on the N-bound aminoethyl substituent is selectively deprotected allowing cyclization from the head (N-terminus) to the backbone substituent, thereby leading to the desired cyclized tripeptides. A number of representative examples of peptides cyclized by this method have been synthesized and characterized by NMR. Protecting groups that allow the incorporation of side chain functionalized amino acids have been found. Thus, the route provides access to generic libraries of conformationally restricted peptide sequences expressing a range of proteinogenic pharmacophores.

Cinnamic amides of (S)-2-(aminomethyl)pyrrolidines are potent H3 antagonists.

New imidazole-free H3 antagonists have been found in a series of cinnamic amides of (S)-(aminomethyl)pyrrolidines. The influence of the substituent on the aromatic moiety on the potency and the inhibition of three cytochrome P450 subtypes are also described.