Pharmacokinetics and ex vivo whole blood clot formation of a new recombinant FVIII (N8) in haemophilia A dogs.

N8, a new recombinant factor VIII (rFVIII) compound developed for the treatment of haemophilia A, is produced in Chinese hamster ovary (CHO) cells and formulated without human- or animal-derived materials. The aim of the present study was to compare the pharmacokinetics (PK) and the procoagulant effect, measured by ex vivo whole blood clot formation, of N8 and a commercial rFVIII in a cross-over study in haemophilia A dogs. N8 and Advate® (100 IU kg⁻¹) were administered intravenously to three haemophilia A dogs. Blood was sampled between 0 and 120 h postdose and FVIII:C analysed. PK parameters maximum plasma concentration, area under the curve, half-life (t(½)), clearance, mean residence time (MRT) and volume of distribution and incremental recovery were calculated. Whole blood clotting time (WBCT) and thromboelastography (TEG®) were used to determine the haemostatic potential. No adverse reactions were observed with N8 or Advate ®. N8 and Advate® exhibited similar PK parameters, with t(½) 7.7-11 h and MRT 11-14 h. Both rFVIII compounds corrected the prolonged WBCT (> 48 min) to the range of normal dogs (8-12 min), i.e. N8 to 7.5-10.5 min and Advate® to 7.5-11.5 min. N8 and Advate® also normalized the whole blood clot formation according to TEG®. The native whole blood clotting assays (WBCT, TEG®) appeared to be more sensitive to low concentrations of FVIII than assays in citrated plasma samples. In conclusion, comparison of N8 and Advate ® in haemophilia A dogs revealed similar safety, similar PK and similar effects in whole blood clot formation assays.

Purification and characterization of a new recombinant factor VIII (N8).

SUMMARY: A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Factor VIII with a 237 amino acid B-domain has an extended half-life in F8-knockout mice.

Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths.

Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

Systemically administered trefoil factors are secreted into the gastric lumen and increase the viscosity of gastric contents.

BACKGROUND AND PURPOSE: Trefoil factors (TFFs) secreted by mucus-producing cells are essential for the defence of the gastrointestinal mucosa. TFFs probably influence the viscoelastic properties of mucus, but this has not been demonstrated in vivo. We therefore studied the gastric secretion of systemically administered TFF2 and TFF3, and their influence on the viscosity of the secretions. EXPERIMENTAL APPROACH: Mice and rats under general anaesthesia were injected intravenously with human (h) TFF2, hTFF3 (5 mg kg(-1) to mice and 25 mg kg(-1) to rats), murine (m) (125)I-TFF3, or (125)I-hTFF3 (300,000 cpm, mice only). The appearance of TFFs in the gastric mucosa and luminal secretions was analysed by autoradiography, gamma-counting, and ELISA, and the viscosity by rheometry. KEY RESULTS: (125)I-mTFF3 and (125)I-hTFF3 were taken up by secretory cells of the gastrointestinal tract and detected at the gastric mucosal surface 15 min after injection. Stressing the stomach by carbachol (3.5 microg kg(-1)) and pyloric ligation significantly increased the uptake. Injected hTFF2, hTFF3, and mTFF3 were retrieved from the gastric contents after 4 h. In rats, an approximately seven-fold increase in the viscosity was detected after injection of TFF2 compared to the controls, whereas TFF3 did not increase the viscosity. In mice, TFF2 increased the viscosity approximately 4-fold. CONCLUSIONS: These data indicate that systemically administered TFFs are transferred to the gastric lumen in a biologically active form.

Pancreatic spasmolytic polypeptide: first three-dimensional structure of a member of the mammalian trefoil family of peptides.

BACKGROUND: The trefoil peptides are a rapidly growing family of peptides, mainly found in the gastrointestinal tract. There is circumstantial evidence that they stabilize the mucus layer, and may affect the rate of healing of the mucosal epithelium. RESULTS: We have determined the structure of porcine pancreatic spasmolytic polypeptide (PSP) to 2.5 A resolution. The polypeptide contains two trefoil domains. The domain structure is compact, and is composed of a central short antiparallel beta-sheet with one short helix above and one below it. This is a novel motif. The two domains are related by two-fold symmetry, and each domain contains a cleft. CONCLUSIONS: The cleft within each domain could accommodate a polysaccharide chain, and may therefore be responsible for binding mucin glycoproteins. We suggest that PSP may cross-link glycoproteins, explaining its ability to stabilize the mucus layer.

Molecular forms of katacalcin, calcitonin gene-related peptide and gastrin-releasing peptide, in a human medullary thyroid carcinoma.

Peptides synthesized by a human medullary thyroid carcinoma were purified to homogeneity by reverse-phase high performance liquid chromatography and structurally characterized by determination of amino acid composition, amino acid sequence, and fast atom bombardment mass spectra. The katacalcin-related material in the tumor extract was heterogeneous. Katacalcin (1-21) represented the predominant molecular form but metabolites, identified as katacalcin (1-20), (1-19), (1-15) and (1-13), were also identified in high concentration. Calcitonin gene-related peptide-I was isolated from the tumor but calcitonin gene-related peptide-II was absent. A minor component of calcitonin gene-related peptide-like immunoreactivity was of higher molecular weight and may represent an incompletely processed form of the prohormone. Gastrin-releasing peptide (1-27) and gastrin-releasing peptide (18-27) (neuromedin C) were isolated from the tumor but gastrin-releasing peptide (14-27) and bombesin were absent.

Lipid binding and activating properties of porcine pancreatic colipase split at the Ile79-Thr80 bond.

Porcine colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1-93 of the 95-residue form of colipase (procolipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1-91 of procolipase. Also, two fractions of colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of colipase and of fragment 1-93, respectively, both specifically cleaved at the Ile79-Thr80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. Procolipase split at the 79-80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native procolipase. Also, all forms of procolipase split at the 79-80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native procolipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced procolipase. Treatment of native procolipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79-80 bond of procolipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of colipase split at the 79-80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.

The amino acid sequence of pancreatic spasmolytic polypeptide.

The sequence of porcine pancreatic spasmolytic polypeptide has been established by a variety of techniques including manual as well as automatic sequencing of fragments resulting from the cleavage of reduced and S-carboxymethylated pancreatic spasmolytic polypeptide with trypsin, chymotrypsin, clostripain, cyanogen bromide and formic acid. The N- and C-terminal sequences were established using pyroglutamate amino-peptidase and carboxypeptidase A, respectively. Pancreatic spasmolytic polypeptide contains 106 amino acid residues in a single chain with seven S-S bridges and a pyroglutamyl blocked N-terminal. The alignment of the sequences representing amino acids 14-49 and 63-98 shows pair-wise identical amino acid residues in 18 out of 36 positions, indicating that these two "domains" have been derived from a common gene.

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine.

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.

Revised amino acid sequence of pancreatic spasmolytic polypeptide exhibits greater similarity with an inducible pS2 peptide found in a human breast cancer cell line.

The published amino acid sequence of pancreatic spasmolytic polypeptide (Thim, L., Thomsen, J., Christensen, M. and Jørgensen, K.H. et al. (1985) Biochim. Biophys. Acta 827, 410-418) has been checked by a combination of mass spectroscopy and Edman degradation. The pyroglutamyl blocking group was positively identified, and residue assignments at four positions were corrected: Lys48 (not Ser), Ser63 (not Lys), Cys68 (not Ser) and Ser74 (not Cys). The revised sequence exhibits greater similarity with pS2 peptide, a 60 residue polypeptide which is induced by oestrogen in the human breast cancer cell line MCF-7 and found in malignant but not in non-malignant breast tissue.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

CART, a new anorectic peptide.

Cocaine and amphetamine regulated transcript peptide (CART), is a recently discovered hypothalamic peptide with a potent appetite suppressing activity. In the rat the CART gene encodes a peptide of either 129 or 116 amino acid residues whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a prohormone of 102 or 89 residues. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule. In the central nervous system CART is highly expressed in many hypothalamic nuclei, some of which are involved in regulating feeding behaviour. The CART mRNA is regulated by leptin, and the expressed CART is a potent inhibitor of feeding that even overrides the feeding response induced by neuropeptide Y. The putative CART receptor is therefore a potential therapeutic target for an anti-obesity drug.

Galanin and galanin extended at the N-terminus with seven and nine amino acids are produced in and secreted from the porcine adrenal medulla in almost equal amounts.

Galanin is present in high concentrations in porcine adrenals, but nothing is known about the processing and secretion of other products of the 123-amino acid precursor preprogalanin. Using, in combination, RIA against galanin, a variety of chromatographic procedures, mass spectrometry, and amino acid sequencing, we studied the processed and the secreted products of preprogalanin. From the tissue extracts we isolated in equimolar amounts and sequenced two major pools of galanin immunoreactive peptides: galanin and two N-terminally extended forms, preprogalanin-(24-61) and preprogalanin-(26-61). The same peptides were identified upon gel chromatography and analytical HPLC in effluents collected during electrical stimulation of the intact splanchnic nerve supply of an isolated perfused preparation of porcine adrenals. The processing of preprogalanin in porcine adrenals thus includes the formation and release of galanin, preprogalanin-(24-61), and preprogalanin-(26-61). The signal peptidase cleaves the preprogalanin at either Gly23 or Gly25.

Primary structures of somatostatins from the islet organ of the hagfish suggest an anomalous pathway of posttranslational processing of prosomatostatin-1.

The cyclostomes represent the first class of vertebrate in evolution to develop an endocrine pancreas. Two peptides with somatostatin-like immunoreactivity were isolated from the islet organ of one such cyclostome, the Atlantic hagfish (Myxine glutinosa). The primary structure of the more abundant peptide was established as: Ala-Val-Glu-Arg-Pro5-Arg-Gln-Asp-Gly-Gln10-Val-His-Glu-Pro- Pro15-Gly-Arg-Glu-Arg-Lys20-Ala-Gly-Cys-Lys-Asn25-Phe- Phe-Trp-Lys-Thr30-Phe-Thr-Ser-Cys. The second peptide, comprising 27% of the total immunoreactivity in the islet extract, was identical to mammalian somatostatin-14. The pathway of posttranslational processing of prosomatostatin in the hagfish islet differs markedly from the pathway in the higher vertebrates. In the mammalian pancreas, prosomatostatin is cleaved at the site of the single arginyl residue (corresponding to position 6 in hagfish somatostatin-34) and at the arginine-lysine site (corresponding to positions 19 and 20 in the hagfish peptide) to generate somatostatin-14 and somatostatin-28(1-12)-peptide. In the hagfish islet, Arg6 is not used as a cleavage site and cleavage at Arg19-Lys20 represents only a minor pathway of processing. The data provide further evidence of the strong evolutionary pressure to conserve the complete amino acid sequence of somatostatin-14.

Adenosine is an agonist of the growth hormone secretagogue receptor.

Growth hormone secretagogues (GHSs) are synthetic compounds that induce GH release in several species, including man. The aim of the current study was to identify hypothalamic GHS receptor (GHS-R) agonists. This led to the discovery of adenosine as a GHS-R agonist. We demonstrate that adenosine as well as the A1 adenosine receptor agonist N6-R-phenylisopropyladenosine (R-PIA) induce calcium responses, with EC50 values of 50 nM and 0.5 nM, respectively, in cells which express recombinant human GHS-R. However, neither compound induces a calcium response in nontransfected cells. Binding experiments show that adenosine and the GHS compound MK-0677 bind to membranes from GHS-R expressing cells with nearly identical Bmax values (2.6 +/- 0.1 x 10(-10) mol/mg protein for adenosine and 2.0 +/- 0.3 x 10(-10) mol/mg protein for MK-0677). However, no binding to membranes from nontransfected cells could be detected. Furthermore, we show that the IC50 values for inhibition of the adenosine, R-PIA, and GHS induced calcium responses by the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P are similar. These findings strongly suggest that adenosine and R-PIA are agonists of the GHS-R. Interestingly, neither adenosine nor R-PIA were able to induce GH release from rat pituitary cells in vitro. The implications of the latter finding is discussed.

A new family of growth factor-like peptides. 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins).

Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38-39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic 'trefoil' disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

Secretion of human insulin by a transformed yeast cell.

A yeast expression plasmid encoding a mini-proinsulin molecule was constructed and transformed into Saccharomyces cerevisiae. The plasmid encoded the sequence: B-Arg-Arg-Leu-Gln-Lys-Arg-A in which B represents the B-chain (30 amino acid residues) and A represents the A-chain (21 amino acid residues) of human insulin. The secreted peptides were shown to be a mixture of human insulin and des(B-30)human insulin. Thus, correct disulphide bridges can be established in proinsulin-like molecules devoid of a normal C-peptide region. Furthermore, the specificity of the yeast processing enzymes is so similar to the proinsulin converting enzymes in the human pancreatic beta-cell that it allows the processing of the mini-proinsulin to insulin.

Primary structure of glucagon from the gut of the common dogfish (Scyliorhinus canicula).

The primary structure of glucagon isolated from the intestine of the common dogfish, Scyliorhinus canicula, was established as H S E G T F T S D Y S K Y M D N R R A K D F V Q W L M N T. The peptide shows four substitutions compared with human glucagon: Glu-3 for Gln, Met-14 for Leu, Asn-16 for Ser and Lys-20 for Gln. Glucagon represented the predominant molecular form of the glucagon-like immunoreactivity in the dogfish gut extracts demonstrating that the pathway of posttranslational processing of proglucagon in the gut of this fish differs markedly from the pathway in the mammalian gut.

The isolation and sequencing of human gastric inhibitory peptide (GIP).

Human GIP 1-42 and fragments of human GIP corresponding to GIP 10-42, GIP 11-42, and GIP 17-42 were isolated from acid-ethanol extracts of human small intestines with the aid of an anti-GIP serum specific for the extreme C-terminal portion of the GIP molecule. The full sequence of human GIP has been established by Edman degradation of these peptides and fragments thereof by automatic gas-phase sequencing. Human GIP differs from porcine GIP at residues 18 and 34. The sequence of human GIP is thus: (Formula: see text) Amino acid residues 18 and 34 are Arg and Ser, respectively, in porcine GIP.