Clinical evaluation of an advanced category antihaemophilic factor prepared using a plasma/albumin-free method: pharmacokinetics, efficacy, and safety in previously treated patients with haemophilia A.

The efficacy and safety of an advanced category recombinant antihaemophilic factor produced by a plasma- and albumin-free method (rAHF-PFM) was studied in 111 previously treated subjects with haemophilia A. The study comprised a randomized, double-blinded, crossover pharmacokinetic comparison of rAHF-PFM and RECOMBINATE rAHF (R-FVIII); prophylaxis (three to four times per week with 25-40 IU kg(-1) rAHF-PFM) for at least 75 exposure days; and treatment of episodic haemorrhagic events. Median age was 18 years, 96% of subjects had baseline factor VIII <1%, and 108 received study drug. Bioequivalence, based on area under the plasma concentration vs. time curve and adjusted in vivo recovery, was demonstrated for rAHF-PFM and R-FVIII. Mean (+/-SD) half-life for rAHF-PFM was 12.0 +/- 4.3 h. Among 510 bleeding events, 473 (93%) were managed with one or two infusions of rAHF-PFM and 439 (86%) had efficacy ratings of excellent or good. Subjects who were less adherent to the prophylactic regimen had a higher bleeding rate (9.9 episodes subject(-1) year(-1)) than subjects who were more adherent (4.4 episodes subject(-1) year(-1); P < 0.03). One subject developed a low titre, non-persistent inhibitor (2.0 BU) after 26 exposure days. These data demonstrate that rAHF-PFM is bioequivalent to R-FVIII, and suggest that rAHF-PFM is efficacious and safe, without increased immunogenicity, for the treatment of haemophilia A.

Kinetics of factor IX activity differ from that of factor IX antigen in patients with haemophilia B receiving high-purity factor IX replacement.

Pharmacokinetic studies in haemophilia B have found in vivo recovery of FIX (FIX) to be uniformly lower than the factor VIII recovery in haemophilia A. We hypothesized that this lower recovery could result from rapid binding to high-affinity receptors on platelets and endothelium. To test this hypothesis, we evaluated the kinetics of FIX activity and protein in haemophilia B patients. Twelve patients were enrolled in a double dosing, crossover study with two high-purity FIX concentrates, AlphaNine SD and MonoNine. Subjects were given 40 U kg-1 of FIX concentrate and blood samples were taken at 15, 30, and 60 min. A second infusion of 40 U kg-1 was given after the 60 min blood sample and further blood samples removed at 15, 60, 120, and 360 min after the second dose. Patients were infused with the alternate concentrate at least 7 days later. Plasma samples were assayed for FIX activity by coagulation assay and antigen by RIA. FIX antigen in the infused concentrates was measured and quantified as microg U-1. There was no difference between the two FIX concentrates (AlphaNine vs. MonoNine) in the initial (15 min) activity (57% +/-1 19% vs. 53% +/-1 12%) and antigen (62% +/-1 16% vs. 55% +/-1 19%) recoveries. Recoveries after the second FIX dose were not statistically different than those observed after the first FIX dose. In one patient, a doubling of the initial infusion dose did not increase FIX recovery after the second FIX dose. However, the recovery of FIX antigen was significantly greater than the recovery of FIX activity and the differences became more significant in the post-15 min samples. We calculated a ratio of plasma FIX antigen to FIX activity in microg U-1. Average antigen to activity ratio increased from 5.8 +/-1 1.9 microg U-1 at 15 min to 7.1 +/-1 2.2 microg U-1 at 60 min. At 420 min the ratio increased to 9.3 +/-1 2.4 microg U-1. Although these studies failed to demonstrate a significant FIX receptor pool, they did demonstrate a phenomenon of progressive loss of biologic activity of the FIX protein after infusion of FIX concentrates.

Fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Crotalus basiliscus basiliscus: cleavage site specificity towards the alpha-chain of fibrin.

Zinc metalloproteinases with fibrinolytic activity have been isolated from the venom of Agkistrodon contortrix contortrix (fibrolase) and Crotalus basiliscus basiliscus (basiliscusfibrases 1, 2 and 3). Fibrolase cleaves the A alpha-chain of fibrinogen initially at a single site: Lys413-Leu. Basiliscusfibrases 1, 2 and 3 also cleave at this site as well as others. Since cleavage in Lys-Leu locations is not common among zinc metalloproteinases, we examined the degree to which the Lys-Leu bond determines cleavage specificity and cleavage rates for these enzymes. We employed the oxidized B-chain of insulin and the following synthetic octapeptides: InsA (Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val) which includes the insulin B-chain sequence around the Ala14-Leu15 bond (which is cleaved by all of the enzymes); InsK (Leu-Val-Glu-Lys-Leu-Tyr-Leu-Val) which is identical to InsA apart from the Ala4 to Lys4 substitution; and PA alpha (His-Thr-Glu-Lys-Leu-Val-Thr-Ser) which reproduces the sequence around the Lys413-Leu414 cleavage site of the A alpha-chain of fibrinogen. Results suggest that fibrolase is better adapted for cleaving the A alpha-chain at the Lys413-Leu414 locus and that cleavage specificity for the enzymes tested was not dictated by the Lys-Leu bond per se, but by the surrounding sequence.

Intraperitoneal 5-fluoro-2'-deoxyuridine with escalating doses of leucovorin: pharmacology and clinical tolerance.

In a preceding study, we established the tolerance and pharmacokinetic behavior of 5-fluoro-2'-deoxyuridine (FdUrd) given by the intraperitoneal (IP) route. A dose of 3 g daily x 3 days was found satisfactory for Phase II study and exploration of biochemical modulation. Therefore, the current study was conducted to study the tolerance and pharmacokinetics of such a dose-schedule and route of FdUrd combined with escalating doses of leucovorin (LV). Fourteen patients were entered and 13 were evaluable for tolerance determination. Pharmacologic determinations of IP FdUrd and 5-Fluorouracil (FUra) derived from it and LV were obtained by HPLC methods on 11 occasions. Findings were compared with the preceding study of FdUrd alone. LV did not appear to alter the tolerance of IP FdUrd even in the four patients receiving the highest dose of LV (640 mg). Toxicities included nausea, vomiting, and rarely neutropenia and diarrhea. Pharmacokinetic parameters indicate a parallel rate of egress of FdUrd and LV from the peritoneal cavity. The pharmacologic advantage for FdUrd is at least 3 logs as previously reported and one log for LV. Evidence of antitumor effect was noted particularly among untreated patients with gastrointestinal primaries. We conclude that IP FdUrd 3 g and LV in doses of up to 640 mg x 3 days are well tolerated. Since FdUrd is more potent, has an even greater hepatic clearance and shows greater potential for modulation with LV than FUra, it may be the preferred fluoropyrimidine for subsequent studies via the IP route in the treatment of carcinomas with prominent peritoneal spread. The pharmacologic advantage for leucovorin is limited but it is a good marker for peritoneal clearance since it parallels FdUrd clearance.

Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.

Purification, characterization, and fibrinogen cleavage sites of three fibrinolytic enzymes from the venom of Crotalus basiliscus basiliscus.

Three distinct fibrinolytic enzymes have been purified from the venom of Crotalus basiliscus basiliscus (the Mexican west coast rattlesnake). The high-performance liquid chromatography-based purification comprised the following steps: (a) hydrophobic interaction chromatography; (b) hydroxylapatite chromatography; (c) anion-exchange chromatography. Following hydrophobic interaction chromatography two fibrinolytic activity peaks were detected, Cbfib1 and Cbfib2. Cbfib2 was rendered homogeneous following hydroxylapatite chromatography. Upon hydroxylapatite chromatography Cbfib1 was shown to consist of two components, Cbfib1.1 and Cbfib1.2. Both Cbfib1.1 and Cbfib1.2 were purified to homogeneity using anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis revealed that Cbfib1.1 and Cbfib1.2 had similar molecular weights (approximately 23,500), whereas Cbfib2 displayed a molecular weight of approximately 22,500. The enzymes do not appear to be glycosylated. Tryptic digests of all three enzymes, analyzed by high-performance reverse-phase chromatography, suggest that Cbfib1.1 and Cbfib1.2 are closely related and different from Cbfib2. The latter displayed more similarity with Cbfib1.2 than with Cbfib1.1. Specific fibrinolytic activity for all three enzymes was very similar, but general proteolytic activity varied substantially with Cbfib2 showing a 12-fold higher specific proteolytic activity when compared to Cbfib1.1 and Cbfib1.2. None of these enzymes exhibited hemorrhagic activity when injected (up to 100 micrograms) subcutaneously into mice. Cbfib1.1 and Cbfib1.2 action against fibrinogen was directed equally against both the A alpha- and B beta-chains. Against fibrin the rate of degradation of the alpha-chain was considerably higher than that of the beta-chain. Cbfib2 showed mainly alpha-fibrin(ogen)ase activity with limited activity on the beta-chain. Several fibrinogen cleavage sites on the A alpha-chain have been identified: Cbfib1.1 and Cbfib1.2 cleave at Lys413-Leu414, Ser505-Thr506, and Tyr560-Ser561. Cbfib2 cleaves mainly at Gly254-Ser255 and Pro516-Met517.

Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix).

A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom has been purified by combination of CM-cellulose chromatography, molecular sieve chromatography on Sephadex G-100, p-aminobenzamidine-agarose affinity chromatography, and DEAE-cellulose chromatography. The enzyme, fibrolase, has a molecular weight of 23,000-24,000 and an isoelectric point of pH 6.8. It is composed of approximately 200 amino acids, possesses a blocked NH2-terminus and contains little or no carbohydrate. The enzyme shows no activity against a series of chromogenic p-nitroanilide substrates and is not inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, Trasylol, or p-chloromercuribenzoate. However, the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator). Metal analysis revealed 1 mol of zinc/mol of protein. Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond. Interestingly, unlike some other venom fibrinolytic metalloproteinases, fibrolase exhibits little if any hemorrhagic activity. The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.

HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti.

In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.

A direct-acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix: effects on various components of the human blood coagulation and fibrinolysis systems.

A direct acting fibrinolytic enzyme (fibrolase) has been isolated from venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Time-course experiments established that the venom enzyme cleaves primarily the A alpha-chain of human fibrinogen and fibrin between the Lys-413 and Leu-414 position. The B beta-chain is cleaved more slowly, while the gamma-chain is minimally affected. The cleavage pattern of fibrinogen and fibrin clearly varies from plasmin cleavage of the same molecules. The enzyme does not activate plasminogen or protein c and it is thus different from "Protac", a protein c activator isolated from the same venom.

Enzymes of the contact phase of blood coagulation: kinetics with various chromogenic substrates and a two-substrate assay for the joint estimation of plasma prekallikrein and factor XI.

Kinetic constants (Km and kcat) of kallikrein and factor XIa for the chromogenic substrates H-D-L-prolyl-L-phenylanyl-L-arginine-p-nitroanilide (S-2302) and L-pyroglutamyl-L-propyl-L-arginine-p-nitroanilide (S-2366) were determined. The determined constants allow the use of S-2302 and S-2366 in an assay that leads to the joint estimation of factor XI and prekallikrein in activated plasma. The assay reports approximately 3.1 micrograms/ml factor XIa and 34.5 micrograms/ml kallikrein in kaolin-activated plasma (kaolin content 2 mg/ml). The dual-substrate amidolytic assay shows good correlation with the coagulant assay of both factors (0.92 with the prekallikrein assay and 0.98 with the factor XI assay). It is capable, through the joint estimation of factor XI and prekallikrein levels, of differentiating among plasma samples deficient in components of the contact phase of blood coagulation. Kinetic constants of factor beta-XIIa (factor XII fragment) for these substrates and for N-benzol-L-isoleusyl-L-glutamyl-glycyl-L-arginine-P-nitro ani lide (S-2222) were determined, and they allowed the assessment of the contribution of this factor to this assay and its estimation in the activated phase.

Effects of chemical modifications on the surface- and protein-binding properties of the light chain of human high molecular weight kininogen.

The light chain of kallikrein-cleaved human high molecular weight kininogen is solely responsible for its cofactor activity in blood clotting. Sequencing of the NH2-terminal region of the light chain reported herein identified the third kallikrein cleavage site of high molecular weight kininogen as Arg-437. The co-factor activity of high molecular weight kininogen consists of the capacity to bind to negatively charged surfaces and to factor XI or prekallikrein. Chemical modification of the histidines by either photooxidation or ethoxyformic anhydride affected the equivalent of 14-16 of 23 histidines available and resulted in over 90% loss in procoagulant activity. The modified protein had drastically reduced surface- and zinc-binding capacity, but it bound successfully to either factor XI or prekallikrein. In contrast, modification of two carboxyl groups, which led to approximately 80-90% loss of procoagulant activity, seriously compromised protein binding but left surface binding unaffected. All 3 tryptophans were modified at pH 4.0 with N-bromosuccinimide with a 70% reduction in procoagulant activity, but only 1 tryptophan was available for reaction at pH 7.35, resulting in a 50% loss in activity. Tryptophan modification at acidic pH affected protein binding but did not modify surface or zinc binding. Modification of both available tyrosine and 9 of 18 available lysine residues did not have a significant effect on the procoagulant activity of the light chain. These studies indicate that histidines participate in surface binding and that free carboxyl groups and tryptophan side chains are involved in binding of high molecular weight kininogen to other clotting factors.

Chemical basis of the electrophoretic variation observed at the alcohol dehydrogenase locus of Drosophila melanogaster.

The amino acid substitution responsible for the different electrophoretic mobility of the ADHs alleloenzyme and the ADHf alleloenzyme of the alcohol dehydrogenase from a Nigerian population of Drosophila melanogaster has been established as lysine (ADHs) for threonine (ADHf). This result is discussed with reference to the charge state model of electrophoretic variation, in conjunction with other know substitutions at this locus. It is concluded that electrophoretic methods should be capable of distinguishing many alleloenzymes which have identical isoelectric points without recourse to explanations involving conformational variability.