Characterization of high molecular weight plasma protein complexes induced by clotting factor rFXIII-treatment in the cynomolgus monkey.

BACKGROUND: In cynomolgus monkeys, suprapharmacological doses of clotting recombinant factor XIII (rFXIII) cause a generalized coagulopathy, associated with formation of circulating high molecular weight protein complexes (HMEX). HMEX consist of plasma protein substrates cross-linked by FXIII transglutaminase activity. OBJECTIVE: To characterize HMEX, with a view to develop safety biomarker assays. METHODS: Cynomolgus monkeys received single i.v. injections with vehicle or rFXIII at 1, 3 and 10 mg kg(-1). Plasma HMEX were analyzed by sodium dodecylsulfate-polyacrylmide gel electrophoresis, silver staining, Western blotting and quantitative dissociation-enhanced lanthanide fluoroimmunoassay. Plasma FXIII antigen was analyzed by quantitative ELISA. Human HMEX were made in vitro, by spiking plasma with thrombin-activated rFXIII. RESULTS: Maximal circulating HMEX levels were reached within 1 h of rFXIII treatment, and remained stable over 24 h. HMEX above 250 kDa contained fibrinogen alpha-chains and fibronectin. Fibrinogen gamma-chain was detected only in HMEX below 250 kDa. The total plasma concentration of HMEX was in the low microg mL(-1) range, distributed on less than 20 main species. Human and cynomolgus HMEX were similar. HMEX formation increased with rFXIII dose in a disproportionate manner, with 3-fold and fortyfold increases in HMEX exposure associated with rFXIII dose increments from 1 to 3 and 3 to 10 mg kg(-1), respectively. CONCLUSIONS: The disproportionate HMEX formation parallels the steep toxicity dose response previously reported for rFXIII in cynomolgus monkeys, supporting a mechanistical role for HMEX in the generalized coagulopathy seen in rFXIII toxicity. Our findings support that HMEX constitute candidate (potential) safety biomarkers in rFXIII treatment.

Preclinical toxicity biomarkers for combination treatment with clotting factors rFXIII and rFVIIa.

Combination treatment with the clotting factors recombinant activated factor VII (rFVIIa), serine protease, and recombinant factor XIII (rFXIII), protransglutaminase, is being explored for haemostatic therapy. We performed a single-dose toxicology study in the cynomolgus monkey, with four dose groups receiving 0.1 + 0.34 mg kg(-1) (group 1), 0.33 + 1.12 mg kg(-1) (group 2), 1.67 + 5.60 mg kg(-1) (group 3) and 5.00 + 16.80 mg kg(-1) (group 4) of a rFVIIa + rFXIII combination. In the three lower dose groups, no clinical, histopathological or blood chemistry changes were observed. In group 4, the animals died at 4 h post-dosing, with histopathology revealing a systemic coagulopathy resembling, but distinct from, disseminated intravascular coagulation. Due to the absence of toxicity warning signs, toxicity biomarkers were identified by a Western blot-based screening of approximately 20 plasma proteins known to be involved in the clotting cascade. Three of the examined proteins were specifically affected by rFVIIa + rFXIII treatment. Fibronectin and fibrinogen exhibited dose-dependent reductions from less than 10% reduction (group 2) to more than 90% reduction (group 4). These reductions were reversible, and specific. For vitronectin, a dose-dependent conversion to the 65-kDa form was found to occur in groups 3 and 4. Thus, fibrinogen, fibronectin and vitronectin represent the first biomarkers for clotting factor toxicity.

Combination of 'omics' data to investigate the mechanism(s) of hydrazine-induced hepatotoxicity in rats and to identify potential biomarkers.

To gain novel insight into the molecular mechanisms underlying hydrazine-induced hepatotoxicity, mRNAs, proteins and endogenous metabolites were identified that were altered in rats treated with hydrazine compared with untreated controls. These changes were resolved in a combined genomics, proteomics and metabonomics study. Sprague-Dawley rats were assigned to three treatment groups with 10 animals per group and given a single oral dose of vehicle, 30 or 90 mg kg(-1) hydrazine, respectively. RNA was extracted from rat liver 48 h post-dosing and transcribed into cDNA. The abundance of mRNA was investigated on cDNA microarrays containing 699 rat-specific genes involved in toxic responses. In addition, proteins from rat liver samples (48 and 120/168 h post-dosing) were resolved by two-dimensional differential gel electrophoresis and proteins with changed expression levels after hydrazine treatment were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting. To elucidate how regulation was reflected in biochemical pathways, endogenous metabolites were measured in serum samples collected 48 h post-dosing by 600-MHz 1H-NMR. In summary, a single dose of hydrazine caused gene, protein and metabolite changes, which can be related to glucose metabolism, lipid metabolism and oxidative stress. These findings support known effects of hydrazine toxicity and provide potential new biomarkers of hydrazine-induced toxicity.

Pharmacokinetics and allometric scaling of levormeloxifene, a selective oestrogen receptor modulator.

The pharmacokinetics of a new selective oestrogen receptor modulator levormeloxifene was investigated in mice, rats, cynomolgus monkeys and humans by compartmental pharmacokinetics. Levormeloxifene was administered as an oral solution in all studies. Allometric scaling was used to predict human pharmacokinetic parameters and the performance of the approach was evaluated. Mean values of clearance confounded by F(CL/F) were 0.073, 0.29, 3.18 and 2.4 l/h in mice, rats, monkeys and humans, respectively. Values of distribution volume at steady state confounded by F(V(ss)/F) were 0.073 and 7.5 l in mice and rats. In monkeys, values of the central volume F(V(c)/F) and volume at steady state F(V(ss)/F) were 28.9 and 57.9 l, respectively. In humans, values of V(c)/F and V(ss)/F were 106 and 587 l, respectively. Predicted CL/F and V(ss)/F showed a linear relationship when plotted vs BW on a log-log scale; for CL/F, r was 0.95-0.98 and for V(ss)/F, r was 0.99. Using allometric scaling the predicted human V(ss)/F deviated 3-fold from the experimentally determined values. Observed values of CL/F deviated 21-25 fold from the predicted, the latter depending on the scaling method. Confidence intervals for the predicted parameters showed major lack of precision for all the allometric scaling methods.

Pharmacokinetics of levormeloxifene in young versus elderly postmenopausal women.

BACKGROUND: Physiologic changes of aging may affect processes of drug absorption and distribution, in some cases necessitating age-dependent dose adjustment. OBJECTIVE: The possibility of age dependence in the pharmacokinetic behavior and tolerability of levormeloxifene was investigated in a single-center, open-label study. METHODS: The study comprised 2 groups of healthy postmenopausal women: group A included younger subjects (50-60 years) and group B included elderly subjects (> or = 66 years). All subjects received a single 40-mg tablet of levormeloxifene base. Blood samples were collected immediately before drug intake and at several points after administration, through day 34. Peak plasma concentration, time to maximum plasma concentration, area under the plasma concentration-time curves from zero to the last quantifiable value and to infinity, and terminal half-life were calculated for levormeloxifene and compared between age groups. RESULTS: Of 29 subjects enrolled, 28 (15 group A, 13 group B) completed the study. The ages of the women in group A ranged from 50 to 58 years and in group B from 66 to 79 years. No serious adverse events were reported. Ten subjects experienced 17 adverse events, of which 2 (abdominal pain and vaginal hemorrhage) were judged to be possibly related to study drug. There was no noticeable difference between age groups in the frequency of adverse events or laboratory abnormalities. The plasma concentration-time curves of levormeloxifene were indistinguishable between age groups up to 48 hours after dosing. From 72 hours onward, the mean plasma concentration-time curve was approximately 20% higher and the area under the curve was approximately 19% greater in the older subjects compared with the younger subjects. However, no statistically significant differences were observed between groups in any of the pharmacokinetic parameters, except for the elimination rate constant. The difference in mean elimination half-life was 25 hours (group A, 126 hours; group B, 151 hours). CONCLUSION: The findings suggest that it is not necessary to adjust the dose of levormeloxifene on the basis of age in postmenopausal women. However, these results need confirmation in a multiple-dose setting under steady-state conditions--that is, as the drug is intended to be used clinically.

Metabolism, disposition, excretion, and pharmacokinetics of levormeloxifene, a selective estrogen receptor modulator, in the rat.

The tissue distribution, pharmacokinetics, metabolism, and excretion of the selective estrogen receptor modulator levormeloxifene have been investigated after oral administration of [(14)C]-levormeloxifene to male and female Sprague-Dawley rats. The quantitative distribution of radiolabeled levormeloxifene and/or metabolites was confirmed by whole body autoradiography. Levormeloxifene was absorbed from the gastrointestinal tract and was widely distributed into tissues, with peak radioactive concentrations generally being observed 4 h after administration in the intestine, liver, lung, kidney, spleen, pancreas, adrenals, and ovary (females). Fecal elimination was the major excretion route of radioactivity. In a separate pharmacokinetic study, plasma C(max) was generally observed 6 h after dose administration and the half-life of elimination was long (24 h) and a doubling in dose resulted in an approximate doubling in exposure. The majority of the drug was excreted as norlevormeloxifene; the 7-desmethyl metabolite of levormeloxifene, via the formation of phase II metabolites (glucuronides) and excretion into the bile. Unchanged drug was also excreted, mainly from 0 to 24 h, and accounted for about 6 to 12% of the dose. Together these two components accounted for approximately 50% of the radioactivity excreted. Additional metabolites isolated and identified by liquid chromatography-tandem mass spectrometry, and accounting for 1 to 5% of the excreted radioactivity in rat feces during the first 24 h, included two monohydroxylevormeloxifene species, a pyrrolidinone ring-opened metabolite of levormeloxifene, and desmethylnorlevormeloxifene.

Assay for levormeloxifene, a selective estrogen receptor modulator, in human and monkey plasma employing high-performance liquid chromatography and solid-phase extraction.

Assays for levormeloxifene, a new selective estrogen receptor modulator, and its 7-desmethyl metabolite in human and cynomolgus monkey plasma are described. Plasma was extracted on mixed-mode bonded sorbent material (C8/SCX) and the extracts were analysed by high-performance liquid chromatography with fluorescence detection. Recoveries of levormeloxifene and the metabolite exceeded 70%. Within and total assay precision calculated as a coefficient of variation (C.V.) were <8% for both compounds at all concentration levels, except at the limit of quantitation (LOQ) where the C.V. was 15%. Within and total-assay accuracy calculated as a percentage of the nominal value were between 90 and 114% for both analytes. The LOQ was for levormeloxifene and 7-desmethyllevormeloxifene, respectively, 1.5 and 2.5 ng/ml (man) and 5.2 and 6.9 ng/ml (monkey). In the monkey plasma assay, human plasma could substitute monkey plasma as blank plasma.

Albumin binding and time action of acylated insulins in various species.

Insulins acylated with fatty acids at the epsilon-amino group of LysB29 constitute a new class of insulin analogs, which are prolonged-acting due to albumin binding. In the present study it is shown that the affinity of fatty acid acylated insulins for albumin varies considerably (> 50-fold) among species. The relative affinities of acylated insulin for albumin in human, pig, and rabbit serum are about 1:1:5:35. The several fold higher binding affinity in rabbit serum than in pig serum is reflected in a relatively more protracted effect after sc injection in rabbits than in pigs. Due to the similar binding affinities in pig serum and human serum, the pig model should provide a useful estimate of the degree of protraction of acylated insulin in humans. The results emphasize that species differences in ligand binding can be of major importance in the preclinical evaluation of highly albumin bound drugs.

Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo.

Albumin is a multifunctional transport protein that binds a wide variety of endogenous substances and drugs. Insulins with affinity for albumin were engineered by acylation of the epsilon-amino group of LysB29 with saturated fatty acids containing 10-16 carbon atoms. The association constants for binding of the fatty acid acylated insulins to human albumin are in the order of 10(4)-10(5) M-1. The binding apparently involves both non-polar and ionic interactions with the protein. The acylated insulins bind at the long-chain fatty acid binding sites, but the binding affinity is lower than that of the free fatty acids and depends to a relatively small degree on the number of carbon atoms in the fatty acid chain. Differences in affinity of the acylated insulins for albumin are reflected in the relative timing of the blood-glucose-lowering effect after subcutaneous injection into rabbits. The acylated insulins provide a breakthrough in the search for soluble, prolonged-action insulin preparations for basal delivery of the hormone to the diabetic patient. We conclude that the biochemical concept of albumin binding can be applied to protract the effect of insulin, and suggest that derivatization with albumin-binding ligands could be generally applicable to prolong the action profile of peptide drugs.

The cobalt(III)-insulin hexamer is a prolonged-acting insulin prodrug.

The insulin hexamer has two high-affinity metal ion binding sites, each involving three HisB10 residues, one from each dimer. Insulin hexamers containing Co2+ at both these sites were oxidized to form a stable Co(3+)-insulin complex. It is shown that the Co(3+)-coordinated insulin monomers are released extremely slowly in aqueous solution at pH 8.0, and that the hexamer does not spontaneously dissociate into subunits at nanomolar concentrations of insulin. The Co(3+)-insulin hexamer is not recognized by the insulin receptor in vitro but the complex shows a protracted action profile following subcutaneous (s.c.) injection into rabbits. The Co(3+)-insulin hexamer provides a novel prodrug approach to a soluble, prolonged-acting insulin preparation of potential use for basal insulin delivery in the treatment of diabetes.