| Sign In to gain access to subscriptions and/or personal tools. |
Preclinical Safety and Pharmacokinetics of Recombinant Human Factor XIII
1 ZymoGenetics, Inc., Seattle, Washington 98102, USA Correspondence: Address correspondence to: Dr. Rafael Ponce, ZymoGenetics, Inc., 1201 Eastlake Ave. E., Seattle, WA 98102, USA; e-mail:reap{at}zgi.com
Factor XIII (FXIII) is a thrombin-activated protransglutaminase responsible for fibrin clot stabilization and longevity. Deficiency in FXIII is associated with diffuse bleeding and wound-healing disorders in humans. This report summarizes results from several studies conducted in adult cynomolgus monkeys (M. fascicularis) to evaluate the safety and pharmacokinetics of recombinant human factor XIII A2 dimer (rFXIII). Intravenous slow bolus injection of rFXIII resulted in the expected formation of the heterotetramer rA2cnB2, prolonged circulating half-life (5–7 days), and increased plasma transglutaminase activity. Recombinant FXIII was well tolerated as a single dose up to 20 mg/kg rFXIII (2840 U/kg), as repeated daily doses up to 6 mg/kg (852 U/kg) for 14 days, and as 3 repeated doses of 8 mg/kg (1136 U/kg) separated by 14 days. Overt toxicity occurred after a single intravenous injection of  22.5 mg/kg rFXIII (3150 U/kg), or with 2 doses of =12.5 mg/kg (1775 U/kg) administered within 72 hours. The rFXIII-mediated toxicity was expressed as an acute systemic occlusive coagulopathy. Evaluation of plasma samples from dosed animals demonstrated formation of cross-linked fibrin/fibrinogen oligomers and higher-order protein aggregates, which are hypothesized to be responsible for the observed vessel occlusion and associated embolic sequelae. These results demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support an initial clinical dose range of 0.014 to 0.35 mg/kg (2–50 U/kg).
Key Words: Factor XIII • Recombinant Factor XIII • preclinical safety • cynomolgus monkey • pharmacokinetics • coagulation factor Abbreviations: rFXIII, rA2, recombinant human FXIII [A2] dimer • FXIII, pFXIII, A2B2, plasma FXIII [A2B2] heterotetramer • cFXIII, cellular, placental, or platelet FXIII [A2]dimer • A2, factor XIII A-dimer • rA2cnB2, de-lineates the complex formed when mixing rFXIII and cynomolgus FXIII-B subunit • FXIIIa, all or any forms of active FXIII • FXIIIa*, proteolytically activated FXIII • FXIIIa°, nonproteolytically activated FXIII • ZGI, ZymoGenetics, Inc. (Seattle, WA) • SA-HRP, streptavidin-horseradish per-oxidase • NHS-LC biotin, succinimidyl-6-(biotinamido) hexanoate • OPD, (ortho-phenylenediamine dihydrochloride) • TMB, 3,3'5,5' tetramethylbenzidine
Congenital Factor XIII (FXIII) deficiency is a rare inherited autosomal recessive blood disorder estimated to affect 1 in 5 million persons worldwide (Gootenberg, 1998; Di Paola et al., 2001). Congenital FXIII deficiency usually presents as delayed bleeding from the umbilical stump or circumcision site in homozygous newborn infants. The majority of FXIII deficient patients not receiving prophylactic therapy with FXIII-containing plasma products develop spontaneous hemorrhages, subcutaneous hematomas, superficial ecchymoses, spontaneous bleeding, epistaxis bleeding, and delayed bleeding from sites of trauma (Board et al., 1993; Shaikh and Khurshid, 1993; Egbring et al., 1996). Because of the high mortality associated with intracranial hemorrhage, prophylaxis with FXIII-containing blood components or human plasma concentrates is recommended in these patients (Board et al., 1993; Miloszewski and Losowsky, 1991; Gootenberg, 1998). In addition to congenital deficiency, acquired FXIII deficiency can occur subsequent to blood loss, chronic coagulation system activation, or FXIII inhibitor development. Factor XIII-containing plasma products and human plasma-derived concentrates have been shown to restore clot stability and increase plasma FXIII activity as measured by in vitro assays. Multiple long-term observational studies have demonstrated that monthly prophylaxis (every 4–6 weeks) with FXIII-containing plasma, cryoprecipitate, or human plasma-derived concentrates have virtually eliminated bleeding episodes, including intracranial hemorrhage, among those who are congenitally deficient (Miloszewski and Losowsky, 1991; Board et al., 1993; Emrich et al., 1993). Recombinant FXIII (rFXIII) is being developed as a safe and effective alternative to FXIII-containing preparations derived from human plasma. Factor XIII is present in plasma and in platelets, monocytes, and other cells of monocytic lineage. Plasma FXIII (pFXIII) circulates as a heterotetramer composed of 2 FXIII-A and 2 FXIII-B subunits (A2B2). Enzymatic activity resides with the FXIII-A subunit. The FXIII-B subunit circulates in excess in plasma and acts as a carrier, thus conferring a longer circulating half-life to the FXIII-A subunit. The cellular form of FXIII (cFXIII) lacks the FXIII-B subunits and is a homodimer of FXIII-A subunits (A2). Recombinant FXIII (rFXIII) is produced in Saccharomyces cerevisiae as a nonglycosylated FXIII A2 homodimer that is virtually identical to human cFXIII. Both human cFXIII and rFXIII readily form heterotetrameric A2B2 complexes in the presence of human FXIII-B subunit (Radek et al., 1993). Studies on the binding of rFXIII to cynomolgus FXIII-B subunit conducted to support selection of species for toxicology studies confirm appropriate formation of the heterotetramer (data not shown). The activity of rFXIII on human fibrin is similar to native FXIII (Bishop et al., 1990).
Factor XIII functions as a transglutaminase and forms N
Summarized here are results from several studies examining the preclinical safety and pharmacokinetics of single and repeated intravenous rFXIII injection to adult cynomolgus monkeys. Cynomolgus monkeys were used as a study model because their coagulation parameters are similar to those of humans (Seaman and Malinow, 1968) and they demonstrate pharmacologic response to rFXIII including the expected in vitro plasma fibrin cross-linking activity and formation of the Factor XIII heterotetramer complexes between endogenous FXIII-B subunit and rFXIII (i.e., rA2cnB2). Administration of rFXIII at supraphysiological levels results in saturation of available uncomplexed FXIII-B subunit and leaves excess, uncomplexed rFXIII in circulation; uncomplexed FXIII-A2 dimer is not observed under normal physiological conditions (nor is it expected to occur under conditions of therapeutic rFXIII administration). Evaluation of plasma samples after rFXIII treatment revealed evidence of rFXIII activation and the associated formation of high molecular weight protein complexes comprised of expected substrates for FXIII transglutaminase activity. These high molecular weight complexes appeared to have a relatively long persistence and their increased burden presumably underlies the observed occlusive coagulopathy that is characteristic of rFXIII toxicity.
Drug Product Composition The drug product used in the preclinical safety studies was manufactured by ZymoGenetics, Inc. (Seattle, WA) and supplied as 5 mL vials containing an isotonic 5 mg/mL (710 U/mL) rFXIII solution formulated in 3% sucrose, 20 mM histidine, 200 mM glycine, and 0.01% polysorbate 20 at pH 8.0 suitable for intravenous injection. The concentration of recombinant FXIII (mg/kg) can be expressed as units of enzymatic activity (U/kg) by using the following conversion: 1 mg rFXIII = 142 U, where 1 U equals the amount of transglutaminase activity in 1 mL of normal human plasma. The composition and purity of the test article was characterized by high-performance liquid chromatography (content, identity, % aggregate), anion exchange high-performance liquid chromatography (rFXIII-related charge heterogeneity), potency (total activity, % activated rFXIII), and for endotoxin content. The control article was of the same formulation without rFXIII. The rFXIII protein was extensively characterized by amino acid analysis, N-terminal sequencing, peptide mapping by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry-mass spectrometry, whole mass measurement by electrospray ionization-MS (ESI-MS), size exclusion chromatography-multiangle light scattering (SEC-MALS), and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Amino acid analysis and peptide mapping demonstrated that the protein had an amino acid composition predicted from the human wild-type cDNA sequence. An average protein mass of 166 kD measured by SEC-MALS is consistent with the molecular weight of a FXIII [A2] dimeric protein. N-terminal sequencing showed no evidence of proteolytically activated FXIII, internal cleavages, or contaminating sequences. There was no evidence of N- or C-terminal heterogeneity, or unexpected posttranslational modifications.
In Vivo Study Design The safety of rFXIII administered as a single dose or repeated dose intravenous slow bolus injection (=2 mL/min) was evaluated in cynomolgus monkeys as indicated in Table 1.
Animal Dosing Within studies, animals were randomized to dosing with the goal of balancing mean body weights across dose groups. Control article dosing volumes were matched against the largest test article volumes used in any given study. Animals were dosed by slow bolus intravenous injection into a saphenous or cephalic vein. Animals were routinely observed at least twice daily for clinical observations beginning at least 7 days prior to test article administration and continuing through the end of study. Continuous clinical monitoring was conducted through 6 hr after test article administration.
Cardiovascular Safety Pharmacology
Pathology
Clinical Pathology Comprehensive serum chemistry analysis was performed on a Beckman Synchrotron CX7 automated chemistry analyzer (Beckman Instruments, Palo Alto, CA) using approximately 0.5 mL serum isolated from whole blood collected from a femoral or saphenous vein via butterfly catheter into a syringe. Endpoints measured included sodium, calcium, potassium, phosphorus, chloride, urea nitrogen (BUN), carbon dioxide, creatinine, total bilirubin, total protein, alkaline phosphatase (AP), albumin, lactate dehydrogenase (LDH), globulin, aspartate aminotransferase (AST), albumin/globulin ratio, alanine aminotransferase (ALT), glucose, gamma-glutamyltransferase (GGT), cholesterol, C-reactive protein (CRP), and triglycerides. For single-dose studies, serum chemistry analyses were conducted on all available animals once prior to dosing and 24 hours, 72 hours, and 168 hours postdose. For repeated dose studies, periodic serum chemistry analyses were obtained on all animals so as to establish baseline levels prior to dosing, and to evaluate responses during the dosing period (at least weekly) and chronic effects or recovery from induced changes during an extended (up to 4-week) dose-free period. Coagulation analyses of activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen were conducted on an AMAX CS-190 Coagulation Analyzer (Sigma Diagnostics, St. Louis, MO). Plasma was isolated from whole blood generally collected from a femoral or saphenous vein via butterfly catheter into a syringe containing sodium citrate as the anticoagulant. For single-dose studies, coagulation analyses were conducted on all available animals 1–2 times prior to dosing, and 2–4 hours, 24 hours, 72 hours, and 168 hours postdose, with additional collections at 30 minutes, 8 hours, and 312 hours for some animals. For repeated-dose studies, periodic coagulation analyses were obtained on all animals to establish baseline levels prior to dosing, and to evaluate responses during the dosing period (at least weekly) and chronic effects or recovery from induced changes during an extended (up to 4-week) dose-free period. When possible, blood was collected for clinical pathology analyses from moribund animals. Additional clinical pathology analyses were also collected at the request of the clinical veterinarian.
Pharmacokinetics Validated enzyme linked immunosorbent assays (ELISA) were developed to monitor each of the possible FXIII molecular species in plasma including FXIII-B subunit, endogenous A2B2, newly formed rA2cnB2, and uncomplexed rA2. These assays included the Total A2 assay for the combined detection of all molecular forms containing the FXIII-A sub-unit (cnA2cnB2, rA2cnB2, and rA2), the Free B subunit assay for detection of uncomplexed cynomolgus FXIII-B subunit (cnB) and the A2B2 Tetramer assay for specific detection of heterotetrameric FXIII species (i.e., endogenous FXIII (cnA2cnB2) and the formed heterotetramer (rA2cnB2)). The capture-detection configuration, performance characteristics, and molecular species detected by each ELISA assay are presented in Table 2. Plates were read on a Spectramax 190 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA) or equivalent.
Protein standards established for these assays by ZGI included rFXIII, and highly purified and well-characterized human FXIII A2B2 protein and human FXIII-B subunit protein. The human FXIII-B protein standard was derived from partially purified pFXIII (Enzyme Research Laboratories, South Bend, IN) treated with thrombin to cleave the FXIII-A subunit, and then Ca++ to separate the subunits. The mixture was centrifuged to pellet the cleaved FXIII-A, and the supernatant was purified by anion exchange chromatography. Purified human pFXIII A2B2 protein was obtained from partially purified pFXIII (Enzyme Research Laboratories) by size-exclusion high performance liquid chromatography (SE-HPLC) to isolate the heterotetramer from other protein species. Protein standards were extensively analyzed by N-terminal sequencing, reduced and nonreduced SDS-PAGE, SE-HPLC, and amino acid analysis to confirm identity, purity and concentration. Enzymatic activity and ability to form the expected heterotetrameric complex (in the case of the purified A2- and B-subunits) were also demonstrated. While in principle, the amount of FXIII measured by the Total A2 ELISA should quantify all FXIII regardless of whether it is in the dimeric A2 or heterotetrameric A2B2 state, the Total A2 assay underdetects the heterotetrameric form of FXIII. Thus mass balance between the assays is not possible. Despite this caveat, the analytical methods do allow for an estimation of the pharmacokinetic parameters for the heterotetrameric species alone and allow for the monitoring of uncomplexed B species of FXIII. This information is important in establishing the relationship between dosed rFXIII, formed heterotetramer (rFXIII complexed with cnB subunit), and the possible induction of cnB. The data obtained from the Total A2 and FXIII A2B2 tetramer ELISA data were subjected to noncompartmental toxicokinetic analysis using WinNonlin (Pharsight Inc., Cary, NC). In all cases, the postdose plasma concentrations were corrected for individual baseline FXIII plasma levels prior to toxicokinetic analysis.
Anti-FXIII Antibody Analyses The anti-FXIII antibody assay used rFXIII A2 on the solid phase to capture any anti-FXIII Ig present in the test sample. Affinity purified rabbit-anti-rFXIII antibodies spiked into cynomolgus serum was used as the quality control sample. Plates were washed to remove unbound protein, and protein G-HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), which recognizes cynomolgus monkey Ig, was used to detect immunoglobulin captured from the test or rabbit-anti-rFXIII antibodies in quality control samples. The plates were washed and TMB (BioFX Laboratories, Owens Mills, MD) was added as a detection reagent. Plates were read on a Spectramax 190 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). The polyclonal rabbit anti-rFXIII antibody was produced by ZGI by immunizing rabbits with rFXIII. The assay, validated for use with cynomolgus monkey sera or plasma, has an interassay precision of <10%, an intra-assay precision of <5%, is robust to multiple analysts. Sample stability was demonstrated through multiple freeze-thaw cycles and through storage at 4°C for up to four days. Assay sensitivity was established by spiking 5 µg/mL of affinity-purified rabbit anti-rFXIII antibody into naïve cynomolgus sera or plasma, which quantified at a titer of 2.3 (or a dilution between 1:100 and 1:500). By comparison, analysis of plasma and sera from rFXIII-treated animals yielded detectable titers between 2.1 and 2.6 in plasma, suggesting a sufficient assay sensitivity.
Evaluation of Fibrinogen Cross-Linking
Identification of Cross-Linked Proteins
Pharmacokinetics Following a Single Dose of rFXIII Using the available bioanalytical assays, plasma concentration versus time profiles were generated for the free FXIII-B subunit, the Total A2 (including both the free rA2 and the complexed rA2) and the FXIII A2B2 tetramer (Figure 2). As a rule, pharmacokinetic analyses of control animals demonstrated no change in FXIII molecular species.
Free FXIII-B Subunit Following slow bolus intravenous injection of saturating quantities of rFXIII A2 (e.g., 5 mg/kg), the free B subunit bound to the rFXIII A2 and no free B subunit was detected in the plasma until the 24 or 72 hours postdose sampling time. The time to recovery of free B subunit was directly related to administered dose, taking longer in animals that received higher rFXIII doses. For example, in animals dosed with 5 mg/kg rFXIII, average free B subunit concentrations of approximately 1 µg/mL at 72 hours postdose (and later) were comparable to those observed at baseline (0 hours), but no free B subunit was detected at 30 minutes, 2 hours, or 8 hours postdose, and reduced concentrations were observed at 24 hours postdose (Figure 2). The return of free B subunit to baseline concentrations by 72 hours in these animals and a Cmax, A2B2 observed between 24 and 72 hours determined by the FXIII A2B2 tetramer assay suggests that by 72 hours all remaining rFXIII was in the form of the rA2 dimer complexed with cynomolgus B subunit, and suggests induction of B subunit production following rFXIII dose administration. Using population-based PK modeling, the turnover for free B subunit in cynomolgus monkeys was estimated to be 10.5 ug kg–1 hr–1 (Dodds et al., 2004a).
Kinetics of rFXIII A2 Complexed with Cynomolgus FXIII-B Subunit
The profile resulting from analysis with the FXIII A2B2 ELISA demonstrated a time to maximum concentration (Tmax) of approximately 24–72 hours followed by monoexponential decay for all tested doses. Terminal half-lives were estimated to be 125–225 hours. This is consistent with the range of terminal half-lives estimated for the Total A2 ELISA results and thus confirms that the terminal half-life represents the elimination of the rA2cnB2 in both the total A2 and the FXIII A2B2 assays.
Pharmacokinetics of rFXIII Following Repeated Doses
Formation of Anti-rFXIII Antibodies
Single-Dose Toxicity Studies Moribund animals had a consistent decrease in platelet count, with critically low platelet counts (<50,000/µL) observed in some animals. Serum chemistry changes in animals with a declining health condition were consistent with a histologically confirmed coagulopathy. Serum chemistry alterations indicative of stress and organ/tissue damage included increased blood urea nitrogen (>30 mg/dL), creatinine (>1.1 mg/dL), lactate dehydrogenase (LDH, > 882 U/L), aspartate aminotransferase (AST, >94 U/L), alanine aminotransferase (ALT, >127 U/L) levels, and C-reactive protein (CRP >3 mg/dL). Prolongations in apparent prothrombin time (APTT, >42.3 seconds) or prothrombin time (PT, >14.3 seconds) were observed only as late-stage events occurring immediately prior to humane sacrifice in some animals. Cardiovascular safety pharmacology data consisting of blood pressure, body temperature and heart rate were collected during the toxicology studies of rFXIII. Cardiovascular safety pharmacology was evaluated because of the pharmacological activity of rFXIII as the terminal enzyme in the clotting cascade involved in stabilizing fibrin clots. No general trends in measured blood pressure, body temperature, or heart rate were observed related to rFXIII dose level or time after dose. However, among acutely moribund animals it was often difficult to collect blood samples immediately prior to animal euthanasia, suggesting low blood pressure during the more advanced stages of toxicity. Gross pathology observations generally consisted of red discoloration or red foci indicative of focal hemorrhage in a variety of tissues including adrenal glands, lung, kidneys, heart, liver, and the gastrointestinal tract. Most of the histologic findings (e.g., intravascular congestion, embolisation, and subsequent coagulative necrosis) correlated with the expected sequelae of overexposure to FXIII. Microscopic evidence of a generalized embolisation and/or ischemia was found with varying degrees of severity in many organs and tissues including adrenal glands, kidneys, eye, lung, heart, gastrointestinal tract, pancreas, spleen, liver, brain, neurohypophysis (pituitary), and bone marrow. The primary target tissues most severely and consistently affected by this occlusive coagulopathy were adrenal gland, kidney, eye, lung, and heart. Acellular granular emboli were observed within small blood vessels throughout the adrenal glands and kidneys, most prominently in the glomerular capillaries of the kidney (Figure 3a). These emboli were frequently associated with multifocal to coalescing hemorrhage (Figure 3b) and coagulative necrosis. In the eye, vessel occlusion within capillaries and small vessels of the retina and choroid were mixtures of classic fibrin thrombi and emboli formed of acellular granular material (Figure 3c) and in the heart, the acellular granular emboli found within the capillaries of the myocardium were frequently associated with adjacent areas of hemorrhage and coagulative necrosis (Figure 3d). Distribution in all tissues varied from multifocal, coalescing to diffuse.
Repeated Dose Studies Four experiments evaluated the effect of repeated slow bolus intravenous rFXIII injection (Table 1). No clinical or anatomic pathologies were observed in animals that received twice-weekly slow bolus injections of 5.0 mg/kg rFXIII for 4 weeks or daily intravenous slow bolus injections of up to 6.0 mg/kg rFXIII for 14 days. Similarly, intravenous administration of three doses of control (0.0 mg/kg) or rFXIII (5.0, 8.0, or 12.5 mg/kg) each separated by 14 days was well tolerated in adult male and female cynomolgus monkeys, and no effects were seen in clinical observations, clinical pathology evaluations, or gross evaluations of tissues and organs. Minor histological changes observed in the glomeruli in 1 of 6 animals dosed with 12.5 mg/kg and sacrificed two days after the last dose (but not 4 weeks later) suggested possible formation of transient emboli. This glomerulopathy was characterized by loss of defined glomerular capillaries due to increased eosinophilic material and cellularity in the glomerular tuft, and in some glomeruli, periglomerular fibrosis and cellular infiltrates. In contrast to these results, 2 intravenous injections of 12.5–17.5 mg/kg rFXIII separated by 72 hours resulted in toxicity within 1 day after the second dose despite the absence of any indication of effect after the first dose. The clinical observations in the moribund animals after repeated rFXIII dose administration was similar to that observed after a single dose, and consisted of a generalized occlusive coagulopathy diagnosed through clinical and anatomic pathology examinations.
Development of Cross-Linked Protein Complexes
The accumulated evidence of both kinetic and dynamic activity of rFXIII in cynomolgus monkeys consistent with the enzymatic properties of a transglutaminase justify the use of cynomolgus monkeys as a suitable species for nonclinical studies of rFXIII. The in vitro evidence that rFXIII correctly cross-links cynomolgus monkey fibrin to form gamma-dimers following thrombin-stimulated clotting, and the additional evidence of observed circulating cross-linked fibrin (and other higher-order aggregates) after intravenous injection of high doses of rFXIII to cynomolgus monkeys demonstrates pharmacodynamic activity of rFXIII in cynomolgus monkeys. The in vivo pharmacology of FXIII has been investigated in a limited number of animal models. These include animal models of FXIII deficiency (Lauer and Dickneite, 2001; Lee et al., 2001), in vivo biochemistry models (Shainoff and Dardik, 1990; Uchino et al., 1991), and models of exogenous FXIII administration in specific disease states (e.g., Isogai et al., 1990; Hirahara et al., 1993; D’Argenio et al., 2000). The available studies demonstrate a conserved biologic activity of FXIII across a number of mammalian species including mice, rats, rabbits, guinea pigs, nonhuman primates, and humans. The in vitro and in vivo evidence that rFXIII A2 binds to cynomolgus FXIII-B subunit resulting in a molecule of the correct molecular weight and circulating half-life shows comparability between the rFXIII kinetics in cynomolgus monkeys and endogenous FXIII in humans. Recombinant FXIII when complexed with endogenous B subunit has a terminal half-life of 5–9 days in cynomolgus monkeys, which is consistent with the terminal half-life estimated in congenitally deficient patients receiving the plasma purified FXIII product Fibrogammin (~6–12 days). Uncomplexed rFXIII (circulating in excess of free B subunit) has a short circulating half-life of 4–7 hours (data not shown). This observation is consistent with the theory that the FXIII-B subunit serves to stabilize the heterotetramer. However, in all cases where rFXIII is dosed in excess of available free B subunit, our estimates for Vss, totalA2 are larger than the typical blood volume for a 3–4 kg cynomolgus monkey (approx. 270 mL). There appears to be a well-controlled feedback with regards to FXIII-A2 and Free B subunit levels. It is possible that the rapid clearance and large volume of distribution of total A2 may be associated with the activation of excess rA2 and its binding to other cellular components and/or compartmentalization out of circulating plasma. Importantly, completed clinical trials in both healthy volunteers and congenitally deficient patients show that when therapeutically relevant doses of rA2 were administered (not in excess of available of free B subunit), the volume of distribution was similar to total blood volume (Lovejoy et al., 2004; Reynolds et al., 2004). Population-based models have been developed to describe the pharmacokinetics of rFXIII in both healthy volunteers and congenitally deficient patients (Dodds et al., 2004b).
The steep dose-response associated with rFXIII toxicity is consistent with its activity as a central mediator between coagulation and fibrinolysis, and can be understood in the context of the homeostatic controls that regulate the balance between bleeding and thrombosis. The likely sequence of events leading to death of animals undergoing acute rFXIII toxicity was an initial formation of acellular granular emboli possibly consisting of aggregated polymers of fibrin, fibrinogen, Hematological changes consistent with disseminated intravascular coagulation (DIC) were detected in samples taken from moribund animals shortly before death. These changes included decreased platelet counts, prolongations in PT and APTT, and increases in D-dimer and fibrin degradation products. Therefore, DIC was most likely a terminal event. The histological picture and proposed sequence of events are consistent with the mechanism of action of rFXIII and the collected clinical pathology data. Thus, the widespread embolisation resulting in death of animals at high doses represents exaggerated pharmacology of the test article. The observed occlusive coagulopathy following rFXIII toxicity is distinct from that of DIC. Classical DIC ultimately involves loss of control over the production of systemic thrombin and systemic plasmin via imbalances in the clotting and fibrinolytic systems (Bick et al., 1999). In contrast, the rFXIII-mediated toxicity is attributed to the formation of cross-linked protein complexes (including fibrinogen polymers) in plasma by activated rFXIII that ultimately occludes small vessels and results in a secondary systemic coagulation cascade activation. Evidence in support of this hypothesis includes the lack of an observed procoagulant activity of rFXIII added to plasma in vitro and evidence of high molecular weight complexes in the absence of histopathologically observed emboli/vessel occlusion. Thus, although both rFXIII-and thrombin-mediated plasma coagulation can ultimately cause comparable clinical and morphological pathology in vivo, the pathogenesis of coagulopathy is distinct. Moreover, diagnostic efforts to identify thrombin activation or consumption of enzymes involved in clotting system activation would not be expected to detect early rFXIII-mediated alterations in the disease process because the rFXIII-mediated coagulopathy does not appear to involve traditional mechanisms of clotting system activation.
One aspect of the observed rFXIII toxicity is the persistence of circulating cross-linked protein complexes. Siebenlist and Mosesson (1996) have observed low levels of cross-linked fibrinogen and of fibrinogen-
Activated FXIII (FXIIIa), whether endogenous or recombinant, is unlikely to be present in the systemic circulation. Once activated, rFXIII binds strongly to fibrin (Procyk et al., 1993) and binds to a variety of cells in the vasculature, similar to endogenous FXIII. Two published studies report efforts to detect FXIIIa in plasma. Nelson and Lerner (1978) claimed to have detected circulating FXIIIa, but the technical details of their assay suggest that pFXIII was being activated during their assay, making their results difficult to interpret. Subsequently, Rodeghiero et al. (1981) reported only trace FXIIIa in both plasma from normal patients and plasma from patients with disseminated intravascular coagulation. In the absence of a direct detection assay for FXIIIa in plasma, detection of circulating cross-linked protein complexes involving fibrinogen, fibronectin, or Given the potency of activated FXIII, the toxicity of rFXIII could be mediated by a small fraction of the administered drug that became activated. The primary physiological control over FXIII activity appears to be through the regulation of thrombin generation rather than through a specific biological inhibitor. While EDTA can be used to inhibit FXIII activity ex vivo, there is no evidence of an endogenous physiological inhibitor of FXIIIa, and FXIIIa remains active even when bound within a clot (Muszbek et al., 1999; Robinson et al., 2000). Likewise, FXIIIa remains active when bound to thrombin-stimulated platelets (Greenberg and Shuman, 1984). The normal decay in FXIII activity probably results from oxidation of the active site cysteine (Robinson et al., 2000). The pathway that results in the activation of rFXIII at high doses is open to speculation. While thrombin is understood to be the typical regulator of FXIII activity during coagulation system activation, a nonproteolytic activation can occur in the presence of elevated level of intracellular calcium (Muszbek et al., 1999) and salt concentrations (Polgar et al., 1990). FXIII-B plays a protective role with respect to the nonproteolytic activation of the cFXIII, and cellular FXIII A2 is more readily activated through the nonproteolytic pathway than pFXIII A2B2 (Polgar et al., 1990). Consequently, activity associated with non-proteolytic activation of rFXIII in vivo likely occurs when uncomplexed rFXIII is present in excess, such as the conditions provoked with supraphysiological rFXIII administration and depletion of free FXIII-B subunit. The results presented here represent novel finding regarding the pharmacokinetics and pharmacodynamics of rFXIII in cynomolgus monkeys. We demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support clinical dosing of 0.014 to 0.35 mg/kg (2–50 U/kg) in healthy volunteers and congenital FXIII-deficient patients.
The authors would like to gratefully acknowledge the efforts of Elizabeth Gribble for assistance in preparation of the manuscript, and David Adler and Margo Rogers for their assistance with preparation of the figures.
Bick, RL, Arun, B, & Frenkel, EP. (1999). Disseminated intravascular coagulation. Clinical and pathophysiological mechanisms and manifestations. Haemostasis, 29, 111-34[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Bishop, PD, Teller, DC, Smith, RA, Lasser, GW, Gilbert, T, & Seale, RL. (1990). Expression, purification, and characterization of human Factor XIII in Saccharomyces cerevisiae. Biochemistry, 29, 1861-9[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Blomback, B, Procyk, R, & Hessel, B. (1987). Alternative pathways in blood coagulation. Dev Biol Stand, 67, 157-62[Medline] [Order article via Infotrieve] Board, PG, Losowsky, MS, & Miloszewski, KJA. (1993). Factor XIII: inherited and acquired deficiency. Blood Rev, 7, 229-42[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Brummel, KE, Butenas, S, & Mann, KG. (1999). An integrated study of fibrinogen during blood coagulation. J Biol Chem, 274, 22862-70 D’Argenio, G, Grossman, A, Cosenza, V, Valle, ND, Mazzacca, G, & Bishop, PD. (2000). Recombinant Factor XIII improves established experimental colitis in rats. Dig Dis Sci, 45, 987-97[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Di Paola, J, Nugent, D, & Young, G. (2001). Current therapy for rare factor deficiencies. Haemophilia, 7, 16-22 Dodds, MG, Visich, JE, & Vicini, P. (2004a). Population pharmacokinetics of recombinant Factor XIII in cynomolgus monkeys. Submitted to the AAPS Journal. Dodds, MG, Visich, JE, & Vicini, P. (2004b). A population pharmacokinetics model for recombinant Factor XIII in healthy and congenitally deficient subjects. Submitted to Clin Pharm Therap. Egbring, R, Kroniger, A, & Seitz, R. (1996). Factor XIII deficiency: pathogenic mechanisms and clinical significance. Semin Thromb Hemost, 22, 419-25[Medline] [Order article via Infotrieve] Emrich, HG, Egbring, R, Seitz, R, Lerch, L, Fuchs, G, & Maasberg, M. (1993). Long-term interval Factor XIII substitution in congenital (homozygote) Factor XIII deficiency: Factor XIII increase and recovery studies in homozygous patients after substitution with Factor XIII concentrate. In McDonagh, J, Seitz, R, & Egbring, R (Eds.). Factor XIII - Second International Conference, July 9–10, 1991, Marburg, Schattauer-Verlag: New York Gootenberg, JE. (1998). Factor concentrates for the treatment of Factor XIII deficiency. Curr Opin Hematol, 5, 372-5[Medline] [Order article via Infotrieve] Greenberg, CS, & Shuman, MA. (1984). Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets. J Biol Chem, 259, 14721-7 Hirahara, K, Shinbo, K, Takahashi, M, & Matsuishi, T. (1993). Suppressive effect of human blood coagulation Factor XIII on the vascular permeability induced by anti-guinea pig endothelial cell antiserum in guinea pigs. Thromb Res, 71, 139-48[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Isogai, N, Ueda, Y, Kurozumi, N, & Kamiishi, H. (1990). Wound healing at the site of microvascular anastomosis: fibrin-stabilizing Factor XIII administration and its effects. Microsurgery, 11, 40-6[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Lauer, P, & Dickneite, G. (2001). Increased bleeding and abnormalities in structural characteristics of clot formation in transgenic coagulation Factor XIII deficient mice. Ann Hematol, 80, A40 Lee, SY, Chang, SK, Lee, IH, Kim, YM, & Chung, SI. (2001). Depletion of plasma Factor XIII prevents disseminated intravascular coagulation-induced organ damage. Thromb Haemost, 85, 464-9[Web of Science][Medline] [Order article via Infotrieve] Lewis, KB, Teller, DC, Fry, J, Lasser, GW, & Bishop, PD. (1997). Crosslinking kinetics of the human transglutaminase, Factor XIII[A2], acting on fibrin gels and gamma-chain peptides. Biochemistry, 36, 995-1002[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Lorand, L. (1972). Fibrinoligase: the fibrin-stabilizing factor system of blood plasma. Ann N Y Acad Sci, 202, 6-30[Web of Science][Medline] [Order article via Infotrieve] Lovejoy, A, Reynolds, T, Visich, J, Butine, M, Young, G, Belvedere, M, Blain, R, Pederson, S, Ishak, L, & Nugent, D. (2004). Safety and pharmacokinetics of recombinant Factor XIII administration in subjects with congenital Factor XIII deficiency. Submitted to Blood. McDonagh, J, & Fukue, H. (1996). Determinants of substrate specificity for Factor XIII. Semin Thromb Hemost, 22, 369-76[Medline] [Order article via Infotrieve] Miloszewski, KJA, & Losowsky, MS. (1991). Safety of long-term prophylaxis in inherited Factor XIII deficiency. In McDonagh, J, Seitz, R, & Egbring, R (Eds.). In Factor XIII-Second International Conference, July 9–10, Marburg, Schattauer-Verlag, 1993 Muszbek, L, Yee, VC, & Hevessy, Z. (1999). Blood coagulation Factor XIII: structure and function. Thromb Res, 94, 271-305[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Nelson, JC, & Lerner, RG. (1978). Detection of Factor XIIIa (active fibrin-stabilizing factor) in normal plasma. Blood, 52, 581-91 Polgar, J, Hidasi, V, & Muszbek, L. (1990). Non-proteolytic activation of cellular protransglutaminase (placenta macrophage Factor XIII). Biochem J, 267, 557-60[Web of Science][Medline] [Order article via Infotrieve] Procyk, R, Bishop, PD, & Kudryk, B. (1993). Fibrin-recombinant human Factor XIII a-subunit association. Thromb Res, 71, 127-38[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Radek, JT, Jeong, JM, Wilson, J, & Lorand, L. (1993). Association of the A subunits of recombinant placental Factor XIII with the native carrier B subunits from human plasma. Biochemistry, 32, 3527-34[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Reynolds, TC, Butine, MD, Visich, JE, Gunewardena, KA, MacMahon, M, Zuckerman, LA, Bishop, PD, & Morton, KM. (2005). Safety, pharmacokinetics, and immunogenicity of single dose rFXIII administration to healthy volunteers. Submitted to J Thromb Haemost. in press. in press. in press. Robinson, BR, Houng, AK, & Reed, GL. (2000). Catalytic life of activated Factor XIII in thrombi. Implications for fibrinolytic resistance and thrombus aging. Circulation, 102, 1151-7 Rodeghiero, F, Morbin, M, & Barbui, T. (1981). Failure to measure plasma activated Factor XIII during disseminated intravascular coagulation. Ric Clin Lab, 11, 333-6[Web of Science][Medline] [Order article via Infotrieve] Seaman, AJ, & Malinow, MR. (1968). Blood clotting in nonhuman primates. Lab Anim Care, 18, 80-4[Web of Science][Medline] [Order article via Infotrieve] Shaikh, AN, & Khurshid, M. (1993). Factor XIII deficiency in Pakistan. J Pak Med Assoc, 43, 67-9[Medline] [Order article via Infotrieve] Shainoff, JR, & Dardik, BN. (1990). Effects of cross-linking on clearance of circulating alpha-fibrin monomer and its complexes. J Lab Clin Med, 115, 314-23[Web of Science][Medline] [Order article via Infotrieve] Siebenlist, KR, Meh, DA, & Mosesson, MW. (2001). Protransglutaminase (Factor XIII) mediated crosslinking of fibrinogen and fibrin. Thromb Haemost, 86, 1221-8[Web of Science][Medline] [Order article via Infotrieve] Siebenlist, KR, & Mosesson, MW. (1996). Evidence for intramolecular cross-linked A alpha. gamma chain heterodimers in plasma fibrinogen. Biochemistry, 35, 5817-21[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Siebenlist, KR, Mosesson, MW, Meh, DA, DiOrio, JP, Albrecht, RM, & Olson, JD. (2000). Coexisting dysfibrinogenemia (gammaR275C) and factor V Leiden deficiency associated with thromboembolic disease (fibrinogen Cedar Rapids). Blood Coagul Fibrinolysis, 11, 293-304[Web of Science][Medline] [Order article via Infotrieve] Uchino, R, Cardinali, M, & Chung, SI. (1991). Regulation of extravascular fibrinolysis by Factor XIII. Fibrinolysis, 5, 93-8[CrossRef][Web of Science]
Toxicologic Pathology, Vol. 33, No. 4,
495-506 (2005) This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
Top
Abstract
Introduction
(
-glutamyl)lysyl cross-links between a glutamine residue of 1 protein and a lysine of another. Factor XIII circulates in plasma in a nonactive form (zymogen or proenzyme). 
-chains of fibrin, and other proteins such as 
