- Email: [email protected]
Reversal of heparin-induced increases in aPTT in the rat by PM102, a novel heparin antagonist
European Journal of Pharmacology 635 (2010) 165–170
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Cardiovacular Pharmacology
Reversal of heparin-induced increases in aPTT in the rat by PM102, a novel heparin antagonist Daniel J. Cushing a,⁎, Warren D. Cooper a, Marlene L. Cohen b, Julie R.S. McVoy c, Michael Sobel d, Robert B. Harris c a
Prism Pharmaceuticals, Inc., King of Prussia, PA, United States Creative Pharmacology Solutions, Carmel, IN, United States Commonwealth Biotechnologies, Inc., Richmond, VA, United States d University of Washington, Seattle, WA, United States b c
a r t i c l e
i n f o
Article history: Received 12 August 2009 Received in revised form 18 February 2010 Accepted 4 March 2010 Available online 20 March 2010 Keywords: Heparin Protamine Anticoagulation PM102
a b s t r a c t Protamine is the only agent approved to reverse heparin-induced anticoagulation. Due to the significant adverse effects of protamine there is an important need for an alternative agent with an improved safety profile. The pharmacodynamics of PM102, a novel peptide-based heparin antagonist, was evaluated and compared to protamine in a rat model. Rats were dosed with intravenous heparin (50 U/kg) and 4 min later with protamine (0.25, 0.75 mg/kg single intravenous bolus) or PM102 (0.1, 0.3, 1, 3, 30 mg/kg single intravenous bolus). Blood samples were collected though 60 min for assessment of activated partial thromboplastin time (aPTT) and plasma concentration of PM102. Both doses of protamine markedly lowered the elevated aPTT to baseline values within 1 to 5 min after administration. PM102 (0.3–30 mg/kg) also rapidly and completely reversed heparin-induced increases in aPTT within 1 to 5 min. The effects of PM102 administered as an infusion over 10 min also reversed aPTT with similar potency to that observed for bolus administration. The onset of reversal with infusion was delayed relative to the same total dose given as a bolus; however, the maximum effect was similar. PM102 rapidly (Tmax 1–2.6 min) appeared in plasma after dosing. Concentrations of PM102 generally declined rapidly after reaching Tmax with a mean T1/2 of 4 to 31 min. PM102 is a novel synthetic peptide that effectively reverses the anticoagulant effect of heparin. It's utility as a bolus injection as well as infusion, its rapid efficacy and its rapid clearance make this an ideal candidate for clinical development. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Unfractionated, intravenous heparin is commonly used as an anticoagulant in cardiovascular surgery and other vascular interventional procedures. In cardiopulmonary bypass surgery high-dose heparin is used and the intense degree of anticoagulation must be rapidly reversed at the conclusion of cardiopulmonary bypass to avoid excess bleeding. Protamine is the only agent currently approved to reverse heparin anticoagulation (A.P.P. Pharmaceuticals, 2008). Although an effective reversal agent, protamine is associated with adverse effects that range from minor hemodynamic instability to serious fatal cardiovascular collapse, which has been reported to occur in 2.6% of cardiac surgery patients (Weiss et al., 1989; Weiler et al., 1990; Kimmel et al., 1998; Kimmel et al., 2002; Panos et al., 2003). In fact, protamine-induced hemodynamic effects are associated with increased morbidity and mortality in patients undergoing cardiopul⁎ Corresponding author. 1150 First Avenue; Suite 1050, King of Prussia, PA 19406, United States. Tel.: + 1 610 986 1024. E-mail address: [email protected] (D.J. Cushing). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.03.016
monary bypass surgery (Welsby et al., 2005). Thus, improvement in the pharmacological management of heparin-induced anticoagulation is highly desired. Several alternative approaches to heparin reversal have been reported and include the use of Platelet Factor IV (Levy et al., 1995), heparinase I (Stafford-Smith et al., 2005), methylene blue, hexadimethrine, vancomycin (Kikura et al., 1996), protamine-like peptides (Wakefield et al., 1996), and concatameric heparin-binding peptides (Schick et al., 2004). Each of these approaches has limitations and none has been approved for clinical use. In anticipation of minimizing the adverse hemodynamic effects of protamine, Shenoy et al. (1999) reported a rationale synthetic design approach leading to the preparation of a series of low molecular weight helix peptides, which, by design, were promising as heparin-reversal agents. Of those agents, the Tris-Arg Helix #3 peptide (designated in this study as PM102, also termed HepArrest®; developed at Commonwealth Biotechnologies Inc. and licensed by Prism Pharmaceuticals Inc), was selected for further nonclinical and clinical development. PM102 is peptide of molecular weight 5829 in which three identical amino acid helix segments are joined by an organic tether (Shenoy et al., 1999). The tether provides the
166
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
necessary physical constraints for creating a heparin binding channel from two of the helix segments while the third helix segment forms contact with heparin once it is bound within the channel, thus securing the binding of heparin (Harris and Sobel, 1999, Harris and Sobel, 2001). The binding constant of PM102 for heparin is approximately 10 nM. The purpose of the current study was to assess the dose–response of intravenous PM102 on heparin-induced anticoagulation in an in vivo model that resembles a typical cardiovascular application of heparin. This study also compared the efficacy of PM102 to that of protamine, and examined the pharmacokinetics of PM102 after intravenous administration in the rat. 2. Materials and methods 2.1. Ethics All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication, 1996). The protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Covance Laboratories (Madison, WI), where the studies were performed. 2.2. Animals Male Sprague Dawley rats (Harlan Sprague Dawley) between the ages of 13 and 17 weeks (weight range 374–409 g) were surgically prepared with jugular and femoral vein cannulas by the supplier prior to arrival in the experimental facility (Covance Laboratories, Madison, WI). Rats were acclimated for 3 to 5 days prior to experimentation. During acclimation and experimentation, animals were individually housed in suspended, stainless steel wire-mesh cages. Certified Rodent Diet #8728C (Harlan Teklad, Inc.) and water were provided ad libitum and animals were not fasted during the course of the study. 2.3. Surgical procedures The jugular and femoral veins were cannulated and locked with a heparinized glycerol solution and patency was confirmed approximately 2 to 4 h prior to heparin administration by flushing with 2 ml 0.9% Sodium Chloride for Injection, USP (normal sterile saline, NSS) after removal of approximately 0.3 ml of locking solution (cannula void volume = approximately 0.2 ml). Cannula patency was continually maintained during the course of the study by flushing with NSS. At the end of the experiment, animals were sacrificed via overdose of isoflurane anesthesia. 2.4. Study drugs Unfractionated pharmaceutical heparin (Heparin Sodium for Injection, Baxter Laboratories) and protamine (sulfate salt, Sigma) were supplied as stock solutions (10,000 USP U/ml) and stored at room temperature. Subsequent to conducting the study, a number of heparin lots were reported by Kishimoto et al. (2008) to be contaminated with oversulfated chondroitin sulphate; the lot number of heparin used to conduct this study was not affected. PM102 was supplied as a dry powder and stored protected from light in desiccant at approximately −20 °C. For heparin administration, aliquots of the stock solution were diluted daily using NSS to the appropriate dosing concentration(s) and were gently inverted to mix and obtain clear solutions for administration. For protamine administration, the powder was weighed and diluted daily in NSS to achieve the appropriate dosing concentration(s) and the vials were gently inverted to mix and obtain clear solutions for administration.
PM102 was manufactured cGMP conditions by American Peptide Co, Inc. (Vista, CA) and stored desiccated at room temperature. For PM102 administration, a stock solution in sterile water for injection (SWFI) was prepared daily by weighing the dry powder into a volumetric flask. An appropriate volume of SWFI was then added, and the flask was gently inverted to mix and obtain a clear solution. Individual doses of PM102 were prepared by diluting appropriate aliquots of the stock solution in NSS to achieve the target dose concentrations. Dose formulations were gently inverted to mix and obtain clear solutions for administration. NSS was used as the control vehicle for heparin, protamine and PM102. Individual doses were calculated based on rat body weights on the day of dosing. Intravenous doses of compounds were administered as bolus injections (i.e. injection over less than one second) via the femoral venous catheter followed by flushing of the catheter with approximately 1 ml of NSS. For the infusion experiments, PM102 vehicle and PM102 were administered to animals as a 10 min infusion via the femoral vein catheter using an infusion pump (Medfusion Model 2001; Medex, Inc.). To complete dose administration, the cannula was flushed with approximately 1 ml of NSS. 2.5. Experimental design Fig. 1 provides a schematic representation of the timeline of events for dose administration and blood draws to measure activated partial thromboplastin time (aPTT) as the pharmacodynamic measurement of coagulation in all animals, or to measure plasma PM102 concentrations in some animals for pharmacokinetic assessment. Heparin or heparin vehicle pre-treatment occurred at Time 0 = 0 min, and protamine, PM102, or applicable vehicle was dosed intravenously at 4 min. 2.6. Blood sampling and measurement of activated partial thromboplastin time (aPTT) as the pharmacodynamic estimate of anticoagulant activity The aPTT was chosen as a widely accepted clinically relevant measure the anticoagulant effect of intravenous heparin. For measurement of aPTT, blood (approximately 0.35 ml) was collected via the jugular vein catheter from each animal approximately 5 min prior to heparin or heparin vehicle administration and at 1, 5, 10, 15, 30, and 60 min following heparin or heparin vehicle administration and was transferred into tubes containing sodium citrate anticoagulant. In some animals (animals not receiving drugs), blood (approximately 0.35 ml) was collected via direct jugular venipuncture prior to heparin administration and transferred into tubes containing sodium citrate anticoagulant. Blood samples collected for aPTT assessment were processed for plasma and analyzed for aPTT, using an AMAX Destiny Coagulation Analyzer (Trinity Biotech). Freshly prepared plasma was used for aPTT analyses. 2.7. Blood sampling and measurement of PM102 plasma concentration For pharmacokinetic measurement, blood (approximately 0.25 ml) was collected from rats receiving PM102 predose (approximately 5 min prior to heparin administration) and at 1, 5, 10, 15, 30, and 60 min following the administration of PM102 either as a bolus or infusion. Blood samples for pharmacokinetics were maintained at 32 °C prior to centrifugation to obtain plasma, within 30 min of collection. For each sample, an aliquot (99 µl) of plasma was harvested and placed into an individual 1.4 ml Matrix screw top tube (Matrix Part No. 3741) which contained approximately 1 µL of 50× protease inhibitor cocktail solution (Sigma-Aldrich Catalog No. P2714). The tube was vortexed briefly to ensure mixing of the plasma and inhibitor. A 50 µL aliquot of the mixture was then removed and placed into a second Matrix screw top tube, and both samples were
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
167
Fig. 1. Schematic representation of the study protocol. PD = pharmacodynamic measurement of the activated partial thromboplastin time (aPTT), and PK = the measurement of plasma PM102 concentrations in some animals for pharmacokinetic assessment. Heparin or heparin vehicle pre-treatment occurred at Time 0 = 0 min, and protamine, PM102, or applicable vehicle was dosed intravenously at 4 min.
flash frozen in liquid nitrogen. Subsequent to flash freezing, the tubes were placed on dry ice prior to storage at approximately −70 °C. To establish lack of exposure to PM102, and to serve as true negative controls in the assay, a single (approximately 0.25 ml) blood sample was collected from one animal in the PM102 vehicle dose group at 60 min after PM102 vehicle-only administration. Blood was collected via the jugular vein catheter and transferred into tubes containing potassium (K2) EDTA anticoagulant. Individual plasma samples were analyzed for concentrations of PM102 with an enzyme-linked immunosorbent assay (ELISA). This antigen capture competition ELISA is based upon the ability of PM102 in the solution phase to block the capture of anti-PM102 antibodies by PM102 which has been passively immobilized in the wells of a 96 well microtiter plate in a concentration-dependent manner. The assay was conducted per Standard Operating Procedure and was validated by Commonwealth Biotechnologies, Inc (CBI) for use in detecting and quantitating PM102 in rat serum and plasma. Anti-PM102 antibodies were raised in hens and purified per standard protocols. Noncompartmental analysis (NCA) was applied to individual PM102 plasma concentration-time data (Gibaldi and Perrier, 1982). The following parameters were estimated whenever possible: Cmax Tmax Tlast
AUC0 − t
AUC0 − ∞
maximum concentration in plasma. time to maximum concentration. time of last measurable plasma concentration. Cmax, Tmax, and Tlast were determined by visual inspection of the concentration–time data. area under the plasma concentration–time curve from hour 0 up to the last measurable sampling time, estimated by linear trapezoidal rule. area under the plasma concentration–time curve from hour 0 to infinity, calculated as follows:
AUC0−t + Ct = λz where Ct is the last measurable concentration in plasma and λz is the elimination rate constant estimated using log-linear regression during the terminal elimination phase. The number of points used in λz
calculation was determined by visual inspection of the data describing the terminal phase. At least the last three time points with measurable values were used in λz calculation. T1/2
terminal elimination half-life, determined by ln(2)/λz.
CL
systemic clearance, determined by dose/AUC0 - ∞.
Vz
volume of distribution, determined by CL/λz.
Vss
volume of distribution at steady-state, determined by CL × MRT; where MRT is mean residence time and calculated using standard procedures.
Pharmacokinetic parameters were calculated by using WinNonlin Professional Edition (Pharsight Corporation, Version 5.2). NCA Model 201 was used to analyze the applicable PM102 plasma concentration profiles after intravenous bolus administration, and NCA Model 202 was used for a constant intravenous infusion. The calculated doses administered (mg PM102/kg) were used in the calculations. The 10 min infusion time was used in the NCA Model 202 calculations. Calculations conducted for actual doses administered and descriptive statistics (mean, standard deviation, standard error of the mean) were performed using Excel Version 11.0.
3. Results 3.1. Establishment of the optimal heparin dose for subsequent experiments Initially, it was necessary to establish an intravenous bolus dose of heparin that would induce a sufficient increase in aPTT so that inhibition could be quantitatively documented after administration of protamine or PM102. For this purpose, an aPTT of 60 to 120 s was targeted to define the appropriate bolus dose for heparin to use in all subsequent experiments. Single intravenous bolus injections of heparin (35, 50, and 70 U/kg) safely induced marked increases in aPTT, generally within approximately 1 min of administration (Fig. 2). Heparin at 50 U/kg resulted in an aPTT increase of approximately 85 s with low variability. Thus, this dose was selected for all future
168
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
3.3. Comparison of PM102 to protamine reversal of heparin anticoagulation Having established a viable ex vivo assay for inhibition of heparininduced increases in aPTT, the effects of multiple doses of intravenously administered PM102 were examined and compared to protamine (0.25 mg/kg). As observed earlier, protamine (0.25 mg/ kg) reversed heparin-induced increases in aPTT (Fig. 4). The 0.1 mg/ kg dose of PM102 did not inhibit heparin-induced increases in aPTT (Fig. 4). However, higher doses of PM102 (0.3, 1, 3, and 30 mg/kg) produced dose-dependent reversal of heparin-induced increases in aPTT (Fig. 4). Qualitatively, the time course of the effect of PM102 was similar to that of protamine, with PM102 lowering aPTT to control levels within 5 min after administration. PM102 (1 mg/kg) produced a similar magnitude reduction in aPTT as protamine (0.25 mg/kg). There was no evidence of a rebound or intrinsic anticoagulation with PM102. Fig. 2. Dose–response for heparin-induced anticoagulation (mean ± SEM.; n = 4/ group). The dose of 50 U/kg heparin was chosen for subsequent experiments.
experiments to measure the inhibitory effect of protamine and PM102 on heparin-induced increases in aPTT.
3.2. Effect of protamine on reversal of heparin anticoagulation To establish the viability of the assay and to provide a comparative estimate of inhibitory effectiveness, we first examined the ability of intravenously administered protamine to inhibit heparin-induced increases in aPTT. Protamine (0.25 and 0.75 mg/kg) or vehicle (NSS) was administered via bolus injection to rats (n = 5 rats/group) previously dosed with heparin (50 U/kg). The heparin-induced increase in aPTT was similar to that observed previously (Fig. 3). As anticipated, both doses of protamine reversed the prolongation of the aPTT to control values within approximately 1 to 5 min of administration (Fig. 3). Thus, this ex vivo assay using plasma samples from heparinized animals treated with protamine and measuring clotting at various times after administration was highly effective in detecting the inhibitory effect of protamine on heparin-induced increases in clotting time.
Fig. 3. Effect of protamine (mean ± SEM.; n = 5/group) on heparin-induced (50 U/kg) anticoagulation.
3.4. Effect of PM102 infusion on reversal of heparin anticoagulation Because of its toxicity and frequent adverse effects, in clinical usage protamine is often administered as a short intravenous infusion rather than a bolus. Thus, the effects of a 10 min infusion of PM102 at 0.1, 0.3, and 1 mg/kg on aPTT were investigated (Fig. 5). Similar to bolus administration, the 0.1 mg/kg infusion of PM102 did not reduce heparin-induced increases in aPTT. In contrast to the bolus administration, the 0.3 mg/kg infused dose also did not appear to produce a clear effect. However, PM102 (1 mg/kg) infused over approximately 10 min produced complete reversal of heparin-induced increases in aPTT by the end of the infusion (Fig. 5). Of note, bolus administration of the same total dose of PM102 (1 mg/kg) also produced complete reversal, but more rapidly (Fig. 5). 3.5. Pharmacokinetic evaluation of intravenous PM102 A summary of the mean pharmacokinetic parameters obtained for PM102 following intravenous bolus or infusion is shown in Table 1. Graphical representations of the plasma concentrations are shown in Figs. 6 and Fig. 7. Following single 0.1, 0.3, 1, 3, and 30 mg/kg PM102 bolus administration the mean plasma Tmax for PM102 was 1 to 2.6 min (Table 1). Concentrations of PM102 generally declined in a rapid fashion after reaching Tmax with a mean T1/2 of 4.30 to
Fig. 4. Comparison of intravenous bolus PM102 (mean ± SEM; n = 5/group) to protamine reversal of heparin-induced (50 U/kg) anticoagulation.
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
169
Fig. 5. Effect of PM102 (mean ± S.E.M.; n = 5/group) administered as an intravenous infusion over 10 min on heparin-induced (50 U/kg) anticoagulation.
Fig. 6. Plasma concentration profile (mean ± SEM; n = 5/group) of PM102 administered as an intravenous bolus in animals treated with heparin (50 U/kg).
31.5 min (Table 1). Mean Cmax and AUC0 − t values for PM102 increased proportionally with the increase in dose for bolus administration from 0.1 to 0.3 mg/kg, but then generally increased in a less than proportional fashion for the higher doses.
rapid and inexpensive approach to estimate and compare the in vivo activity of PM102 to protamine. Heparin produced rapid and dosedependent increases in aPTT which were rapidly reversed after administration of protamine, confirming the applicability of this animal model to evaluate the effects of PM102. In this study protamine was effective at a dose of 0.25 mg/kg and produced complete reversal at 0.75 mg/kg. These data are consistent with that previously reported by Racanelli and Fareed (1992) for protamine doses of 0.32–0.65 mg/kg. These data are also consistent with data obtained in the guinea pig where 0.35 mg/kg protamine produced complete reversal of heparininduced increases in aPTT (Shenoy et al., 1999). PM102, administered as an intravenous bolus, produced dosedependent reversal of heparin-induced anticoagulation. Doses of 1 mg/kg, 3 mg/kg and, 30 mg/kg produced complete reversal of heparin-induced increases in aPTT. The onset of aPTT reversal was evident at 1 min after dosing and was near maximal between 1 to 5 min after dosing. There was no apparent rebound during the 60 min observation period, even with the highest dose used. The dose of PM102 that produced complete reversal in the rat was less than that previously reported for the guinea pig (7.5 mg/kg) and Yorkshire pig (10 mg/kg) (Shenoy et al., 1999). The greater sensitivity of the rat model may be due to differences in heparin dose, differential binding of PM102 to plasma proteins, or differences in hemostasis in the rat. Also, weight-based dosages may not be directly translatable from
4. Discussion As a potential alternative to protamine for deactivation of heparin, the current study describes the ability of PM102, a novel synthetic peptide, to reverse heparin-induced anticoagulation in the rat. Heparin reversal by PM102 was rapid, complete, and dose-dependent. The rapid, safe, and effective reversal of heparin-induced anticoagulation in the setting of cardiovascular surgery is necessary to avoid excessive bleeding. Protamine which possesses cardiovascular safety limitations (e.g. severe hypotension) is currently the only available agent for this purpose. While several alternative approaches have been contemplated none has yet been proven to be safe and effective for human use (Levy et al., 1995; Stafford-Smith et al., 2005; Kikura et al., 1996; Wakefield et al., 1996; Schick et al., 2004). The purpose of this study was to assess the pharmacodynamic effect of PM102, a novel synthetic peptide rationally designed to bind and inactivate heparin. To evaluate and compare the effectiveness of PM 102 to protamine, a rat model for heparin-induced anticoagulation was developed where ex vivo changes in the aPTT as a function of dose and time were assessed. The rat was chosen since it represents a preferred and commonly used species for nonclinical toxicology and provided a
Table 1 Mean selected pharmacokinetic parameters for PM102 in plasma following single intravenous doses to rats pretreated with heparin. Tmax AUC0 − t t1/2 Vz CL Vss Dose Cmax (mg/kg) (µg/ml) (min) (min·µg/ml) (min) (ml/kg) (ml/min/kg) (ml/kg) PM102 bolus injection 0.1 11.4 1.00 0.3 37.9 1.82 1 36.0 1.00 3 60.0 1.00 30 42.9 2.60
76.9 217 243 589 530
4.30 4.50 8.57 17.1 31.5
7.44 8.61 53.8 119 2327
1.20 1.36 4.28 4.93 49.0
6.90 6.57 35.7 70.4 1689
PM102 infusion over 10 min 0.1 3.7 11.40 41.3 0.3 6.2 11.00 73.5 1 10.8 11.20 142
4.67 6.08 9.52
14.7 31.3 98.5
2.13 3.86 7.26
21.3 39.6 98.3
Note: each animal in each group (n = 5/group) was pretreated approximately 4 min prior to the start of PM102 administration with a single 50 Units/kg bolus injection of Heparin.
Fig. 7. Plasma concentration profile (mean ± SEM.; n = 5/group) of PM102 administered as an intravenous infusion over 10 min in animals treated with heparin (50 U/kg).
170
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
small to large animals. An intravenous infusion of PM102 was also studied, to compare it with another common clinical strategy used for protamine, and to determine if an infusion might reduce the total amount of drug required for reversal. Infusion of PM102 over 10 min reversed heparin anticoagulation with similar potency to that observed for bolus administration. The onset of action was delayed relative to the same total dose given as a bolus; however, the maximum effect was similar. Therefore, there is no apparent advantage of an infusion compared with bolus. The appearance of PM102 in plasma was rapid following bolus and infusion dosing with peak appearance within 1 to 3 min after bolus dosing and 11 min after the 10 min infusion dose. Plasma concentrations decreased rapidly after dosing with a T1/2 of 4 to 30 min. AUC and Cmax generally increased with increasing dose. Increases in AUC above approximately 75 min-µg/ml appeared to align with the inhibition of aPTT, although no specific PK/PD modeling was performed. The current study has some limitations. While this study was conducted in the rat it should be noted that while the rat model is widely used in early nonclinical development it is not a commonly used animal for hemostatic evaluation. Therefore the observed differences in coagulation between the rat and other species should be carefully considered. Future studies in the primate are planned to fully define the effective and initial doses to be used for human studies. The current study used aPTT to assess anticoagulation. It should be noted that the measurement of anticoagulant effect during cardiac bypass is also performed using activated clotting time (ACT) which was not measured in this study. A safe, rapid and effective reversal agent for heparin has long been sought. These current data, combined with previous in vitro and animal studies, suggest that PM102 may be a promising new drug for the reversal of heparin anticoagulation. Its rapid action and clearance make this an ideal candidate for clinical development. Acknowledgements The authors wish to thank Dr. Philip Teitelbaum, Dr. Marina SemeNelson, Mr. Todd Oppeneer, and Ms. Kelly Hitz of Covance Laboratories and Ms. Jamie Eftekhari of Commonwealth Biotechnologies Inc. for their technical assistance in conducting the experiments.
References A.P.P. Pharmaceuticals, 2008. Protamine Sulfate Injection USP. Package Insert, Schaumburg, IL. Gibaldi, M., Perrier, D., 1982. Pharmacokinetics, Second Ed. New York, Marcel Dekker. Harris, R.B., Sobel, M., 1999. Heparin-binding peptides. US Patent No. 5,877,153. Harris, R.B., Sobel, M., 2001. Heparin-binding peptides. US Patent No. 6,200,955. Kikura, M., Lee, M.K., Levy, J.H., 1996. Heparin neutralization with methylene blue, hexadimethrine, or vancomycin after cardiopulmonary bypass. Anesth. Analg. 83, 223–227. Kimmel, S.E., Sekeres, M.A., Berlin, J.A., Goldberg, L.R., Strom, B.L., 1998. Adverse events after protamine administration in patients undergoing cardiopulmonary bypass: risks and predictors of under-reporting. J. Clin. Epidemiol. 51, 1–10. Kimmel, S.E., Sekeres, M., Berlin, J.A., Ellison, N., 2002. Mortality and adverse events after protamine administration in patients undergoing cardiopulmonary bypass. Anesth. Analg. 94, 1402–1408. Kishimoto, T.K., Viswanathan, K., Ganguly, T., Elankumaran, S., Smith, S., Pelzer, K., Lansing, J.C., Sriranganathan, N., Zhao, G., Galcheva-Gargova, Z., Al-Hakim, A., Bailey, G.S., Fraser, B., Roy, S., Rogers-Cotrone, T., Buhse, L., Whary, M., Fox, J., Nasr, M., Dal Pan, G.J., Shriver, Z., Langer, R.S., Venkataraman, G., Austen, K.F., Woodcock, J., Sasisekharan, R., 2008. Contaminated heparin associated with adverse clinical events and activation of the contact system. N. Engl. J. Med. 358, 2457–2467. Levy, J.H., Cormack, J.G., Morales, A., 1995. Heparin neutralization by recombinant platelet factor 4 and protamine. Anesth. Analg. 81, 35–37. NIH Publication, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press. Panos, A., Orrit, X., Chevalley, C., Kalangos, A., 2003. Dramatic post-cardiotomy outcome, due to severe anaphylactic reaction to protamine. Eur. J. Cardiothorac. Surg. 24, 325–327. Racanelli, A., Fareed, J., 1992. Neutralization of the antithrombotic effects of heparin and Fraxiparin by protamine sulfate. Thromb. Res. 68, 211–222. Schick, B.P., Maslow, D., Moshinski, A., San Antonio, J.D., 2004. Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo. Blood 103, 1356–1363. Shenoy, S., Sobel, M., Harris, R.B., 1999. Development of heparin antagonists with focused biological activity. Curr. Pharm. Des. 5, 965–986. Stafford-Smith, M., Lefrak, E.A., Qazi, A.G., Welsby, I., Barber, L., Hoeft, A., Dorenbaum, A., Mathias, J., Rochon, J.J., Newman, M.F., 2005. Efficacy and safety of heparinase I versus protamine in patients undergoing coronary artery bypass grafting with and without cardiopulmonary bypass. Anesthesiology 103, 229–240. Wakefield, T.W., Stanley, J.C., Andrews, P.C., 1996. Peptides for heparin and low molecular weight heparin anticoagulation reversal. United States Patent No. 5,534,619. Weiler, J.M., Gellhaus, M.A., Carter, J.G., Meng, R.L., Benson, P.M., Hottel, R.A., Schillig, K.B., Vegh, A.B., Clarke, W.R., 1990. A prospective study of the risk of an immediate adverse reaction to protamine sulfate during cardiopulmonary bypass surgery. J. Clin. Immunol. 85, 713–719. Weiss, M.E., Nyhan, D., Peng, Z.K., Horrow, J.C., Lowenstein, E., Hirshman, C., Adkinson Jr., N.F., 1989. Association of protamine IgE and IgG antibodies with life-threatening reactions to intravenous protamine. N. Engl. J. Med. 320, 886–892. Welsby, I., Newman, M.F., Phillips-Bute, B., Messier, R.H., Kakkis, E.D., Stafford-Smith, M., 2005. Hemodynamic changes after protamine administration. Anesthesiology 103, 229–240.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Cardiovacular Pharmacology
Reversal of heparin-induced increases in aPTT in the rat by PM102, a novel heparin antagonist Daniel J. Cushing a,⁎, Warren D. Cooper a, Marlene L. Cohen b, Julie R.S. McVoy c, Michael Sobel d, Robert B. Harris c a
Prism Pharmaceuticals, Inc., King of Prussia, PA, United States Creative Pharmacology Solutions, Carmel, IN, United States Commonwealth Biotechnologies, Inc., Richmond, VA, United States d University of Washington, Seattle, WA, United States b c
a r t i c l e
i n f o
Article history: Received 12 August 2009 Received in revised form 18 February 2010 Accepted 4 March 2010 Available online 20 March 2010 Keywords: Heparin Protamine Anticoagulation PM102
a b s t r a c t Protamine is the only agent approved to reverse heparin-induced anticoagulation. Due to the significant adverse effects of protamine there is an important need for an alternative agent with an improved safety profile. The pharmacodynamics of PM102, a novel peptide-based heparin antagonist, was evaluated and compared to protamine in a rat model. Rats were dosed with intravenous heparin (50 U/kg) and 4 min later with protamine (0.25, 0.75 mg/kg single intravenous bolus) or PM102 (0.1, 0.3, 1, 3, 30 mg/kg single intravenous bolus). Blood samples were collected though 60 min for assessment of activated partial thromboplastin time (aPTT) and plasma concentration of PM102. Both doses of protamine markedly lowered the elevated aPTT to baseline values within 1 to 5 min after administration. PM102 (0.3–30 mg/kg) also rapidly and completely reversed heparin-induced increases in aPTT within 1 to 5 min. The effects of PM102 administered as an infusion over 10 min also reversed aPTT with similar potency to that observed for bolus administration. The onset of reversal with infusion was delayed relative to the same total dose given as a bolus; however, the maximum effect was similar. PM102 rapidly (Tmax 1–2.6 min) appeared in plasma after dosing. Concentrations of PM102 generally declined rapidly after reaching Tmax with a mean T1/2 of 4 to 31 min. PM102 is a novel synthetic peptide that effectively reverses the anticoagulant effect of heparin. It's utility as a bolus injection as well as infusion, its rapid efficacy and its rapid clearance make this an ideal candidate for clinical development. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Unfractionated, intravenous heparin is commonly used as an anticoagulant in cardiovascular surgery and other vascular interventional procedures. In cardiopulmonary bypass surgery high-dose heparin is used and the intense degree of anticoagulation must be rapidly reversed at the conclusion of cardiopulmonary bypass to avoid excess bleeding. Protamine is the only agent currently approved to reverse heparin anticoagulation (A.P.P. Pharmaceuticals, 2008). Although an effective reversal agent, protamine is associated with adverse effects that range from minor hemodynamic instability to serious fatal cardiovascular collapse, which has been reported to occur in 2.6% of cardiac surgery patients (Weiss et al., 1989; Weiler et al., 1990; Kimmel et al., 1998; Kimmel et al., 2002; Panos et al., 2003). In fact, protamine-induced hemodynamic effects are associated with increased morbidity and mortality in patients undergoing cardiopul⁎ Corresponding author. 1150 First Avenue; Suite 1050, King of Prussia, PA 19406, United States. Tel.: + 1 610 986 1024. E-mail address: [email protected] (D.J. Cushing). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.03.016
monary bypass surgery (Welsby et al., 2005). Thus, improvement in the pharmacological management of heparin-induced anticoagulation is highly desired. Several alternative approaches to heparin reversal have been reported and include the use of Platelet Factor IV (Levy et al., 1995), heparinase I (Stafford-Smith et al., 2005), methylene blue, hexadimethrine, vancomycin (Kikura et al., 1996), protamine-like peptides (Wakefield et al., 1996), and concatameric heparin-binding peptides (Schick et al., 2004). Each of these approaches has limitations and none has been approved for clinical use. In anticipation of minimizing the adverse hemodynamic effects of protamine, Shenoy et al. (1999) reported a rationale synthetic design approach leading to the preparation of a series of low molecular weight helix peptides, which, by design, were promising as heparin-reversal agents. Of those agents, the Tris-Arg Helix #3 peptide (designated in this study as PM102, also termed HepArrest®; developed at Commonwealth Biotechnologies Inc. and licensed by Prism Pharmaceuticals Inc), was selected for further nonclinical and clinical development. PM102 is peptide of molecular weight 5829 in which three identical amino acid helix segments are joined by an organic tether (Shenoy et al., 1999). The tether provides the
166
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
necessary physical constraints for creating a heparin binding channel from two of the helix segments while the third helix segment forms contact with heparin once it is bound within the channel, thus securing the binding of heparin (Harris and Sobel, 1999, Harris and Sobel, 2001). The binding constant of PM102 for heparin is approximately 10 nM. The purpose of the current study was to assess the dose–response of intravenous PM102 on heparin-induced anticoagulation in an in vivo model that resembles a typical cardiovascular application of heparin. This study also compared the efficacy of PM102 to that of protamine, and examined the pharmacokinetics of PM102 after intravenous administration in the rat. 2. Materials and methods 2.1. Ethics All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication, 1996). The protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Covance Laboratories (Madison, WI), where the studies were performed. 2.2. Animals Male Sprague Dawley rats (Harlan Sprague Dawley) between the ages of 13 and 17 weeks (weight range 374–409 g) were surgically prepared with jugular and femoral vein cannulas by the supplier prior to arrival in the experimental facility (Covance Laboratories, Madison, WI). Rats were acclimated for 3 to 5 days prior to experimentation. During acclimation and experimentation, animals were individually housed in suspended, stainless steel wire-mesh cages. Certified Rodent Diet #8728C (Harlan Teklad, Inc.) and water were provided ad libitum and animals were not fasted during the course of the study. 2.3. Surgical procedures The jugular and femoral veins were cannulated and locked with a heparinized glycerol solution and patency was confirmed approximately 2 to 4 h prior to heparin administration by flushing with 2 ml 0.9% Sodium Chloride for Injection, USP (normal sterile saline, NSS) after removal of approximately 0.3 ml of locking solution (cannula void volume = approximately 0.2 ml). Cannula patency was continually maintained during the course of the study by flushing with NSS. At the end of the experiment, animals were sacrificed via overdose of isoflurane anesthesia. 2.4. Study drugs Unfractionated pharmaceutical heparin (Heparin Sodium for Injection, Baxter Laboratories) and protamine (sulfate salt, Sigma) were supplied as stock solutions (10,000 USP U/ml) and stored at room temperature. Subsequent to conducting the study, a number of heparin lots were reported by Kishimoto et al. (2008) to be contaminated with oversulfated chondroitin sulphate; the lot number of heparin used to conduct this study was not affected. PM102 was supplied as a dry powder and stored protected from light in desiccant at approximately −20 °C. For heparin administration, aliquots of the stock solution were diluted daily using NSS to the appropriate dosing concentration(s) and were gently inverted to mix and obtain clear solutions for administration. For protamine administration, the powder was weighed and diluted daily in NSS to achieve the appropriate dosing concentration(s) and the vials were gently inverted to mix and obtain clear solutions for administration.
PM102 was manufactured cGMP conditions by American Peptide Co, Inc. (Vista, CA) and stored desiccated at room temperature. For PM102 administration, a stock solution in sterile water for injection (SWFI) was prepared daily by weighing the dry powder into a volumetric flask. An appropriate volume of SWFI was then added, and the flask was gently inverted to mix and obtain a clear solution. Individual doses of PM102 were prepared by diluting appropriate aliquots of the stock solution in NSS to achieve the target dose concentrations. Dose formulations were gently inverted to mix and obtain clear solutions for administration. NSS was used as the control vehicle for heparin, protamine and PM102. Individual doses were calculated based on rat body weights on the day of dosing. Intravenous doses of compounds were administered as bolus injections (i.e. injection over less than one second) via the femoral venous catheter followed by flushing of the catheter with approximately 1 ml of NSS. For the infusion experiments, PM102 vehicle and PM102 were administered to animals as a 10 min infusion via the femoral vein catheter using an infusion pump (Medfusion Model 2001; Medex, Inc.). To complete dose administration, the cannula was flushed with approximately 1 ml of NSS. 2.5. Experimental design Fig. 1 provides a schematic representation of the timeline of events for dose administration and blood draws to measure activated partial thromboplastin time (aPTT) as the pharmacodynamic measurement of coagulation in all animals, or to measure plasma PM102 concentrations in some animals for pharmacokinetic assessment. Heparin or heparin vehicle pre-treatment occurred at Time 0 = 0 min, and protamine, PM102, or applicable vehicle was dosed intravenously at 4 min. 2.6. Blood sampling and measurement of activated partial thromboplastin time (aPTT) as the pharmacodynamic estimate of anticoagulant activity The aPTT was chosen as a widely accepted clinically relevant measure the anticoagulant effect of intravenous heparin. For measurement of aPTT, blood (approximately 0.35 ml) was collected via the jugular vein catheter from each animal approximately 5 min prior to heparin or heparin vehicle administration and at 1, 5, 10, 15, 30, and 60 min following heparin or heparin vehicle administration and was transferred into tubes containing sodium citrate anticoagulant. In some animals (animals not receiving drugs), blood (approximately 0.35 ml) was collected via direct jugular venipuncture prior to heparin administration and transferred into tubes containing sodium citrate anticoagulant. Blood samples collected for aPTT assessment were processed for plasma and analyzed for aPTT, using an AMAX Destiny Coagulation Analyzer (Trinity Biotech). Freshly prepared plasma was used for aPTT analyses. 2.7. Blood sampling and measurement of PM102 plasma concentration For pharmacokinetic measurement, blood (approximately 0.25 ml) was collected from rats receiving PM102 predose (approximately 5 min prior to heparin administration) and at 1, 5, 10, 15, 30, and 60 min following the administration of PM102 either as a bolus or infusion. Blood samples for pharmacokinetics were maintained at 32 °C prior to centrifugation to obtain plasma, within 30 min of collection. For each sample, an aliquot (99 µl) of plasma was harvested and placed into an individual 1.4 ml Matrix screw top tube (Matrix Part No. 3741) which contained approximately 1 µL of 50× protease inhibitor cocktail solution (Sigma-Aldrich Catalog No. P2714). The tube was vortexed briefly to ensure mixing of the plasma and inhibitor. A 50 µL aliquot of the mixture was then removed and placed into a second Matrix screw top tube, and both samples were
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
167
Fig. 1. Schematic representation of the study protocol. PD = pharmacodynamic measurement of the activated partial thromboplastin time (aPTT), and PK = the measurement of plasma PM102 concentrations in some animals for pharmacokinetic assessment. Heparin or heparin vehicle pre-treatment occurred at Time 0 = 0 min, and protamine, PM102, or applicable vehicle was dosed intravenously at 4 min.
flash frozen in liquid nitrogen. Subsequent to flash freezing, the tubes were placed on dry ice prior to storage at approximately −70 °C. To establish lack of exposure to PM102, and to serve as true negative controls in the assay, a single (approximately 0.25 ml) blood sample was collected from one animal in the PM102 vehicle dose group at 60 min after PM102 vehicle-only administration. Blood was collected via the jugular vein catheter and transferred into tubes containing potassium (K2) EDTA anticoagulant. Individual plasma samples were analyzed for concentrations of PM102 with an enzyme-linked immunosorbent assay (ELISA). This antigen capture competition ELISA is based upon the ability of PM102 in the solution phase to block the capture of anti-PM102 antibodies by PM102 which has been passively immobilized in the wells of a 96 well microtiter plate in a concentration-dependent manner. The assay was conducted per Standard Operating Procedure and was validated by Commonwealth Biotechnologies, Inc (CBI) for use in detecting and quantitating PM102 in rat serum and plasma. Anti-PM102 antibodies were raised in hens and purified per standard protocols. Noncompartmental analysis (NCA) was applied to individual PM102 plasma concentration-time data (Gibaldi and Perrier, 1982). The following parameters were estimated whenever possible: Cmax Tmax Tlast
AUC0 − t
AUC0 − ∞
maximum concentration in plasma. time to maximum concentration. time of last measurable plasma concentration. Cmax, Tmax, and Tlast were determined by visual inspection of the concentration–time data. area under the plasma concentration–time curve from hour 0 up to the last measurable sampling time, estimated by linear trapezoidal rule. area under the plasma concentration–time curve from hour 0 to infinity, calculated as follows:
AUC0−t + Ct = λz where Ct is the last measurable concentration in plasma and λz is the elimination rate constant estimated using log-linear regression during the terminal elimination phase. The number of points used in λz
calculation was determined by visual inspection of the data describing the terminal phase. At least the last three time points with measurable values were used in λz calculation. T1/2
terminal elimination half-life, determined by ln(2)/λz.
CL
systemic clearance, determined by dose/AUC0 - ∞.
Vz
volume of distribution, determined by CL/λz.
Vss
volume of distribution at steady-state, determined by CL × MRT; where MRT is mean residence time and calculated using standard procedures.
Pharmacokinetic parameters were calculated by using WinNonlin Professional Edition (Pharsight Corporation, Version 5.2). NCA Model 201 was used to analyze the applicable PM102 plasma concentration profiles after intravenous bolus administration, and NCA Model 202 was used for a constant intravenous infusion. The calculated doses administered (mg PM102/kg) were used in the calculations. The 10 min infusion time was used in the NCA Model 202 calculations. Calculations conducted for actual doses administered and descriptive statistics (mean, standard deviation, standard error of the mean) were performed using Excel Version 11.0.
3. Results 3.1. Establishment of the optimal heparin dose for subsequent experiments Initially, it was necessary to establish an intravenous bolus dose of heparin that would induce a sufficient increase in aPTT so that inhibition could be quantitatively documented after administration of protamine or PM102. For this purpose, an aPTT of 60 to 120 s was targeted to define the appropriate bolus dose for heparin to use in all subsequent experiments. Single intravenous bolus injections of heparin (35, 50, and 70 U/kg) safely induced marked increases in aPTT, generally within approximately 1 min of administration (Fig. 2). Heparin at 50 U/kg resulted in an aPTT increase of approximately 85 s with low variability. Thus, this dose was selected for all future
168
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
3.3. Comparison of PM102 to protamine reversal of heparin anticoagulation Having established a viable ex vivo assay for inhibition of heparininduced increases in aPTT, the effects of multiple doses of intravenously administered PM102 were examined and compared to protamine (0.25 mg/kg). As observed earlier, protamine (0.25 mg/ kg) reversed heparin-induced increases in aPTT (Fig. 4). The 0.1 mg/ kg dose of PM102 did not inhibit heparin-induced increases in aPTT (Fig. 4). However, higher doses of PM102 (0.3, 1, 3, and 30 mg/kg) produced dose-dependent reversal of heparin-induced increases in aPTT (Fig. 4). Qualitatively, the time course of the effect of PM102 was similar to that of protamine, with PM102 lowering aPTT to control levels within 5 min after administration. PM102 (1 mg/kg) produced a similar magnitude reduction in aPTT as protamine (0.25 mg/kg). There was no evidence of a rebound or intrinsic anticoagulation with PM102. Fig. 2. Dose–response for heparin-induced anticoagulation (mean ± SEM.; n = 4/ group). The dose of 50 U/kg heparin was chosen for subsequent experiments.
experiments to measure the inhibitory effect of protamine and PM102 on heparin-induced increases in aPTT.
3.2. Effect of protamine on reversal of heparin anticoagulation To establish the viability of the assay and to provide a comparative estimate of inhibitory effectiveness, we first examined the ability of intravenously administered protamine to inhibit heparin-induced increases in aPTT. Protamine (0.25 and 0.75 mg/kg) or vehicle (NSS) was administered via bolus injection to rats (n = 5 rats/group) previously dosed with heparin (50 U/kg). The heparin-induced increase in aPTT was similar to that observed previously (Fig. 3). As anticipated, both doses of protamine reversed the prolongation of the aPTT to control values within approximately 1 to 5 min of administration (Fig. 3). Thus, this ex vivo assay using plasma samples from heparinized animals treated with protamine and measuring clotting at various times after administration was highly effective in detecting the inhibitory effect of protamine on heparin-induced increases in clotting time.
Fig. 3. Effect of protamine (mean ± SEM.; n = 5/group) on heparin-induced (50 U/kg) anticoagulation.
3.4. Effect of PM102 infusion on reversal of heparin anticoagulation Because of its toxicity and frequent adverse effects, in clinical usage protamine is often administered as a short intravenous infusion rather than a bolus. Thus, the effects of a 10 min infusion of PM102 at 0.1, 0.3, and 1 mg/kg on aPTT were investigated (Fig. 5). Similar to bolus administration, the 0.1 mg/kg infusion of PM102 did not reduce heparin-induced increases in aPTT. In contrast to the bolus administration, the 0.3 mg/kg infused dose also did not appear to produce a clear effect. However, PM102 (1 mg/kg) infused over approximately 10 min produced complete reversal of heparin-induced increases in aPTT by the end of the infusion (Fig. 5). Of note, bolus administration of the same total dose of PM102 (1 mg/kg) also produced complete reversal, but more rapidly (Fig. 5). 3.5. Pharmacokinetic evaluation of intravenous PM102 A summary of the mean pharmacokinetic parameters obtained for PM102 following intravenous bolus or infusion is shown in Table 1. Graphical representations of the plasma concentrations are shown in Figs. 6 and Fig. 7. Following single 0.1, 0.3, 1, 3, and 30 mg/kg PM102 bolus administration the mean plasma Tmax for PM102 was 1 to 2.6 min (Table 1). Concentrations of PM102 generally declined in a rapid fashion after reaching Tmax with a mean T1/2 of 4.30 to
Fig. 4. Comparison of intravenous bolus PM102 (mean ± SEM; n = 5/group) to protamine reversal of heparin-induced (50 U/kg) anticoagulation.
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
169
Fig. 5. Effect of PM102 (mean ± S.E.M.; n = 5/group) administered as an intravenous infusion over 10 min on heparin-induced (50 U/kg) anticoagulation.
Fig. 6. Plasma concentration profile (mean ± SEM; n = 5/group) of PM102 administered as an intravenous bolus in animals treated with heparin (50 U/kg).
31.5 min (Table 1). Mean Cmax and AUC0 − t values for PM102 increased proportionally with the increase in dose for bolus administration from 0.1 to 0.3 mg/kg, but then generally increased in a less than proportional fashion for the higher doses.
rapid and inexpensive approach to estimate and compare the in vivo activity of PM102 to protamine. Heparin produced rapid and dosedependent increases in aPTT which were rapidly reversed after administration of protamine, confirming the applicability of this animal model to evaluate the effects of PM102. In this study protamine was effective at a dose of 0.25 mg/kg and produced complete reversal at 0.75 mg/kg. These data are consistent with that previously reported by Racanelli and Fareed (1992) for protamine doses of 0.32–0.65 mg/kg. These data are also consistent with data obtained in the guinea pig where 0.35 mg/kg protamine produced complete reversal of heparininduced increases in aPTT (Shenoy et al., 1999). PM102, administered as an intravenous bolus, produced dosedependent reversal of heparin-induced anticoagulation. Doses of 1 mg/kg, 3 mg/kg and, 30 mg/kg produced complete reversal of heparin-induced increases in aPTT. The onset of aPTT reversal was evident at 1 min after dosing and was near maximal between 1 to 5 min after dosing. There was no apparent rebound during the 60 min observation period, even with the highest dose used. The dose of PM102 that produced complete reversal in the rat was less than that previously reported for the guinea pig (7.5 mg/kg) and Yorkshire pig (10 mg/kg) (Shenoy et al., 1999). The greater sensitivity of the rat model may be due to differences in heparin dose, differential binding of PM102 to plasma proteins, or differences in hemostasis in the rat. Also, weight-based dosages may not be directly translatable from
4. Discussion As a potential alternative to protamine for deactivation of heparin, the current study describes the ability of PM102, a novel synthetic peptide, to reverse heparin-induced anticoagulation in the rat. Heparin reversal by PM102 was rapid, complete, and dose-dependent. The rapid, safe, and effective reversal of heparin-induced anticoagulation in the setting of cardiovascular surgery is necessary to avoid excessive bleeding. Protamine which possesses cardiovascular safety limitations (e.g. severe hypotension) is currently the only available agent for this purpose. While several alternative approaches have been contemplated none has yet been proven to be safe and effective for human use (Levy et al., 1995; Stafford-Smith et al., 2005; Kikura et al., 1996; Wakefield et al., 1996; Schick et al., 2004). The purpose of this study was to assess the pharmacodynamic effect of PM102, a novel synthetic peptide rationally designed to bind and inactivate heparin. To evaluate and compare the effectiveness of PM 102 to protamine, a rat model for heparin-induced anticoagulation was developed where ex vivo changes in the aPTT as a function of dose and time were assessed. The rat was chosen since it represents a preferred and commonly used species for nonclinical toxicology and provided a
Table 1 Mean selected pharmacokinetic parameters for PM102 in plasma following single intravenous doses to rats pretreated with heparin. Tmax AUC0 − t t1/2 Vz CL Vss Dose Cmax (mg/kg) (µg/ml) (min) (min·µg/ml) (min) (ml/kg) (ml/min/kg) (ml/kg) PM102 bolus injection 0.1 11.4 1.00 0.3 37.9 1.82 1 36.0 1.00 3 60.0 1.00 30 42.9 2.60
76.9 217 243 589 530
4.30 4.50 8.57 17.1 31.5
7.44 8.61 53.8 119 2327
1.20 1.36 4.28 4.93 49.0
6.90 6.57 35.7 70.4 1689
PM102 infusion over 10 min 0.1 3.7 11.40 41.3 0.3 6.2 11.00 73.5 1 10.8 11.20 142
4.67 6.08 9.52
14.7 31.3 98.5
2.13 3.86 7.26
21.3 39.6 98.3
Note: each animal in each group (n = 5/group) was pretreated approximately 4 min prior to the start of PM102 administration with a single 50 Units/kg bolus injection of Heparin.
Fig. 7. Plasma concentration profile (mean ± SEM.; n = 5/group) of PM102 administered as an intravenous infusion over 10 min in animals treated with heparin (50 U/kg).
170
D.J. Cushing et al. / European Journal of Pharmacology 635 (2010) 165–170
small to large animals. An intravenous infusion of PM102 was also studied, to compare it with another common clinical strategy used for protamine, and to determine if an infusion might reduce the total amount of drug required for reversal. Infusion of PM102 over 10 min reversed heparin anticoagulation with similar potency to that observed for bolus administration. The onset of action was delayed relative to the same total dose given as a bolus; however, the maximum effect was similar. Therefore, there is no apparent advantage of an infusion compared with bolus. The appearance of PM102 in plasma was rapid following bolus and infusion dosing with peak appearance within 1 to 3 min after bolus dosing and 11 min after the 10 min infusion dose. Plasma concentrations decreased rapidly after dosing with a T1/2 of 4 to 30 min. AUC and Cmax generally increased with increasing dose. Increases in AUC above approximately 75 min-µg/ml appeared to align with the inhibition of aPTT, although no specific PK/PD modeling was performed. The current study has some limitations. While this study was conducted in the rat it should be noted that while the rat model is widely used in early nonclinical development it is not a commonly used animal for hemostatic evaluation. Therefore the observed differences in coagulation between the rat and other species should be carefully considered. Future studies in the primate are planned to fully define the effective and initial doses to be used for human studies. The current study used aPTT to assess anticoagulation. It should be noted that the measurement of anticoagulant effect during cardiac bypass is also performed using activated clotting time (ACT) which was not measured in this study. A safe, rapid and effective reversal agent for heparin has long been sought. These current data, combined with previous in vitro and animal studies, suggest that PM102 may be a promising new drug for the reversal of heparin anticoagulation. Its rapid action and clearance make this an ideal candidate for clinical development. Acknowledgements The authors wish to thank Dr. Philip Teitelbaum, Dr. Marina SemeNelson, Mr. Todd Oppeneer, and Ms. Kelly Hitz of Covance Laboratories and Ms. Jamie Eftekhari of Commonwealth Biotechnologies Inc. for their technical assistance in conducting the experiments.
References A.P.P. Pharmaceuticals, 2008. Protamine Sulfate Injection USP. Package Insert, Schaumburg, IL. Gibaldi, M., Perrier, D., 1982. Pharmacokinetics, Second Ed. New York, Marcel Dekker. Harris, R.B., Sobel, M., 1999. Heparin-binding peptides. US Patent No. 5,877,153. Harris, R.B., Sobel, M., 2001. Heparin-binding peptides. US Patent No. 6,200,955. Kikura, M., Lee, M.K., Levy, J.H., 1996. Heparin neutralization with methylene blue, hexadimethrine, or vancomycin after cardiopulmonary bypass. Anesth. Analg. 83, 223–227. Kimmel, S.E., Sekeres, M.A., Berlin, J.A., Goldberg, L.R., Strom, B.L., 1998. Adverse events after protamine administration in patients undergoing cardiopulmonary bypass: risks and predictors of under-reporting. J. Clin. Epidemiol. 51, 1–10. Kimmel, S.E., Sekeres, M., Berlin, J.A., Ellison, N., 2002. Mortality and adverse events after protamine administration in patients undergoing cardiopulmonary bypass. Anesth. Analg. 94, 1402–1408. Kishimoto, T.K., Viswanathan, K., Ganguly, T., Elankumaran, S., Smith, S., Pelzer, K., Lansing, J.C., Sriranganathan, N., Zhao, G., Galcheva-Gargova, Z., Al-Hakim, A., Bailey, G.S., Fraser, B., Roy, S., Rogers-Cotrone, T., Buhse, L., Whary, M., Fox, J., Nasr, M., Dal Pan, G.J., Shriver, Z., Langer, R.S., Venkataraman, G., Austen, K.F., Woodcock, J., Sasisekharan, R., 2008. Contaminated heparin associated with adverse clinical events and activation of the contact system. N. Engl. J. Med. 358, 2457–2467. Levy, J.H., Cormack, J.G., Morales, A., 1995. Heparin neutralization by recombinant platelet factor 4 and protamine. Anesth. Analg. 81, 35–37. NIH Publication, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press. Panos, A., Orrit, X., Chevalley, C., Kalangos, A., 2003. Dramatic post-cardiotomy outcome, due to severe anaphylactic reaction to protamine. Eur. J. Cardiothorac. Surg. 24, 325–327. Racanelli, A., Fareed, J., 1992. Neutralization of the antithrombotic effects of heparin and Fraxiparin by protamine sulfate. Thromb. Res. 68, 211–222. Schick, B.P., Maslow, D., Moshinski, A., San Antonio, J.D., 2004. Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo. Blood 103, 1356–1363. Shenoy, S., Sobel, M., Harris, R.B., 1999. Development of heparin antagonists with focused biological activity. Curr. Pharm. Des. 5, 965–986. Stafford-Smith, M., Lefrak, E.A., Qazi, A.G., Welsby, I., Barber, L., Hoeft, A., Dorenbaum, A., Mathias, J., Rochon, J.J., Newman, M.F., 2005. Efficacy and safety of heparinase I versus protamine in patients undergoing coronary artery bypass grafting with and without cardiopulmonary bypass. Anesthesiology 103, 229–240. Wakefield, T.W., Stanley, J.C., Andrews, P.C., 1996. Peptides for heparin and low molecular weight heparin anticoagulation reversal. United States Patent No. 5,534,619. Weiler, J.M., Gellhaus, M.A., Carter, J.G., Meng, R.L., Benson, P.M., Hottel, R.A., Schillig, K.B., Vegh, A.B., Clarke, W.R., 1990. A prospective study of the risk of an immediate adverse reaction to protamine sulfate during cardiopulmonary bypass surgery. J. Clin. Immunol. 85, 713–719. Weiss, M.E., Nyhan, D., Peng, Z.K., Horrow, J.C., Lowenstein, E., Hirshman, C., Adkinson Jr., N.F., 1989. Association of protamine IgE and IgG antibodies with life-threatening reactions to intravenous protamine. N. Engl. J. Med. 320, 886–892. Welsby, I., Newman, M.F., Phillips-Bute, B., Messier, R.H., Kakkis, E.D., Stafford-Smith, M., 2005. Hemodynamic changes after protamine administration. Anesthesiology 103, 229–240.