Pharmacokinetics and Pharmacology of Hirulog-like Peptide

Abstract

Hirulog-like peptide (HLP), a new thrombin peptide inhibitor, effectively reduces neointimal formation or restenosis in rat and rabbit vascular injury models. The present study investigated the pharmacokinetics and pharmacology of HLP in Sprague-Dawley (SD) rats. The input response of HLP in rats was studied using radioisotopic tracing method. Male SD rats were intravenously injected with a single dose of ¹²⁵I-HLP (3.2, 6.4, or 12.8 mg/kg) for pharmacokinetic analysis. The concentration-time curve of ¹²⁵I-HLP following bolus injection fitted a 3-compartment model. The half-life of HLP in rats was between 25 and 31 min following 3.2 to 12.8 mg/kg of bolus injection. Radioactivity of ¹²⁵I-HLP was detected in all tested tissues and was most abundant in kidneys or stomachs. Blood pressure, respiratory frequency, and heart rates were not significantly altered during continuous intravenous infusion with saline or 1.6 to 3.2 mg/kg/h of HLP for 4 h. Bleeding time and activated partial thromboplastin time were significantly prolonged in rats infused with HLP compared to vehicle. ADP-induced platelet aggregation was significantly reduced in the HLP-treated groups compared with controls. The results suggest that HLP possesses first-order kinetic characteristics. HLP is secreted mainly through kidneys. Beside its anticoagulant activity, no other adverse effect was detected in SD rats receiving HLP.

INTRODUCTION

Thrombin-specific inhibitors have been successfully applied in the prevention of heparin-induced thrombocytopenia and post-angioplasty thrombosis in clinical practice.¹ Commercially available thrombin-specific peptide inhibitors, recombinant hirudins, or hirulog-1 (bivaliurdin, Hirulog, or Angiomax) were reported to increase the frequency of major hemorrhagic complications in clinical studies.^2,3 A novel thrombin inhibitor, hirulog-like peptide (HLP), was designed to improve the safety profile of thrombin-specific inhibitors and to prevent restenosis induced by vascular procedures. HLP reduces balloon catheter-induced neointima formation in rat carotid arteries and the expression of platelet-derived growth factor, but it induces less elongation of bleeding time or activated partial thromboplastin time (aPTT) compared to hirulog-1 or heparin.⁴ HLP attenuates restenosis in carotid arteries in diet-induced atherosclerotic rabbits and the expression of tissue factor or transforming growth factor-β in neointima and results in less increase in bleeding tendency compared to heparin.⁵ The combination of the findings suggests that HLP potentially helps to prevent vascular procedure-induced restenosis. The clearance, distribution, secretion, or cardiovascular pharmacology of HLP has not been systematically examined. The present study characterized the pharmacokinetics and pharmacology of HLP in Sprague-Dawley (SD) rats.

METHODS

Labeling and Purification of ¹²⁵I-HLP

HLP (FPESKATNATLDPRPGGGGNGDFEEIPEEYLQ) was designed on the basis of the sequences of hirulog-1, hirudin, and the thrombin receptor.^6,7 Synthesized HLP was purified by high-performance liquid chromatography (HPLC).⁴ Na¹²⁵I was purchased from Gaotong Isotope Co Ltd, Chengtu, China. HLP was radiolabeled with ¹²⁵I-Na using iodogen method.⁸ Labeled HLP was separated using Sephadex G-15 (1 × 25 cm) column and drenched with 0.01 M phosphate-buffered saline (pH 7.4). The elution fractions were collected and free ¹²⁵I in the fraction was removed using ultrafiltration tubes with a molecular weight cutoff of 1 kD (Pall Life Sciences).

The purity of ¹²⁵I-HLP was assessed using HPLC method and SDS-PAGE. For HPLC analysis, a nonlinear gradient with 0% to 100% acetonitrile against 0.1% trifluoroacetic acid at a flow rate of 1 mL/min for 20 min was used to elute the peptide from a Hypersil C₁₈ column (300Å, 5 μm, 4.6 × 250 mm). The radioactivity was measured using γ-counter. The size of labeled HLP was verified on 15% SDS-PAGE gel and detected using Coomassie brilliant blue staining and radioactivity counting. The radiochemical purity stability of ¹²⁵I-HLP was measured at stable at 4°C (95%) and 37°C (93%) after 53 h.

Measurement of Plasma Concentrations

The quantification of radioactive HLP in plasma was achieved by using standard curves generated by plotting peak heights versus concentrations of the peptide. The curves were linear over the concentration ranges. Fifteen male SD rats (239 ± 13 g, mean ± SD) were randomly divided into 3 groups (n = 5/group). The animals were intravenously injected with single bolus of ¹²⁵I-HLP (3.2, 6.4, or 12.8 mg/kg). Blood was collected at 0, 2, 4, 6, 10, 15, 20, 30, 45, 60, 90, or 120 min after the injection. The method of solid phase extract (SPE) was used to separate ¹²⁵I-HLP from free ¹²⁵I in plasma.⁸ Fifty microliters of rat plasma were loaded on SPE column (Agilent Technologies) equilibrated with 1 mL methanol and 1 mL deionized water at the speed of 0.5 mL/min. The column was washed using 1 mL phosphate buffer (0.02 M, pH 5.0) to elute free ¹²⁵I. The radioactivity remaining in SPE columns were measured using γ-counter.

Tissue Distribution

Male SD rats (210 ± 20 g) were randomized to 3 groups (n = 5). Rats in all groups received a single dosage of ¹²⁵I-HLP (6.4 mg/kg) via intravenous route. The animals were euthanized at 15, 45, or 90 min after a saline perfusion (100 mL at 50 mm Hg). Tissues were collected and weighed for measuring radioactivity.

Excretion

Five male SD rats (221 ± 4 g) received an intravenous bolus injection of ¹²⁵I-HLP at 6.4 mg/kg. Urine and feces were collected during following time intervals after the injection: 0-1 h, 1-2 h, 2-4 h, 4-8 h, 8-12 h, 12-24 h, 24-36 h, and 36-48 h. The radioactivity of cumulative excretion of labeled HLP in urine and feces was counted. The radioactivity of thyroid was measured at 48 h after the injection. All the rats were housed individually in wire-bottom metabolic cages with free access to chow and water under controlled humidity (68%) and temperature (21 ± 2°C), with lights on from 6:00 AM to 6:00 PM.

Pharmacological Analyses

Thirty-two male SD rats were randomized into 4 groups (n = 8/group). HLP was dissolved in saline and intravenously infused at 1.6, 2.3, or 3.2 mg/kg/h via femoral vein using a peristaltic pump with a speed of 0.5 mL/h for 4 h. Control animals received saline at equal volume and speed via matching route. After anaesthetization with 20% urethan (1 g/ kg, IP), tail-cuff systolic blood pressure (SBP) was recorded using a Power Lab system at 30 min after the start of the infusion. Electrocardiogram (EKG) was recorded via needle electrodes connected to an AD Instruments Bioamp2 system at 45 min after the start of the infusion. Blood samples were collected at 10 min after the end of the infusion through a puncture of abdominal artery with 23-gauge needles rinsed with 38 g/L sodium citrate (9:1, v/v). Platelet-rich plasma (PRP) was prepared by low-speed centrifugation at 150 ×g for 15 min at 22°C as described.⁹ ADP-induced platelet aggregation in platelet-rich plasma was performed with 4-channel aggregometer (Chrono-log, Havertown, PA) according to the manufacturer's instructions. Platelets in PRP were counted, and the levels of platelets were justified (approximately 200,000 to 250,000/mm³) using autologous platelet-poor plasma. In a pretest, several concentrations of ADP (5, 10, 15, 20, and 40 μM in final concentrations) were applied to optimize the concentration of ADP for the test. A final concentration of 10 μM ADP was used in platelet aggregation described in the present study.

Plasma was prepared by a centrifugation at 3000 × g for 15 min at 4°C for the following tests. Prothrombin time (PT), aPTT, and fibrinogen (Fg) were mesured using an ACL 3000 plus system (Beckman Coulter Inc, Miami, FL) in Haematology Laboratory in Ruijin Hospital.

Bleeding time was measured by cutting the tail-tip as described.¹⁰ The tail was cut 2 mm from the end using a sharp pair of surgical scissors at 15 min after the start of the infusion of HLP or saline. The resultant wound was gently blotted with filter paper immediately after the cut and each 30 seconds thereafter until bleeding stopped. Periods between cutting and no blood blotted onto filter paper were defined as bleeding time.¹¹

Statistics

One-way ANOVA analysis was performed for comparisons among multiple groups. Student t test was used for the determination of probabilities between 2 groups. The level of significance was defined as P < 0.05.

RESULTS

Pharmacokinetics of ¹²⁵I-HLP

The plasma concentration-time profile of HLP following bolus intravenous injection of 3.2, 6.4, or 12.8 mg/kg ¹²⁵I-HLP was best fitted to a 3-compartment model [concentration(time) = Ae^−αt + Be^−βt + Ce^−γt] using a 3p87 pharmacokinetic program (Fig. 1). The mean values of distribution phase half-life (T1/2α) for HLP at tested dosages were 8.15 to 9.69 min, and the apparent elimination half-lives (T1/2β) were 24.74 to 30.49 min in SD rats. No significant difference was detected in exponents (α, β, γ) in plasma concentration (time) relation, T1/2α, T1/2β, T1/2γ, volume of central compartment [V(c)], intercompartmental distribution rate constants (K), clearance (CL), or mean residence time (MRT) among the 3 dosages. Area under concentration-time curve (AUC) in rats receiving 12.8 mg/kg bolus injection of HLP was significantly greater than that receiving 3.2 or 6.4 mg/kg (P < 0.05, Table 1).

TABLE 1: Pharmacokinetic Variables of ¹²⁵I-HLP in Rats

FIGURE 1: Concentration-time curve of ¹²⁵I-HLP in rats. Male SD rats received bolus intravenous injection of 3.2, 6.4, or 12.8 mg/kg of ¹²⁵I -HLP or saline (n = 5/group). Blood was collected at 0, 2, 4, 6, 10, 15, 20, 30, 45, 60, 90, or 120 min after the injection. Fifty microliters of duplicate plasma samples from each collection were loaded on solid phase extract (SPE) column. The radioactivity remaining in SPE columns were measured using γ-counter. Values were expressed in μg/mL with log-scale (average of duplicate measurements).

Excretion

The mean cumulative excretion rate of ¹²⁵I-HLP at 48 h after administration was 86.4 ± 2.5 %. The cumulative excretion of HLP in feces or thyroid (<5%) was substantially lower than that in urine (>70%)(Fig. 2). Therefore, the excretion of HLP in rats is predominantly through kidneys.

FIGURE 2: Cumulative excretion of ¹²⁵I -HLP in rats. Five male SD rats received an intravenous bolus injection of 6.4 mg/kg ¹²⁵I-HLP. Urine and feces were collected at time intervals of 0-1 h, 1-2 h, 2-4 h, 4-8 h, 8-12 h, 12-24 h, 24-36 h, and 36-48 h after the injection. The radioactivity of cumulative excretion of labeled HLP in urine and feces was counted. The radioactivity in thyroid was detected at 48 h after the injection. Values were expressed in % of cumulative excretion (mean ± SD, n = 5).

Tissue Distribution

Radioactivity of ¹²⁵I-HLP (6.4 mg/kg) in blood and tissues were examined at 5, 30, 60, and 90 min after bolus injection, and the data were expressed in percentage of dose per gram (%D/g) of tissue. The radioactivity of ¹²⁵I-HLP was detected in all examined tissues. At 15 min after the injection, kidneys contained most abundant of HLP. At 45 min after the injection, almost equal amounts of HLP were detected in kidneys and stomachs. At 90 min after the injection, HLP in stomachs was greater than that in other organs or tissues. Substantial amounts of HLP were also detected in small intestine, spleen, liver, lung, reproductive glands, adipose tissue, and muscle, but amounts were low in hearts or brains (Table 2).

TABLE 2: Tissue Distribution of Bolus Injection of ¹²⁵I-HLP (6.4 mg/kg) in Male SD Rats After Intravenous Administration. Values Were Expressed in % of Dosage/gram of Tissue (%D/g, Mean ± SD, N = 5).

Pharmacological Analyses

Bleeding time was significantly longer in rats received 1.6, 2.3, or 3.2 mg/kg/h of HLP compared to vehicle control. aPTT were significantly prolonged in rats treated with 2.3 mg/kg/h of HLP compared to saline control. ADP-induced platelet aggregation was significantly reduced in rats treated with all 3 dosages of HLP compared to controls. The levels of PT and Fg in rats receiving HLP were not significantly different from controls(Table 3). No significant difference in SBP, heart rates, respiration frequency, or EKG waves was detected between rats receiving HLP and with controls (data not shown).

TABLE 3: Effect of HLP on Platelet Aggregation and Coagulation Parameters in Rats.

DISCUSSION

The present study examined for the first time the pharmacokentics of HLP, a new thrombin peptide inhibitor.¹²⁵I labeled isotopic tracer method offers a sensitive and efficient method for the pharmacokinetic analysis of peptides. HLP is a small peptide with molecular weight of 3200, which is too small to separate labeled peptide from free isotope or unlabelled peptide using routine trichloride acetic acid precipitation.¹² SPE is a common separation method, but it is rarely used in the separation of radioisotope for isotopic tracing.¹³ SPE has been successfully applied in our study for separating labeled HLP from radioiodide or unlabelled small molecular polypeptides.⁸ The minimal detection level of labeled HLP is 52.8 ng/mL with a recovery >92%,⁸ which provides a sensitive and reliable tool for the pharmacokinetic study of HLP.

The elimination half-lives of ¹²⁵I-HLP after 3.2 to 12.8 mg/kg of bolus injection were 24.7 to 30.5 min, which were shorter than low molecular weight heparin (112 min), hirudin (72 min), or hirulog-1 (36 min).^14-16 HLP preserves the “PRP” structure as hirulg-1, which allows it to become a reversible thrombin inhibitor. HLP does not contain an artificial amino acid, “D-F”, at the N-terminal of peptide as hirulog-1,⁶ which may contribute to the relatively shorter half-life of HLP. The structural and pharmacokinetic properties of HLP may partially explain its better safety profile in terms of bleeding.^4,5 HLP possesses a first-order kinetic characteristic as that of hirudin. The clearance of HLP is mainly through urinary system similar to hirudin¹⁵ but not hirulog-1, which is through a nonrenal metabolism.¹⁷ Previous studies have demonstrated that the clearance of hirudin was greatly prolonged in end-stage renal failure patients.¹⁸ The results of the present study suggest that the clearance of HLP is potentially impaired in patients with renal dysfunction, although it remains to be determined in subsequent clinical studies. It is rather unexpected that stomach contains high levels of HLP. The isoelectric point of HLP is around 4. The high level of HLP in stomach may partially result from acidic environment in the organ.

There is no significant difference in tail SBP, respiratory frequencies, or heart rates in rats during continuous intravenous infusion with saline or 1.6 to 3.2 mg/kg/h of HLP for 4 h. Bleeding time and aPTT were significantly prolonged in HLP-treated rats compared to saline-infused controls. ADP-induced platelet aggregation was significantly shortened in the HLP-treated groups compared to controls. Our previous studies indicate that HLP infusion at 1.6 mg/kg/h for 4 h prevents or reduces vascular injury or induced neointimal formation or restenosis in rats or atherosclerotic rabbits with significantly less prolonged aPTT or bleeding time compared to heparin or hirulog-1.^4,5 Since most vascular procedures are elective, safety becomes an important criteria for medication used for preventing restenosis. The favorable safety profile of HLP may have a positive impact on this drug in terms of being considered as a potential alternative for preventing vascular procedure-induced restenosis.

CONCLUSION

The results of the present study suggest that HLP possesses a shorter half-life compared to other available thrombin inhibitors. Its main metabolic route is through kidney. Beside moderate anticoagulant activity, no other side effects of HLP at effective dosages have been detected in rats in the present study. The findings imply that HLP is a relatively safe thrombin inhibitor. The administration of HLP is required through intravenous route. Receivers with kidney dysfunction need to be cautious for HLP treatment. It should be pointed out that the results of the present study are generated from animal experiments. Pharmacokinetic and pharmacological properties of HLP are required to be determined in humans before the medication to be applied in clinical practice.