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A Phase 3, Randomized, Double-Blind Comparative Study of the Efficacy and Safety of Topical Recombinant Human Thrombin and Bovine Thrombin in Surgical Hemostasis
A Phase 3, Randomized, Double-Blind Comparative Study of the Efficacy and Safety of Topical Recombinant Human Thrombin and Bovine Thrombin in Surgical Hemostasis William C Chapman, MD, FACS, Neil Singla, MD, Yuri Genyk, MD, FACS, James W McNeil, MD, FACS, Kenneth L Renkens Jr, MD, FACS, Thomas C Reynolds, MD, PhD, Aileen Murphy, MPH, Fred A Weaver, MD, FACS Plasma-derived bovine thrombin is used as a topical agent to improve surgical hemostasis, but development of antibodies to bovine hemostatic proteins has been associated with increased bleeding and thrombotic complications. Recombinant human thrombin could reduce the risk of these complications. STUDY DESIGN: The objective of this randomized, double-blind, comparative trial was to compare the efficacy, safety, and antigenicity of recombinant human thrombin (rhThrombin) and bovine thrombin as adjuncts to hemostasis in liver resection, spine, peripheral arterial bypass, and dialysis access surgery. Blinded study drug was applied topically to bleeding sites with an absorbable gelatin sponge. The primary efficacy end point was time to hemostasis, summarized as the incidence of hemostasis within 10 minutes. Safety analyses were conducted for 1 month after operation, and the development of antibodies to rhThrombin or to the bovine product was evaluated. RESULTS: Four hundred one patients completed this trial. Hemostasis was achieved at the time-tohemostasis evaluation site within 10 minutes in 95% of patients in each treatment group. Overall complications, including operative mortality, adverse events, and laboratory abnormalities, were similar between groups. Forty-three (21.5%) patients receiving bovine thrombin developed antibodies to the product; three patients (1.5%; p ⬍ 0.0001) in the rhThrombin group developed antibodies to rhThrombin. None of the three patients who developed antirhThrombin antibodies had abnormal coagulation laboratory results or bleeding, thromboembolic, or hypersensitivity events. CONCLUSIONS: Results of this trial suggest that rhThrombin has comparable efficacy, a similar safety profile, and is considerably less immunogenic than bovine thrombin when used for surgical hemostasis. (J Am Coll Surg 2007;205:256–265. © 2007 by the American College of Surgeons) BACKGROUND:
Hemostasis, a key tenet of any surgical procedure, is usually established with suture ligation of larger vessels and electrocautery of small vessels. But certain surgical procedures can result in bleeding that is not amenable to these techniques, including cases in which there may be
diffuse raw surface bleeding (eg, liver resection) or where cautery and suture ligation are not feasible (eg, bone and epidural venous plexus exposed in spinal surgery, or needle hole bleeding from anastomotic graft sites in vascular surgery). For these situations, topical agents continue to be investigated to enhance hemostatic control.
Competing Interests Declared: Authors Murphy and Reynolds were employees of ZymoGenetics, the sponsor of the trial; during the entire study. They are no longer employees of ZymoGenetics. Funding for this trial was provided by ZymoGenetics, Inc. Trial Registration: This study is registered with ClinicalTrials.gov with the identifier NCT00245336.
adena, CA (Singla); USC Healthcare Consultation Center, University of Southern California, Los Angeles, CA (Genyk); Vascular Surgery Associates, Baton Rouge, LA (McNeil); Indiana Spine Group, Indianapolis, IN (Renkens); ZymoGenetics Inc, Seattle, WA (Reynolds, Murphy); USC Healthcare Consultation Center, University of Southern California and LA County Hospital, Los Angeles, CA (Weaver). Correspondence address: William C Chapman, MD, Washington University, Department of Surgery, Section of Transplantation, 660 South Euclid Ave, Campus Box 8109, Room 6107 Queeny Tower, St Louis, MO 63110.
Received February 27, 2007; Accepted March 5, 2007. From the Department of Surgery, Washington University, St Louis School of Medicine, St Louis, MO (Chapman); Huntington Memorial Hospital, Pas-
© 2007 by the American College of Surgeons Published by Elsevier Inc.
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Abbreviations and Acronyms
bThrombin INR PAB PT rhThrombin TTH
⫽ ⫽ ⫽ ⫽ ⫽ ⫽
bovine thrombin international normalized ratio peripheral arterial bypass prothrombin time recombinant human thrombin time to hemostasis
Knowledge of the coagulation system has facilitated development of targeted strategies for improved hemostasis in surgery. One critical component of the clotting cascade is thrombin, a plasma protein that is activated by both the intrinsic and extrinsic coagulation pathways. Activated thrombin converts fibrinogen to fibrin, which, along with factor XIII and platelets, forms a fibrin clot. On the basis of this mechanism of action, topical thrombin has been developed for direct application to bleeding sites during surgical procedures. Currently, the only commercially available stand-alone thrombin is derived from bovine plasma and is used in more than1 million patients undergoing surgical procedures in the US each year.1 Concerns have been raised about the antigenicity of topical bovine thrombin and the potential for antibodies directed against the product to be harmful. Among patients treated with various bovine thrombin preparations, approximately 20% or more have been reported to develop antibodies directed against bovine coagulation factors (eg, thrombin and factor V).2,3 Such antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in coagulation laboratory parameters, hypersensitivity reactions, or severe bleeding or thrombosis.4-11 The use of thrombin derived from plasma sources also carries a potential risk of transmitting blood-borne pathogens. Because of these safety concerns, recombinant human thrombin (rhThrombin, ZymoGenetics, Inc), which contains no human plasma components, was developed to provide an alternative to bovine thrombin. Previously reported phase 2 clinical results suggested that rhThrombin was safe when used as a topical hemostatic agent in surgery.12 In that prospective, randomized, double-blind evaluation, antibody development after exposure to rhThrombin was not increased when compared with the placebo control. Based on those findings, a phase 3 investigation of rhThrombin versus bovine thrombin for surgical hemostasis was performed in pa-
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tients undergoing liver, spine, peripheral vascular, or vascular access procedures. Results of the trial are reported here. The primary objective of the trial was to evaluate the relative efficacy of rhThrombin and bovine thrombin, and the secondary objectives were to evaluate safety and antigenicity. METHODS This double-blind comparative study was conducted at 34 medical centers in the US. Thrombin-JMI (bThrombin) or rhThrombin was applied topically to bleeding site(s) in combination with an absorbable gelatin sponge. The primary efficacy end point was time to hemostasis (TTH), summarized as the incidence of hemostasis within 10 minutes. Secondary end points were the incidence and severity of adverse events, the incidence and grade of clinical laboratory abnormalities, and the incidence of antiproduct antibodies. An independent data monitoring committee comprising experts in surgery, hematology, and statistics monitored the study data at prespecified milestones based on projected patient accrual and made recommendations about safety, futility, and comparable efficacy. This study used a group sequential design with a flexible sample size based on statistical information13 to enroll between 400 and 600 patients. The group sequential design allowed up to three interim analyses; two were conducted. To provide an adequate safety evaluation, at least 400 patients were to be enrolled so that a minimum of approximately 200 patients would be exposed to rhThrombin. The maximum number of patients was computed to maintain 99.5% power to declare comparable efficacy when the two treatments were equivalent. At the second interim analysis, it was determined that sufficient statistical information was available and the independent data monitoring committee recommended that the study continue as planned until 400 patients with evaluable efficacy data were enrolled. The first patient was treated with study drug in October 2005, and the last patient completed followup evaluations in July 2006. All eligible patients were aged 18 years or older, and had no history of heparininduced thrombocytopenia and no known sensitivity to Thrombin-JMI components, bovine materials, or porcine collagen. Patients were ineligible if they had ever been treated in a clinical study of rhThrombin, had undergone a therapeutic surgical procedure or been treated with any experimental agent within 30 days, or had re-
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ceived blood products within 24 hours before operation. For spinal surgery, patients undergoing cervical, thoracic, or lumbar discectomy; corpectomy; laminectomy; or lateral or interbody fusion (including both anterior and posterior approaches) in the cervical region were eligible. For hepatic resection, patients undergoing wedge or anatomic resection of one to five contiguous hepatic segments (open or hand-assisted laparoscopic), with or without involvement of the gallbladder or bile duct, were eligible, as were patients undergoing livingrelated liver donation. For vascular surgery, patients undergoing peripheral arterial bypass (PAB) surgery or AV graft formation with synthetic conduit for hemodialysis using a polytetrafluoroethylene graft, including revision procedures with graft-graft anastomoses, were eligible. Protocol deviations (eg, failure to meet eligibility criteria) were balanced between groups and did not necessitate changes to analyses or affect the validity of study conclusions. The institutional review boards of participating medical centers approved the protocol and associated materials, and all patients gave written informed consent before study-specific procedures began. Once a patient was deemed eligible, and before surgery, a designated unblinded pharmacist at the trial site called an interactive voice response system, obtained a randomization assignment, and provided blinded study drug (bThrombin or rhThrombin) to the surgical staff. A central computer-generated randomization scheme was used to assign patients in a 1:1 ratio to receive bThrombin or rhThrombin. Dynamic allocation was used to attain an approximately equal number of patients randomized to each treatment for each surgeon and surgical indication. Enrollment was monitored during the study to ensure that patients undergoing vascular procedures comprised approximately 40% and patients undergoing spinal or hepatic surgery each comprised approximately 30% of the total population. Patients who were randomized but not treated with blinded study drug (eg, because of a lack of appropriate TTH evaluation site) were withdrawn from the study and replaced; the same treatment was assigned to the next patient in the stratum from which the patient was withdrawn. Blinded bThrombin (Thrombin-JMI; GenTrac, Inc; 1,000 U/mL) or rhThrombin (1,000 U/mL) was applied topically to bleeding site(s) in combination with an absorbable gelatin sponge (Gelfoam [Pharmacia & Upjohn] for spinal surgery and Surgifoam [Johnson &
J Am Coll Surg
Johnson Wound Management] for other types of surgery). A gauze pad was placed on top of the sponge(s) and held in place with gentle pressure. The pad was to be changed at least every 30 seconds until hemostasis was achieved. Bleeding appropriate for TTH evaluation was defined as mild to moderate bleeding, either on its own or remaining after brisk bleeding had been controlled by standard surgical modalities. For spinal and hepatic surgery, the first appropriate bleeding site identified during the operation was to be designated as the TTH evaluation site. If multiple appropriate bleeding sites presented simultaneously, the larger or more hemostatically challenging site was to be selected. For vascular procedures, needle hole bleeding at the proximal (PAB) or arterial (AV graft) anastomotic site was required for TTH evaluation. If application of blinded study drug did not result in hemostasis within 10 minutes, use of other hemostatic measures was to follow a tiered approach to avoid confounding the safety and antigenicity evaluations. Additional blinded study drug or surgical measure (eg, suture, cautery, or ligature) was attempted first, followed by a nonthrombin-containing topical hemostatic agent, then by an alternative agent if needed. At any time after initiating the TTH evaluation, blinded study drug also could be used at additional appropriate bleeding sites. The incidence of hemostatic success at these additional sites was not considered in the primary efficacy analysis. The primary efficacy analysis (comparing the incidence of hemostasis within 10 minutes) included all patients who received blinded study drug at one of four prespecified bleeding site types (ie, epidural venous plexus, hepatic resection site, PAB proximal anastomosis, and AV graft arterial anastomosis). Time to hemostasis, which was timed with a stopwatch and assessed for up to 10 minutes, was defined as the time from application of blinded study drug through the occurrence of hemostasis (ie, when no more blood from the TTH evaluation site was observed seeping through or around the gelatin sponge). If bleeding from the TTH evaluation site was believed to pose a severe risk to the patient, the evaluation was to be stopped early and TTH data for the patient would be recorded as failure to achieve hemostasis within 10 minutes. The primary analysis was conducted to provide approximately 99.5% power to exclude an absolute difference in the incidence of hemostasis within 10 minutes of ⬎ 15% at an overall 0.025 (one-sided) level of significance. Exclusion of a ⬎
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15% difference in incidence rates was indicated by the lower limit of a 95% confidence interval adjusted for interim analyses. The linear model for the primary end point included terms for treatment group, bleeding site type, and interaction between group and bleeding site type. Each of the four bleeding site types was weighted by patient enrollment. The bias adjusted mean estimator14 was used to adjust the treatment effect to account for interim analyses and the sample mean ordering method15 was used to adjust confidence intervals to maintain a nominal level of coverage probability. The method described by Hall and Liu16 was to be used to handle over-running data. Incidence of hemostasis within 10 minutes is a binary outcome. Because of the mean variance relationship for binary data, robust standard errors17 were derived for the linear regression model to deal with potential heteroscedasticity. The study was not powered to evaluate comparability for each individual type of operation. Additionally, a Kaplan-Meier analysis of TTH was performed by treatment group; no inferential tests were prespecified around the KaplanMeier analysis. Safety analyses were conducted on all patients who received study drug. Patients were followed for safety for 1 month after operation. Safety evaluations included assessment of adverse events and clinical laboratory data, including summaries for certain categories of adverse events (bleeding events, thromboembolic events, cardiac events, hypersensitivity, infections) that were prospectively defined based on their clinical significance or potential relationship to thrombin’s mechanism of action, risks associated with gelatin sponges, or the development of antiproduct antibodies. Coagulation parameters, including prothrombin time (PT), aPTT, and international normalized ratio (INR), were examined closely because bThrombin, rhThrombin, or antibodies to either product could potentially affect the coagulation cascade. Safety data were summarized descriptively. The presence of antibodies to either bThrombin or rhThrombin was evaluated using assays that were developed at ZymoGenetics, Inc. Plasma from blood samples collected at the baseline and at 1-month time points were tested in sequential enzyme-linked immunosorbent assays18: (1) The first assay was used to screen for antibodies binding to bThrombin or rhThrombin according to treatment assignment. The assays detected binding to any component found in the products. For example, a positive antibThrombin response could be
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from antibodies binding to bovine thrombin, bovine factor V (a contaminant found in the product), or other contaminating plasma proteins. (2) Samples that tested positive in the binding assay were titered to determine relative antibody levels in a second assay. (3) Samples with antibody titers were tested in a confirmatory assay and reported as specific, nonspecific, or nonreactive (if specificity could not be reliably determined). Patients were considered to have developed antibodies if specific seroconversion occurred (ie, pretreatment samples were nonreactive and the 1-month sample had a specific and measurable titer above the lower limit of quantification) or if a change greater than one-titer unit (10-fold increase) from baseline was observed at the 1-month time point. In addition, samples from patients developing specific antibodies to rhThrombin were evaluated for their ability to neutralize the activity of native human thrombin. Fisher’s exact test was used to compare the incidence of antiproduct antibodies between treatment groups. Exact binomial confidence intervals were created for an analysis of the potential relationship between postbaseline antiproduct antibodies and adverse clinical outcomes. RESULTS Four hundred sixty-three patients were enrolled and randomized; 52 patients were not treated because of a lack of appropriate TTH evaluation site or other reasons (Fig. 1). Of the 411 patients who received blinded study drug (206 bThrombin; 205 rhThrombin), 401 (98%) completed the study. Baseline demographics and disease characteristics were well balanced between treatment groups (Table 1). The randomization scheme effectively met the goal of assigning approximately equal numbers of patients to bThrombin and rhThrombin by surgeon: approximately 90% of surgeons had no more than one patient difference between treatment arms. Patients undergoing vascular procedures comprised approximately 40% and patients undergoing spinal or hepatic operations each comprised approximately 30% of the total population, as designed. The primary efficacy analysis included 401 patients (203 bThrombin; 198 rhThrombin). The incidence of hemostasis at the TTH evaluation site within 10 minutes after application of blinded study drug was comparable between treatment groups (95.1%, bThrombin; 95.4%, rhThrombin, Fig. 2). The lower bound of the 95% confidence interval of the 0.3% treatment effect
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J Am Coll Surg
Randomized N = 463
Received Study Drug N = 411
bThrombin N = 206
52 Not Treated: bThrombin (26) rhThrombin (26)
Efficacy Evaluable N = 203
rhThrombin N = 205
Reasons: lack of appropriate TTH evaluation site non-PTFE graft used surgery cancelled
Efficacy Evaluable N = 198
6 Discontinued:
4 Discontinued:
Death (2) Lost to follow-up (4)
Death (1) Lost to follow-up (1) Withdrew consent (1) Other (1)
Completed Study N = 200
Completed Study N = 201
Figure 1. Patient disposition. Safety analyses were conducted for all 411 patients who received blinded study drug. Efficacy analyses were conducted for the 401 patients who received blinded study drug at 1 of 4 prespecified bleeding site types (epidural venous plexus, hepatic resection site, peripheral arterial bypass proximal anastomosis, and arteriovenous graft arterial anastomosis). bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin; TTH, time to hemostasis.
was ⫺3.73%, which excluded the prespecified value of ⫺15%, so the treatments had comparable efficacy. Both treatments had similar efficacy in the various surgical settings. The treatment effect for rhThrombin ranged from ⫺0.7% (95% CI, 16.0 to 14.6) in PAB surgery to 1.6% (95% CI, 3.78 to 6.91) in hepatic surgery. Time-to-hemostasis curves showed that patients in both treatment groups achieved hemostasis rapidly, with a trend toward faster TTH in favor of rhThrombin (Fig. 3). Five percent of patients in each treatment group did not achieve hemostasis at the TTH evaluation site within 10 minutes after initial treatment with blinded study drug. Blinded study drug was the most frequently administered additional hemostatic treatment, and was successful in achieving hemostasis 85% of the time (83% [5 of 6 patients] in the bThrombin group; 86% [6 of 7 patients] in the rhThrombin group). Approximately 30% of patients were treated with blinded study
drug at one or more additional bleeding sites (28%, bThrombin; 32%, rhThrombin). The incidence of hemostasis at these sites was comparable between treatment groups and similar to that seen at the TTH evaluation sites (79 of 83 bleeding sites [95%], bThrombin; 96 of 100 bleeding sites [96%] rhThrombin). Table 1. Demographics and Baseline Characteristics Characteristic
Median age (range), y Gender, n (%) Female Male Operation, n (%) Spine Liver Peripheral arterial bypass Arteriovenous graft
bThrombin (n ⴝ 206)
rhThrombin (n ⴝ 205)
59.5 (22–89)
60.0 (21–88)
94 (46) 112 (54)
101 (49) 104 (51)
61 (30) 63 (31) 44 (21) 38 (18)
61 (30) 62 (30) 44 (21) 38 (19)
bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
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Figure 2. Incidence of hemostasis within 10 minutes. (A) Absolute incidence of hemostasis within 10 minutes. Closed squares indicate bThrombin; open squares indicate rhThrombin. (B) Difference (rhThrombin – bThrombin) and 95% confidence intervals. Overall results are adjusted for interim analyses; results by operation type are unadjusted. bThrombin, bovine thrombin; PAB, peripheral arterial bypass; rhThrombin, recombinant human thrombin.
creases in hemoglobin, increased white blood cell counts, and decreased absolute lymphocyte counts were observed. The incidence of abnormal values and the magnitude of changes in chemistry parameters were similar between treatment groups. Coagulation parameters (PT, aPTT, and INR) were generally similar between treatment groups at all time points tested.
100
Cumulative Percent Achieving Hemostasis
Four hundred eleven patients (206 bThrombin; 205 rhThrombin) received blinded study drug and were evaluated for safety. Overall, safety profiles were as expected after operations and were similar between treatment groups (Table 2). Nearly 100% of patients experienced at least one adverse event within the 1-month followup period. The severity of adverse events was similar between treatment groups, with investigators assessing most adverse events as moderate in severity. Three patients died within 30 days of study drug administration (two bThrombin [one cerebrovascular accident; one unknown cause]; one rhThrombin [severe sepsis]). The deaths were not considered by the investigators to be related to the study drug. Prospectively defined categories of clinically significant or plausibly related adverse events occurred with a similar incidence between treatment groups: bleeding (11%, bThrombin; 13%, rhThrombin), thromboembolic events (5%, bThrombin; 6%, rhThrombin), cardiac events (18%, bThrombin; 20%, rhThrombin), hypersensitivity (17%, bThrombin; 14%, rhThrombin), postoperative wound infections (10%, bThrombin; 9%, rhThrombin), and other infections (15%, bThrombin; 12%, rhThrombin). The incidence of common adverse events was similar between treatment groups (Table 2). In general, laboratory results for both groups were as expected for a postoperative population: modest de-
90 80 70 60
rhThrombin (N=198) 50
bThrombin (N=203)
40 30 20 10 0 0
1
2
3
4
5
6
7
8
9
10
Time (Minutes)
Figure 3. Cumulative incidence of hemostasis over time. Three observations in each treatment arm were censored early; observations for 13 additional patients (7 bThrombin; 6 rhThrombin) were censored at 10 minutes because hemostasis was not achieved. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
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the rhThrombin group had greater than one-titer unit postbaseline increases in antibody titer. The incidence of posttreatment antiproduct antibody development (ie, seroconversion or a greater than a onetiter unit change) was considerably lower in the rhThrombin group (43 of 200 patients [21.5%], bThrombin versus 3 of 198 patients [1.5%], rhThrombin, p ⬍ 0.0001). Samples for the three patients in the rhThrombin group who seroconverted did not neutralize native human plasma thrombin activity. Because of the reported association between antibodies to bovine thrombin and adverse clinical outcomes, a post hoc evaluation of the potential association of postbaseline antiproduct antibodies with certain adverse clinical outcomes was performed (Table 4). Patients who developed antibThrombin antibodies had a numerically increased incidence of bleeding and thromboembolic events, hypersensitivity, and abnormal aPTT relative to patients in the bThrombin group without antibodies. Abnormalities in PT and INR were balanced between patients with and without antibThrombin antibodies. None of the three patients who developed antirhThrombin antibodies had abnormal coagulation parameters (PT, INR, or aPTT) or experienced bleeding, thromboembolic, or hypersensitivity events.
Table 2. Overview of Adverse Events
Parameter
Patients with any adverse event Maximum severity of adverse events* Mild Moderate Severe Life-threatening Death Patients with any serious adverse event† Patients with a treatment-related event‡ Patients with common adverse events§ Incision site complication Nausea Procedural pain Constipation Pyrexia Vomiting Peripheral edema Anemia Hypokalemia Insomnia
bThrombin (n ⴝ 206) n %
rhThrombin (n ⴝ 205) n %
205
100
204
100
18 116 65 4 2 46 2
9 56 32 2 1 22 1
14 106 73 10 1 36 7
7 52 36 5 ⬍1 18 3
130 73 71 52 40 30 21 22 28 19
63 35 34 25 19 15 10 11 14 9
129 58 59 46 41 27 32 29 19 25
63 28 29 22 20 13 16 14 9 12
J Am Coll Surg
*Summarized by patient. † Serious adverse events include those that necessitate hospitalization, are lifethreatening, or result in significant disability or death. ‡ Considered by the investigator to be possibly, or probably; or definitely related to study drug. § Events with ⱖ 10% incidence overall. Adverse events were coded using the Medical Dictionary for Regulatory Activities, version 9. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
DISCUSSION The primary objective of this study was to evaluate the relative efficacy and safety of bThrombin and rhThrombin. The study demonstrated that both agents had comparable efficacy, with 95% of patients in each treatment group achieving hemostasis within 10 minutes. Overall complications, including adverse events and operative mortality, were similar between treatment groups. But there was a statistically significant difference in the development of antibodies to the bThrombin and rhThrombin products.
Three hundred ninety-eight patients were evaluable for the presence of antibodies to either the bThrombin or the rhThrombin product (Table 3). Antiproduct antibodies were detectable at baseline in 13 patients (10 patients [5%], bThrombin; 3 patients [1.5%], rhThrombin). Hemostasis at the TTH site was achieved within 10 minutes for all 13 patients. Of these patients, 8 of 10 in the bThrombin group and 0 of 3 in Table 3. Development of Antiproduct Antibodies* bThrombin Variable
Detectable at baseline Antibody positive* Seroconversion† ⱖ 1.0 titer unit change‡
n
rhThrombin %
10/200 5.0 43/200 21.5 35/190 (18.4%) 8/10 (80.0%)
n
%
3/198 3/198 3/195 0/3
1.5 1.5 1.5 0.0
*Seroconversion or ⱖ 1 unit increase in titer postbaseline. † Denominators include only patients who had a postbaseline observation and no detectable antiproduct antibodies at baseline. ‡ Denominators include only patients who had a postbaseline observation and had detectable antiproduct antibodies at baseline. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
p Value
⬍0.0001
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Table 4. Association of Posttreatment Antiproduct Antibodies with Clinical Outcomes Anti-bThrombin Negative Positive
Patients with bleeding or thromboembolic event* n (%) 95% CI, % Patients with hypersensitivity event* n (%) 95% CI, % Patients with high aPTT* n (%) 95% CI, %
Anti-rhThrombin Negative Positive
20/157 (13) 8–19
8/43 (19) 8–33
30/195 (15) 11–21
0/3 (0) 0–71
25/157 (16) 11–23
12/43 (28) 15–44
27/195 (14) 9–20
0/3 (0) 0–71
21/155 (14)† 9–20
14/43 (33)† 19–49
33/191 (17)† 12–23
0/2 (0)† 0–84
*Events occurring within the 1-month followup period. † Smaller denominators are from missing aPTT values at the 1-month time point. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
Many hemostatic agents have been developed to enhance surgical hemostasis, including gauze sponges, oxidized cellulose, absorbable gelatin sponges, and fibrin sealants. The choice of one agent over another in various surgical settings is highly variable and probably most dependent on availability or surgeon preference because the indications for and efficacy of these agents overlap greatly. The incidence of hemostasis within 10 minutes in the current trial (95% in each treatment group) is similar to that reported for other hemostatic agents such as CoStasis (Cohesion Technologies) and Floseal (Baxter) (97% and 96%, respectively).19,20 But more detailed comparisons are difficult because of differences in surgical technique, study populations, and other variables. In the absence of well controlled clinical trials such as this one, knowledge of the relative efficacy and safety of various hemostatic agents will likely remain limited. Based on the results of this trial, rhThrombin appears to be safe for use as a topical hemostatic agent. Both treatments were well tolerated, with similar adverse event profiles and changes in laboratory parameters that were consistent with the complex surgical procedures performed. Three patients (two bThrombin, one rhThrombin) died within 30 days of study drug administration, with no suspected association with the study drug. The most commonly observed adverse events were those expected in a postoperative population, and the incidence was generally similar between treatment groups. Perhaps the most important finding in this trial was the substantially lower incidence of development of antirhThrombin antibodies compared with antibThrombin antibodies (1.5%, rhThrombin versus 21.5%,
bThrombin; p ⬍ 0.0001). Because the amino acid sequence, structure, and biology are consistent with those of human plasma thrombin,21 rhThrombin was not expected to provoke a robust antibody response, and indeed, this was the case. This finding is in agreement with the phase 2 clinical trial results,12 in which 1 of 88 patients (1.2%) exposed to rhThrombin developed antirhThrombin antibodies during followup. Although the number of antibody-positive patients analyzed for the association of antiproduct antibodies with adverse clinical outcomes in this study is quite small, the data did not show a correlation of antibodies to rhThrombin with altered coagulation, bleeding, or thromboembolic events, or hypersensitivity. But in a posthoc analysis, the presence of antibThrombin antibodies was associated with a numeric increase in the incidence of these events, consistent with other reports.4,5,7-9,11 It should be noted, however, that confidence intervals were broad and overlapping, and that underlying patient comorbidities, concomitant medications, and blood product usage confound an unambiguous interpretation of these findings. The current trial had several inherent limitations, as in all clinical investigations. This study was designed to evaluate whether bThrombin and rhThrombin have comparable efficacy, and no placebo group was included. No conclusions can be drawn about the efficacy of either treatment relative to placebo in this study, although a trend toward faster hemostasis with rhThrombin relative to placebo was observed in the phase 2 safety evaluation. Although rigorous comparisons of bThrombin to placebo have not been previously reported, bThrombin has been used historically as a comparator for numerous products in other studies. Direct compar-
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ison of rhThrombin with bThrombin, which is widely used for surgical hemostasis, seems appropriate and was the standard used in this study. The comparability margin was selected in consultation with regulatory authorities. Of note, the lower 95% confidence bound for the observed treatment effect (–3.73) is well within the comparability margin (–15%), providing added assurance that the two treatments have comparable efficacy. Proper conduct of comparability trials is critical, as with other study designs. The comparability margin was prespecified in the protocol, randomization by surgeon was effective, and good clinical practices were followed throughout the study. This study demonstrated that bThrombin and rhThrombin have comparable efficacy when used in conjunction with a gelatin sponge as a topical adjunct for surgical hemostasis. Although the safety profiles were similar between the bThrombin and rhThrombin treatment groups, there was a notably decreased rate of antigenicity associated with rhThrombin. Author Contributions Study conception and design: Chapman, Singla, Renkens, Reynolds, Murphy, Weaver Acquisition of data: Chapman, Singla, Genyk, McNeil, Renkens, Weaver Analysis and interpretation of data: Chapman, Singla, Genyk, McNeil, Renkens, Reynolds, Murphy, Weaver Drafting of manuscript: Chapman, Murphy, Weaver Critical revision: Chapman, Singla, Genyk, McNeil, Renkens, Reynolds, Murphy, Weaver Acknowledgment: Members of the independent data monitoring committee were Stanton Nolan, University of Virginia, Charlottesville, VA; Dan Gillen, University of California, Irvine, Irvine, CA; Stephen Lowry, Robert Wood Johnson Medical School, New Brunswick, NJ; Reginald Knight, Orthopedics International, Kirkland, WA; and Michael Tarantino, University of Illinois College of Medicine – Peoria, Peoria, IL. Additional principal investigators in this trial were Reid Adams, University of Virginia, Charlottesville, VA; Thomas Biehl, Virginia Mason Medical Center – Buck Pavilion, Seattle, WA; Voytek Bosek, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL; Ravi Chari, Vanderbilt University Medical Center, Nashville, TN; Joseph Cheng, Vanderbilt University Medical Center, Nashville, TN; Randall Chesnut, Harborview Medical Center Department of Neurologic Surgery, Seattle, WA; Michael Choti, The Johns
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Hopkins Medical Institutions, Baltimore, MD; Michael D’Angelica, Memorial Sloan Kettering Cancer Center, New York, NY; Sander Florman, Tulane University Hospital and Clinic Center for Abdominal Transplant, New Orleans, LA; Reed Fogg, The Intermountain Spine Institute, Murray, UT; Kenneth Granke, John D Dingell Veteran Affairs Medical Center, Detroit, MI; John Gray, Duke University Medical Center, Durham, NC; Navyash Gupta, University of Pittsburgh Medical Center at Presbyterian University Hospital and Shadyside Hospital, Pittsburgh, PA; John Harlan, Cardio Thoracic Surgeons, PC, at Medical Center East Hospital, Birmingham, AL; Patrick Hartsell, Peripheral Vascular Associates at St Luke’s Baptist Hospital and Peripheral Vascular Associates at Methodist Specialty and Transplant Hospital, San Antonio, TX; William Jordan, University of Alabama at Birmingham, Birmingham, AL; Alan Koffron, Northwestern University Feinburg School of Medicine, Chicago, IL; Harry Lockstadt, Bluegrass Orthopaedics, Lexington, KY; Robert Martin, University of Louisville Norton Healthcare Pavilion, Louisville, KY; William Muir, The Intermountain Spine Institute, Murray, UT; Don Peska, University of North Texas Health Science Center at Fort Worth and Plaza Medical Center, Fort Worth, TX; William Pearce, Northwestern University Vascular Surgery, Chicago, IL; Carlton Randleman, Cardio Thoracic Surgeons, PC, Baptist Health System Montclair, Birmingham, AL; Gilberto Russo, University of Alabama at Birmingham, Birmingham, AL; Mark Sarfati, University of Utah, Salt Lake City, UT; Myron Schwartz, Mt Sinai School of Medicine-RMTI, New York, NY; Cynthia Shortell, Duke University Medical Center, Durham, NC; Sonia Singla, Glendale Adventist Medical Center, Glendale, CA; and Layne Yonehiro, Baptist Hospital and Baptist Clinical Research, Pensacola, FL. ZymoGenetics sponsored the clinical trial. Linda Zuckerman, an employee of ZymoGenetics, provided immunologic expertise. Roberta Connelly provided assistance with article preparation under the sponsorship of ZymoGenetics.
REFERENCES 1. Lawson JH. The clinical use and immunologic impact of thrombin in surgery. Semin Thromb Hemost 2006;32(Suppl 1):98– 110. 2. Nelson PA, Powers JN, Estridge TD, et al. Serological analysis of patients treated with a new surgical hemostat containing bovine proteins and autologous plasma. J Biomed Mater Res 2001;58: 710–719. 3. Winterbottom N, Kuo JM, Nguyen K, et al. Antigenic responses to bovine thrombin exposure during surgery: a prospective study of 309 patients. J Applied Res 2002;2:1–11. 4. Thrombin-JMI [package insert]. Bristol, VA: Jones Pharma Incorporated; 2005.
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5. Lawson JH, Lynn KA, Vanmatre RM, et al. Antihuman factor V antibodies after use of relatively pure bovine thrombin. Ann Thorac Surg 2005;79:1037–1038. 6. Ortel TL, Mercer MC, Thames EH, et al. Immunologic impact and clinical outcomes after surgical exposure to bovine thrombin. Ann Surg 2001;233:88–96. 7. Sands JJ, Nudo SA, Ashford RG, et al. Antibodies to topical bovine thrombin correlate with access thrombosis. Am J Kidney Dis 2000;35:796–801. 8. Sarfati MR, Dilorenzo DJ, Kraiss LW, et al. Severe coagulopathy following intraoperative use of topical thrombin. Ann Vasc Surg 2004;18:349–351. 9. Savage WJ, Kickler TS, Takemoto CM. Acquired coagulation factor inhibitors in children after topical bovine thrombin exposure. Pediatr Blood Cancer 2006 [Epub ahead of print]. 10. Schoenecker JG, Hauck RK, Mercer MC, et al. Exposure to topical bovine thrombin during surgery elicits a response against the xenogeneic carbohydrate galactose alpha1-3galactose. J Clin Immunol 2000;20:434–444. 11. Streiff MB, Ness PM. Acquired FV inhibitors: a needless iatrogenic complication of bovine thrombin exposure. Transfusion 2002;42:18–26. 12. Chapman WC, Lockstadt H, Singla N, et al. Phase 2, randomized, double-blind, placebo-controlled, multicenter clinical evaluation of recombinant human thrombin in multiple surgical indications. J Thromb Haemost 2006;4:2083–2085.
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13. Burington BE, Emerson SS. Flexible implementations of group sequential stopping rules using constrained boundaries. Biometrics 2003;59:770–777. 14. Whitehead J. The design and analysis of sequential clinical trials (revised second edition). Chichester, West Sussex, England: John Wiley & Sons Ltd.; 1997. 15. Emerson S, Fleming T. Parameter estimation following group sequential hypothesis testing. Biometrika 1990;7:875–892. 16. Hall WJ, Liu A. Sequential tests and estimators after overrunning based on maximum-likelihood ordering. Biometrika 2002; 89:699–707. 17. Huber PJ. The behavior of maximum likelihood estimates under nonstandard conditions. Math Statist Prob 1967;1:221–233. 18. Mire-Sluis AR, Barrett YC, Devanarayan V, et al. Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J Immunol Methods 2004;289:1–16. 19. CoStasis Multi-center Collaborative Writing Committee. A novel collagen-based composite offers effective hemostasis for multiple surgical indications: Results of a randomized controlled trial. Surgery 2001;129:445–450. 20. Weaver FA, Hood DB, Zatina M, et al. Gelatin-thrombin-based hemostatic sealant for intraoperative bleeding in vascular surgery. Ann Vasc Surg 2002;16:286–293. 21. Bishop PD, Lewis KB, Schultz J, et al. Comparison of recombinant human thrombin and plasma-derived human alphathrombin. Semin Thromb Hemost 2006;32(Suppl 1):86–97.
Hemostasis, a key tenet of any surgical procedure, is usually established with suture ligation of larger vessels and electrocautery of small vessels. But certain surgical procedures can result in bleeding that is not amenable to these techniques, including cases in which there may be
diffuse raw surface bleeding (eg, liver resection) or where cautery and suture ligation are not feasible (eg, bone and epidural venous plexus exposed in spinal surgery, or needle hole bleeding from anastomotic graft sites in vascular surgery). For these situations, topical agents continue to be investigated to enhance hemostatic control.
Competing Interests Declared: Authors Murphy and Reynolds were employees of ZymoGenetics, the sponsor of the trial; during the entire study. They are no longer employees of ZymoGenetics. Funding for this trial was provided by ZymoGenetics, Inc. Trial Registration: This study is registered with ClinicalTrials.gov with the identifier NCT00245336.
adena, CA (Singla); USC Healthcare Consultation Center, University of Southern California, Los Angeles, CA (Genyk); Vascular Surgery Associates, Baton Rouge, LA (McNeil); Indiana Spine Group, Indianapolis, IN (Renkens); ZymoGenetics Inc, Seattle, WA (Reynolds, Murphy); USC Healthcare Consultation Center, University of Southern California and LA County Hospital, Los Angeles, CA (Weaver). Correspondence address: William C Chapman, MD, Washington University, Department of Surgery, Section of Transplantation, 660 South Euclid Ave, Campus Box 8109, Room 6107 Queeny Tower, St Louis, MO 63110.
Received February 27, 2007; Accepted March 5, 2007. From the Department of Surgery, Washington University, St Louis School of Medicine, St Louis, MO (Chapman); Huntington Memorial Hospital, Pas-
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Abbreviations and Acronyms
bThrombin INR PAB PT rhThrombin TTH
⫽ ⫽ ⫽ ⫽ ⫽ ⫽
bovine thrombin international normalized ratio peripheral arterial bypass prothrombin time recombinant human thrombin time to hemostasis
Knowledge of the coagulation system has facilitated development of targeted strategies for improved hemostasis in surgery. One critical component of the clotting cascade is thrombin, a plasma protein that is activated by both the intrinsic and extrinsic coagulation pathways. Activated thrombin converts fibrinogen to fibrin, which, along with factor XIII and platelets, forms a fibrin clot. On the basis of this mechanism of action, topical thrombin has been developed for direct application to bleeding sites during surgical procedures. Currently, the only commercially available stand-alone thrombin is derived from bovine plasma and is used in more than1 million patients undergoing surgical procedures in the US each year.1 Concerns have been raised about the antigenicity of topical bovine thrombin and the potential for antibodies directed against the product to be harmful. Among patients treated with various bovine thrombin preparations, approximately 20% or more have been reported to develop antibodies directed against bovine coagulation factors (eg, thrombin and factor V).2,3 Such antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in coagulation laboratory parameters, hypersensitivity reactions, or severe bleeding or thrombosis.4-11 The use of thrombin derived from plasma sources also carries a potential risk of transmitting blood-borne pathogens. Because of these safety concerns, recombinant human thrombin (rhThrombin, ZymoGenetics, Inc), which contains no human plasma components, was developed to provide an alternative to bovine thrombin. Previously reported phase 2 clinical results suggested that rhThrombin was safe when used as a topical hemostatic agent in surgery.12 In that prospective, randomized, double-blind evaluation, antibody development after exposure to rhThrombin was not increased when compared with the placebo control. Based on those findings, a phase 3 investigation of rhThrombin versus bovine thrombin for surgical hemostasis was performed in pa-
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tients undergoing liver, spine, peripheral vascular, or vascular access procedures. Results of the trial are reported here. The primary objective of the trial was to evaluate the relative efficacy of rhThrombin and bovine thrombin, and the secondary objectives were to evaluate safety and antigenicity. METHODS This double-blind comparative study was conducted at 34 medical centers in the US. Thrombin-JMI (bThrombin) or rhThrombin was applied topically to bleeding site(s) in combination with an absorbable gelatin sponge. The primary efficacy end point was time to hemostasis (TTH), summarized as the incidence of hemostasis within 10 minutes. Secondary end points were the incidence and severity of adverse events, the incidence and grade of clinical laboratory abnormalities, and the incidence of antiproduct antibodies. An independent data monitoring committee comprising experts in surgery, hematology, and statistics monitored the study data at prespecified milestones based on projected patient accrual and made recommendations about safety, futility, and comparable efficacy. This study used a group sequential design with a flexible sample size based on statistical information13 to enroll between 400 and 600 patients. The group sequential design allowed up to three interim analyses; two were conducted. To provide an adequate safety evaluation, at least 400 patients were to be enrolled so that a minimum of approximately 200 patients would be exposed to rhThrombin. The maximum number of patients was computed to maintain 99.5% power to declare comparable efficacy when the two treatments were equivalent. At the second interim analysis, it was determined that sufficient statistical information was available and the independent data monitoring committee recommended that the study continue as planned until 400 patients with evaluable efficacy data were enrolled. The first patient was treated with study drug in October 2005, and the last patient completed followup evaluations in July 2006. All eligible patients were aged 18 years or older, and had no history of heparininduced thrombocytopenia and no known sensitivity to Thrombin-JMI components, bovine materials, or porcine collagen. Patients were ineligible if they had ever been treated in a clinical study of rhThrombin, had undergone a therapeutic surgical procedure or been treated with any experimental agent within 30 days, or had re-
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ceived blood products within 24 hours before operation. For spinal surgery, patients undergoing cervical, thoracic, or lumbar discectomy; corpectomy; laminectomy; or lateral or interbody fusion (including both anterior and posterior approaches) in the cervical region were eligible. For hepatic resection, patients undergoing wedge or anatomic resection of one to five contiguous hepatic segments (open or hand-assisted laparoscopic), with or without involvement of the gallbladder or bile duct, were eligible, as were patients undergoing livingrelated liver donation. For vascular surgery, patients undergoing peripheral arterial bypass (PAB) surgery or AV graft formation with synthetic conduit for hemodialysis using a polytetrafluoroethylene graft, including revision procedures with graft-graft anastomoses, were eligible. Protocol deviations (eg, failure to meet eligibility criteria) were balanced between groups and did not necessitate changes to analyses or affect the validity of study conclusions. The institutional review boards of participating medical centers approved the protocol and associated materials, and all patients gave written informed consent before study-specific procedures began. Once a patient was deemed eligible, and before surgery, a designated unblinded pharmacist at the trial site called an interactive voice response system, obtained a randomization assignment, and provided blinded study drug (bThrombin or rhThrombin) to the surgical staff. A central computer-generated randomization scheme was used to assign patients in a 1:1 ratio to receive bThrombin or rhThrombin. Dynamic allocation was used to attain an approximately equal number of patients randomized to each treatment for each surgeon and surgical indication. Enrollment was monitored during the study to ensure that patients undergoing vascular procedures comprised approximately 40% and patients undergoing spinal or hepatic surgery each comprised approximately 30% of the total population. Patients who were randomized but not treated with blinded study drug (eg, because of a lack of appropriate TTH evaluation site) were withdrawn from the study and replaced; the same treatment was assigned to the next patient in the stratum from which the patient was withdrawn. Blinded bThrombin (Thrombin-JMI; GenTrac, Inc; 1,000 U/mL) or rhThrombin (1,000 U/mL) was applied topically to bleeding site(s) in combination with an absorbable gelatin sponge (Gelfoam [Pharmacia & Upjohn] for spinal surgery and Surgifoam [Johnson &
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Johnson Wound Management] for other types of surgery). A gauze pad was placed on top of the sponge(s) and held in place with gentle pressure. The pad was to be changed at least every 30 seconds until hemostasis was achieved. Bleeding appropriate for TTH evaluation was defined as mild to moderate bleeding, either on its own or remaining after brisk bleeding had been controlled by standard surgical modalities. For spinal and hepatic surgery, the first appropriate bleeding site identified during the operation was to be designated as the TTH evaluation site. If multiple appropriate bleeding sites presented simultaneously, the larger or more hemostatically challenging site was to be selected. For vascular procedures, needle hole bleeding at the proximal (PAB) or arterial (AV graft) anastomotic site was required for TTH evaluation. If application of blinded study drug did not result in hemostasis within 10 minutes, use of other hemostatic measures was to follow a tiered approach to avoid confounding the safety and antigenicity evaluations. Additional blinded study drug or surgical measure (eg, suture, cautery, or ligature) was attempted first, followed by a nonthrombin-containing topical hemostatic agent, then by an alternative agent if needed. At any time after initiating the TTH evaluation, blinded study drug also could be used at additional appropriate bleeding sites. The incidence of hemostatic success at these additional sites was not considered in the primary efficacy analysis. The primary efficacy analysis (comparing the incidence of hemostasis within 10 minutes) included all patients who received blinded study drug at one of four prespecified bleeding site types (ie, epidural venous plexus, hepatic resection site, PAB proximal anastomosis, and AV graft arterial anastomosis). Time to hemostasis, which was timed with a stopwatch and assessed for up to 10 minutes, was defined as the time from application of blinded study drug through the occurrence of hemostasis (ie, when no more blood from the TTH evaluation site was observed seeping through or around the gelatin sponge). If bleeding from the TTH evaluation site was believed to pose a severe risk to the patient, the evaluation was to be stopped early and TTH data for the patient would be recorded as failure to achieve hemostasis within 10 minutes. The primary analysis was conducted to provide approximately 99.5% power to exclude an absolute difference in the incidence of hemostasis within 10 minutes of ⬎ 15% at an overall 0.025 (one-sided) level of significance. Exclusion of a ⬎
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15% difference in incidence rates was indicated by the lower limit of a 95% confidence interval adjusted for interim analyses. The linear model for the primary end point included terms for treatment group, bleeding site type, and interaction between group and bleeding site type. Each of the four bleeding site types was weighted by patient enrollment. The bias adjusted mean estimator14 was used to adjust the treatment effect to account for interim analyses and the sample mean ordering method15 was used to adjust confidence intervals to maintain a nominal level of coverage probability. The method described by Hall and Liu16 was to be used to handle over-running data. Incidence of hemostasis within 10 minutes is a binary outcome. Because of the mean variance relationship for binary data, robust standard errors17 were derived for the linear regression model to deal with potential heteroscedasticity. The study was not powered to evaluate comparability for each individual type of operation. Additionally, a Kaplan-Meier analysis of TTH was performed by treatment group; no inferential tests were prespecified around the KaplanMeier analysis. Safety analyses were conducted on all patients who received study drug. Patients were followed for safety for 1 month after operation. Safety evaluations included assessment of adverse events and clinical laboratory data, including summaries for certain categories of adverse events (bleeding events, thromboembolic events, cardiac events, hypersensitivity, infections) that were prospectively defined based on their clinical significance or potential relationship to thrombin’s mechanism of action, risks associated with gelatin sponges, or the development of antiproduct antibodies. Coagulation parameters, including prothrombin time (PT), aPTT, and international normalized ratio (INR), were examined closely because bThrombin, rhThrombin, or antibodies to either product could potentially affect the coagulation cascade. Safety data were summarized descriptively. The presence of antibodies to either bThrombin or rhThrombin was evaluated using assays that were developed at ZymoGenetics, Inc. Plasma from blood samples collected at the baseline and at 1-month time points were tested in sequential enzyme-linked immunosorbent assays18: (1) The first assay was used to screen for antibodies binding to bThrombin or rhThrombin according to treatment assignment. The assays detected binding to any component found in the products. For example, a positive antibThrombin response could be
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from antibodies binding to bovine thrombin, bovine factor V (a contaminant found in the product), or other contaminating plasma proteins. (2) Samples that tested positive in the binding assay were titered to determine relative antibody levels in a second assay. (3) Samples with antibody titers were tested in a confirmatory assay and reported as specific, nonspecific, or nonreactive (if specificity could not be reliably determined). Patients were considered to have developed antibodies if specific seroconversion occurred (ie, pretreatment samples were nonreactive and the 1-month sample had a specific and measurable titer above the lower limit of quantification) or if a change greater than one-titer unit (10-fold increase) from baseline was observed at the 1-month time point. In addition, samples from patients developing specific antibodies to rhThrombin were evaluated for their ability to neutralize the activity of native human thrombin. Fisher’s exact test was used to compare the incidence of antiproduct antibodies between treatment groups. Exact binomial confidence intervals were created for an analysis of the potential relationship between postbaseline antiproduct antibodies and adverse clinical outcomes. RESULTS Four hundred sixty-three patients were enrolled and randomized; 52 patients were not treated because of a lack of appropriate TTH evaluation site or other reasons (Fig. 1). Of the 411 patients who received blinded study drug (206 bThrombin; 205 rhThrombin), 401 (98%) completed the study. Baseline demographics and disease characteristics were well balanced between treatment groups (Table 1). The randomization scheme effectively met the goal of assigning approximately equal numbers of patients to bThrombin and rhThrombin by surgeon: approximately 90% of surgeons had no more than one patient difference between treatment arms. Patients undergoing vascular procedures comprised approximately 40% and patients undergoing spinal or hepatic operations each comprised approximately 30% of the total population, as designed. The primary efficacy analysis included 401 patients (203 bThrombin; 198 rhThrombin). The incidence of hemostasis at the TTH evaluation site within 10 minutes after application of blinded study drug was comparable between treatment groups (95.1%, bThrombin; 95.4%, rhThrombin, Fig. 2). The lower bound of the 95% confidence interval of the 0.3% treatment effect
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Randomized N = 463
Received Study Drug N = 411
bThrombin N = 206
52 Not Treated: bThrombin (26) rhThrombin (26)
Efficacy Evaluable N = 203
rhThrombin N = 205
Reasons: lack of appropriate TTH evaluation site non-PTFE graft used surgery cancelled
Efficacy Evaluable N = 198
6 Discontinued:
4 Discontinued:
Death (2) Lost to follow-up (4)
Death (1) Lost to follow-up (1) Withdrew consent (1) Other (1)
Completed Study N = 200
Completed Study N = 201
Figure 1. Patient disposition. Safety analyses were conducted for all 411 patients who received blinded study drug. Efficacy analyses were conducted for the 401 patients who received blinded study drug at 1 of 4 prespecified bleeding site types (epidural venous plexus, hepatic resection site, peripheral arterial bypass proximal anastomosis, and arteriovenous graft arterial anastomosis). bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin; TTH, time to hemostasis.
was ⫺3.73%, which excluded the prespecified value of ⫺15%, so the treatments had comparable efficacy. Both treatments had similar efficacy in the various surgical settings. The treatment effect for rhThrombin ranged from ⫺0.7% (95% CI, 16.0 to 14.6) in PAB surgery to 1.6% (95% CI, 3.78 to 6.91) in hepatic surgery. Time-to-hemostasis curves showed that patients in both treatment groups achieved hemostasis rapidly, with a trend toward faster TTH in favor of rhThrombin (Fig. 3). Five percent of patients in each treatment group did not achieve hemostasis at the TTH evaluation site within 10 minutes after initial treatment with blinded study drug. Blinded study drug was the most frequently administered additional hemostatic treatment, and was successful in achieving hemostasis 85% of the time (83% [5 of 6 patients] in the bThrombin group; 86% [6 of 7 patients] in the rhThrombin group). Approximately 30% of patients were treated with blinded study
drug at one or more additional bleeding sites (28%, bThrombin; 32%, rhThrombin). The incidence of hemostasis at these sites was comparable between treatment groups and similar to that seen at the TTH evaluation sites (79 of 83 bleeding sites [95%], bThrombin; 96 of 100 bleeding sites [96%] rhThrombin). Table 1. Demographics and Baseline Characteristics Characteristic
Median age (range), y Gender, n (%) Female Male Operation, n (%) Spine Liver Peripheral arterial bypass Arteriovenous graft
bThrombin (n ⴝ 206)
rhThrombin (n ⴝ 205)
59.5 (22–89)
60.0 (21–88)
94 (46) 112 (54)
101 (49) 104 (51)
61 (30) 63 (31) 44 (21) 38 (18)
61 (30) 62 (30) 44 (21) 38 (19)
bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
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Figure 2. Incidence of hemostasis within 10 minutes. (A) Absolute incidence of hemostasis within 10 minutes. Closed squares indicate bThrombin; open squares indicate rhThrombin. (B) Difference (rhThrombin – bThrombin) and 95% confidence intervals. Overall results are adjusted for interim analyses; results by operation type are unadjusted. bThrombin, bovine thrombin; PAB, peripheral arterial bypass; rhThrombin, recombinant human thrombin.
creases in hemoglobin, increased white blood cell counts, and decreased absolute lymphocyte counts were observed. The incidence of abnormal values and the magnitude of changes in chemistry parameters were similar between treatment groups. Coagulation parameters (PT, aPTT, and INR) were generally similar between treatment groups at all time points tested.
100
Cumulative Percent Achieving Hemostasis
Four hundred eleven patients (206 bThrombin; 205 rhThrombin) received blinded study drug and were evaluated for safety. Overall, safety profiles were as expected after operations and were similar between treatment groups (Table 2). Nearly 100% of patients experienced at least one adverse event within the 1-month followup period. The severity of adverse events was similar between treatment groups, with investigators assessing most adverse events as moderate in severity. Three patients died within 30 days of study drug administration (two bThrombin [one cerebrovascular accident; one unknown cause]; one rhThrombin [severe sepsis]). The deaths were not considered by the investigators to be related to the study drug. Prospectively defined categories of clinically significant or plausibly related adverse events occurred with a similar incidence between treatment groups: bleeding (11%, bThrombin; 13%, rhThrombin), thromboembolic events (5%, bThrombin; 6%, rhThrombin), cardiac events (18%, bThrombin; 20%, rhThrombin), hypersensitivity (17%, bThrombin; 14%, rhThrombin), postoperative wound infections (10%, bThrombin; 9%, rhThrombin), and other infections (15%, bThrombin; 12%, rhThrombin). The incidence of common adverse events was similar between treatment groups (Table 2). In general, laboratory results for both groups were as expected for a postoperative population: modest de-
90 80 70 60
rhThrombin (N=198) 50
bThrombin (N=203)
40 30 20 10 0 0
1
2
3
4
5
6
7
8
9
10
Time (Minutes)
Figure 3. Cumulative incidence of hemostasis over time. Three observations in each treatment arm were censored early; observations for 13 additional patients (7 bThrombin; 6 rhThrombin) were censored at 10 minutes because hemostasis was not achieved. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
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the rhThrombin group had greater than one-titer unit postbaseline increases in antibody titer. The incidence of posttreatment antiproduct antibody development (ie, seroconversion or a greater than a onetiter unit change) was considerably lower in the rhThrombin group (43 of 200 patients [21.5%], bThrombin versus 3 of 198 patients [1.5%], rhThrombin, p ⬍ 0.0001). Samples for the three patients in the rhThrombin group who seroconverted did not neutralize native human plasma thrombin activity. Because of the reported association between antibodies to bovine thrombin and adverse clinical outcomes, a post hoc evaluation of the potential association of postbaseline antiproduct antibodies with certain adverse clinical outcomes was performed (Table 4). Patients who developed antibThrombin antibodies had a numerically increased incidence of bleeding and thromboembolic events, hypersensitivity, and abnormal aPTT relative to patients in the bThrombin group without antibodies. Abnormalities in PT and INR were balanced between patients with and without antibThrombin antibodies. None of the three patients who developed antirhThrombin antibodies had abnormal coagulation parameters (PT, INR, or aPTT) or experienced bleeding, thromboembolic, or hypersensitivity events.
Table 2. Overview of Adverse Events
Parameter
Patients with any adverse event Maximum severity of adverse events* Mild Moderate Severe Life-threatening Death Patients with any serious adverse event† Patients with a treatment-related event‡ Patients with common adverse events§ Incision site complication Nausea Procedural pain Constipation Pyrexia Vomiting Peripheral edema Anemia Hypokalemia Insomnia
bThrombin (n ⴝ 206) n %
rhThrombin (n ⴝ 205) n %
205
100
204
100
18 116 65 4 2 46 2
9 56 32 2 1 22 1
14 106 73 10 1 36 7
7 52 36 5 ⬍1 18 3
130 73 71 52 40 30 21 22 28 19
63 35 34 25 19 15 10 11 14 9
129 58 59 46 41 27 32 29 19 25
63 28 29 22 20 13 16 14 9 12
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*Summarized by patient. † Serious adverse events include those that necessitate hospitalization, are lifethreatening, or result in significant disability or death. ‡ Considered by the investigator to be possibly, or probably; or definitely related to study drug. § Events with ⱖ 10% incidence overall. Adverse events were coded using the Medical Dictionary for Regulatory Activities, version 9. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
DISCUSSION The primary objective of this study was to evaluate the relative efficacy and safety of bThrombin and rhThrombin. The study demonstrated that both agents had comparable efficacy, with 95% of patients in each treatment group achieving hemostasis within 10 minutes. Overall complications, including adverse events and operative mortality, were similar between treatment groups. But there was a statistically significant difference in the development of antibodies to the bThrombin and rhThrombin products.
Three hundred ninety-eight patients were evaluable for the presence of antibodies to either the bThrombin or the rhThrombin product (Table 3). Antiproduct antibodies were detectable at baseline in 13 patients (10 patients [5%], bThrombin; 3 patients [1.5%], rhThrombin). Hemostasis at the TTH site was achieved within 10 minutes for all 13 patients. Of these patients, 8 of 10 in the bThrombin group and 0 of 3 in Table 3. Development of Antiproduct Antibodies* bThrombin Variable
Detectable at baseline Antibody positive* Seroconversion† ⱖ 1.0 titer unit change‡
n
rhThrombin %
10/200 5.0 43/200 21.5 35/190 (18.4%) 8/10 (80.0%)
n
%
3/198 3/198 3/195 0/3
1.5 1.5 1.5 0.0
*Seroconversion or ⱖ 1 unit increase in titer postbaseline. † Denominators include only patients who had a postbaseline observation and no detectable antiproduct antibodies at baseline. ‡ Denominators include only patients who had a postbaseline observation and had detectable antiproduct antibodies at baseline. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
p Value
⬍0.0001
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Table 4. Association of Posttreatment Antiproduct Antibodies with Clinical Outcomes Anti-bThrombin Negative Positive
Patients with bleeding or thromboembolic event* n (%) 95% CI, % Patients with hypersensitivity event* n (%) 95% CI, % Patients with high aPTT* n (%) 95% CI, %
Anti-rhThrombin Negative Positive
20/157 (13) 8–19
8/43 (19) 8–33
30/195 (15) 11–21
0/3 (0) 0–71
25/157 (16) 11–23
12/43 (28) 15–44
27/195 (14) 9–20
0/3 (0) 0–71
21/155 (14)† 9–20
14/43 (33)† 19–49
33/191 (17)† 12–23
0/2 (0)† 0–84
*Events occurring within the 1-month followup period. † Smaller denominators are from missing aPTT values at the 1-month time point. bThrombin, bovine thrombin; rhThrombin, recombinant human thrombin.
Many hemostatic agents have been developed to enhance surgical hemostasis, including gauze sponges, oxidized cellulose, absorbable gelatin sponges, and fibrin sealants. The choice of one agent over another in various surgical settings is highly variable and probably most dependent on availability or surgeon preference because the indications for and efficacy of these agents overlap greatly. The incidence of hemostasis within 10 minutes in the current trial (95% in each treatment group) is similar to that reported for other hemostatic agents such as CoStasis (Cohesion Technologies) and Floseal (Baxter) (97% and 96%, respectively).19,20 But more detailed comparisons are difficult because of differences in surgical technique, study populations, and other variables. In the absence of well controlled clinical trials such as this one, knowledge of the relative efficacy and safety of various hemostatic agents will likely remain limited. Based on the results of this trial, rhThrombin appears to be safe for use as a topical hemostatic agent. Both treatments were well tolerated, with similar adverse event profiles and changes in laboratory parameters that were consistent with the complex surgical procedures performed. Three patients (two bThrombin, one rhThrombin) died within 30 days of study drug administration, with no suspected association with the study drug. The most commonly observed adverse events were those expected in a postoperative population, and the incidence was generally similar between treatment groups. Perhaps the most important finding in this trial was the substantially lower incidence of development of antirhThrombin antibodies compared with antibThrombin antibodies (1.5%, rhThrombin versus 21.5%,
bThrombin; p ⬍ 0.0001). Because the amino acid sequence, structure, and biology are consistent with those of human plasma thrombin,21 rhThrombin was not expected to provoke a robust antibody response, and indeed, this was the case. This finding is in agreement with the phase 2 clinical trial results,12 in which 1 of 88 patients (1.2%) exposed to rhThrombin developed antirhThrombin antibodies during followup. Although the number of antibody-positive patients analyzed for the association of antiproduct antibodies with adverse clinical outcomes in this study is quite small, the data did not show a correlation of antibodies to rhThrombin with altered coagulation, bleeding, or thromboembolic events, or hypersensitivity. But in a posthoc analysis, the presence of antibThrombin antibodies was associated with a numeric increase in the incidence of these events, consistent with other reports.4,5,7-9,11 It should be noted, however, that confidence intervals were broad and overlapping, and that underlying patient comorbidities, concomitant medications, and blood product usage confound an unambiguous interpretation of these findings. The current trial had several inherent limitations, as in all clinical investigations. This study was designed to evaluate whether bThrombin and rhThrombin have comparable efficacy, and no placebo group was included. No conclusions can be drawn about the efficacy of either treatment relative to placebo in this study, although a trend toward faster hemostasis with rhThrombin relative to placebo was observed in the phase 2 safety evaluation. Although rigorous comparisons of bThrombin to placebo have not been previously reported, bThrombin has been used historically as a comparator for numerous products in other studies. Direct compar-
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ison of rhThrombin with bThrombin, which is widely used for surgical hemostasis, seems appropriate and was the standard used in this study. The comparability margin was selected in consultation with regulatory authorities. Of note, the lower 95% confidence bound for the observed treatment effect (–3.73) is well within the comparability margin (–15%), providing added assurance that the two treatments have comparable efficacy. Proper conduct of comparability trials is critical, as with other study designs. The comparability margin was prespecified in the protocol, randomization by surgeon was effective, and good clinical practices were followed throughout the study. This study demonstrated that bThrombin and rhThrombin have comparable efficacy when used in conjunction with a gelatin sponge as a topical adjunct for surgical hemostasis. Although the safety profiles were similar between the bThrombin and rhThrombin treatment groups, there was a notably decreased rate of antigenicity associated with rhThrombin. Author Contributions Study conception and design: Chapman, Singla, Renkens, Reynolds, Murphy, Weaver Acquisition of data: Chapman, Singla, Genyk, McNeil, Renkens, Weaver Analysis and interpretation of data: Chapman, Singla, Genyk, McNeil, Renkens, Reynolds, Murphy, Weaver Drafting of manuscript: Chapman, Murphy, Weaver Critical revision: Chapman, Singla, Genyk, McNeil, Renkens, Reynolds, Murphy, Weaver Acknowledgment: Members of the independent data monitoring committee were Stanton Nolan, University of Virginia, Charlottesville, VA; Dan Gillen, University of California, Irvine, Irvine, CA; Stephen Lowry, Robert Wood Johnson Medical School, New Brunswick, NJ; Reginald Knight, Orthopedics International, Kirkland, WA; and Michael Tarantino, University of Illinois College of Medicine – Peoria, Peoria, IL. Additional principal investigators in this trial were Reid Adams, University of Virginia, Charlottesville, VA; Thomas Biehl, Virginia Mason Medical Center – Buck Pavilion, Seattle, WA; Voytek Bosek, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL; Ravi Chari, Vanderbilt University Medical Center, Nashville, TN; Joseph Cheng, Vanderbilt University Medical Center, Nashville, TN; Randall Chesnut, Harborview Medical Center Department of Neurologic Surgery, Seattle, WA; Michael Choti, The Johns
J Am Coll Surg
Hopkins Medical Institutions, Baltimore, MD; Michael D’Angelica, Memorial Sloan Kettering Cancer Center, New York, NY; Sander Florman, Tulane University Hospital and Clinic Center for Abdominal Transplant, New Orleans, LA; Reed Fogg, The Intermountain Spine Institute, Murray, UT; Kenneth Granke, John D Dingell Veteran Affairs Medical Center, Detroit, MI; John Gray, Duke University Medical Center, Durham, NC; Navyash Gupta, University of Pittsburgh Medical Center at Presbyterian University Hospital and Shadyside Hospital, Pittsburgh, PA; John Harlan, Cardio Thoracic Surgeons, PC, at Medical Center East Hospital, Birmingham, AL; Patrick Hartsell, Peripheral Vascular Associates at St Luke’s Baptist Hospital and Peripheral Vascular Associates at Methodist Specialty and Transplant Hospital, San Antonio, TX; William Jordan, University of Alabama at Birmingham, Birmingham, AL; Alan Koffron, Northwestern University Feinburg School of Medicine, Chicago, IL; Harry Lockstadt, Bluegrass Orthopaedics, Lexington, KY; Robert Martin, University of Louisville Norton Healthcare Pavilion, Louisville, KY; William Muir, The Intermountain Spine Institute, Murray, UT; Don Peska, University of North Texas Health Science Center at Fort Worth and Plaza Medical Center, Fort Worth, TX; William Pearce, Northwestern University Vascular Surgery, Chicago, IL; Carlton Randleman, Cardio Thoracic Surgeons, PC, Baptist Health System Montclair, Birmingham, AL; Gilberto Russo, University of Alabama at Birmingham, Birmingham, AL; Mark Sarfati, University of Utah, Salt Lake City, UT; Myron Schwartz, Mt Sinai School of Medicine-RMTI, New York, NY; Cynthia Shortell, Duke University Medical Center, Durham, NC; Sonia Singla, Glendale Adventist Medical Center, Glendale, CA; and Layne Yonehiro, Baptist Hospital and Baptist Clinical Research, Pensacola, FL. ZymoGenetics sponsored the clinical trial. Linda Zuckerman, an employee of ZymoGenetics, provided immunologic expertise. Roberta Connelly provided assistance with article preparation under the sponsorship of ZymoGenetics.
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