Clinical Toxicology ISSN: 1556-3650 (Print) 1556-9519 (Online) Journal homepage: https://www.tandfonline.com/loi/ictx20 Comparison of F(ab’)2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial Sean P. Bush, Anne-Michelle Ruha, Steven A. Seifert, David L. Morgan, Brandon J. Lewis, Thomas C. Arnold, Richard F. Clark, William J. Meggs, Eric A. Toschlog, Stephen W. Borron, Gary R. Figge, Dawn R. Sollee, Farshad M. Shirazi, Robert Wolk, Ives de Chazal, Dan Quan, Walter García-Ubbelohde, Alejandro Alagón, Richard D. Gerkin & Leslie V. Boyer To cite this article: Sean P. Bush, Anne-Michelle Ruha, Steven A. Seifert, David L. Morgan, Brandon J. Lewis, Thomas C. Arnold, Richard F. Clark, William J. Meggs, Eric A. Toschlog, Stephen W. Borron, Gary R. Figge, Dawn R. Sollee, Farshad M. Shirazi, Robert Wolk, Ives de Chazal, Dan Quan, Walter García-Ubbelohde, Alejandro Alagón, Richard D. Gerkin & Leslie V. Boyer (2015) Comparison of F(ab’)2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial, Clinical Toxicology, 53:1, 37-45, DOI: 10.3109/15563650.2014.974263 To link to this article: https://doi.org/10.3109/15563650.2014.974263 © 2015 Informa UK Ltd. Published online: 31 Oct 2014. Submit your article to this journal Article views: 5229 View related articles View Crossmark data Citing articles: 27 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ictx20 Clinical Toxicology (2015), 53, 37–45 iSSn: 1556-3650 print / 1556-9519 online Doi: 10.3109/15563650.2014.974263 CRitiCAl CARe Comparison of F(ab’)2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial SeAn P. BuSh,1 Anne-MiChelle RuhA,2 Steven A. SeiFeRt,3 DAviD l. MoRgAn,4 BRAnDon J. lewiS,5 thoMAS C. ARnolD,6 RiChARD F. ClARk,7 williAM J. MeggS,1 eRiC A. toSChlog,1 StePhen w. BoRRon,8 gARy R. Figge,9 DAwn R. Sollee,10 FARShAD M. ShiRAzi,11 RoBeRt wolk,12 iveS De ChAzAl,12 DAn QuAn,13 wAlteR gARCÍA-uBBelohDe,14 AleJAnDRo AlAgÓn,15 RiChARD D. geRkin,2 and leSlie v. BoyeR16 1East Carolina University Brody School of Medicine/Vidant Medical Center, Greenville, NC, USA 2Banner Good Samaritan Medical Center, Phoenix, AZ, USA 3University of New Mexico Health Sciences Center, Albuquerque, NM, USA 4Scott and White Memorial Hospital, Temple, TX, USA 5Saint Josephs Hospital, Bryan, TX, USA 6Louisiana State University Health Sciences Center, Shreveport, LA, USA 7University of California Medical Center, San Diego, CA, USA 8Paul L. Foster School of Medicine/Texas Tech University Health Sciences Center, El Paso, TX, USA 9Northwest Medical Center, Tucson, AZ, USA 10UF Health Jacksonville, Jacksonville, FL, USA 11Arizona Poison & Drug Information Center, Tucson, AZ, USA 12Tucson Medical Center, Tucson, AZ, USA 13Maricopa Medical Center, Phoenix, AZ, USA 14Instituto Bioclon, México Distrito Federal, Mexico 15UNAM, Avenida Universidad 2001, Cuernavaca, Mexico 16University of Arizona Health Sciences Center, Tucson, AZ, USA Background. Crotalidae Polyvalent immune Fab (ovine) has been the only antivenom commercially available in the uS since 2007 for treatment of Crotalinae envenomation. late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. there have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab’)2 immunoglobulin derivatives have longer plasma half life than do Fab. we hypothesized that F(ab’)2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods. we conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab’)2 with maintenance doses [F(ab’)2/ F(ab’)2], or F(ab’)2 with placebo maintenance doses [F(ab’)2/placebo], versus Fab with maintenance doses [Fab/Fab]. the primary efficacy endpoint was coagulopathy (platelet count ? 150 k/mm3, fibrinogen level ? 150 mg/dl) between end of maintenance dosing and day 8. Results. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p ? 0.05) in the F(ab’)2/F(ab’)2 cohort and 2/38 (5.3%, p ? 0.05) in the F(ab’)2/placebo cohort. the lowest heterologous protein exposure was with F(ab’)2/placebo. no serious adverse events were related to study drug. in each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions. in this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab’)2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation. Keywords Snakes; Antivenins; toxinology Introduction Received 5 August 2014; accepted 4 october 2014. Background Address correspondence to Sean P. Bush, MD, east Carolina university Brody School of Medicine/vidant Medical Center, 600 Moye Blvd, Mailstop Snakebites are an important threat to public health. #625, greenville nC 27834, uSA. e-mail: seanbushmd@gmail.com worldwide, more than 5 million bites result in as many There is an accompanying commentary that discusses this paper. as 94,000 deaths annually. Almost one-fourth of global Please refer to the issue Table of Contents. snakebite incidence occurs in America, although mortality © 2015 informa uk ltd. this is an open-access article distributed under the is relatively low compared with that in Africa and Asia.1 terms of the CC-By-nC-nD 3.0 license which permits users to download in the uS, thousands of bites are reported each year, but and share the article for non-commercial purposes, so long as the article is 2,3 reproduced in the whole without changes, and provided the original source is fewer than half a dozen are fatal. Snakebite mortality credited. has decreased through the years and in certain regions of 37 38 S. P. Bush et al. the world, in part because of the development of safe and C. oreganus oreganus, C. horridus, and Bothrops effective antivenoms.4,5 Antivenoms bind and neutralize alternatus*; viperidae) envenomation with a Fab anti- venom toxins, and facilitate redistribution away from tar- venom.23,26–30 [* - deaths] get tissues and elimination from the body.6,7 Antivenom production involves collecting venom from a medically Goals of this investigation significant organism and inoculating a suitable domestic animal.8 Sublethal amounts generate immunity, and anti- Recurrence of coagulopathy appear, at least in part, to be bodies are obtained from the plasma of the immunized caused by a pharmacokinetic mismatch between venom and animal.9 Adverse drug reactions to early, unrefined antise- antivenom, probably because a depot of venom remains at rums were frequent, often severe and sometimes fatal.5,10,11 the bite site, distributing into the surrounding tissues and Physicians were, therefore, hesitant to use antivenom, and circulation according to the properties and kinetics of its var- fear of dangerous side effects led to delays in administra- ious toxins. 9 igg, F(ab’)2, and Fab differ in molecular mass, 9 tion and to insufficient dosing. Most modern antivenoms volume of distribution, and elimination half-life. A Phase 2 undergo further modification,9,12,13 including plasma frac- clinical trial explored the relationship of venom antigenemia tionation, protein precipitation, ion-exchange or affinity (“venonemia”) with recurrent coagulopathy, showing a clear chromatography, ultra- and nanofiltration, addition of difference between the patterns with F(ab’)2 versus Fab anti- preservative, lyophilization, and the regulation via good venom during the 2 weeks following treatment of enveno- 18 Manufacturing Practices for quality control and minimi- mation. no recurrence was observed in patients treated zation of microbial contamination. Further, many manu- with F(ab’)2 in preliminary Phase 2 study, but larger studies facturers enzymatically reduce the crude immunoglobulin were necessary for confirmation or negation of this finding. g (igg) molecule to yield antigen-binding fragments. the we hypothesized that F(ab’)2 antivenom would be superior y-shaped igg is cleaved with pepsin to yield a v-shaped to Fab in the prevention of late coagulopathy following F(ab’)2 or with papain to generate two Fab fragments. treatment of envenomation. to test this hypothesis, we the Fc portion (the stem of the y), suspected for causing conducted a prospective, multicenter, blinded, randomized, most of the adverse reactions, is removed along with other controlled Phase 3 clinical trial comparing F(ab’)2 to Fab for non-neutralizing components. immune reactions are now the treatment of patients with Crotalinae envenomation. markedly less problematic than those with earlier and less ameliorated preparations.4,5 Methods Study design Importance in this prospective, blinded, randomized, controlled clinical however, another kind of clinical problem emerged as trial, prevention of late coagulopathy using F(ab’) versus Fab antivenom gained widespread use in the uS,7 where it 2Fab antivenom was compared in treatment of Crotalinae has been the only commercially available antidote for pit envenomation at 18 clinical sites in the uS. the institutional viper (Crotalinae) envenomation since 2007.14 Rattlesnake Review Board at each site approved the study protocol. (Crotalus and Sistrurus spp.) envenomation commonly the study was registered at Clinicaltrials.gov, number causes thrombocytopenia and hypofibrinogenemia.15–17 nCt00636116. these venom-induced coagulopathies usually improve, but an estimated one-third to more than one-half of them recur in the days to weeks following treatment with Materials Fab antivenom.18,19 Recurrent coagulopathy is almost the Fab antivenom was Crotalidae polyvalent immune always as severe as the presenting coagulopathy and Fab (ovine) (CroFab®, “Fab”), a commercial product. immu- sometimes worse.20 in addition, new-onset late coagul- nizing venoms used to produce it are from opathies develop in many instances despite a full course C. adamanteus, C. atrox, C. scutulatus, and Agkistrodon of Fab antivenom treatment. when exactly and if coagu- piscivorus. this preparation is purified via sodium sulfate lation abnormalities will develop and nadir are difficult precipitation and affinity chromatography. Protein content of to predict.21,22 Furthermore, coagulopathies sometimes each vial is “up to 1 g,” according to the manufacturer.12 persist, even with repeated doses of Fab antivenoms, the F(ab’)2 antivenom was Crotalidae equine immune and transfusions are ineffective.9,23 Studies in Asia F(ab’)2 [Anavip ®, “F(ab’)2”], an investigational new drug. and Africa conclude that more often repeated doses of immunizing venoms for this product are from B. asper and Fab antivenoms are not required with antigen-binding C. simus. this preparation is purified via ammonium sulfate fragment antivenoms, but that F(ab’)2 is more effective precipitation and filtration. F(ab’)2 contains approximately than Fab at restoring blood coagulability.24,25 Frequent 120 mg of protein per vial.13 A relative potency of one vial monitoring and follow-up are necessary with Fab antiven- of Fab to two vials of F(ab’)2 was assigned based on a oms, along with additional intervention, hospitalization Phase 2 clinical trial and mouse eD50 comparisons. 18,31 and cost.22 Serious bleeding complications and deaths Study drugs and monitoring were provided by sponsors have resulted from late coagulopathies after treatment instituto Bioclon, SA de Cv, and Rare Disease therapeu- of viper (e.g., Crotalus adamanteus*, C. atrox, C. ruber*, tics, inc. Clinical toxicology vol. 53 no. 1 2015 F(ab’)2 vs Fab antivenom for pitviper envenomation 39 “Study drug” was whichever antivenom and/or placebo Measurements combination the subject was randomized to receive. Patients had the following assessments and measurements recorded at baseline: history, medications, vital signs, Selection of participants physical examination, and a urine or blood pregnancy test; blood was drawn for complete blood count (CBC) Pt/Ptt, eligible patients were males and females aged 2–80 years fibrinogen, venom, and antivenom levels; the subfamily, presenting for emergency treatment of a Crotalinae bite and genus, and species of the snake were identified if possible; able to provide informed consent. Signs of envenomation the physician or study nurse drew a line on the patient’s (as judged by clinician investigators) were swelling, tender- skin indicating the proximal leading edge of the lesion; ness, redness, ecchymosis, or blebs emanating from the bite and Snakebite Severity Score (SSS)32 was assessed. on site; decreased platelets or fibrinogen; and hypotension, completion of the initial infusion, the physician or study bleeding beyond the puncture site, refractory vomiting or nurse drew another line on the patient’s skin to compare diarrhea, angioedema, or neurotoxicity. exclusion criteria with the previous line and to facilitate subsequent assess- included pregnancy; breast-feeding; current treatment with ments of ongoing local injury. lesion assessment and repeat antivenom or treatment with antivenom within the last labs (CBC, Pt/inR (international normalized Ratio), month; participation in a clinical trial within the previous fibrinogen, and venom/antivenom levels) were drawn month; allergy to horse serum, sheep serum, or papaya; no 2 hours after the start of the initial infusion and 2 hours clinical indications of snakebite requiring antivenom for after the start of each additional infusion until initial con- treatment (i.e., no local tissue injury other than tooth marks trol was achieved. vitals were repeated 2 hours after start and no lab abnormalities or systemic abnormalities consis- of each maintenance dose, and lab tests were repeated tent with snake venom toxicity); and underlying medical 2 hours after start of the third maintenance dose. At each conditions or medications that significantly alter platelet follow-up visit on days 5, 8, and 15, patients had vitals taken, count or fibrinogen. the study took place from May 2008 lab results obtained, and an assessment for any symptoms through September 2011, and patients were followed for or signs suggestive of recurrent venom effect or adverse 22 days after enrollment. event (Ae). the reason for this periodicity of follow-up was based on the documented range of recurrence chronology. Interventions During the follow-up phone call on day 22, concomitant Randomization was done using Statistical Analysis Soft- medications and Aes were reviewed with the patient. ware (Cary, nC). three treatment arms [F(ab’)2 with F(ab’)2 maintenance therapy, F(ab’)2 with placebo maintenance Outcomes therapy, and Fab with Fab maintenance therapy] were estab- the primary efficacy endpoint was defined as coagulopa- lished, in a 1:1:1 ratio with a block size of 6. After informed thy between the end of maintenance dosing and study day consent was granted, a study pharmacist contacted an inter- 8 (?/? 1 day). Coagulopathy was defined as platelet count active voice Response System (Covance, inc, Princeton nJ) less than 150,000/mm3, fibrinogen less than 150 mg/dl, or to learn the group assignment. Study drug was reconstituted use of antivenom to treat a coagulation abnormality between and diluted to 250 ml with normal saline (nS). Placebo con- the end of maintenance dosing and study day 5. Second- sisted of 250 ml of nS only. to ensure blinding of all other ary efficacy endpoints included platelet counts, fibrinogen participants, study drug was provided to clinicians with no levels, and venom levels. reference to group assignment. Study subjects, all investiga- Safety monitoring included specific overall assessment tors and care providers, the inD sponsor, and study monitors for acute and delayed serum reactions, clinical evidence of remained blinded until completion of the study. bleeding or coagulopathy, and other individual Aes regard- Study drug was infused intravenously over 1 hour. ini- less of apparent cause. Ae seriousness, frequency, intensity, tial dose(s) consisted of 10 vials of F(ab’)2 or 5 vials of Fab relationship to study drug, action taken, and outcome were administered every 2 hours until “initial Control” of the recorded and clinically judged by investigators. Aes were envenomation was achieved, meaning that (1) the leading monitored for 22 days. edge of local injury was not progressing more than 1 inch per hour, and (2) platelet count, serum fibrinogen level, pro- thrombin time (Pt), and partial thromboplastin time (Ptt) Data analysis were either normal or returning toward normal. After initial Sample size calculations were based on predicted rates of control, maintenance dosing of 4 vials of F(ab’) [for the coagulopathy during follow-up of 35 percent for Fab and 2 F(ab’) /F(ab’) group] or 250 ml of nS [for the F(ab’) / 5 percent for F(ab’)2. this difference in proportions would 2 2 2 placebo group] or 2 vials of Fab (for the Fab/Fab group) was yield a moderate effect size. under these assumptions, a administered every 6 hours for 3 doses. At any time during sample size of 36 patients per treatment group gave greater the study, patients with ongoing signs of envenomation could than 80% power to detect a difference in coagulopathy receive an additional 4 vials of F(ab’)2 or 2 vials of Fab, in rates between treatments. the total number (108) was later accordance with group assignment, at physician discretion, amended to 120, to allow a reasonable power for analyses maintaining the blind. even if up to 10% of patients withdrew from the study or were Copyright © informa healthcare uSA, inc. 2014 40 S. P. Bush et al. eliminated because of protocol deviations. the trial ended Fisher’s exact test; continuous outcomes were analyzed when the enrollment goal was reached. the primary efficacy using analysis of variance (AnovA). Statistical analyses analysis was based on the intent-to-treat (itt) population. were performed using iBM SPSS Statistics for Macintosh, the null hypothesis assumed no difference in coagulopa- 21.0, Armonk, ny, and power calculation was performed thy incidence between treatments, the alternative hypothesis using PASS 2005, nCSS, llC, kaysville, ut. is that F(ab’)2 is superior to Fab. Any fibrinogen level less than 60 was analyzed as 60 mg/dl, favoring the null hypoth- esis. For the primary efficacy endpoint, separate pairwise Results comparisons between each F(ab’)2 group versus the Fab group were performed using Fisher’s exact test. the Bonfer- Characteristics of study subjects roni–holm method was used to preserve a family-wise type A total of 123 patients were randomized, 41 in each group, i error rate of 0.05.33 For all other analyses, a similar method of which 121 received antivenom and 114 completed the was employed. Categorical outcomes were analyzed using study (Fig. 1). there were no differences in gender, eth- Fig. 1. ConSoRt flow diagram (colour version of this figure can be found in the online version at www.informahealthcare.com/ctx). Clinical toxicology vol. 53 no. 1 2015 F(ab’)2 vs Fab antivenom for pitviper envenomation 41 nicity, baseline mean platelet count or fibrinogen level, than 50,000/mm3 (n ? 2) after initial control were in the Fab SSS, or time to antivenom administration among groups group. Post-hoc review of venom and antivenom levels by (table 1). the F(ab’)2/F(ab’)2 group included more children instituto Bioclon suggested that analyte degradation during and was younger on average than the Fab/Fab group. eigh- transport and prolonged storage at -20 may have reduced teen study sites in 8 states participated in the study: Az, CA, signal intensity to such an extent that comparison of results Fl, kS, lA, nC, nM, and tX. Protocol deviations related across groups was invalidated. For this reason, these results to study drug type or dose occurred in 6 cases. the identified were excluded from this analysis. snake species included C. oreganus helleri, C. scutulatus, none of the children ? 10 years old (n ? 18; 15.8%) C. mitchellii, C. atrox, C. cerastes, C. ruber, C. viridis, C. experienced late coagulopathy. Six extra doses were given molossus, C. horridus, S. miliarius barbouri, A. contortrix, to F(ab’)2/F(ab’)2 recipients, eleven to F(ab’)2/placebo and a single A. piscivorus. Copperhead bites affected 6 in the recipients, and eighteen to Fab/Fab recipients. of 6 patients F(ab’)2/F(ab’)2 group, 7 in F(ab’)2/placebo group, and 8 in treated with F(ab’)2 who experienced late coagulopathy, all the Fab/Fab group. one cottonmouth bite was identified in manifested at a single site in inland southern California; the Fab/Fab group. the rest were rattlesnake or unidentified the species, identified by the first author, were C. oreganus bites (n ? 102). helleri (n ? 2), C. mitchellii, C. ruber, C. atrox, and one unknown Crotalus sp. late coagulopathy occurred in Fab Efficacy patients at 7 sites: in California (n ? 8), Arizona (n ? 5), new Mexico (n ? 2), Florida (n ? 1), and north Carolina (n ? 1). Coagulopathy was less frequent in the F(ab’)2/F(ab’)2 and Removal of protocol deviation cases from the itt analysis the F(ab’)2/placebo groups compared with that in the Fab/ did not affect significance of results. Fab group, and there was an absolute risk reduction in both F(ab’)2 groups compared with that in Fab group (table 2). the lowest platelet counts were lower in the Fab group compared Adverse events and safety with those in both the F(ab’)2 groups. the lowest fibrinogen overall, fewer Aes were reported in the F(ab’)2/placebo levels were lower in the Fab group compared with those in group than in the F(ab’)2/F(ab’)2 or Fab/Fab (table 3). the the F(ab’)2/placebo group. the only patients with fibrinogen majority of events reported in all three of the study groups levels less than 60 mg/dl (n ? 2) and platelet counts less were assessed as mild. Table 1. Demographic and baseline characteristics of the patients.* F(ab’)2/F(ab’)2 F(ab’)2/Placebo Fab/Fab variable (N ? 41) (N ? 40) (N ? 40) Age – yr Mean 32.9†? 22.26 40.3 ? 21.02 45.6 ? 16.52 Median 36 43 48 Range 2–80 7–77 5–80 Age ? 10 yrs – no. (%) 12‡ (29.3) 5 (12.5) 1 (2.5) Female gender- no. (%) 11 (26.8) 10 (25.0) 12 (30.0) ethnicity- no. (%) white 29 (70.7) 31 (77.5) 26 (65.0) hispanic 8 (19.5) 7 (17.5) 10 (25.0) Black 3 (7.3) 1 (2.5) 2 (5.0) native American 1 (2.4) 1 (2.5) 2 (5.0) Baseline platelet count – k/mm3 Mean 197.1 ? 100.7 223.6 ? 85.7 189.5 ? 79.3 Median 185.5 221.0 199.5 Range 15–400 26–382 35–348 Baseline fibrinogen level – mg/dl Mean 275.8 ? 129.2 276.5 ? 98.5 268.4 ? 86.6 Median 256.0 293.0 267.0 Range 60–718 20–475 60–438 Snakebite Severity Score31 Mean 3.8 ? 2.10 3.0 ? l.51 3.8 ? 2.33 Median 3.0 3.0 3.0 Range 1–11 1–7 1–11 Bite-to-antivenom start time – hrs Mean 6.4 ? 6.22 4.9 ? 3.44 8.7 ? 10.93 Median 4.4 3.5 4.5 Range 2.1–35.5 1.8–18.7 1.6–58.7 *Plus-minus values are means? SD. †p ? 0.05, one-way AnovA, compared with Fab/Fab. ‡p ? 0.05, Fisher’s exact test, compared with Fab/Fab. Copyright © informa healthcare uSA, inc. 2014 42 S. P. Bush et al. Table 2. efficacy endpoints. F(ab’)2/F(ab’)2 F(ab’)2/placebo Fab/Fab (N ? 39) (N ? 38) (N ? 37) experienced late coagulopathy—no. (%) 4 (10.3)‡ 2 (5.3)‡ 11 (29.7) Absolute risk reduction (95% Cl)* 0.195 (0.014–0.367) 0.245 (0.073–0.410) – number needed to treat (patients)* Approximately 5 Approximately 4 – Mean platelet count, 1,000 s per mm ? SD (range) Day 5 265.2 ? 81.4 (121–434) 259.5 ? 61.4 (132–374) 227.1 ? 70.0 (77–389) Day 8 266.8 ? 84.7 (125–447) 265.0 ? 73.5 (114–424) 234.1 ? 80.8 (40–415) Mean fibrinogen, mg/dl ? SD (range) Day 5 369.4 ? 75.1 (223–530) 394.7 ? 97.7 (173–650) 364 ? 116.5 (81–564) Day 8 344.7 ? 70.9 (208–477) 387.1 ? 96.0 (195–693) 334.8 ? 114.4 (105–589) lowest platelet count, 1,000 s per mm3 ? SD (range) 253.4†? 80.3 (121–434) 247.5†? 62.6 (114–374) 208.9 ? 76.9 (40–389) lowest fibrinogen, mg/dl ? SD (range) 340.0 ? 70.0 (208–477) 367.8†? 93.9 (173–650) 309.0 ? 116.4 (? 60–564) *Compared with Fab/Fab . †p ? 0.05, one-way AnovA, compared to Fab/Fab. ‡p ? 0.05, Fisher’s exact test, compared to Fab/Fab. nine patients experienced Serious Aes (SAes), all protein exposure was 3.5–6 times greater in the Fab group assessed as unrelated to study drug. one patient in the than that in either of the F(ab’)2 groups. F(ab’)2/F(ab’)2 group died on Day 7 from injuries related to a traffic collision. one SAe in the Fab/Fab group was related to multicomponent coagulopathy recurrence and Discussion persistence, with major extension of ecchymosis on day 2, this study supports the hypothesis that management of bleeding from an intrajugular intravenous site on day 6, uS Crotalinae envenomation using an F(ab’) antivenom and a 10-g drop in hemoglobin during an 11-day hospital 2reduces subacute coagulopathies following treatment in course. comparison with that using Fab antivenom. An absolute one patient in each arm of the study experienced an risk reduction of 19.5–24.5 percent would suggest that acute serum reaction; however, all three tolerated sub- 4–5 patients would need to be treated with F(ab’)2 to result sequent doses after resolution of the Aes. one patient in in fewer cases of late coagulopathy. Serious bleeding com- each group experienced a pattern of symptoms suggestive plications after treatment with Fab occurred in our study, of serum sickness during follow-up. no immune reaction consistent with past reports.23,26–30 this implies that use was severe. of F(ab’)2 antivenom could reduce medically significant Mean antivenom dose in terms of vials was similar for late bleeding after snakebite, and that the need for repeated the F(ab’)2/placebo (16.1 ? 7.89, range: 5–38) and Fab/Fab blood testing after treatment could be reduced. in addition, (14.2 ? 5.66, range: 7–38) groups, but greater in the F(ab’)2/ our study suggests that maintenance dosing is not required F(ab’)2 (27.2 ? 7.25, range: 22–46) group. Maximum total with F(ab’)2 to prevent late coagulopathy. Table 3. Adverse events (Safety population). F(ab’)2/F(ab’)2* F(ab’)2/Placebo* Fab/Fab event (N ? 43) (N ? 37) (N ? 41) number of Aes 130 72 137 Patients reporting at least 1 Ae—no. (%) 35 (81.4) 24 (64.9) 33 (80.5) itching (pruritus)—no. (%) 20 (46.5) 14 (37.8) 20 (48.8) easy bruising, gingival bleeding, petechiae or melena—no. (%) 4 (9.3) 3 (8.1) 10 (24.4) nausea, vomiting—no. (%) 8 (18.7) 6 (16.2) 7 (17.0) Rash —no. (%) 5 (11.6) 5 (13.5) 5 (12.2) Arthralgia—no. (%) 4 (9.3) 4 (10.8) 7 (17.1) Myalgia—no. (%) 3 (7.0) 3 (8.1) 8 (19.5) Dehydration—no. (%) 2 (4.7) 0 (0.0) 4 (9.8) Chest pain—no. (%) 0 (0.0) 1 (2.7) 2 (4.9) other (fever, headache, cellulitis, diarrhea, pain, fatigue or blisters)—no. (%) 18 (41.9) 11 (29.7) 17 (41.5) Serious adverse events—no. (%) 6 (14.6) 1 (2.7) 2 (4.9) immune reactions Acute serum reaction—no. (%) 1 (2.3) 1 (2.7) 1 (2.4) Serum sickness—no. (%) 1 (2.3) 1 (2.7) 1 (2.4) * no pairwise comparison with Fab/Fab was significant using Fisher’s exact test. Clinical toxicology vol. 53 no. 1 2015 F(ab’)2 vs Fab antivenom for pitviper envenomation 43 the specific F(ab’)2 and Fab antivenoms compared in this Conclusions study differ in many ways, most obviously not only in the structure and mass of the molecule but also in the venom For envenomation by north American Crotalinae snakes immunogens used, affinity purification, protein content, capable of causing unexpected bleeding, this study found and pharmacokinetics. each of these variables could have that management with longer-half-life F(ab’)2 antivenom influenced efficacy, and so each is considered. First, Fab is reduces the risk of post-treatment recurrence and late-onset produced using venoms from snake species indigenous to coagulopathies following treatment when compared with the uS, whereas venoms for manufacturing F(ab’)2 come management with Fab. until such time as F(ab’)2 is com- from latin American species. venom composition and mercially utilized in the uS, patients with Crotalinae enven- antigenicity can vary considerably with species34 and geo- omation will be at continued risk for medically significant graphic35 origin of a snake; it is commonly assumed that late bleeding complications (including death) in the days and venom immunogens derived from local species produce weeks following treatment with Fab. more regionally effective antivenom.24 our study showed the opposite, suggesting that this was less important than Author contributions and responsibilities other factors. Second, Fab is affinity purified while F(ab’)2 lvB, wg-u, and AA designed the study; SPB, A-MR, lvB, is not. effective affinity purification should favor the per-mg SAS, DlM, BJl, tCA, RFC, wJM, eAt, SwB, gRF, DRS, potency and efficacy of Fab, again in contradistinction to FMS, Rw, iDC, and DQ collected the data; SPB, lvB, our results, which favored the lower-protein, non-affinity- A-MR, SAS, RDg analyzed the data; SPB, lvB, A-MR, and purified F(ab’)2 antivenom. Finally, Phase 2 data showed SAS were the primary authors of the manuscript with final that clearance of Fab is associated with recurring venom editing and approval by all coauthors, and all authors vouch antigenemia and an accompanying drop in platelet count for the accuracy and completeness of the reported analyses. and fibrinogen levels, in contrast with F(ab’)2, which cleared more slowly and was not associated with recurrent venom effects.18 this difference is consistent with our Phase 3 find- Acknowledgments ings. therefore, we conclude that differences in incidence of coagulopathy are principally related to antivenom kinet- we thank Joanne Mallie, who assisted with study admin- ics. this is consistent with findings in a comparative study istration and coordinated data management from 4 sites in of F(ab’)2 and Fab antivenoms for Echis ocellatus enveno- tucson; lisbeth gaf, who set up and coordinated central mations in nigeria25 and in a comparison of F(ab’) to Fab data management; Pieter D’Arnaud, who assisted with sta-2 for restoration of blood coagulability after Daboia russellii tistical analysis; Angela Padilla-Jones, Rn, research nurse in Sri lanka.24 and study coordinator at Banner good Samaritan Medical we found no tradeoffs in safety related to immune reac- Center; Frank watkins, study coordinator at vidant Medical tions or miscellaneous Aes. the plasma source for Fab is Center; “team venom” at loma linda university Medical ovine, whereas for F(ab’) it is equine. it has been postu- Center, Susan D. Smith, tammy h. Phan, Sarah R. Pearl, 2 lated that immunizing sheep rather than horses could reduce Sarang kim, and Alyssa Flores-Cuevas; and Ramona gee. risk associated with exposure to equine-produced allergenic In memoriam: the authors acknowledge with respectful components,36 but immune reactions in all groups were low memory the work of John F. haynes, Jr., MD, whose partici- in this study, without discernible differences between the pation as clinical investigator was essential to this study. two antivenoms. Safety as it relates to coagulopathy, how- ever, was better with F(ab’)2, consistent with results from Phase 2.18 Declaration of interest Rare Disease therapeutics, inc. (RDt) sponsored this Limitations study through contracts with the authors’ affiliated insti- tutions, and instituto Bioclon, SA de Cv manufactures limitations of the study arose from the inclusion of a dis- the F(ab’)2 antivenom in this study. Drs. Bush, Ruha, and proportionate number of children in the F(ab’)2/F(ab’)2 Quan report receiving remuneration for educational pre- group compared with that in the Fab/Fab group. none of the sentations related to the medical management of north 17 children treated with F(ab’)2 experienced late coagul- American Crotalinae bites with Fab antivenom from Btg opathy; however, late coagulopathies, some with medically international inc (Btg). Drs. Bush, Ruha, and Seifert par- significant late bleeding, have been reported in children less ticipated in an expert panel sponsored by Btg that devel- than 10 years old, suggesting that the confounding effect oped a treatment algorithm for pit viper envenomation. of this group should be minimal.26,37 this study was not Drs. Bush, Ruha, and Siefert report being investigators in powered to demonstrate the interaction of different snake phase 2 and/or 3 clinical trials of a black widow spider venoms or site-specific populations with the primary antivenom manufactured by instituto Bioclon, and their endpoint, but the difference in geographic distribution of institution(s) contracted with Rocky Mountain Poison coagulopathic cases suggests that future studies should con- & Drug Center (RMPDC) to conduct these studies. Drs. sider the effects of heterologous antivenom(s) on many uS Ruha and Boyer report being investigators in phase 2 and/ Crotalinae species. or 3 clinical trials of a scorpion antivenom manufactured Copyright © informa healthcare uSA, inc. 2014 44 S. P. Bush et al. by instituto Bioclon. Dr. Ruha reports having received 18. Boyer lv, Chase PB, Degan JA, Figge g, Buelna-Romero A, remuneration from RDt for providing presentations about luchetti C, Alagón A. Subacute coagulopathy in a randomized, scorpion antivenom. Dr. Alagón reports that he had grant comparative trial of Fab and F(ab’)2 antivenoms. toxicon 2013; 74:101–108. support from instituto Bioclon at the time of the study, as 19. Ruha AM, Curry SC, Beuhler M, katz k, Brooks De, graeme kA, well as past consultancy for instituto Bioclon. Dr. garcia et al. initial post marketing experience with Crotalidae polyvalent im- reports being a full-time employee of instituto Bioclon. mune Fab for treatment of rattlesnake envenomation. Ann emerg Med Dr. Boyer reports that she had grant support from RDt 2002; 39:609–615. for management of patients in the study, as well as a grant 20. Bush SP, Seifert SA, oakes J, Smith SD, Phan th, Pearl SR, Reibling et. Continuous iv Crotalidae Polyvalent immune Fab from instituto Bioclon for development of north African (ovine) (FabAv) for selected north American Rattlesnake bite antivenom. the authors alone are responsible for the con- patients. toxicon 2013; 69:29–37. tent and writing of the paper. 21. Ruha AM, Curry SC, Albrecht C, Riley B, Pizon A. late hema- tologic toxicity following treatment of rattlesnake envenomation with Crotalidae polyvalent immune Fab antivenom. toxicon 2011; References 57:53–59. 22. lavonas eJ, Ruha AM, Banner w, Bebarta v, Bernstein Jn, 1. kasturiratne A, wickremasinghe AR, de Silva n, gunawardena nk, Bush SP, et al.; Rocky Mountain Poison and Drug Center, Denver Pathmeswaran A, Premaratna A, et al. the global burden of snake- health and hospital Authority. unified treatment algorithm for bite: a literature analysis and modelling based on regional estimates of the management of crotaline snakebite in the united States: results envenoming and deaths. PloS Med 2008; 5:e218. of an evidence-informed consensus workshop. BMC emerg Med 2. Bronstein AC, Spyker DA, Cantilena lR Jr, Rumack Bh, Dart RC. 2011; 11:2. 2011 Annual report of the American Association of Poison Control 23. Bush SP, Seifert SA, Pearl SR. Continuous infusion of crotalidae Centers’ national Poison Data System (nPDS): 29th Annual Report. polyvalent Fab (ovine). in A Patient with Rattlesnake envenoma- Clin toxicol (Phila) 2012; 50:911–1164. tion. Presented at Biology of the Rattlesnakes, tucson Az, 2011. Pub- 3. langley Rl, Morrow we. Deaths resulting from animal attacks in the lished in Bush SP: Case study – rattlesnake envenomation. July 2012. united States. wilderness environ Med 1997; 8:8–16. in: Crotaline Snakebite Management: the role of CroFab Cro- 4. Boyer l, Degan J, Ruha AM, Mallie J, Mangin e, Alagón A. Safety of talidae Polyvalent immune Fab (ovine). Btg international, inc. intravenous equine F(ab’)2: insights following clinical trials involving 2013. 1534 recipients of scorpion antivenom. toxicon 2013; 76:386–393. 24. Ariaratnam CA, Sjostrom l, Raziek z, kularatne SA, Arachchi Rw, 5. Dart RC, Mcnally J. efficacy, safety, and use of snake antivenoms in Sheriff Mh, et al. An open, randomized comparative trial of two the united States. Ann emerg Med 2001; 37:181–195. antivenoms for the treatment of envenoming by Sri lankan Russell’s 6. norris Rl, Bush SP, Smith J. Bites by venomous reptiles in Canada, viper (Daboia russelii russelii). trans Roy Soc trop Med and hyg the u.S. and Mexico. in: Auerbach PS, ed. wilderness Medicine. 2001; 95:74–80. 6th ed. Philadelphia: Saunders; 2012; 1011–1039. 25. Meyer wP, habib Ag, onayade AA, yakubu A, Smith DC, nasidi A, 7. gold BS, Dart RC, Barish RA. Bites of venomous snakes. neJM et al. First clinical experiences with a new ovine Fab echis ocellatus 2002; 347:347–356. snake bite antivenom in nigeria: a randomized comparative trial with 8. landon J, Smith DS. Merits of sheep antisera for antivenom manufac- institute Pasteur Serum (iPSeR) Africa antivenom. Am J trop Med ture. J toxicol 2003; 22:15–22. hyg 1997; 56:291–300. 9. who guidelines for the Production, Control and Regulation of 26. lavonas eJ, khatri v, Daugherty C, Bucher-Bartelson B, king t, Snake Antivenom immunoglobulins. Available at: http://www.who. Dart RC. Medically significant late bleeding after treated crotaline en- int/neglected_diseases/diseases/snakebites/en/ (Accessed June 19, venomation: a systematic review. Ann emerg Med 2014; 63:71–78.e1. 2014). 27. kitchens C, eskin t. Fatality in a case of envenomation by Crotalus 10. litovitz tl, Schmitz BF, Bailey kM. 1989 annual report of the Amer- adamanteus initially successfully treated with polyvalent ovine ican Association of Poison Control Centers national Data Collection antivenom followed by recurrence of defibrinogenation syndrome. System. Am J emerg Med 1990; 8:394. J Med toxicol 2008; 4:180–183. 11. litovitz tl, holm kC, Bailey kM, Schmitz BF. 1991 annual report 28. Amaral versus loma linda university. [Civ S801200] Superior Court of the American Association of Poison Control Centers national Data of San Bernadino County, CA; 2011. Collection System. Am J emerg Med 1992; 10:452. 29. Balow versus university of Cincinatti, 2009-ohio-5448. 12. CroFab®. Prescribing information. Brentwood, tn 37027: Protherics 30. levine M, Ruha AM, Padilla-Jones A, gerkin R, thomas Sh. inc., 2012. Bleeding following rattlesnake envenomation in patients with pre- 13. Anavip®. Prescribing information [pending FDA approval at the envenomation use of antiplatelet or anticoagulant medications. Acad time of this writing]. Mexico D.F., Mexico: instituto Bioclon S.A. emerg Med 2014; 21:301–307. de Cv. 31. Sanchez ee, galan JA, Perez JC, Rodriguez-Acosta A, Chase PB, 14. lavonas eJ, Schaeffer th, kokko J, Mlynarchek Sl, Bogdan gM. Perez JC. the efficacy of two antivenoms against the venom of north Crotaline Fab antivenom appears to be effective in cases of severe American snakes. toxicon 2003; 41:357–365. north American pit viper envenomation: An integrative review. BMC 32. Dart RC, hurlbut kM, garcia R, Boren J. validation of a severity emergency Medicine 2009; 9:13. score for the assessment of Crotalid snakebite. Ann emerg Med 1996; 15. Boyer lv, Seifert SA, Clark RF, Mcnally Jt, williams SR, et al. 27:321–326. Recurrent and persistent coagulopathy following pit viper envenoma- 33. holm S. A simple sequentially rejective multiple test procedure. Scand tion. Arch intern Med 1999; 159:706–710. J Stat 1979; 6:65–70. 16. Boyer lv, Seifert SA, Cain JS. Recurrence phenomena after immu- 34. de Roodt AR, Clement h, Dolab A, litwin S, hajos Se, Boyer l, noglobulin therapy for snake envenomations: Part 2. guidelines for Alagón A. Protein content of antivenoms and relationship with clinical management with crotaline Fab antivenom. Ann emerg Med their immunochemical reactivity and neutralization assays. Clinical 2001; 37:196–201. toxicology 2014; 52:594–603. 17. Seifert SA, Boyer lv. Recurrence phenomena after immunoglobulin 35. French wJ, hayes wk, Bush SP, Cardwell MD, Bader Jo, Rael eD. therapy for snake envenomations: Part 1. Pharmacokinetics and phar- Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattle- macodynamics of immunoglobulin antivenoms and related antibod- snake): molecular and geographic characterization. toxicon 2004; ies. Ann emerg Med 2001; 37:189–195. 44:781–791. Clinical toxicology vol. 53 no. 1 2015 F(ab’)2 vs Fab antivenom for pitviper envenomation 45 36. Dart RC, Seifert SA, Carroll l, Clark RF, hall e, Boyer-hassen lv, 37. Miller AD, young MC, DeMott MC, ly Bt, Clark RF. Recurrent et al. Affinity-purified, mixed monospecific crotalid antivenom ovine coagulopathy and thrombocytopenia in children treated with crotali- Fab for the treatment of crotalid venom poisoning. Ann emerg Med dae polyvalent immune fab: a case series. Pediatr emerg Care 2010; 1997; 30:33–39. 26:576–582. Notice of Correction Since the online publication of this article the following has been corrected in table 1, “3.oil.51” has been changed to “3.0±1.51. Copyright © informa healthcare uSA, inc. 2014