A. A. Marchiondo, T. N. TerHune, and R. L. Herrick Target Animal Safety and Tolerance Study of Pyrantel Pamoate Paste (19.13% w/w Pyrantel Base) Administered Orally to Horses* Alan A. Marchiondo, MS, PhD a Terry N. TerHune, DVM, PhD b Robert L. Herrick, MS b a IVX Animal Health (formerly Phoenix Scientific) 3915 S. 48 th Street Terrace St. Joseph, MO 64503 b HMS Veterinary Development 3346 Avenue 248 Tulare, CA 93274 CLINICAL RELEVANCE Pyrantel pamoate paste (19.13% w/w pyrantel base) for the treatment of Anoplocephala spp tapeworms was evaluated for safety and tolerance in horses treated orally at 0, 1, 3, 5, and 10 times the clinical dose of 13.2 mg pyrantel base/kg bodyweight administered daily for six consecutive days. Parameters evaluated included clinical signs, food and water consumption, bodyweights, physical examinations, clinical pathology (hematology, coagulation, serum chemistry, urinalyses, and fecal examinations), complete necropsy, organ weights, and histopathology. No adverse events or test article related effects were observed in any treatment group during daily clinical observations of the test animals. Statistically significant changes (P <.05) lacked a dose- and/or time-dependent trend and were considered incidental. Administration of pyrantel pamoate paste did not produce any macroscopic or microscopic tissue effects in any dose group of either sex. The no-observed-effect-level (NOEL) for pyrantel pamoate paste, when administered orally to horses once daily for 6 consecutive days, was determined to be 132 mg/kg/day. Pyrantel pamoate paste (19.13% w/w pyrantel base) can be safely administered orally to horses at 13.2 mg of pyrantel base/kg for the treatment of Anoplocephala infestations. INTRODUCTION Pyrantel pamoate administered at the recommended nematocidal dose of 6.6 mg pyrantel base/kg bodyweight has shown partial cestocidal activity of 0% to 100%, with average *Funding for this study was provided by IVX Animal Health (formally Phoenix Scientific), St. Joseph, MO. efficacies of 70% to 87% against Anoplocephala perfoliata. 1 4 At the elevated (2 ) dose of 13.2 mg pyrantel base/kg, pyrantel pamoate has exhibited 93% to 97.8% efficacy against A. perfoliata. 4 7 Thus, the dose of 13.2 mg pyrantel base/kg (i.e., doubling the nematocidal single dose) has become a widely accepted target 311
Veterinary Therapeutics Vol. 6, No. 4, Winter 2005 Dose Calculation Formula Bodyweight (kg) Dose (mg/kg) = Concentration (mg active agent/g paste) dose for treating A. perfoliata infestations in horses. 6 An equine paste containing pyrantel pamoate (19.13% w/w pyrantel base) has recently been developed to provide a single oral dose of 13.2 mg pyrantel base/kg; results of two dose-confirmation studies and clinical field efficacy studies in five geographic locations in the United States have demonstrated an efficacy range of 95% to 98.4% against Anoplocephala spp. 8,9 This unique formulation of pyrantel pamoate paste for equines contains 20.3% less volume at a 25% higher concentration than two syringes of 15.25% w/w pyrantel pamoate paste. The purpose of this study was to evaluate the target animal safety and tolerance of pyrantel pamoate paste (19.13% w/w pyrantel base) when administered orally according to bodyweight at 0 (0 mg/kg), 1 (13.2 mg/kg), 3 (39.6 mg/kg), 5 (66 mg/kg), and 10 (132 mg/kg) the proposed clinical dose daily for 6 consecutive days. This study was conducted to satisfy the safety requirements of a new animal drug in accordance with the U.S. FDA. MATERIALS AND METHODS Horses and Treatments Forty clinically healthy adult horses (20 castrated males and 20 open [i.e., not pregnant] females), approximately 2 to 10 years of age and weighing 351 to 592 kg on treatment day (TD) 1, were selected on TD 1 based on bodyweight specifications, acceptable physical examination, and clinical pathology (as determined on TD 14; see below); breeds included quarter horse, Appaloosa, Thoroughbred, palomino, Arabian, and Arabian quarter horse cross. Horses were randomly assigned to one of five Test Article Dose treatment groups (0, 1, (g of paste) 3, 5, or 10 ; blinded by randomly assigning a color code to each) of eight horses (four males and four females) each. Treatment group mean bodyweights (TD 1) within sex were determined to be equivalent using analysis of variance (ANOVA) with P <.05. Four dosing periods of two horses (one male and one female) each from each of the five treatment groups were stagger-started to have a manageable number of necropsies (10/day) at the end of the treatment periods. Horses were identified by a unique ID number and wore two forms of identification (leg band and halter tag). The testing facility consisted of an open-sided metal truss building with ceiling lighting and metal panel stalls (approximately 128 sq ft); each stall was equipped with a plastic feeder and a trough waterer. Animals received 4.5 kg alfalfa hay and 0.50 kg grain supplement twice daily. Each horse had ad libitum access to a measured volume of water. Temperatures and relative humidity in the animal area were measured daily. Horses were acclimated to the testing facility for a minimum of 7 days (TDs 21 to 14). Following acclimation, baseline measurements for hematology, coagulation profiles, serum chemistry analysis, urinalysis, fecal analysis (frank and occult blood, visual color and consistency, and parasite flotation and microscopic examination), bodyweight, and physical examination findings (body condition; appearance of eyes, skin, and haircoat; cardiovascular, respiratory, gastrointestinal, genitourinary, and neurologic/locomotion condition; and overall appearance) were collected on TDs 14 and 1; food and water consumption were measured during a pretreatment period of 14 days (TDs 14 to 1). Horse care procedures conformed to Institutional Ani- 312
A. A. Marchiondo, T. N. TerHune, and R. L. Herrick mal Care and Use Committee guidelines of the test facility. Stagger-started treatments were initiated on TD 0 and ended on TD 8, with terminal sacrifice and necropsy performed daily on TDs 6 through 9. Horses within their respective groups were treated daily for six consecutive days with one of the following doses (calculated for each animal according to the formula shown on page 312): 0 (control): placebo vehicle paste at the volume administered to the 5 group 1 : 13.2 mg pyrantel base/kg 3 : 39.6 mg pyrantel base/kg 5 : 66 mg pyrantel base/kg 10 : 132 mg pyrantel base/kg Treatments were given by the same dose administrator throughout the study. Before each treatment, the horse s mouth was checked to ensure that no food was present. The nozzle end of the dosing syringe(s) was inserted into the horse s mouth through the interdental space and directed caudally, and the paste was deposited onto the dorsum of the tongue. The horse s head was raised for a few seconds after completion of the dosing. If the test article was refused or lost, that amount was approximated and readministered. Clinical Evaluations Physical examinations were conducted on TDs 14 and 1 and approximately 24 hours after administration of each dose. Horses were weighed on a calibrated scale on TDs 21, 14, 7, and 1 and approximately 24 hours after administration of each dose. Food and water consumption were measured (in 0.05-kg increments) daily during TDs 14 to 1 and during the treatment periods. Daily clinical health observations including categorical and ordinal measurements of appetite, body condition, eyes, respiration, nasal discharge, locomotion/musculature, skin and haircoat, behavioral attitude, feces, urine, and body temperature were conducted on TDs 21 through 1 and at approximately 0, 1, 2, 4, 8, 12, and 24 hours after each dose administration. Blood samples were collected from a jugular vein in evacuated glass tubes from all horses on TDs 14 and 1 and approximately 24 hours after each dose administration for determination of serum chemistry (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, amylase, γ-glutamyl transferase, total protein, albumin, globulin, albumin:globulin ratio, glucose, blood urea nitrogen, creatinine, total and direct bilirubin, calcium, chloride, phosphorus, potassium, and sodium), hematology (total leukocyte count, absolute and relative [%] differential, erythrocyte count, hematocrit, hemoglobin, mean corpuscular hemoglobin concentration and volume, and fibrinogen), and coagulation profile (prothrombin time and activated partial thromboplastin time). Urine samples were collected from all animals on TDs 14 and 1 (using individual plastic catch cups) and approximately 24 hours after the last dose administration or at necropsy. Urinalysis variables determined included color, clarity, volume (amount submitted), blood, ph, glucose, protein, ketones, specific gravity by refractometer, urobilinogen, bilirubin, and microscopic examination of urine sediments (necropsy sample only). Fecal samples were collected from all animals on TDs 14 and 1 and approximately 24 hours after the last dose administration or at necropsy. Fecal analysis included blood (frank [grossly visible] and occult), visual color (green to brown), visual consistency (dry/scant, loose, diarrhea), and presence of parasite eggs (fecal flotation and microscopic evaluation on TD 14). 313
Veterinary Therapeutics Vol. 6, No. 4, Winter 2005 TABLE 1. Summary of Bodyweight, Feed Consumption, Water Consumption, and Body Temperature of Horses Treated with Pyrantel Pamoate Paste (19.13% w/w Pyrantel Base) at 0, 1, 3, 5, and 10 the Target Dose of 13.2 mg Pyrantel Base/kg Bodyweight 0 1 Parameter (F) (M) (F) (M) Mean bodyweight (kg) b 456 416 462 c 436 c Bodyweight range (kg) b 407 519 360 446 447 500 430 482 Mean feed consumption (kg) d 10 9.9 10 10 Feed consumption range (kg) d 10 10 8.9 10 10 10 10 10 Mean water consumption (kg) b 37 39 41 43 Water consumption range (kg) b 27 40 24 45 30 48 30 49 Mean body temperature ( F) b 99.4 99.5 99.5 99.6 Body temperature range ( F) b 97.4 100.5 98.0 100.6 98.0 100.3 98.0 102.5 a Eight horses (four castrated males and four nonpregnant females) per treatment group. b Treatment days (TDs) 1 7. c Significant difference on TDs 1 7 versus 0 (P =.046). Animals were fasted at least 12 hours before termination and were humanely euthanatized approximately 24 hours after the final dosing day. Horses were sedated with xylazine (100 mg/ml; 3 ml total dose IV) and euthanatized with pentobarbitol (390 mg/ml; 100 ml total dose IV) using AVMA-approved methods. 10 Complete gross necropsies were performed on each animal, and tissues were preserved in 10% neutral buffered formalin. Tissues by sex included adrenal glands, aorta, brain (including cerebrum, thalamus, cerebellum, and medulla oblongata), cecum, cervix, colon, duodenum, esophagus, eyes with optic nerve, heart, ileum, jejunum, kidneys, lacrimal gland, liver, lung, lymph nodes (mandibular, mediastinal, and mesenteric), mammary gland, bone marrow smear from sternum, muscle (semimembranosus), ovaries, pancreas, peripheral nerve (sciatic), pituitary gland, rectum, salivary gland (mandibular), skin, spinal cord (cervical, thoracic, and lumbar sections), spleen, sternum, stomach, thymus, thyroid gland/parathyroid, tongue, trachea, urinary bladder, uterus, and vagina. In addition, all gross lesions were collected; however, processing of gross lesions for microscopic evaluation was not conducted if such lesions were interpreted by the study pathologist to be unrelated to treatment. Examples of lesions that often fall into this category included matting or staining of hair, alopecia, excoriations, animal identification wounds, irregular teeth, overgrown hooves, and abrasions. Collected tissues were preserved in 10% neutral buffered formalin on all listed tissues from the 0, 5, and 10 treatment groups and were processed into hematoxylin and eosin stained microscopy slides. Prepared microscopy slides were examined by a board-certified pathologist and, where appropriate, all findings were assigned a severity score of normal/unremarkable, minimal, mild, moderate, or marked. If the 314
A. A. Marchiondo, T. N. TerHune, and R. L. Herrick Treatment Group a 3 5 10 (F) (M) (F) (M) (F) (M) 418 441 480 446 464 448 385 468 344 499 388 555 407 481 421 508 385 494 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 38 42 43 45 44 44 28 48 26 50 29 49 31 49 31 49 26 50 99.3 99.4 99.6 99.5 99.5 e 99.4 97.5 100.4 97.7 100.5 98.1 100.5 97.4 101.6 98.0 101.4 97.5 100.4 d TDs 1 6 e Significant difference on TD 3 versus 0 (P =.026). F = mares; M = geldings. histopathologic findings revealed target organs of toxicity, those organs were also evaluated in the 1 and 3 treatment groups. Organ weights were recoded at necropsy for the brain, heart, kidneys, liver, ovaries, and spleen. The study was conducted in compliance with the Good Laboratory Practice Regulations as set forth in Title 21 of the Code of Federal Regulations Part 58, Food and Drug Code of Federal Regulations of the United States of America. Statistical Analysis Statistical comparisons of main effects and interactions were performed at the significance level of P <.1 for treatment and treatment by time interaction and P <.05 for treatment by sex and treatment by time and sex using SAS PROC MIXED Version 8.02 (SAS Institute, Cary, NC). Each horse was an experimental unit, and horses were blocked to location by sex. All continuous variables, including organ weights, were analyzed using a repeated measures analysis of variance and covariance (mean baseline of the pretreatment observations). Least squares means were used to compare treatment to the vehicle treatment group including both the unadjusted P values and Dunnett s 11 adjusted P value, when appropriate, only for significant treatment main effects. The Kenward-Roger approximation for denominator degrees of freedom was used. Statistical analysis of histopathologic lesion incidence data was limited to those lesions with incidences in a treatment group that differed from the vehicle treatment group by 2 or more (with sexes combined). RESULTS Horses and Treatments All test animals received their intended dose on each scheduled treatment day. The number of syringes used to administer the doses varied from a minimum of one for the 1 treatment (text continues on page 320) 315
TABLE 2. Summary of Hematologic Values a of Horses Treated with Pyrantel Pamoate Paste (19.13% w/w Pyrantel Base) at 0, 1, 3, 5, and 10 the Target Dose of 13.2 mg Pyrantel Base/kg Bodyweight Reference 0 1 Parameter Range b (F) (M) (F) (M) Erythrocytes 5 10 8.3 8.1 9.4 d 8.7 d 10 6 /µl (7.1 8.6) (6.8 9.6) (8.0 11.0) d (6.5 10.5) d Packed cell volume 24% 35.5 37.5 43.4 e 36.9 e (hematocrit) 46% (30.7 40.3) (32.5 43.9) (37.8 50.1) e (29.1 43.0) e Hemoglobin 8 15 12.9 13.6 15.8 f 13.6 f g/dl (11.2 15.0) (11.8 15.5) (13.3 18.5) f (10.3 15.9) f Fibrinogen 110 400 300 250 213 254 mg/dl (225 353) (225 275) (150 275) (220 325) Mean corpuscular 40 60 43.2 46.2 46.4 42.7 volume fl (41.2 45.8) (44.2 48.2) (44.4 49.4) (39.1 45.9) Mean corpuscular 11 17 15.7 16.7 16.9 15.7 hemoglobin pg (15.0 16.5) (16.0 17.5) (16.1 17.9) (14.4 17.0) Mean corpuscular 30 36 36.2 36.2 36.4 36.8 hemoglobin g/dl (35.2 37.3) (35.2 36.8) (35.4 37.6) (36.0 37.6) concentration Leukocytes 2 12 8.5 7.0 7.4 6.5 10 3 /µl (6.2 12.4) (5.1 8.8) (5.9 11.2) (4.9 9.3) Absolute lymphocytes 2,500 2,764 2,754 2,381 3,012 7,500 (1,753 3,327) (1,774 2,927) (1,552 2,484) (1,735 3,619) Lymphocytes 45% 33.0 39.8 33.0 47.6 75% (22.0 51.0) (23.6 56.7) (21.0 58.0) (21.4 68.1) Absolute segmented 600 5,201 3,820 4,434 3,118 neutrophils 4,000 (4,313 5,571) (3,509 4,153) (3,793 4,942) (2,206 3,514) Segmented neutrophils 15% 60.9 54.3 58.9 47.0 45% (39.2 75.6) (31.1 75.3) (41.7 75.5) (24.4 73.8) Banded absolute 0 120 4.7 2.7 0 0 neutrophils (0 28) (0 16) Banded neutrophils 0% 0.05 0.05 0 g 0 h 2% (0 0.3) (0 0.3) Prothrombin time 7.8 8.8 8.5 8.8 8.9 8.8 sec (7.8 9.7) (8.2 9.5) (8.2 9.7) (8.2 9.4) Activated partial 35.2 44.9 45.1 47.5 44.6 42.7 thromboplastin time sec (38.1 49.2) (37.6 60.5) (38.4 50.6) (31.9 71.7) a Blood samples collected approximately 24 hr after administration of the first through the sixth doses. b Normal equine reference ranges from Aiello (ed): The Merck Veterinary Manual, ed 8. Whitehouse Station, NJ, Merck & Co, 1998; and IDEXX Veterinary Services, West Sacramento, CA. c Values reported as geometric mean (range). d Significant difference (P =.0845) compared to 0 for treatment days (TDs) 1 6. No biologic significance as values are within normal ranges.
Treatment Group c 3 5 10 (F) (M) (F) (M) (F) (M) 9.9 8.6 8.7 7.9 8.4 8.3 (8.7 11.6) (7.7 9.6) (7.7 10.0) (6.9 9.2) (6.5 10.1) (6.5 9.5) 40.6 36.5 38.4 34.4 36.5 36.5 (35.7 45.3) (32.8 40.2) (31.2 43.5) (29.6 38.9) (30.0 43.6) (28.1 40.4) 14.9 13.4 14.1 12.5 13.4 13.3 (12.8 16.9) (12.2 14.7) (12.6 15.8) (10.9 14.3) (11.0 15.9) (10.3 15.6) 271 221 217 233 250 213 (175 350) (175 275) (175 250) (200 300) (200 300) (175 250) 41.2 42.5 44.3 43.6 44.1 44.1 (37.3 45.1) (41.3 43.8) (42.0 46.5) (41.4 45.0) (41.5 46.8) (42.8 45.5) 15.0 15.5 16.2 15.8 16.1 16.1 (13.9 16.5) (15.1 16.1) (15.1 17.2) (15.0 16.7) (15.2 16.9) (15.6 16.7) 36.7 36.6 36.7 36.2 36.6 36.4 (36.0 37.3) (35.8 37.3) (36.4 37.6) (35.1 37.1) (36.1 37.5) (35.4 37.1) 6.9 6.8 7.0 6.2 7.0 6.4 (5.2 10.3) (4.1 9.9) (3.3 9.0) (4.5 8.4) (5.6 10.1) (5.2 7.9) 2,129 3,091 3,598 2,863 3,093 2,821 (1,410 2,855) (2,099 3,539) (2,177 4,442) (1,833 3,194) (1,785 3,676) (2,028 3,835) 37.7 45.3 50.7 44.5 44.1 44.1 (17.2 62.9) (23.6 66.6) (22.0 86.2) (26.9 70.1) (14.5 70.2) (27.6 71.7) 3,843 3,370 2,939 2,977 3,495 3,182 (3,163 5,073) (2,567 4,562) (2,557 3,578) (2,404 3,860) (3,039 4,433) (2,593 3,813) 55.7 50.3 36.9 48.9 50.8 48.8 (31.2 78.6) (28.1 73.2) (9.3 72.5) (21.0 69.9) (25.7 67.4) (21.5 60.4) 0 0 0 9.2 0 2.2 (0 54.5) (0 13) 0 g 0 h 0 g 0.13 h,i 0 g 0.05 h,i (0 1.7) h,i (0 0.3) h,i 9.2 8.8 8.3 8.7 8.5 8.7 (8.1 14.5) (8.0 9.6) (7.7 9.4) (8.1 9.4) (7.9 9.0) (8.2 9.5) 45.9 45.2 42.2 45.1 43.2 45.9 (37.8 71.7) (41.0 58.4) (41.0 58.4) (35.5 51.2) (38.7 48.6) (38.3 58.3) e Significant difference (P =.0382) compared to 0 for TDs 1 6. f Significant difference (P =.0894) compared to 0 for TDs 1 6. g Significant difference compared to 0 on TD 2 for 1 (P =.0348), 3 (P =.0289), 5 (P =.0348), and 10 (P =.0348). h Significant difference compared to 0 on TD 1 for 1 (P =.0518), 3 (P =.0348), 5 (P =.0518), and 10 (P =.0518). i Significant difference compared to 0 on TD 3 for 5 (P<.0001) and 10 (P =.0243). F = mares, M = geldings.
TABLE 3. Summary of Serum Biochemistry Values a of Horses Treated with Pyrantel Pamoate Paste (19.13% w/w Pyrantel Base) at 0, 1, 3, 5, and 10 the Target Dose of 13.2 mg Pyrantel Base/kg Bodyweight Reference 0 1 Parameter Range b (F) (M) (F) (M) Alanine amino 2.7 20.5 11.7 7.6 10.1 6.8 transferase IU/L (3.3 22.5) (6.3 9.8) (5.5 17.3) (3.7 9.2) Amylase 46.7 188.1 6.0 6.8 5.6 5.2 IU/L (1.2 8.7) (5.2 9.6) (0.8 8.6) (1.1 8.8) Alkaline 70.1 226.8 226.8 129.7 166.7 139.3 phosphatase IU/L (95.5 385) (100.8 165) (80 272.9) (84.3 199.2) Aspartate amino 115.7 287 347.0 279.5 271.8 227.3 transferase IU/L (173.4 591.4) (180.1 448.8) (224.1 341.1) (183.3 273.8) Total bilirubin 0.3 3.0 1.3 1.3 1.6 d 2.4 d mg/dl (0.7 2.2) (1.0 2.2) (1.0 2.6) d (1.0 4.5) d Direct bilirubin 0.2 0.5 0.4 0.3 0.4 0.4 mg/dl (0.2 0.5) (0.2 0.4) (0.3 0.5) (0.2 0.5) Calcium 10.4 13.4 12.0 12.4 12.1 11.8 mg/dl (10.8 13.5) (11.4 14.9) (11.3 14.5) (11.2 13.3) Chloride 97.2 110.1 94.7 93.6 95.0 95.0 meq/l (92.2 96.8) (89.8 97.5) (91.8 99.2) (92.5 99.8) Creatinine 0.9 2.0 1.0 1.0 1.1 e 1.0 e mg/dl (0.8 1.1) (0.8 1.1) (1.0 1.3) e (0.9 1.1) e γ-glutamyl 2.7 22.4 11.2 10.8 11.1 10.3 transferase IU/L (1.9 18.1) (7.5 17.1) (9.3 14.1) (5.5 14.9) Glucose 62.2 114 83.6 83.9 80.9 81.5 mg/dl (60.8 107.7) (65.6 103.6) (58.6 98.4) (60.4 97.3) Potassium 2.8 4.7 3.5 3.7 3.8 f 3.5 meq/l (3.1 4.0) (3.1 4.3) (3.4 4.4) f (2.6 4.0) Sodium 133.3 147.3 138.0 137.2 138.3 137.3 meq/l (136.3 144.8) (134.0 140.2) (134.4 140.0) (132.5 142.0) Total protein 5.7 7.9 7.0 7.1 7.2 6.9 g/dl (6.2 7.5) (6.5 7.7) (6.9 9.1) (6.4 7.7) Albumin 2.5 3.8 3.3 3.4 3.5 3.4 g/dl (3.1 3.5) (3.1 3.6) (3.1 4.2) (3.1 3.8) Globulin 2.4 4.6 3.7 3.7 3.7 3.5 g/dl (3.1 4.2) (3.1 4.2) (3.4 4.2) (3.0 4.2) Albumin:globulin 0.8 1.3 0.9 1.0 1.0 1.0 ratio (0.79 1.02) (0.8 1.12) (0.78 1.14) (0.77 1.22) a Blood samples collected approximately 24 hr after administration of the first through the sixth doses. b Normal equine reference ranges from Aiello (ed): The Merck Veterinary Manual, ed 8. Whitehouse Station, NJ, Merck & Co, 1998; and IDEXX Veterinary Services, West Sacramento, CA. c Values reported as geometric mean (range). d Significant difference (P =.0008) compared to 0 on treatment days (TDs) 1 6.
Treatment Group c 3 5 10 (F) (M) (F) (M) (F) (M) 10.0 9.3 8.0 7.4 8.4 8.6 (6.4 15.2) (6.0 11.8) (5.2 11.3) (5.9 9.5) (3.6 13.6) (5.3 11.8) 4.7 5.8 5.1 5.6 6.0 5.7 (2.0 7.5) (2.3 10.2) (3.2 10.5) (3.2 10.5) (3.5 13.3) (2.0 9.3) 156.4 127.1 169.2 146.7 146.7 143.1 (128.7 191.3) (88.8 174) (92.8 266.6) (99.2 201.1) (139.5 166.2) (125.7 155.4) 391.2 249.3 263.0 261.7 241.5 291.2 (181.0 477.3) (212.6 293.5) (230.1 326.1) (197.9 335.2) (194.6 294.0) (249.7 332.8) 1.4 1.9 1.4 1.5 1.1 1.4 (1.1 2.0) (1.0 2.9) (0.8 2.3) (0.8 2.4) (0.7 2.3) (0.7 2.3) 0.4 0.3 0.3 0.3 0.4 0.3 (0.2 0.5) (0.2 0.4) (0.1 0.5) (0.2 0.4) (0.2 0.6) (0.2 0.5) 12.0 11.8 12.0 11.5 11.6 11.8 (11.0 13.3) (10.9 13.4) (11.4 14.2) (10.5 13.1) (10.9 13.8) (11.3 14.0) 93.7 94.2 94.9 93.2 94.7 93.3 (91.3 97.1) (91.2 97.2) (92.2 98.4) (91.5 95.8) (91.8 97.8) (90.4 96.2) 1.0e 1.0 e 1.0 1.0 1.0 e 1.1 e (0.9 1.1)e (0.8 1.1) e (0.9 1.1) (0.9 1.1) (0.9 1.1) e (1.0 1.2) e 12.8 9.7 10.9 10.4 12.8 9.9 (7.8 16.5) (6.7 12.5) (9.3 13.9) (6.9 13.6) (10.0 18.0) (5.9 14.2) 86.7 87.3 85.3 86.0 88.0 83.9 (63.1 121.7) (69.5 125.7) (56.3 119.3) (72.4 98.4) (72.2 107.1) (66.1 109.1) 3.9 f 3.6 3.5 3.7 3.7 3.8 (3.4 4.4) f (3.1 4.1) (3.1 4.0) (2.9 4.1) (3.1 4.0) (3.5 4.0) 136.3 138.5 137.8 136.3 137.0 136.8 (132.4 140.6) (135.2 141.9) (134.5 140.2) (133.5 139.8) (133.8 140.2) (133.2 140.2) 6.9 6.5 6.9 6.7 6.8 6.9 (6.7 7.3) (6.0 6.8) (6.3 7.8) (6.3 7.3) (6.4 7.7) (6.3 7.1) 3.5 3.4 3.5 3.4 3.2 g 3.4 g (3.2 3.8) (3.1 3.6) (3.1 4.0) (3.3 3.7) (2.9 3.5) g (3.2 3.7) g 3.4 3.1 3.5 3.2 3.6 3.5 (2.9 3.8) (2.7 3.3) (2.8 4.3) (2.9 3.7) (3.1 4.3) (2.9 4.1) 1.1 1.2 1.1 1.1 0.9 1.0 (0.86 1.18) (1.01 1.35) (0.71 1.34) (0.95 1.17) (0.70 1.15) (0.83 1.14) e Significant difference compared to 0 on TDs 1 6 for 1 (P =.0004), 3 (P =.0270), and 10 (P =.0444). Creatinine values showed statistically signficant differences but were within normal ranges and thus of no biologic significance. f Significant difference compared to 0 on TDs 1 6 for 1 (P =.0532) and 3 (P =.0243). g Significant difference (P =.0472) compared to 0 on TDs 1 6 for 10. F = mares, M = geldings.
Veterinary Therapeutics Vol. 6, No. 4, Winter 2005 TABLE 4. Summary of Urinalysis Values a of Horses Treated with Pyrantel Pamoate Paste (19.13% w/w Pyrantel Base) at 0, 1, 3, 5, and 10 the Target Dose of 13.2 mg Pyrantel Base/kg Bodyweight Reference 0 1 Parameter Range b (F) (M) (F) (M) Specific gravity 1.025 1.030 1.030 1.026 1.030 1.060 (1.022 1.036) (1.026 1.036) (1.019 1.035) (1.015 1.033) ph 8.1 8.9 8.4 8.4 8.4 8.4 (8.0 8.9) (8.0 8.9) (8.0 8.9) (8.0 8.9) a Urine samples collected approximately 24 hours after administration of the first through the sixth doses. b Normal equine reference ranges from Aiello (ed): The Merck Veterinary Manual, ed 8. Whitehouse Station, NJ, Merck & Co, 1998; and IDEXX Veterinary Services, West Sacramento, CA. group to a maximum of 14 in the 10 group. Prefilled product syringes were calibrated with four weight-mark increments to deliver 1,800 mg pyrantel base/136.4 kg/increment for the tapeworm dosage of 13.2 mg pyrantel base/kg up to an animal weight of 545 kg/syringe. The commercial syringe is also separately calibrated with eight weight-mark increments to deliver 900 mg pyrantel base/136.4 kg/increment for the nematocidal dosage of 6.6 mg pyrantel base/kg, thus allowing for the treatment of tapeworms and nematodes with a single syringe of paste. Nematodes include large strongyles (Strongylus vulgaris, Strongylus edentatus, Strongylus equinus), small strongyles, pinworms (Oxyuris equi ), and large roundworms (Parascaris equorum). There were no adverse reactions observed on any of the treatment days in any of the treatment groups. Clinical Evaluations No treatment-related physical examination findings were observed, and no mortalities occurred during the study. All test animals were necropsied approximately 24 hours after the last day of the six-treatment-day regimen. No abnormal daily clinical observations were recorded during the study. Significant differences (P =.046) in the bodyweights of both female and male horses in the 1 group were detected on TDs 1 through 6 versus the 0 group (Table 1). Significant differences (P =.026) were also detected in the body temperatures of female horses on TD 3 in the 10 group versus the female horses in the 0 group (Table 1), but these recorded changes lacked a dose- and/or time-dependent trend. No treatment-related feed consumption or water consumption changes were observed during the study (Table 1). Hematologic values are summarized in Table 2. Significantly higher values (P =.0845) in the 1 group versus the 0 group were observed in both female and male horses on TDs 1 through 6 for erythrocyte counts. In addition, significantly higher values in hematocrit (P =.0894) and hemoglobin (P =.0382) were observed in both female and male horses in the 1 group compared with the 0 group on TDs 1 through 6. Female horses in the 1 (P =.0348), 3 (P =.0289), 5 (P =.0348), and 10 (P =.0348) groups had significantly low- 320
A. A. Marchiondo, T. N. TerHune, and R. L. Herrick Treatment Group c 3 5 10 (F) (M) (F) (M) (F) (M) 1.028 1.029 1.028 1.030 1.027 1.029 (1.020 1.033) (1.021 1.033) (1.020 1.036) (1.023 1.033) (1.017 1.034) (1.024 1.033) 8.4 8.5 8.4 8.4 8.4 8.4 (8.0 8.9) (8.0 8.9) (8.0 8.9) (8.0 8.9) (8.0 8.9) (8.0 8.9) c Values reported as geometric mean (range). F = mares, M = geldings. er banded neutrophil values than females in the 0 group on TD 2. On TD 1, male horses in the 1 (P =.0518), 3 (P =.0348), 5 (P =.0518), and 10 (P =.0518) groups had significantly lower banded neutrophil values than males in the 0X group. Finally, on TD 3, male horses in treatment groups 5 and 10 had significantly higher banded neutrophil values than males in the 0 treatment group (P <.0001 and P =.0243, respectively). These differences were not dose or time dependent, were in the range of pretest values, and/or were of such small magnitude that they were negligible and considered incidental with no test article related hematology changes. Significant differences were seen in serum biochemistry values for albumin, creatinine, total bilirubin, and potassium (Table 3). Total bilirubin values in female and male horses in the 1 group were significantly (P =.0008) higher than in horses in the 0 group. Creatinine values in female and male horses in the 1, 3, and 10 groups were significantly higher (P =.0004, P =.0270, and P =.0444, respectively) on TDs 1 through 6 than in female and male horses in the 0 group. Potassium values in female horses in the 1 and 3 groups had significantly (P =.0532) higher values than females in the 0 group. Female and male horses in the 10 group had significantly lower values (P =.0472) on TDs 1 through 6. Amylase values were below the published reference range during the pretest and dosing period, and no significant differences were observed among treatment groups. These differences were not dose or time dependent, were in the range of pretest values, and/or were of such small magnitude that they were negligible and considered incidental, with no test article related serum biochemistry changes. There were no test article related urinalysis changes (Table 4). Urinalysis values for protein, glucose, and ketones were negative. Urinalysis values of specific gravity were within normal limits for equine urine, and ph values were within pretest limits. Also, no test article related fecal examination changes were observed (data not provided). Equine organ weights were within the 0 treatment group limits, even though the liver values of both female and male horses were significantly higher (P =.0399) in the 10 treat- 321
Veterinary Therapeutics Vol. 6, No. 4, Winter 2005 TABLE 5. Summary of Organ Weights (in grams) of Horses Treated with Pyrantel Pamoate Paste (19.13% w/w Pyrantel Base) at 0, 1, 3, 5, and 10 the Target Dose of 13.2 mg Pyrantel Base/kg Bodyweight 0 1 Organ (F) (M) (F) (M) Brain 569 572 591 540 (487 612) (543 605) (532 657) (504 572) Heart 3,175 2,882 3,259 3,200 (2,805 3,798) (2,455 3,460) (2,870 3,840) (2,809 3,625) Kidneys 1,802 1,743 1,832 1,554 (1,371 2,156) (1,580 1,961) (1,613 1,977) (1,338 1,806) Liver 5,809 4,974 5,964 5,265 (4,231 7,003) (4,371 5,735) (5,661 6,519) (4,845 5,722) Spleen 7,110 5,440 6,186 5,796 (5,027 9,765) (1,332 9,880) (1,990 9,288) (1,860 13,152) Ovaries 176 NA 184 NA (136 226) (141 258) a Significant difference (P =.0399) for 10 compared with 0. F = mares, M = geldings. ment group (Table 5). There were no gross pathologic or histopathologic correlates for the weight difference, and the organ weight difference was of small magnitude and, thus, was considered incidental. Daily administration of pyrantel pamoate paste at 1, 3, 5, and 10 times the proposed clinical dose of 13.2 mg of pyrantel base/kg for 6 consecutive days did not produce any macroscopic or microscopic tissue effects in any dose group of either sex. DISCUSSION Pyrantel, a member of the tetrahydropyrimidine class of anthelmintics, is a cholinergic agonist that acts as a depolarizing neuromuscular blocking agent against target parasites. Pyrantel is available in the pamoate (also known as embonate), hydrochloride, and citrate and tartrate salt forms. Pyrantel pamoate is a yellow to tan solid and is poorly absorbed from the gastrointestinal tract. 12 The objective of this study was to assess the safety of pyrantel pamoate when administered to horses daily for 6 consecutive days at 0, 1, 3, 5, and 10 times the proposed clinical dose of 13.2 mg of pyrantel base/kg administered. The study noted statistically significant differences between control and treated test animals in bodyweight, body temperature, hematocrit, hemoglobin, erythrocyte counts, banded neutrophils, albumin, creatinine, total bilirubin, potassium, and absolute liver weight. These differences were neither dose nor time dependent, were within the range of normal or pretest values, and/or were of such small magnitude that they were negligible. Therefore, all these differences from control were considered incidental. Based on these findings, 322
A. A. Marchiondo, T. N. TerHune, and R. L. Herrick Treatment Group 3 5 10 (F) (M) (F) (M) (F) (M) 586 611 615 601 584 577 (540 633) (532 671) (560 699) (551 700) (555 621) (531 620) 2,924 3,569 3,192 3,214 3,240 3,181 (2,633 3,401) (2,804 3,985) (2,460 3,914) (2,634 3,928) (2,421 3,883) (2,652 3,551) 1,499 1,725 1,661 1,711 1,649 1,606 (1,271 1,778) (1,510 1,823) (1,369 2,016) (1,471 1,951) (1,567 1,704) (1,503 1,732) 4,917 5,418 6,095 5,886 6,205 a 6,149 a (3,834 5,419) (4,642 6,612) (5,466 7,476) (5,813 6,008) (5,559 6,817) a (5,809 6,842) a 5,907 8,803 6,701 9,439 7,295 4,458 (1,401 10,536) (1,953 15,999) (1,250 12,508) (3,513 18,292) (2,313 11,892) (2,321 6,545) 121 NA 167 NA 171 NA (111 131) (82 238) (150 216) the no-observed-effect-level (NOEL) for 19.3% w/w pyrantel pamoate paste, when administered orally to horses once daily for 6 consecutive days, was determined to be 132 mg/kg/day. Dose confirmation and clinical field efficacy studies have determined that the dose of 13.2 mg of pyrantel base/kg administered as a single treatment provided 95.5% to 98.4% and 95% efficacy, respectively, against A. perfoliata in naturally infected horses. 8,9 In conclusion, this study has shown that pyrantel pamoate can be safety administered to horses according to the proposed clinical dose of 13.2 mg of pyrantel base/kg. ACKNOWLEDGMENT The authors thank Philip H. Long, DVM, PhD, DACVP, and George A. Milliken, PhD, for their expert assistance during this study. REFERENCES 1. Lyons ET, Drudge JH, Tolliver SC: Critical tests of three salts of pyrantel against internal parasites of the horse. Am J Vet Res 35:1515 1522, 1974. 2. Slocombe JOD: Prevalence and treatment of tapeworms in horses. Can Vet J 20:136 140, 1979. 3. Lyons ET, Drudge JH, Tolliver SC, et al: Determination of the efficacy of pyrantel pamoate at the therapeutic dose rate against the tapeworm Anoplocephala perfoliata in equids using a modification of the critical test method. Vet Parasitol 31:13 18, 1989. 4. Lyons ET, Tolliver SC, Drudge JH: Further evaluation of pyrantel pamoate at the therapeutic dose rate (6.6 mg base/kg) against Anoplocephala perfoliata in horses. J Helminthol Soc Wash 64:285 287, 1997. 5. Lyons ET, Drudge JH, Tolliver SC: Pyrantel pamoate: Evaluating its activity against equine tapeworms. Vet Med 81:280 285, 1986. 6. Slocombe JOD, De Gannes R, Lake M: Effectiveness of pyrantel pamoate for Parascaris resistant to macrocyclic lactones [abstract]. Proc 49 th AAVP/79 th ASP 49: 46, 2004. 323
Veterinary Therapeutics Vol. 6, No. 4, Winter 2005 7. Höglund J, Nilsson O, Ljunström B-L, et al: Epidemiology of Anoplocephala perfoliata infection in foals on a stud farm in south-western Sweden. Vet Parasitol 75:71 79, 1998. 8. Reinemeyer CR, Hutchens DE, Marchiondo AA: Dose confirmation studies of the cestocidal activity of pyrantel pamoate paste in horses. Vet Parasitol, submitted for publication, 2005. 9. Marchiondo AA, White GW, Smith LL, et al: Clinical field efficacy and safety of pyrantel pamoate paste (19.13% w/w pyrantel base) against Anoplocephala spp. in naturally-infected horses. Vet Parasitol, submitted for publication, 2005. 10. 2000 Report of the AVMA Panel on Euthanasia. JAV- MA 218(5):669 696, 2001. 11. Westfall PH, Tobias RD, Rom D, et al: Multiple comparison and multiple tests: Using the SAS System. Cary, NC, SAS Institute, 1999. 12. Aiello SE (ed): The Merck Veterinary Manual, ed 8. Whitehouse Station, NJ, Merck & Co, 1998. 324