Efficacy of anti-tumor necrosis factor therapy in the treatment of spontaneous endometriosis in baboons

FERTILITY AND STERILITY威 VOL. 81, SUPPL. 1, MARCH 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.

Efficacy of anti-tumor necrosis factor therapy in the treatment of spontaneous endometriosis in baboons Breton F. Barrier, M.D.,a G. Wright Bates, M.D.,a M. Michelle Leland, D.V.M.,b D. Alan Leach, M.D.,a Randal D. Robinson, M.D.,a and Anthony M. Propst, M.D.a Southwest Foundation for Biomedical Research, San Antonio, Texas

Received August 3, 2003; revised and accepted September 26, 2003. Supported by Immunex Corp. (a wholly owned subsidiary of Amgen Inc.), Seattle, Washington. Presented at the 50th Annual Meeting of the Society for Gynecologic Investigation, Washington, D.C., March 26 –30, 2003. The views expressed in this article are those of the authors and do not reflect the official opinion of the Department of Defense or the United States Air Force. Reprint requests: Breton Barrier, M.D., Department of Obstetrics and Gynecology, 2200 Bergquist Drive, Suite 1, Lackland AFB, Texas 78236 (FAX: 210-292-6158; E-mail: breton.barrier@ lackland.af.mil). a Department of Obstetrics and Gynecology, San Antonio Uniformed Services Health Education Consortium, Wilford Hall Medical Center, Lackland AFB, Texas. b Department of Physiology and Medicine, Southwest Foundation for Biomedical Research. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 09.034

Objective: To determine the efficacy of anti-tumor necrosis factor therapy (etanercept) for treating endometriosis in baboons. Design: A randomized, placebo-controlled, blinded study using the baboon endometriosis model. Setting: Southwest National Primate Research Center. Animal(s): Twelve female baboons with spontaneous peritoneal endometriosis. Intervention(s): Etanercept (n ⫽ 8) or sterile water (n ⫽ 4) was administered subcutaneously three times per week. Main Outcome Measure(s): After 8 weeks, the number, color, and surface area of peritoneal lesions was evaluated. Revised American Society for Reproductive Medicine staging was used. Result(s): A statistically significant decrease in red lesion surface area in the treatment group was observed. A trend toward a decrease in the absolute number of red lesions was noted in the treatment group. White and black lesion number and total surface area slightly increased in both groups but failed to achieve statistical significance. Endometriosis was diagnosed in 60% of captive-born baboons with primary infertility. Conclusion(s): These results indicate that etanercept effectively reduces the amount of spontaneously occurring active endometriosis in the baboon. (Fertil Steril威 2004;81(Suppl 1):775–79. ©2004 by American Society for Reproductive Medicine.) Key Words: Endometriosis, baboon, infertility, etanercept, anti-TNF therapy, peritoneal lesions

Immunologic factors and inflammatory mediators are thought to be significantly involved in the pathophysiology of endometriosis. Studies have shown that women with endometriosis have higher levels of tumor necrosis factor (TNF) in peritoneal fluid (1– 4). Tumor necrosis factor is a proinflammatory cytokine and is known to stimulate the expression of matrix metalloproteinases by endometriotic tissues (5, 6). Matrix metalloproteinases actively participate in the invasion and matrix remodeling of endometriotic lesions (7– 9). Tumor necrosis factor also stimulates the expression of prostaglandin synthase-2, which in turn increases the production of prostaglandin E2 (10). New evidence suggests that prostaglandin E2 might up-regulate the expression of aromatase in endometriotic lesions, leading to endogenous steroid synthesis and support of the lesions (11).

Despite the clear link between aberrant inflammatory processes and endometriosis, current treatment options are limited to suppression of hormonal stimulation. Although the induction of a hypoestrogenic state effectively reduces pain, the adverse effects decrease compliance and limit long-term usage (12). Moreover, the disease often recurs after discontinuation of current therapies (13). Anti-TNF therapy is a potential, nonhormonal therapeutic option for treatment of endometriosis. In one study (14), endometriosis was prevented in baboons whose pelvic cavity was inoculated with menstrual aspirate that was treated with recombinant TNF receptor-1. Although the control animals developed characteristic endometriotic lesions, treated animals whose menstrual aspirate was depleted of TNF did not develop any lesions. This effect was identical to that produced by treatment of the 775

animals with leuprolide acetate, a commonly used GnRH agonist. The baboon has emerged as an ideal animal model for endometriosis (15). Baboons are afflicted with spontaneous endometriosis of differing stages, and in these animals the disease is often progressive (16). Both induced and spontaneous endometriosis in baboons cause macroscopic lesions that show remarkable similarity to the lesions observed in the human disease (17). The purpose of this study was to explore the efficacy of etanercept for treating established endometriosis in baboons. Etanercept, a fusion protein consisting of human recombinant soluble TNF receptor-2 (p75) conjugated to a human Fc antibody subunit, neutralizes TNF activity and is currently used to reduce signs and symptoms of rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis.

MATERIALS AND METHODS Use of the animals in this study was approved by the Southwest National Primate Research Center’s Institutional Animal Care and Use Committee (San Antonio, TX). Twenty healthy female baboons with primary infertility and three additional animals with a past history of endometriosis found at cesarean section underwent selection for diagnostic laparoscopy. The mean (⫾SD) age of animals with primary infertility was 10.8 years ⫾ 2.3 years, and all had been exposed to fertile male baboons throughout their reproductive lives without successful pregnancy. Once an animal had been scheduled for surgery, it was separated from the colony, sedated with ketamine, intubated, and administered general anesthesia. Vaginal examination was performed under anesthesia. Animals with significant vaginal stenosis that prevented cannulation of the cervix for uterine manipulation were excluded from the study (n ⫽ 5). Laparoscopy of the remaining animals was performed with a rigid laparoscope. A high-resolution photographic printer and compact disk-writing hardware (Stryker Endoscopy, San Jose, CA) were used with video recordings to document findings. Of the 15 laparoscopically screened animals with primary infertility, 9 animals (60%) were diagnosed with endometriosis and enrolled in the study, whereas 6 had no evidence of endometriosis. All baboons had spontaneous cyclic menses, although detailed cycle information was not recorded before or after the study. The medical histories of the animals included no autoimmune diseases or prior exposure to immunomodulatory substances. At the time of laparoscopy, a representative depiction of the lesion size and shape was documented on a standard pelvic diagram. The color of the lesion, location, and any associated adhesions were recorded. Lesion color was noted to be one of three types: red, white, or black. Red lesions appeared grossly as red or pink nonblanching lesions or 776

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vesicular (clear) lesions. White lesions were opaque, nonpigmented lesions. Black lesions appeared grossly as yellowbrown, black, or blue pigmented lesions. Lesion surface area was calculated from measurements made with 5-mm trocars with tips scored at 1-mm increments. Because most lesions were circular, lesion surface area was calculated with the formula ␲r2 and expressed as millimeters squared. For polygonal lesions, the length was multiplied by the width to obtain lesion surface area. The lesion depth was roughly estimated based on lesion surface area to facilitate modification of revised American Society for Reproductive Medicine (rASRM) staging criteria for the baboon, as previously described (16). Once the laparoscopic procedure was complete, the animal was allowed to recover in an individual cage and remained there for the duration of the 8-week study. Treatment and control groups were randomly assigned to receive either etanercept (Enbrel; Immunex Corp., Seattle, WA) or placebo (vehicle: sterile water for injection) administered as a subcutaneous injection three times per week for 8 weeks. Animals were randomized by a third party not participating directly in the study; folded pieces of paper were drawn from a container with the assigned group named on them. All laparoscopies were performed and scored by the same surgeon (B.F.B.), who was blinded to the randomization results until all data were collected and ready for processing. Because of the small number of animals, to maximize power in the drug group for detecting a pre- to posttreatment difference, a 2:1 treatment-to-control random allocation format was used. Treatment was begun the Monday after laparoscopic diagnosis (if diagnosed on a Monday, treatment was started the same day). Veterinary medical staff monitored the animals daily for general health and for any signs of significant local inflammation at the injection site or anaphylactoid reactions. No local or systemic reactions or respiratory tract infections were noted in the treatment animals. Eight baboons received etanercept at a dose level of 2 mg/kg. Placebo was administered at a similar volume and frequency to four baboons. To ensure adequate serum concentrations of etanercept beyond 4 weeks, a higher dosage and an increased frequency of dosing (three times per week) than the standard dose given to humans (16 mg twice weekly) was used in this study. This decision was based on data from a previous study of cynomolgus monkeys and etanercept, in which a higher renal clearance and the development of neutralizing antibodies within 4 weeks led to a more rapid clearance of study drug than is seen in humans. The weights of the baboons used in this study ranged from 12.4 kg to 26.3 kg, for a total dosage of 25 mg to 53 mg three times weekly subcutaneously. In women with rheumatoid arthritis, the standard dosage is 25 mg twice weekly subcutaneously. Therefore, the dosage used in baboons on a Vol. 81, Suppl 1, March 2004

TABLE 1 Summary of lesion changes and impact on rASRM staging. Number/total surface area (mm2) of peritoneal lesionsa Pretreatment Animal ID no. 9490 9502 9265 9321 1660 9936 9316 7561 9774 8540 9907 a b

rASRM score (stage)b

Posttreatment

Mode

R

W

B

R

W

B

Pretreatment

Posttreatment

Etanercept Etanercept Etanercept Etanercept Etanercept Etanercept Etanercept Vehicle Vehicle Vehicle Vehicle

4/4.1 9/68.9 8/33.8 4/3.3 10/136.2 7/15.8 5/19.1 2/6.7 2/12.8 13/32.4 3/1.6

1/0.8 3/10.4 8/24.8 7/1294 2/26.7 3/6.5 1/7.1 0 5/103.5 8/37.9 3/4.1

0 0 0 0 3/20.4 0 6/82.5 0 2/1.6 1/7.1 0

0 2/28.3 0 0 7/56.5 4/3.7 0 1/0.8 10/54.6 8/36.1 6/11.7

2/3.9 4/26.7 4/38.5 3/1260 4/55.8 6/16.5 3/13.4 2/4.1 6/111.2 7/94.8 4/16.9

0 1/1.8 0 0 8/88 0 6/78.5 0 3/3.4 0 1/0.2

6 (II) 10 (II) 12 (II) 7 (II) 60 (IV) 14 (II) 130 (IV) 13 (II) 9 (II) 12 (II) 10 (II)

7 (II) 10 (II) 6 (II) 6 (II) 36 (III) 14 (II) 130 (IV) 11 (II) 24 (III) 12 (II) 18 (III)

Surface area of lesions is rounded to the nearest whole number. Red, white, and black lesions are in “R,” “W,” and “B” columns, respectively. Revised ASRM staging modified for the baboon as described by D’Hooghe et al. (16).

Barrier. Anti-TNF therapy for endometriosis. Fertil Steril 2004.

per-kilogram basis per week was approximately sevenfold higher than that used in humans, but this was necessary to obtain similar serum levels. Blood samples were taken at predetermined times on days 8, 10, 12, 15, 22, 24, 26, 29, 36, 43, 50, 52, and 54. The blood samples were taken immediately before administration of the next dose; these samples represented the concentration trough values. The presence of anti-etanercept antibodies was analyzed on days 15, 29, and 54. Blood samples were allowed to clot, after which they were centrifuged, and the serum was removed and frozen before shipment. Samples were shipped and analyzed by the etanercept manufacturer (Immunex Corp.) for the presence of drug and the presence of antibodies formed to the drug. Laparoscopies were timed for the late luteal phase, although one repeat laparoscopy occurred out of phase (midcycle) because of cycle irregularity. Repeat laparoscopy was performed within 1 week of the last subcutaneous injection. At the time of repeat laparoscopy, biopsies of any remaining endometriotic lesions were performed for histologic confirmation of disease by an independent, blinded veterinary pathologist. Pretreatment and posttreatment data (rASRM score, surface area, and absolute number of lesions) were not normally distributed and were therefore evaluated with the Wilcoxon signed rank test (with significance defined as ⬍.025) and Mann-Whitney U test (with significance defined as ⬍.05), as indicated for paired and multiple comparisons, respectively. One animal was removed from the drug group during the first week of treatment because of anorexia and weight loss. Physical examination suggested the presence of a trichoFERTILITY & STERILITY威

bezoar (“hairball”) in the stomach, and this condition was confirmed at laparotomy. At the time of surgical removal of the bezoar, the endometriotic lesions were removed as well, allowing for early histologic confirmation of lesions in this animal. These symptoms were attributed to the bezoar and not believed to have resulted from administration of the study drug.

RESULTS A summary of individual lesions is given in Table 1. A statistically significant pretreatment to posttreatment decrease in red lesion surface area was noted within the etanercept group (Wilcoxon signed rank test, P⫽.018) (Fig. 1). To ensure that this decrease in red lesion surface area was attributable to etanercept, the percent change before and after treatment in the etanercept group was compared with that of the control group, and a significant difference was confirmed (Mann-Whitney U test, P⫽.042) (Fig. 2). Although no statistically significant change in white or black lesion surface area was noted in the etanercept group, there was a trend toward an increase in white lesion surface area within the placebo group (P⫽.068). The placebo group contained an increase in red peritoneal lesion surface area at the time of repeat laparoscopy, but this did not achieve significance (P⫽.273). When the absolute number of red, white, and black lesions was compared between pre- and posttreatment, a trend toward a decrease in the number of red lesions was noted in the etanercept group (P⫽.073), whereas no significant changes were noted in the number of white and black lesions. Revised ASRM staging was used for all animals before and after laparoscopy. This staging was applied to the ba777

FIGURE 1

FIGURE 3

Pre- and posttreatment lesion surface area in animals treated with either etanercept (brown and light brown bars, n ⫽ 7) or vehicle (purple and light purple bars, n ⫽ 4).

Concentration (trough)-time curves of animals treated with etanercept (n ⫽ 7). Each line represents one animal. Only one animal (9490, dark blue diamond) was unable to maintain a measurable serum concentration of etanercept.

Barrier. Anti-TNF therapy for endometriosis. Fertil Steril 2004. Barrier. Anti-TNF therapy for endometriosis. Fertil Steril 2004.

boon as previously described (16). Although some differences between pre- and posttreatment rASRM score and stage were observed (Table 1), the differences were not statistically significant. Biopsies of endometriotic lesions from 8 of the 11 animals were performed for histologic confirmation of endometriosis. The 3 remaining animals had either no material available for biopsy, or the biopsy was technically impossible. Of the biopsies performed, four of five samples from

FIGURE 2 Percent change in red lesion surface area between first and second laparoscopy in animals treated with etanercept (brown bar) and with placebo (purple bar).

treatment animals confirmed the diagnosis of endometriosis, as did all three samples from the control group. One of the confirmed biopsies from the treatment group showed only glandular epithelial cells and no recognizable stroma, which is consistent with, but not diagnostic of, endometriosis. With the exception of one black lesion, only red lesions were biopsied. One representative biopsy per animal was taken. One had a black lesion and six had red lesions that were biopsied. Of the seven biopsies, only one (a red one) returned negative, for a confirmation rate of 86%. No etanercept or anti-etanercept antibodies were detected in serum from the control group (Fig. 3). Most treated animals developed anti-etanercept antibodies, causing a sharp drop in the concentration-time profiles after the second week. After repeated administration of etanercept, a steadystate concentration of etanercept was achieved in most animals. Only one animal (9490) developed a vigorous antibody response that prevented maintenance of an adequate serum concentration. However, this animal, which had rASRM stage II disease composed of four red lesions and one white lesion, seemed to respond to treatment with resolution of all red lesions and persistence of two white lesions (Table 1).

DISCUSSION

Barrier. Anti-TNF therapy for endometriosis. Fertil Steril 2004.

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This study has shown that 8 weeks of treatment with etanercept diminishes the amount of spontaneously occurring active endometriosis in the baboon. It has been hypothesized that red lesions represent active disease, whereas white and black lesions represent the fibrosis and hemosiderin deposition left by “burned out” red lesions (18). The temporal relationship between the disappearance of red lesions in the drug group and a slight increase in size and Vol. 81, Suppl 1, March 2004

number of white and black lesions (Table 1) is consistent with this hypothesis. The improvement noted in rASRM score did not achieve statistical significance because this scoring system does not account for lesion type; red lesions had been replaced with white and black lesions, and the absolute amount of measurable disease did not change appreciably. The number and size of lesions in treatment and placebo groups were different at baseline laparoscopy. This was owing to the large variability in the amount of disease in each animal combined with random allocation to study group. With a larger number of animals, this might even out and approach population norms. Despite these baseline differences, our statistics for comparing the two groups are valid because we compared the proportional change in total red lesion surface area, not the change in absolute number or size of lesions. Initial diagnosis of endometriosis was based on gross appearance of lesions, and biopsy for confirmation was not performed before treatment was begun. Because lesions were typically small, pretreatment biopsies would have compromised the integrity of the study. A previous study reported greater than 90% histologic confirmation of disease for subtle red lesions (17). All animals enrolled in the study had at least two of these lesions present. When an animal with a tricho-bezoar was removed from the study, we found that all of three red lesions removed from the animal confirmed our initial diagnosis. Similarly, 86% of lesions biopsied at the conclusion of the study contained histologic confirmation of endometriosis, as determined by a veterinary pathologist blinded to the results. Current (GnRH agonists) and investigational (aromatase inhibitors, GnRH antagonists) treatments for endometriosis have distinct disadvantages because of their mechanism of action. All of these treatments cause a decrease in E2 concentration, and might potentially lead to a significant loss of bone mineral density with their long-term use. We have demonstrated that etanercept, which has no known serious adverse effects with long-term usage, reduces the amount of active endometriosis in the well-accepted baboon model. Etanercept might offer a safe, nonsurgical, nonhormonal option for the long-term treatment of this disease in humans.

Acknowledgments: The authors thank Michael Strauss, B.S., and Shannon Theriot, B.S., for their expert assistance in the operating room; Dr. Gene Hubbard, D.V.M., for his expert pathologic interpretation of the biopsies;

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and Drs. Karen Rice, Ph.D., and Elaine Winhurst, B.S., for selecting and preparing the animals used in this study. The authors also thank workers at Immunex Corp., including Drs. Mark Rogge, Ph.D., and Jeanine Bussiere, Ph.D., for project oversight; Dr. Charles Foerder, Ph.D., for pharmacokinetic and antibody sample analysis; and Linda Carlock, M.S., for study monitoring.

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