Methodological Issues in Preclinical Mouse Efficacy Studies of Adenomyosis

Adenomyosis is a benign, non-neoplastic gyne- cologic disorder with a poorly understood pathogenesis. Its treatment has been a challenge, with hysterectomy being considered the definitive therapy for severe adenomyosis. More efficacious drugs with better side-effect and cost pro- files are sorely needed. Unfortunately, there is a dearth of registered clinical trials on adenomyosis, and the registered trials are not testing new compounds. This appears to sug- gest a loss in translating discoveries made in preclinical studies to clinical practice. This article presents a systematic review of all published mouse efficacy studies of adeno- myosis aimed at the evaluatio n of potential therapeutic compounds, focusing on methodological quality and de- scribing the limitations of the published studies. The com- plex issues involved in transl ating preclinical studies to clinical practice will be summarized. Lastly, some method- ological issues in mouse efficacy studies of adenomyosis will be highlighted, with the aim to improve such studies in the future.

Adenomyosis . Animal studies . Drug development . Efficacy . Methodological quality . Mouse . Outcome measures

Adenomyosis is a benign, non-neoplastic gynecologic disor- der characterized by the ectopic proliferation of endometrial tissue into the myometrium with smooth muscle hypertro- phy. A common disorder with a prevalence in hysterecto- my specimens ranging from 14 % to 66 % [ 1], its common presenting symptoms include abnormal uterine bleeding, dysmenorrhea, and subfertility [ 2], although a sizeable portion of patients are asymptomatic [ 3]. Dysmen- orrhea is the most debilitating symptom [ 4]. Treatment of adenomyosis has been a challenge, with hysterectomy being considered the definitive therapy for severe adeno- myosis [ 5]. Uterine artery embolization (UAE) also has shown promise, but a recent review noted that the recur- rence risk after UAE is high [ 6]. Although adenomyosis is hormone-sensitive, progesto- genic agents are not very effective, and gonadotrophin- releasing hormone (GnRH) agonists induce suppression of adenomyosis, yet their use is restricted by short duration [ 7]. In addition, the symptoms quickly reappear after discontin- uation of GnRH agonist therapy [ 8]. While the insertion of a levonorgestrel-releasing intrauterine system (LNG-IUS) has been reported to result in a reduction in adenomyosis- associated pain and abnormal bleeding [ 9], side effects such as spotting are reported in one third of the women, with many women experiencing oligomenorrhea [ 9]. Conse- quently, more efficacious drugs with better side-effect and cost profiles are sorely needed. Despite this unmet medical need, a search of clinical trial registries suggests few new agents are being investigated. Compared with 1,679 trials on ovarian cancer, 146 on uterine fibroids, and 132 on endometriosis, only 15 trials on adenomyosis were registered ( www.ClinicalTrials.gov, accessed on June 5, 2012). Among the 15 trials, 1 was not yet recruiting, 1 was active but not recruiting, 3 were com- pleted, 4 were recruiting, and 6 had an “unknown” status, meaning that “the trial status has not been verified in more t h a n2y e a r s .” Of the 15 trials, only 4 trials evaluated S.-W. Guo (*) Shanghai Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, China e-mail: [email protected] Curr Obstet Gynecol Rep (2012) 1:138 –145 DOI 10.1007/s13669-012-0018-3 nonsurgical treatment: one on progestin, one on acupunc- ture, one on LUS-IUD, and one on letrozole, an aromatase inhibitor. There are no novel compounds currently under trial evaluation. The completed trial on letrozole indicated that its efficacy, evaluated 12 weeks after treatment, was comparable with the GnRH agonist [ 10]. Thus, the research and development (R&D) for medical treatment of adeno- myosis seem to be limited. One indispensable component of preclinical research in drug R&D is animal efficacy studies. This review will focus on the methodology of mouse efficacy studies through a systematic review of all published mouse efficacy studies of adenomyosis evaluating potential therapeutic compounds and will describe the limitations in these studies. With the exception of danazol and danazol-containing intrauterine devices (IUDs) that were tested clinically before mouse studies, no compound evaluated preclinically has been test- ed clinically or in clinical trials, at least not within the public domain. The completed trial on letrozole was conducted without an animal study demonstrating its therapeutic po- tential. Many complex issues are involved in translating preclinical studies to clinical practice. Some methodological aspects of preclinical animal studies of adenomyosis will be reviewed, with the aim to improve mouse efficacy studies of adenomyosis.

A systematic and comprehensive search of PubMed was per- formed for all studies published up to June 5, 2012, using the following search terms: “adenomyosis” or “endometriosis”in combination with“mouse,”“ mice,”“ rat,”“ rodent,” or “animal model.” Included studies used rodent models of adenomyosis, not endometriosis and evaluated the therapeutic potential of a compound or compounds. The search was limited to publica- tions written in English. Thus, articles on endometriosis, but not adenomyosis per se, were excluded. The focus on mouse studies is of necessity because no other animal models of adenomyosis have been used for efficacy studies. The search resulted in 14 studies of mouse models of adenomyosis [ 11–20, 21, 22, 23, 24]. Of the 14 studies, four were excluded because one study started the interven- tion from the day of birth [ 24], one reported an intervention that was not effective [ 13], one actually focused on mam- mary tumors and mentioned adenomyosis as a secondary end point [ 15], one that tested the same compound that was evaluated previously [ 16], and one study to test feasibility, but not efficacy, of an intervention [ 20]. The characteristics of the remaining ten studies, along with the major treatment outcome measures in the control group and the group re- ceiving the highest dose, representing the best treatment effect, are listed in Table 1.

of Inducing Adenomyosis in Mouse Studies Greaves and White [ 26] recently reviewed animal models of adenomyosis, including mouse models. Briefly, adenomyo- sis can be induced in mice through various forms of hor- monal imbalance, such as hyperprolactinemia [27, 28], prolonged treatment with or prenatal exposure to estrogenic compounds [ 27, 29], and prolonged treatment with proges- terone [30, 31]. For efficacy studies, however, none of these induction methods have been used. One reason is the long induction period. The other reason is their nonspecific ef- fect, such that in addition to inducing adenomyosis, other pathological changes in the reproductive tract are also in- duced. For example, prenatal exposure to diethylstilbestrol also results in cervix enlargement with extensive mucoid alterations within the muscularis [ 27]. For efficacy studies, some publ ished studies used the SHN mouse strain, which has spontaneous adenomyosis [32]. Others have used one of two adenomyosis induction methods, ectopic grafting of pituitary glands (EGPG) or neonatal feeding with tamoxifen (NFT). In EGPG, adeno- myosis is induced by intrauterine implantation of isographs of anterior pituitary glands [ 33–35]. This induction method has the advantage of a high success rate, reported to be greater than 90 % 50 days after induction [ 16], and the ultrastructural changes in myometrium from mice with in- duced adenomyosis have been documented [ 36]. The limi- tations of this approach include that the induction procedure requires a certain level of surgical skill such that surgical changes might be a confounding effect in efficacy studies. A second limitation is that the success rate is strain dependent, with high rates in SHN and SLN mouse strains but lower rates in other strains [ 34, 35]. A third limitation is the long induction period of 4 months in an strain of ICR mice to reach a success rate of 95 % [ 21] and a 6-month induction period in C3H and F1 hybrids with a variable success rate [35]. Lastly, the induction method appears to be sensitive to treatment with dimethyl sulfoxide (DMSO), often used as a vehicle for many compounds. In Dr. Mori ’s lab, the success rate of adenomyosis induction after EGPG followed by DMSO treatment was reduced to 80 % in one study [ 18] and 40 % in another [ 19]. The anti-inflammatory effect of DMSO may be related to its use to treat intractable intersti- tial cystitis, radiation cystitis, chronic prostatitis, and chron- ic female trigonitis [ 37]. DMSO also is used most as a topical analgesic [ 38]. Thus, if the induction is truly sensi- tive to DMSO, an underestimation of the efficacy of studied compounds may occur when DMSO is used as a solvent. Neonatal feeding with tamoxifen was first reported by Parrott et al. [ 39, 40] in CD-1 mouse. A previous study of neonatal exposure to tamoxifen, delivered through subcuta- neous injection, in the CD-1 mouse resulted in atypical hyperplasia in the endometrium, uterine adenocarcinoma, Curr Obstet Gynecol Rep (2012) 1:138 –145 139 Table 1 Characteristics of published preclinical mouse efficacy studies ID* Study Compound(s) tested Mouse strain

of induction Length of induction, d Treatment duration, d Major outcome measure Randomization mentioned? Dose– response? Total sample size ** Trt*** Ctl COI statement? D1, Singtripop et al. [11] Danazol SHN S NA 35 (+35 d no Tx) P Y es No 8+13 0.375 0.846 No D2a EGPG 0 35 (+35 d no Tx) Y es No 9+11 0.222 1.00 K Mori et al. [ 12] KBG (Chinese herb medicine) SHN S NA 95 P , H Y es No 11+13 0.182 No M Zhou et al. [ 14] Mifepristone (SPRM) SHN EGPG 21 28 P . UWW Y es No 10+9 0.200 0.778 No O1 Mori et al. [ 16] ONO-4817 SHN EGPG 7 42 P , FI, GLCI No Yes 12+12 0.083 0.917 No O2a (MMP inhibitor) EGPG 42 28 No No 10+10 1.00 1.00 C1 Mori et al. [ 17] CP8816 SHN EGPG 0 41 P No No 10+10 0.10 0.80 No C2a CP8863 7 35 No No 12+10 0.083 0.90 (SPRM) T Zhou et al. [ 18] TNP-470 (anti- antiogenic agent) SHN EGPG 1 41 P ,BW,OW,EC Y es No 15+15 0.00 0.80 No P Zhou et al. [ 19] Probucol (hypo- cholesterolemic agent) SHN EGPG 1 41 P ,BW,OW,SL Y es No 10+10 0.00 0.40 No U Zhang et al. [ 21] Danazol-IUD ICR EGPG 120 60 UWW/BW ratio, N, HE Y es Yes 5+5 2.2 (5.6) 2.5 (11.5) No VL i u a n d G u o [ 22] VPA (HDACI) ICR NFT 84 28 UWW/BW ratio, D, HP , TF Y es Yes 12+12 0.0030 0.0047 b Yes 12.1 10.3 H Mao et al. [ 23] l-THP , Andro, VPA ICR NFT 107 21 D, HP , UT, UWW Y es Yes 10+8 1.0 c 2.5 Yes COI conflict of interest, S spontaneous, EGPG ectopic graft of pituitary gland, Tx treatment, P prevalence, KBG Keishi-Bukuryo-Gan, H histology, SPRM selective progesterone receptor modulator, UWW uterine wet weight, MMP matrix metalloproteinase, FI food intake, GLCI graded levels of cell invasiveness, BW body weight, OW organ weight, EC estrous cycle, SL serum levels of lipids, IUD intrauterine device, UWW/BW ratio uterine wet weight vs body weight ratio, N number of nodules, HE hematoxylin eosin staining, VP Avalproic acid, HDACI histone deacetylase inhibitor, NFT neonatal feeding of tamoxifen, D depth of myometrial infiltration, HP hotplate test, TF tail-flick test, l-THP levo-tetrahydropalmatine, Andro andrographolide, UT uterine contractility * Identification of study on graph in Fig. 1. ** Sample size of the treatment and the control groups combined. *** The treatment with the best effect, typically the group receiving the highest dose or joint treatment. a Two experiments were conducted and were thus labeled differently b The first row was the UWW/BW ratio, and the second was the hotplate response latency (in seconds) c The depth of myometrial infiltration 140 Curr Obstet Gynecol Rep (2012) 1:138– 145 and uterine hypoplasia with focal areas of basal cell hyper- plasia in the endometrium, but not adenomyosis [ 41]. Be- cause the ICR mouse is genetically related with the CD-1 mouse, NFT with the ICR mouse yielded identical results in inducing adenomyosis [ 22]. Compared with the EGPG method, NFT is much simpler, requiring no surgical skill. In addition, it also has a high success rate in induction: 3 months after the induction procedure, all mice have adeno- myosis [22, 40]. In fact, by day 42 or 37 days after the NFT, the success rate is 100 % [ 42]. Like the EGPG method, the NFT also has the limitation that the success of the induction is highly strain dependent [ 43]. Features of Published Mouse Efficacy Studies From Table 1, several notable features can be seen. First, the earlier (up to 2004) mouse efficacy studies were conducted exclusively in Japan, specifically, in Dr. Mori ’s lab in the University of Tokyo, which first reported that EGPG resulted in a high rate of development of adenomyosis [44]. In SHN mouse strain, which has a high incidence of mammary tumors, an unusually high incidence of spontane- ous adenomyosis also was reported [ 45]. In this model, an increase in plasma levels of prolactin is noted [ 28]. Diverse compounds have been evaluated for their thera- peutic potential. These included danazol, a Chinese herb concoction, a matrix metalloproteinase (MMP) inhibitor (ONO-4817), several selective progesterone receptor mod- ulators (SPRMs [mifepristone, CP-8816, and CP-8863]), a danazol-containing IUD, a histone deacetylase inhibitor (valproic acid [VPA]), an anti-angiogenic agent (TNP- 470); a hypocholesterolemic agent (probucol), an analgesic (levo-tetrahydropalmatine [l -THP]), and a nuclear factor (NF)-κB inhibitor (andrographolide). Of these, only danazol [46], VPA [ 47, 48], and andrographolide [ 49], have been evaluated in in vitro or clinical studies for endometriosis or adenomyosis. Despite promising results, only danazol has been tested in clinical trials or is in use clinically today. However, adenomyosis usually fails to respond to oral danazol therapy [ 50]. Earlier studies were focused on biological plausibility. For example, because adenomyosis involves cell migration or invasion, which is associated with the degradation and reconstruction of the extracellular matrix in which MMPs are involved, MMP inhibitors were studied. In contrast, more recent, studies have been designed based on previous molecular studies. For example, because adenomyosis may be an epigenetic disease [ 51], it may be treatable by histone deacetylase inhibitors similar to endometriosis [ 52]. Conse- quently, VPA recently has been tested in animal models of adenomyosis [ 22]. Interestingly, clinical studies of oral ad- ministration of VPA in treating symptomatic adenomyosis have yielded promising results [ 48, 53]. Andrographolide is an NF- κB inhibitor that causes covalent modification of reduced cysteine 62 of the p50 subunit of NF- κB[ 54] and may also be therapeutic given the constitutive NF- κB acti- vation in adenomyosis [ 55]. Tetrahydropalmatine (THP) is an alkaloid compound and a main active ingredient of Corydalis yanhusuo W. T. Wang [56], a well-known analge- sic in traditional Chinese medicine (TCM) often used as a constituent herb for treating endometriosis [ 57]. Its levo- enantiomer (l-THP) is an analgesic with remarkable sedative tranquilizing but nonaddictive effects [58]. Listed in the Chinese Pharmacopoeia since 1977, l-THP has been used as a sedative or analgesic for chronic pains in China. The danazol-containing IUD and vaginal ring were tested clini- cally for adenomyosis [ 50, 59, 60] and endometriosis [ 61]. On a molecular level, danazol treatment results in reduced expression of estrogen receptor and bcl-2 in adenomyotic endometrium with concomitant increased apoptosis [ 62]. Over time, the clinical outcome in animal models has evolved from using only the prevalence of adenomyosis to including details such as the ratio of uterine wet weight versus total body weight [ 21], number of nodules [ 21], or depth of myometrial infiltration of endometrial cells [ 22, 23] as outcome measures. Additionally, two recent studies used latency response to noxious thermal stimuli as outcome measures [ 22, 23]. Using adenomyosis prevalence as an outcome measure is useful in determining suppression or slowed development of adenomyosis. However, because women with endometriosis and a prolong history of dys- menorrhea often have adenomyosis [ 63], ideal compounds will reverse adenomyosis and treat associated symptoms. Analysis of Earlier Mouse Studies As seen in Table 1, the seven mouse efficacy studies pub- lished up to 2004 were conducted by one research group using the SHN mouse strain and evaluated different com- pounds. The induction methods and major outcomes were nearly identical. The three later studies from 2008 ( n01) and 2011 ( n02), used different strains of mice or different in- duction methods. The outcome measures also differed from earlier studies. An analysis was conducted to determine whether the magnitude of treatment effect, defined as the difference in prevalence of adenomyosis between the treatment and con- trol groups, was associated with features of the experiment. Comparison of the distributions among two or more groups of continuous variables was made using the Wilcoxon and Kruskal-Wallis tests, respectively. Pearson ’s or Spearman ’s rank correlation coefficient was used when evaluating cor- relations between two variables when both variables were continuous or when at least one variable was ordinal. To see Curr Obstet Gynecol Rep (2012) 1:138 –145 141 whether the age of mice used in the experiment, the length of adenomyosis-induction period, duration of treatment, and t o t a ls a m p l es i z ew e r er e s p o n s i b l ef o rt h er e d u c t i o ni n prevalence of adenomyosis between treatment and control groups, a multiple linear regression model was used. P values of less than 0.05 were considered statistically signif- icant. All computations were made with R statistics software system version 2.15.0 [ 25]. As illustrated in Fig. 1, the treatment effect was inversely correlated with the length of induction period (ie, the time between performing the pituitary graft and the start of treat- ment [ r00.79, P00.02; Fig. 1A]). In other words, better efficacy was reported from experiments using a shorter induction period, or earlier intervention. I m p r o v e de f f i c a c ya l s os e e m e dt ob ea s s o c i a t e dw i t h longer duration of treatment, although the correlation did not reach statistical significance ( r 0−0.38, P 00.38 [Fig. 1B]). Greater reduction in prevalence tended to be reported from experiments that did not mention randomiza- tion in allocating animals, but no statistical significance was reached ( P00.61 [Fig. 1C]). A multiple linear regression analysis using the difference in prevalence of adenomyosis between the treatment and control groups as the independent variable and the age of mice used in the experiment, year of publication, length of induction period, duration of treatment, total sample size, and the mention of randomization as dependent variables revealed that the length of induction period was the only covariate that was significantly associated with the reduc- tion in prevalence ( P00.035, R 2 00.78) indicating that great- er efficacy is associated with earlier intervention. Methodological Qualities of Published Mouse Efficacy Studies The recently proposed Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines [ 64]c o m p r i s e seven core categories that indicate methodological qualities of animal efficacy studies. These categories for the ten reviewed studies are summarized in Table 2. In considering these core elements, none of the reviewed studies provide any justification for number of mice studied or how the sample size was determined. Eight of ten studies mentioned that the animal allocation was carried out randomly, but none described how it was done. None of the reviewed studies reported allocation concealment. None of the ten studies explicitly stated that the outcome measures were evaluated blinded to treatment allocation. Of the ten studies, four used two or more dosages to establish the minimally effective or maximally tolerated doses. Because the two major complaints from women with adenomyosis are pain and heavy menstrual bleeding, one needs to evaluate not Fig. 1 a Scatter plot of the difference in incidence of adenomyosis between treatment and control groups versus length of induction. The alphabets were identifications of experiments listed in Table 1. b Scatter plot of the difference in prevalence of adenomyosis between treatment and control groups versus duration of treatment (in days). The alphabets were identifications of experiments listed in Table 1. The experiments with blue alphabets were done with spontaneous adeno- myosis in SHN mice, while those with red were performed with ectopic graft of pituitary glands. c Box plot of the difference in incidence of adenomyosis between treatment and control groups by the statement of randomization in allocating mice in experiment 142 Curr Obstet Gynecol Rep (2012) 1:138– 145 only the histological outcome of an intervention (ie, number of nodules, depth of myometrial infiltration, with or without proliferation index measurement or immunostaining analysis) but also functional outcome (ie, severity of pain or fertility). While eight of ten studies evaluated at least two disease- related outcomes, only two of those eight studies evaluated functional outcomes (ie, pain behavior). Of the ten studies, only two recently published studies addressed possible con- flict of interest (COI). The lack of a statement regarding COI in the earlier studies may reflect their publication at an earlier time when disclosure regarding COI was not required. The importance of the methodological quality in animal studies has been appreciated in other clinical settings, such as stroke, after some failed clinical trials [ 66–68]. Rigorous, robust, and detailed preclinical evaluation has been recog- nized as a critical step in the development of effective novel therapies [66]. The less than desirable methodological quality of mouse efficacy studies of adenomyosis illustrated in Table2 could have contributes to the lack of progress in developing novel treatments for adenomyosis. The scarcity of registered clinical trials on adenomyosis may be attributable, at least in part, to various deficiencies in mouse efficacy studies. The tendency to alter animal numbers based on scientifi- cally irrelevant issues such as availability or cost has been noted before [ 69]. Inadequate sample sizes would result in underpowered studies, bringing into question the statistical validity of study conclusions. For continuous outcome meas- ures, the rule of thumb for calculating the desired sample size n to achieve the significance level of 0.05 and 80 % power in the treatment and the control group is n – 1016 s 2/d2,w h e r es is the standard deviation, andd is the difference to be detected [70]. In other words, n−1 is proportional to the squared standard deviation, but inversely proportional to the squared difference to be detected. Without knowings and/or d,s a m p l e size determination is impossible. Ideally, pilot experiments would aid in determining s and/or d. In some cases, this approach would result in doing the entire experiment. There- fore, this quandary may explain why in many mouse efficacy studies, sample size justification is not done or is carried out post hoc. Randomization and allocation concealment can be done in mouse efficacy studies. Blinding also can be done, but when there is a shortage of hands, as in experiments for a student’s PhD dissertation, extra care should be taken to meet this requirement. Declaration of potential COI is now mandatory for publication in many biomedical journals. When a preclinical study is sponsored or even conducted by a drug company testing a compound developed by the company itself, there is obvio usly vested interest in the

of the tested compound and, as such, potential COI should disclosed and declared.

Preclinical mouse efficacy studies of adenomyosis appar- ently have deficiencies in methodology. Critical evalua- tion of published data before embarking on animal studies, the use of mouse models that adequately mimic human adenomyosis, the use of at least one functional outcome, and higher methodological quality could greatly increase the chance of a successful translation of mouse experiments into clinical success. Fortunately, the impor- tance of methodological quality of animal efficacy studies is now well recognized, and, as a result, the ARRIVE guidelines for experimental design, execution, and report- ing have been established [65 ]. The use of these guide- lines in future mouse efficacy studies of adenomyosis would strengthen preclinical research. Adenomyosis is a difficult disease to manage [ 65], espe- cially by nonsurgical means. Developing novel, more effi- cacious therapeutics with better safety and cost profiles is a pressing but unmet medical need. On www.ClinicalTrials. gov, the number of registered clinical trials studying adeno- myosis is currently only 15, about one tenth of the number of trials on endometriosis; none are testing novel drugs. The amount of research on adenomyosis indicated by PubMed using the phrase “adenomyosis not endometriosis ” yielded 389 publications, as compared with 18,593 when the search used the word “endometriosis” alone (PubMed, accessed on May 14, 2012). Insufficient breadth of research regarding adenomyosis may hamper compiling information relevant to drug development. Besides the methodological quality as discussed above, proceeding from preclinical studies to clinical trials requires that efficacy is reproduced in two laboratories, one of which is independent of any sponsoring company [ 66]. In addition, the use of second larger species such as cats, dogs, or primates would be highly valuable, but, unfortunately, no such animal model is available now. Only when the preclin- ical mouse efficacy study is carried out with methodological Table 2 Summary of core quality features of ten published mouse efficacy studies of adenomyosis Category Fulfilled, % Sample size justification 0 Randomization 80 Allocation concealment 0 Blinding 0 Does-response relationship 40 Assessment of at least one functional outcome 20 Statement regarding possible conflicting interest 20 Curr Obstet Gynecol Rep (2012) 1:138 –145 143 rigor and careful consideration will there be a better chance to develop a successful drug to treat adenomyosis. Acknowledgment This research was supported in part by grants 09PJD015 and 10410700200 from the Shanghai Science and Technol- ogy Commission, and support from Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases and from the Key Specialty Project of the Ministry of Health, People’ s Republic of China. The author thanks Dr. Pamela Stratton for her constructive comments on an earlier version of this manuscript. Disclosures No potential conflicts of interest relevant to this article were reported.

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