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Studying Sex Differences in Translational Research: Examples from Four Major Disease Areas

Following the introductory presentations on the challenges and opportunities for studying sex differences in neuroscience research, four specific disease areas within neuroscience were discussed in greater detail: depression, pain and pain perception, sleep medicine, and multiple sclerosis and neuroinflammation. These areas were identified by the planning committee as particularly relevant to the discussion with known sex differences and therefore areas with potential for critical advances. In addition, these diseases have very different etiologies and thus allowed a broad overview of many different mechanisms. (Key points of the presentations in each disease area are provided in boxes at the end of each set of panel presentations, Boxes 3-1 through 3-4.)

DEPRESSION

Characterization of Sex Differences in Depression

In science, we seek to define variables on which populations are similar to and differ from one another, said Katherine Wisner, director of Women’s Behavioral HealthCARE at the University of Pittsburgh Medical Center. Two sexes provide a source of “variable partitioning” that creates a natural opportunity for comparative investigation. Disease states have been studied for a long time, considering a variety of different impacting factors (e.g., environment). The task at hand is to look at disease states by sex or gender across the lifecycle, and harness that information for treatment. The



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3 Studying Sex Differences in Translational Research: Examples from Four Major Disease Areas Following the introductory presentations on the challenges and oppor- tunities for studying sex differences in neuroscience research, four specific disease areas within neuroscience were discussed in greater detail: depres- sion, pain and pain perception, sleep medicine, and multiple sclerosis and neuroinflammation. These areas were identified by the planning committee as particularly relevant to the discussion with known sex differences and therefore areas with potential for critical advances. In addition, these dis- eases have very different etiologies and thus allowed a broad overview of many different mechanisms. (Key points of the presentations in each disease area are provided in boxes at the end of each set of panel presentations, Boxes 3-1 through 3-4.) DEPRESSION Characterization of Sex Differences in Depression In science, we seek to define variables on which populations are similar to and differ from one another, said Katherine Wisner, director of Women’s Behavioral HealthCARE at the University of Pittsburgh Medical Center. Two sexes provide a source of “variable partitioning” that creates a natural opportunity for comparative investigation. Disease states have been studied for a long time, considering a variety of different impacting factors (e.g., environment). The task at hand is to look at disease states by sex or gen- der across the lifecycle, and harness that information for treatment. The 

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 SEX DIFFERENCES AND IMPLICATIONS ultimate question is whether treatments will be optimized by incorporating sex and gender principles into interventions. Women are 1.5 to 2.5 times more likely to experience major depression than men, from puberty onward. Women have the highest prevalence of de- pression during the childbearing years and are also at increased risk during the perimenopausal period (the 5-year period before the cessation of menses). According to a World Health Organization study, depression is the leading cause of days lost to disability for women worldwide. Depression is often discussed as if it was a homogeneous illness, but further study is needed into the different subtypes of depression and how they vary by sex and gender. Wisner cited one recent study that included sex-specific analyses is the STAR*D (Sequenced Treatment Alternatives to Relieve Depression) study, a large-scale clinical trial of multiple depression treatments (Marcus et al., 2005). The investigators found differences in symptoms between the two sexes. Depressed women experienced more anxiety, physical somatoform symptoms, and bulimia. For men, the symptoms concurrent with depression tended to be obsessive-compulsive symptoms and substance use. Depressive episodes were longer in women than men, and suicide attempts occurred more frequently. Interestingly, women were more likely than men to have remission (loss of all symptoms) in response to the drugs under investiga- tion (30 percent of women compared to 24 percent of men), and half of women responded (had symptom reduction) compared to 44 percent of men. The question facing clinicians now is how to apply this information. Individualized, personalized treatment for depression and other psy- chiatric illnesses is a primary goal of translational research. In addition to sex and gender, individuals vary with regard to symptoms, comorbidities, clinical factors, personal history, family features, social background, genetic polymorphisms, developmental stage, and characteristics identified from brain imaging or other technologies. Differences in the longitudinal devel- opment of males and females also naturally provide a variety of hormonal conditions under which to study sex differences as well as the hormonal changes that are unique to females: in utero differentiation, menarche, the premenstruum, pregnancy, postpartum, and menopause. When considering a disease state or a process, there is a broad biological- to-societal spectrum of distal health determinants that fluctuate throughout an individual’s lifetime; from basic genetics, to gene–environment interac- tions, to the physical and social environments (e.g., which pollutants or other stressors an individual is subjected to often vary by gender) (Misra et al., 2003). Proximal determinants, including biomedical responses (e.g., nutri- tional status, inflammatory response) and behavioral responses (e.g., al- cohol use, actively practicing a religion) impact the disease process acutely. Health outcomes are influenced by these distal and proximal determinants, as well as by inputs and processes such as health care.

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH Wisner closed noting that the so-called “valleys of death” in clinical and translational research are, in fact, valleys of opportunity. Mechanisms such as the Specialized Centers of Research and Building Interdisciplinary Research Careers in Women’s Health programs are bringing people together to elimi- nate these valleys. Questions to be addressed when translating neuroscience research are whether there is enough of a sex difference to merit changing the way medicine is practiced to accommodate those differences, and if so, how to train individual practitioners to consider these differences in practice. Fetal Antecedents to Sex Differences in Depression Jill Goldstein, director of research at the Connors Center for Women’s Health and Gender Biology at Brigham and Women’s Hospital and pro- fessor of psychiatry and medicine at Harvard Medical School, discussed fetal hormonal programming of sex differences in the brain, and its role in understanding sex differences in depression. The incidence of major depressive disorder has an approximately 2:1, female-to-male ratio. Furthermore, depression is comorbid with several chronic diseases, including the fact that the comorbidity of depression and cardiovascular disease is the fourth leading cause of morbidity and mortality worldwide. Goldstein and colleagues are currently testing the hypothesis that there are shared etiologies associated with understanding sex differences in depression and cardiovascular disease; that they are initi- ated during the sexual differentiation of the brain; and that they involve disruption of the fetal hormonal programming of the brain, which leads to endocrine disruptions throughout life, and sex differences in adulthood in these chronic diseases. Throughout life windows of opportunity are available for studying sex differences in these disorders, Goldstein said. These occur when the brain and the body are flooded differentially with hormones: fetal development, puberty, pregnancy, perimenopause, and menopause. Although depression and cardiovascular disease are, for the most part, adult-onset disorders, they have developmental precursors, and considering this lifespan perspec- tive is important. Some risk factors for depression that have been identified from population- level studies include small for gestational age; low birthweight; obstetric complications (e.g., preeclampsia, oxygen deprivation); second trimester in- fluenza; and second to third trimester famine. Population-level studies in the field of cardiovascular risk and hypertension have identified some of the same fetal risk factors for cardiovascular disease: small for gestational age; low birthweight; preeclampsia; and maternal prenatal famine. Studies on the fetal programming of cardiovascular disease have focused on prenatal and early life stress and the disruption of the hypothalamic pi-

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 SEX DIFFERENCES AND IMPLICATIONS tuitary adrenal (HPA) axis in development. The HPA axis is also the focus of studies on the fetal programming of sex differences in depression. Timing is critical for understanding sex effects, Goldstein said. Population-level studies that have looked at first trimester factors have found fewer sex differences in the incidence of these disorders than those that looked at second and third trimester factors. This may, in part, reflect the fact that hormonal regulation of the sexual differentiation of the brain starts at the beginning of second trimester, when testes begin to secrete testosterone, which has direct and indirect effects (through aromatization into estradiol) on brain sexual differentiation. In addition, as described by Arnold (see Chapter 2), prior to gonad differentiation, genetics play a criti- cal role in sex differentiation. Estrogen and testosterone have major effects on neuronal growth and development. These effects are not all over the brain, but are region specific, in areas such as the hypothalamic and amygdala nuclei, the hippocampus, medial dorsal thalamus, and areas of the cortex. Although much of the pre- vious work on the sexual differentiation of the brain has been conducted in animals, magnetic resonance imaging (MRI) of healthy human brains shows that brain regions affected by sex hormones during development are highly sexually dimorphic (i.e., exhibit sex differences in brain volumes relative to the size of the cerebrum) (Goldstein et al., 2001). Brain imaging studies of depression show crossover between those brain regions that are normally highly sexually dimorphic and those that are implicated as abnormal in depression, including the paraventricular nucleus, lateral hypothalamic area, hippocampus, and areas of the amyg- dala. Imaging studies of central nervous system (CNS) control of the auto- nomic nervous system show that some of those same brain regions are also important for regulation of the heart. This, Goldstein said, is the basis for her studies on shared etiologies for depression and heart disease. Studying the stress response circuitry is a model system for the study of hormonal regulation of the brain and of the impact on major depressive disorder, Goldstein explained. Stress response circuitry crosses over with mood regulation, control of the HPA axis, and brain regions that regulate heart and blood pressure through autonomic nervous system function, such as the hypothalamic paraventricular nucleus and hippocampus. To charac- terize the hormonal phenotype in response to stress using this model, blood was collected and heart rate was monitored while an individual was lying in the MRI scanner, viewing a visual stress response challenge. The responses of the brain to low- and high-arousal pictures (e.g., a cow in a green field versus a serious car crash) were compared. Results showed that the stress response circuitry in the healthy brain activates differently at different points in the menstrual cycle, and those hormonal differences contribute to explaining sex differences in stress response circuitry activation (Goldstein

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH et al., 2005, 2010). The physiology of male and female healthy brains is different, and findings show that male and female brains act differently to maintain homeostasis with regard to one’s response to stress. Stress response circuitry function is abnormal in women with recurrent depression. Furthermore, in depression, there is a lower parasympathetic control, which can be operationalized as the high-frequency component of heart rate variability, and which has been found to be significantly associ- ated with estradiol in women. Thus, brain activity deficits, hormonal defi- cits, heart rate and autonomic nervous system function, and sex differences in the brain are all highly related to each other and increased understand- ing will contribute important new knowledge regarding depression and its comorbidity with major general medical diseases, such as cardiovascular disease. Goldstein is now looking at the shared fetal antecedents to sex differ- ences in depression and risk for cardiovascular disease using the National Collaborative Perinatal Cohort developed in the 1960s. This study initially followed a New England cohort of 17,000 women in Boston and Provi- dence through their pregnancies, and their children for 7 years after birth, until study funding ran out. Over the past 20 years, study participants (who are now adults) have been re-recruited and interviewed, and a subsample have been brought to the brain imaging center, facilitating human studies of fetal antecedents to brain, hormone, and heart regulation phenomenology and risk for different psychiatric and general medical diseases. In a separate study, Goldstein and colleagues are following 300 discor- dant sibling pairs, one of whom has been exposed to fetal growth restriction or to preeclampsia, and the other as the unaffected control. As an example, one initial finding shows that healthy adult males who were exposed to fetal growth restriction or preeclampsia have significantly less parasympathetic control of the heart than females. In conclusion, Goldstein stressed that understanding sex differences in depression and its comorbid conditions is absolutely critical for sex-specific drug discovery and development. One must take a life-course perspective for understanding the medical implications of sex differences for both the healthy brain and for models of disease. Taking a brain–body approach for understanding the impact of sex differences in the brain will be fruitful for new sex-specific drug discovery and other treatment modalities. Finally, clinical and population-level research is critical for informing the develop- ment of basic animal models and vice versa. Sex Differences in Translational Studies of Major Depression Etienne Sibille, associate professor in the Translational Neuroscience Program at the University of Pittsburgh, explained that major depression

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 SEX DIFFERENCES AND IMPLICATIONS is a heterogeneous syndrome that is characterized by chronic low mood. Mechanistically, depression is a chronic, recurrent disease known to be influenced by genes and environment. The higher prevalence of depression in women is cross-cultural, and is probably one of the most robust findings in all of psychiatric epidemiology, Sibille said, and yet this finding often is not considered in basic studies. Perspectives differ on the origin of sexual dimorphism in depression. A societal perspectie focuses on the interaction between increased victim- ization and female character traits. In the Darwinian perspectie on the adaptive role of mood, mood is defined as emotion over time that is less dependent on immediate triggers. Low or high mood is a source of informa- tion about goal achievement and serves as a regulator of effort and energy allocation. For example, behavior inhibition associated with low mood is an adaptive response that saves resources in the face of unachievable goals or potential negative outcomes. Low mood, under normal conditions, is criti- cal in strategy reassessment. Under this definition, based on sexual selection theory, women allocate more effort and energy in long-term reproductive goals and are more sensitive than men to negative outcomes about lifetime strategies in the context of normal mood regulation. In the Darwinian per- spective, depression is a chronic maladaptive state of mood dysregulation. For reasons as yet unknown, the female system is evolutionarily more at risk of a maladaptive state. The biological perspectie seeks to determine if increased female vulnerability to develop depression is due to sex hormones in early development (organizational) or in adulthood (activational). For translational studies, mood states (e.g., anxiety-like and antidepressant- like behaviors) can be modeled in animals, including rodents. Mood regu- lation neural networks are conserved across mammalian species. Still, the primary pathology of depression is poorly characterized because there are numerous limits with current animal models. The models are often over- simplified; there is poor conceptualization of baseline traits versus induced depressive-like states; conceptualization of syndrome versus single behavior is poor; and little consideration is given to sex differences. Specific concerns include differences between behavioral tests, genetic models designed to characterize a trait, and animal models that induce depressive states. The forced swim test as a behavioral animal model of depression, for example, is not a really a model at all, but rather a single behavioral response. Its only value is predictive validity for short-term response to antidepressants. Genetic models are generally very good, but we must recognize that often, what is reported is the impact of the lack of a specific gene on traits. These are not multisystem models, but a single entry into complex disease. Sibille described her work with the unpredictable chronic mild stress (UCMS) model, which induces a depressive-like state in mice that mim- ics, in a naturalistic way, both the role of stress in precipitating depressive

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH pathology and the time frame of therapeutic response to antidepressive treatment. Mice are subjected to an unpredictable regimen of mild psycho- social stressors such as forced bath, predator’s song or smell, tilted cage, or social stress. Over 4 to 6 weeks, they develop a syndrome, or a collection of symptoms, including measurable outcome of behaviors that relate to emo- tions (e.g., increased anxiety, increased depressive-like behavior), increased anhedonia-like behavior). Physiological changes also occur, such as de- creased weight gain, reduced quality of coat, and neuroendocrine changes. One study using this model has also shown cardiovascular changes. After onset, this syndrome can be blocked by chronic application of antidepres- sants. Using the UCMS model, Sibille and colleagues have shown that emo- tionality (expressed as Z score) is much higher after stress in female subjects than male. In another test, female mice genetically altered to express low levels of serotonin transporters are more vulnerable to chronic stress than male counterparts (Joeyen-Waldorf et al., 2009). This model has also been used to test the translational hypothesis that the molecular pathology of altered mood regulation will manifest as conserved gene changes across species. Using large-scale gene expression data from human postmortem brain analysis, researchers have shown that changes in the amygdala of depressed human subjects actually predict what is observed in the amygdala of chronically stressed mice, and vice versa (Sibille et al., 2009). A set of 32 core genes has been identified that form a tight gene network, which is structurally conserved across species. This, Sibille said, suggests that in the context of depression or chronic stress, existing cellular pathways are abnormally recruited. In summary, Sibille said, in the evolutionary context of mood regula- tion, these findings suggest that sexual dimorphism in biological mecha- nisms of depression should be expected. Animal models are associated with considerable limitations, at the levels of both concept and interpretation. UCMS could serve as an appropriate model of the human syndrome, and rodent findings parallel sex differences of human depression, setting the basis for development of realistic studies of sexual dimorphism. Ultimately, evidence shows sexual dimorphism in the primary pathology of depression in humans. Industry Perspective on the Implications of Sex Differences for Translational Research Carla Canuso, senior director of external innovation, Neuroscience Therapeutic Area at Johnson & Johnson, provided an overview of how and when industry considers sex differences, particularly in antidepressant drug development, during each phase of development, from preclinical through postmarketing.

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 SEX DIFFERENCES AND IMPLICATIONS Industry is necessarily concerned with regulations and guidance from the Food and Drug Administration (FDA) and regulatory bodies around the world. The FDA issued guidance in 1993 about the inclusion of women in the clinical evaluation of drugs, lifting the restriction on the participation of women of childbearing potential in Phase I and early Phase II trials (see http://www.nlm.nih.gov/services/ctphases.html), even before the completion of all animal toxicology studies. This placed greater onus on investigators to employ strict inclusion criteria regarding the use of birth control or absti- nence, as well as strict guidance for pregnancy monitoring, and put more re- sponsibility on institutional review boards to monitor clinical protocols. The intent was to have fair balance and representation of both sexes in the study so the data could be analyzed to detect any clinically significant differences. The guidance also addressed the assessment of demographic differences in pharmacodynamics in Phase I and II studies. Interestingly, Canuso said, the guidance specifically noted that the effects of menstrual cycle on pharmaco- kinetics should be evaluated when feasible, but this is not routinely done. The European Medicines Agency (EMEA) does not have specific guid- ance on the inclusion of women, but EMEA did conduct a recent review of International Conference on Harmonisation (ICH) guidelines to deter- mine whether special guidance for inclusion of women was needed. EMEA concluded that the current ICH guidance is sufficient to address the special needs of women and, in a review of recent clinical trials, found that women were adequately represented. Canuso concurred with the previous speakers regarding the limita- tions of animal models, which are necessary for drug development. Animal models show sex differences in depression and stress, and that these differ- ences in stress response are related to differences in corticotropin-releasing factor and serotonin neurotransmission. These are core regulators of mood and the coping response. The vast majority of preclinical studies done in the pharmaceutical industry are done in males, partly because of varia- tion across the estrous cycle in laboratory animals. Nonetheless, studies are rarely replicated in females of the species, Canuso said. Preclinical studies have poor predictability of sex differences with respect to clinical response and toxicology, including reproductive toxicology, teratogenicity, and carcinogenicity. Despite the 1993 FDA guidance, the vast number of participants in Phase I clinical trials are male, Canuso said. Reasons include the logistical challenges of birth control for women participants (e.g., double-barrier methods, the need to be on oral contraceptives for 3 months prior to entry into the study) and the lengthy informed consent process for early phase studies of drugs in women. The pharmacokinetics of drugs differ between women and men, not just because of body weight or volume of distribution, but also hormonal interplay. Also, drug–drug interaction studies must be

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH done for coadministration of antidepressant drugs, which are substrates or inhibitors of the cytochrome P-450 system, and drugs women commonly take that are metabolized by the P-450 system (e.g., oral contraceptives, tamoxifen). Throughout all phases of clinical development, the consideration of sex differences should include designing studies to be appropriately enriched for women; requiring birth control or abstinence; pregnancy reporting; data analysis using sex-specific laboratory ranges; and studying sex-specific pharmacodynamic responses and adverse drug reactions. Other sex-specific factors are considered for proof of concept and pivotal trials conducted for product registration. Products may have sex- specific indications (e.g., premenstrual dysphoric disorder; vasomotor symptoms associated with menopause; postpartum depression), or have been developed for use in only one sex (e.g., a safety concern in the op- posite sex). As a result of the 1993 FDA guidance, inclusion of women in Phase II and III studies is generally adequate, and subgroup analyses by sex is included in labeling. Finally, sex differences also come into play in Phase IV and postmarket- ing studies. Populations of interest are studied further, such as those with comorbidities. Investigator-initiated studies are conducted by academic researchers. Epidemiological studies are used to revise labels as new infor- mation comes to light following widespread use. Pregnancy registries are also used more often. In closing, Canuso offered several ways industry can foster transla- tional research in neuroscience, as follows: • Partner with academia to advance the science of personalized medi- cine, while considering sex and gender in every phase of drug devel- opment so that differential responses in dosing, efficacy, and safety can be fully appreciated. • Partner with academia to develop and validate better preclinical animal models that are truly predictive of the diseases, and then study both sexes of the species in those models. • Identify and evaluate sex-specific endophenotypes and other bio- markers, such as increased stress sensitivity. • Identify moderators and predictors of disease, specifically those that may confer resilience. • Establish multisector collaborations across industry, academia, and the National Institutes of Health (NIH) and create data-sharing mechanisms (e.g., the Psychiatric Genome-wide Association Study [GWAS] Consortium and the North American Antiepileptic Preg- nancy Registry) so that once viable drug targets are identified, there will be large datasets that can be used to assess and validate them.

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0 SEX DIFFERENCES AND IMPLICATIONS BOX 3-1 Key Points: Depression • Major depressive disorder is a leading cause of disability worldwide. • Depression is a significant contributor to other systemic and organ diseases. • While there has been some progress in treatment options, current approaches are inadequate. • Primary pathological mechanisms of depression are poorly characterized. • Women are 1.5 to 2.5 times more likely to experience major depression than men, from puberty onward. Symptomatology is also different between the sexes. • Sex differences must be studied across the lifespan: Natural variation of hormone levels across the lifespan provides oppor- tunities for the study of sex differences in psychiatric and neurological disorders. Adult-onset disorders have developmental precursors. Consideration of comorbid conditions is important. • Current animal models of depressive disorders have significant limitations at the levels of both concept and interpretation. • Sex and gender should be taken into account in every phase of drug develop- ment (Phases I through III and postmarketing studies, as well as preclinical studies in animals). PAIN AND PAIN PERCEPTION Sex Differences in Pain and Pain Perception Studies in humans have shown that females generally experience more clinical pain and often show greater experimental pain responses (i.e., have lower thresholds and less tolerance for pain) than males, said Karen J. Berkley, professor of psychology and neuroscience at Florida State University. That difference, however, can be manipulated by a variety of experimental factors (e.g., stimulus type, pain scale used, testing paradigms, endpoints selected) and impacted by individual factors (e.g., age, reproductive status, general health, blood pressure, food intake, odors, social and cultural factors). Although individuals show significant variability when it comes to al- leviating pain, some generally accepted sex differences in pain are worth considering. First, more painful conditions have a higher prevalence in fe- males than males. In other words, women are more likely to have painful chronic conditions than men. The underlying basis for this disparity is not known, but probably has multiple causes, Berkley said, and is an oppor- tunity for further research. Second, hundreds of therapies are available to

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH alleviate pain, and women use more of them (e.g., drugs, herbal products, complementary alternative medicine) than men.Yet little attention has been paid to how this usage difference affects the efficacy and side effects of various treatments. One of the key questions considered in a 2007 consensus report on studying sex differences in pain and analgesia was “is there enough evi- dence to warrant sex-specific pain interventions?” The authors concluded that “the findings are mixed” and that “the evidence does not appear strong enough to warrant sex-specific pain interventions in most situa- tions” and noted that more studies are required, including clinical trials that should take sex into consideration and report any differences in outcomes (Greenspan et al., 2007, p. 14). The consensus group also expressed concern about the “translation hindering” effects of the “disconnects” among specialties, and between basic and clinical researchers. Berkley also noted that translational research is not unidirectional from animal research to clinical practice, but is really circular, and evidence from human and clinical research should inform animal models. In conclusion, Berkley said that knowledge of statistical sex differences is already beginning to save lives and improve the health of both females and males, but dissemination of this knowledge is key. Better understand- ing of the interplay between social roles and health is needed. These issues are complex, but seemingly small increments in knowledge can have large lifetime impacts. Dissecting Pain and Pain Perception into Sex-Related Endophenotypes Emeran Mayer, director of the University of California–Los Angeles (UCLA) Center for Neurobiology of Stress, studies persistent pain syn- dromes, with a focus on visceral pain from the gastrointestinal and urinary tracts. Based on reported spontaneous symptoms, persistent pain syndromes are more common in women (including irritable bowel syndrome [IBS] and interstitial cystitis). Awareness is growing that persistent pain syndromes (e.g., fibromyalgia, temporomandibular joint disorder, vulvodynia, inter- stitial cystitis, IBS) are not distinct diseases, but rather, they significantly overlap with each other, and with disorders of affect and mood, particularly anxiety, depression, and somatization. Most of these disorders are studied using experimental pain assays to determine if an individual has a high or a normal pain threshold or pain sensitivity. Such measurement would be simple if the relationship between pain perception and a stimulus was lin- ear. But pain perception is a highly complex, modulated system (Figure 3-1). These networks prepare the system and modulate perception of a stimulus

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 SEX DIFFERENCES AND IMPLICATIONS women in terms of the incidence is roughly the same. This may be due to less diagnostic delay in more recent times (which will increase the preva- lence without changing the incidence) and greater survival (which does not necessarily mean greater quality of life). In terms of prognosis relative to relapses and progressive disability, observations vary. A large number of earlier studies suggest that females have a favorable prognosis. Quite a few other studies have shown no sex difference in the prognosis of MS. Although a sex difference is prominent in the incidence in neuromyelitis optica, no sex differences have been observed in progression. With regard to the role of sex hormones in MS, from the clinical point of view it is clear and widely accepted that relapse rate decreases during pregnancy, particularly the third trimester. Then, in the months after de- livery, a rebound brings the relapse rate back to the prepregnancy rate or perhaps even higher. The effect of oral contraceptives is less clear. A lower incidence of MS is seen among women using oral contraceptives, although some studies have found no effect. One study, interestingly, found an in- creased risk in long-term users of oral contraceptives, suggesting the need for further research in this area. Earlier and smaller MRI studies (n 5 50 to 413) of active inflammation found gadolinium-enhancing lesions were more common in women, but they did not control for the differential age and the disease course of pa- tients. Later studies that did control for some of these covariates, enrolling 700 and 1,300 patients, found no sex difference in gadolinium-enhancing lesions. MRI studies of cumulative injury found no sex difference in T2 lesions after covariate adjustment. Studies of T1 lesions observed no sex difference in relapsing–remitting MS, and greater T1 lesion volume in male primary progressive MS. No sex differences in atrophy have been demonstrated in cross-sectional or longitudinal studies. Results are conflicting about gray-matter atrophy; some studies suggest it is greater in men, and others indicate it is equal in both sexes. Studies using advanced imaging metrics of MS tissue injury, including diffusion-weighted imaging and magnetization transfer ratio, have not demonstrated any sex differences. Only a few histopathologic studies of MS have evaluated sex differ- ences. No differences have been observed in studies of active inflammation (equal number of microglial cells and T-cells in MS lesions and normal- appearing white matter in men versus women), tissue damage (equal num- ber of amyloid-precursor, protein-positive spheroids; equal reduction in axonal density; no difference in number, type, or distribution of cortical lesions), or repair (no difference in the pathologic evidence of repair, oli- godendrocyte precursor cells, and other related lineages; no difference in remyelinating lesions between men and women). The only pathologic sex

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH difference available in the literature, Fox said, was a low fiber density in spinal pyramidal tracts in men. All together, evidence showing a sex differ- ence is limited in the histopathology of MS. The vast majority of clinical trials have found no effect of sex on out- come. Many of these trials had a preplanned covariate analysis of sex, and while some reported no effect of sex, others did not report any sex-based results. However, notable exceptions include two studies of interferon in secondary progressive MS, which found that only women had slowed pro- gression of disability. A third interferon study did not find any effect of sex. Whether these studies adjusted for covariates such as age of patient and age at diagnosis was unclear, Fox said. A post-hoc analysis of data from a study of glatiramer acetate and pri- mary progressive MS showed that only men had a slowed progression of disability. This difference persisted after covariate adjustment. Several other glatiramer acetate studies (in relapsing–remitting MS and primary progres- sive MS) did not identify a sex difference. No clinical trial has shown a differential effect of sex on any MRI measure. In summary, Fox said, MS is two to three times more common in women. Onset is later in men and is more likely to be primary progressive MS, making interpretation of studies challenging. Women may have a better prognosis, but the differences decrease after adjusting for age at diagnosis and disease course. Pregnancy decreases disease activity, but whether oral contraceptives have an impact is unclear. Despite the observations of smaller studies, the larger MRI studies fail to suggest an effect of sex on MRI measures of either inflammation or tis- sue injury. Histopathology shows virtually no effect of sex, nor did most clinical trials. A few post-hoc analyses suggest some sex-based effect, but how to interpret these findings is unclear in light of all the other studies that found no impact. Altogether, while a sex difference is clear in the incidence of MS, little evidence is available on sex differences in the clinical course of MS. That does not mean, however, that consideration of sex differences should not be included in planning future studies. MuLTIPLE SCLEROSIS AND NEuROINFLAMMATION: CONSIDERING SEx DIFFERENCES IN DESIGNING THERAPEuTIC AGENTS Multiple sclerosis is just one of a list of autoimmune diseases that show sex differences. Halina Offner, professor of neurology and anesthesiology and perioperative medicine at Oregon Health and Science University (see ohsu.edu), said 78 percent of people affected with autoimmune diseases are women.

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 SEX DIFFERENCES AND IMPLICATIONS Experimental autoimmune encephalomyelitis (EAE) is the prevailing animal model for MS. Disease can be induced in genetically susceptible rodents by immunization with spinal cord homogenates, myelin, or spe- cific myelin peptides in combination with adjuvant (active EAE), and by adoptive transfer of encephalitogenic cells (passive EAE). Typically, EAE is thought to be a Th1/Th17-mediated disease. However, other cellular play- ers such as antigen-presenting cells also play a role in disease progression. Increased numbers of antigen-presenting cells in the CNS increase suscepti- bility to and severity of EAE in female mice. T-cell response to foreign an- tigen increases the numbers of activated T-cells in female versus male CNS. This could be influenced by sex steroids, Offner said. CNS T-cells recruit higher numbers of antigen-presenting cells to the CNS, which enhance the encephalitogenic activity of myelin-specific T-cells, thus producing the “high susceptibility” phenotype found in female mice. MS does have sex-based immune differences. Females mount a stronger immune response, with higher levels of antigen-presenting cells, CD41 Th2 cells, and immunoglobulin responses. Males have severe inflammation and enhanced CD41 Th1 and CD81 T-cell activity. The immune functions of males and females have fundamental differences, which may require differ- ent immunomodulatory strategies in MS. The regulatory balance between the detrimental and beneficial effects of immune cells in MS is very com- plex, Offner explained. In MS, the initiating factors are susceptibility genes and environmental factors. Sex hormones and neuroendocrine factors are very important mod- ulatory factors in the immune and autoimmune responses, Offner said. Some data on MS are rather controversial. Offner cited a report on the recent increase in incidence rates of MS in females compared to males (Debouverie, 2009) and several studies that showed increased ratios of fe- males to males (Eikelenboom et al., 2009; Maghzi et al., 2010; Sadovnick, 2009). One study connected the increase in the sex differences to vitamin D, suggesting that higher levels of vitamin D are associated with a lower incidence of multiple sclerosis only in women (Kragt et al., 2009). MS cases are well known for increasing with geographic distance from the equator, and vitamin D levels decrease with distance from the equator. Decreasing MS cases with increasing ultraviolet (UV) light also has been documented in several countries. UV light catalyzes the first step of vitamin D3 synthesis (the inactive form of the vitamin), and serum D3 levels cor- relate with exposure to the UV light. To evaluate this further, researchers looked at the effects of vitamin D3 in the EAE model. The severity of disease was much lower in females who were fed a vitamin diet, but there was no difference in males in clini- cal disease (Spach and Hayes, 2005). Further studies demonstrated that estrogen was needed for vitamin D3 to inhibit EAE in female mice. Ovari-

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH ectomy eliminated the vitamin D3-mediated inhibition of EAE, and estrogen replacement in these animals restored vitamin D3-mediated inhibition of EAE. If humans have similar gender differences in vitamin D metabolism, Offner said, then sunlight deprivation would increase the MS risk more significantly in women than in men, which could explain higher incidence of MS in females. The current hypothesis is that there may be a female bias in the pro- tective effects of vitamin D3 in MS, and that insufficiency in vitamin D3 may contribute to the higher female/male sex ratio. Lifestyle changes could be linked to insufficient sunlight exposure in recent years (women in the workforce, indoor lifestyle, sunscreen use). Also, declining ovarian function and limited vitamin D3 supplies may be driving the transition of relapsing– remitting to chronic progressive MS. In contrast, Offner described a study in which male and female animals with EAE were treated with a recombinant T-cell receptor ligand (RTL) construct at the onset of disease. The results showed a significant reduction in disease activity in mice treated with RTL, which was comparable in both sexes. Spinal cord imaging of mice at the onset of disease and at 3 days posttreatment with RTL showed reversal of T-cell infiltration, again with no difference between females and males. Based on these preclinical stud- ies with RTL in the EAE model, a Phase I safety study in MS patients was designed. Thirty-four adult subjects (male and female) who received varying doses of RTL injections or placebo were followed for more than 90 days. The primary outcome measure was whether the RTL construct increased MS disease activity, and the conclusion was that the RTL did not increase MS disease activity by any measure in either females or males. In conclusion, Offner said that sex differences matter in many clinical diseases and animal disease models. Disease models such as EAE often have used males, with the assumption that this decreases experimental variability caused by female hormone cycling. There are also misconceptions that dis- ease mechanisms or treatment effects will be the same for both sexes. This is not the case, as demonstrated in the two examples provided, showing that response to vitamin D is sex dependent, while response to RTL is not. Therefore, preclinical studies should always include both sexes. Studying Sex Differences from Bedside to Bench to Bedside When discussing sex-based disparities in health, separating incidence from progression is important. Incidence is akin to susceptibility, or the im- mune system’s overactivation in autoimmune diseases like MS and lupus, whereas progression or disability accumulation can be a reflection of not only the immune system, but the overlay of the CNS reaction to that im-

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 SEX DIFFERENCES AND IMPLICATIONS mune attack. These are very different situations, said Rhonda Voskuhl, di- rector of the Multiple Sclerosis Research and Treatment Program at UCLA. Although sex differences in the progression of MS generally have not been observed, there is a clear difference in incidence. Sex differences in MS could be from the basic differences between males and females, including sex hormones (estrogen and testosterone) or sex chromosomes (XX or XY gene effects). Conditions that occur in one sex, but not in another, such as pregnancy, could also be major factors. In the case of MS, pregnancy reduces relapses by 80 percent, significantly more than most of the currently available drug therapies, which reduce relapses by about 33 percent. (Some reduce relapses by half to two thirds, but these carry with them risks of significant adverse events.) Understanding this effect of pregnancy on MS pathogenesis could aid the discovery and development of better therapies for MS. Voskuhl described a “bedside-to-bench-to-bedside” approach to con- sidering MS. The classic bench-to-bedside approach is somewhat risky, she said, starting with a molecule or a pathway, studying it in vitro and in vivo and then in human trials, often finding that the treatment does not work as predicted in people. Sex differences are known to be clinically important as major disease modifiers. A better approach in the case of MS is to go from what is known clinically, to characterize the cellular and molecular mechanisms, then to go back to the patients with a clinical trial. One example of this approach is the bedside observation of reduced relapses during pregnancy. As discussed, this could be related to any num- ber of things, including hormones or vitamin D. Several laboratories have now shown that estrogens, particularly estriol, are protective in the animal model of MS. Based on this clinical observation, and the subsequent results from animal models, estriol was then administered in pill form to people with MS in a Phase I trial, and reduction in the number and size of lesions was observed. A multicenter Phase II trial is under way. A second example stems from the clinical observation of the sex dif- ference in incidence of MS, and the fact that men are older than women at disease onset. Based on this decreased susceptibility of younger men (when testosterone levels are high), studies of the potentially protective role of tes- tosterone in MS were conducted in the animal model. Results supported the protective role of testosterone, and a pilot study of men with MS found that treatment with testosterone gel slowed brain atrophy and caused immune shifts that are biomarkers for improvement. A Phase II trial of testosterone in men with MS is being planned. As discussed by Arnold (Chapter 2), sex differences are not all about adult hormones. Developmental hormones and sex chromosomes also have effects. Using the four core genotype mouse model describe by Arnold to study EAE and lupus, Voskuhl found sex chromosome clearly had an effect,

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH with the XX genotype promoting disease development in both EAE and lupus (Smith-Bouvier et al., 2008). Voskuhl is also studying developmental hormone effects using the same system. In summary, Voskuhl stressed that clinical observations can lead to promising potential treatments for MS. Research on sex hormones has reached clinical trials, and research on sex chromosome effects are still in the early stages, but may have potential as well. Whether the sex chromo- some effect is an X dosage effect or a Y gene effect—and what that gene is—remains to be seen. Animal studies to elucidate mechanisms, and pilot trials of potential products in humans, are possible, but funding is a major obstacle, Voskuhl said. The small pilot trials described by Voskuhl were funded by the MS Society and the NIH. But the challenge is funding a potentially $30 mil- lion Phase III trial, especially when the drug under study is not a patent- protected product (e.g., estriol). Although estriol is already broadly used and well characterized with regard to safety, the fact that it is not patent protected means that a pharmaceutical company that might otherwise in- vest in developing a new product may not be able to recoup the significant product development costs through future product sales. Open Discussion: Multiple Sclerosis and Neuroinflammation In the open discussion, panelists further considered the implications of sex differences in MS, and how MS is different from the other diseases discussed so far. Participants were interested in further information on the studies discussed, and raised issues about approaches to research. Fox noted that for the other diseases discussed at the workshop, sex differences are apparent in the disease course. However, for MS, although the incidence is different for men and women, the disease course appears similar. Voskuhl noted that although progression may not be different, the immune response is more aggressive in women. The question is why, if im- mune response is different, is progression not different? A difference in the brain must be counter to the difference in the immune system. A question was raised as to whether estriol had been tested only in women, and if there are any obstacles to trying it in both sexes. Voskuhl responded that estriol has been shown to have an effect in male mice with EAE as well. This has not been done in clinical trials. The adverse events could be different, she noted; there would be no concern about endometrial or uterine cancers in men. Precedents have been set for giving estrogens to men with other diseases. A participant suggested that another way to study sex differences is to look at the factors at play when, for example, a male develops a disorder that is common in women. Another participant cautioned that care must

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0 SEX DIFFERENCES AND IMPLICATIONS be taken when looking at clinic populations versus the larger community. People with the most severe conditions are the ones who come to tertiary care. BOX 3-4 Key Points: Multiple Sclerosis (MS) and Neuroinflammation • Clear sex differences are evident in the incidence of MS: MS is two to three times more common in women. Onset occurs later in men, and is more likely to be primary progressive MS. Women appear to have a better prognosis, but differences are less appar- ent after adjusting for age at diagnosis and disease course. Pregnancy decreases disease activity (but the effect of oral contraceptives is unclear). • The clinical course of sex differences shows little evidence of sex differences: Despite observations in smaller studies, large magnetic resonance imaging (MRI) studies suggest no effect of sex on MRI measures of either inflam- mation or tissue injury. Histopathology shows virtually no effect of sex differences. Most clinical trials show no effect of sex differences. • There are sex-based immune differences in MS: Females mount a stronger immune response. Males have more severe inflammation. • Studies suggest a role for ultraviolet light and vitamin D in the pathogenesis of MS. • Clinical observations can lead to potential treatments for MS: Animal studies to elucidate mechanisms of the observed clinical pheno- types and pilot trials of potential products in humans are possible, but funding can be a major obstacle. Preclinical studies should include both sexes. In the community, there are large sex differences in pain, but within the ter- tiary care setting there are not many differences between males and females. OvERARCHING DISCuSSION Following the disease-specific panel discussions of issues related to sex differences in translational research, the four panel moderators and the workshop cochairs assembled to consider overarching issues across major disease areas.

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH Making the Decision to Study Sex Differences The panel first considered when, during development of products to treat disorders of the nervous system, consideration of sex differences would be most appropriate, taking into account financial, time, and mate- rial resource constraints. Richard Nakamura, director of the Division of Intramural Research Programs at the National Institute of Mental Health,1 said that ideally, information should be collected on any sex differences that may emerge, but sex differences are unlikely to be identified in relatively small proof-of- concept trials. The larger studies, such as STAR*D, are the ones that need to focus on sex differences. Investigators should publish any supplementary data on sex differences that emerge from trials, even when the data are not statistically significant. He also noted that large databases, such as the NIH’s GWAS databases, could provide a valuable resource in trying to understand sex differences. In addition, the Veterans Administration and other large healthcare entities that have large electronic health records systems should be encouraged to develop common features and nomenclature so that these systems could be analyzed for information on sex differences. Progress toward a system of universal medical records in the United States would aid this effort as well. From an industrial perspective, Chi-Ming Lee, executive director of Translational Science at AstraZeneca Pharmaceuticals, noted that unex- pected failures in later phases of product development (Phase II or III) are extremely costly. A company usually has a very strong rationale, and has met certain criteria, before moving a drug from early phase into the later phases of development. Data from animal models are considered in these decisions, but experience has shown that some animal models that were relied on failed, in the sense that they did not predict the outcomes later observed in humans. Theoretically, sex differences should be addressed early, Lee said, but there are many considerations. Should every animal model study include both male and female? Simply including females in studies will not neces- sarily provide the answer, and can provide a false sense of security. If either preclinical or clinical evidence suggests that sex makes a difference, then further research on these differences should be conducted. Many factors must be considered, including, but not limited to, hormonal effects, sex chromosome effects, lifespan, psychosocial factors, or species differences. Paul Hoffman, associate chief of staff for Research and Program De- 1 Dr. Nakamura’s comments were based on personal experience over the course of a career, and do not necessarily represent official NIH policy or official NIH endorsement of potential policies.

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 SEX DIFFERENCES AND IMPLICATIONS velopment at North Florida/South Georgia Veterans Health System, con- curred that evidence of an increased prevalence in one sex versus another merits further attention. The question is where the funding is best applied. In clinical trials, sex should be treated as a variable, and studies should be designed with enough power to allow for analyses by sex (rather than sex being a post-hoc analysis). Zorn emphasized the need for a business case before a pharmaceuti- cal company expends significant resources. The current costs of bringing a drug from the bench to the patient are in the range of $1.8 billion. To include sex as a variable in preclinical studies and clinical trial design, a hypothesis based on data is needed. While much of the data presented at the workshop have been very good and could be used to generate hypoth- eses about sex differences, some of those data are not yet clear enough to justify the significant investment required. A stronger investment is needed in basic research, he said, looking at differences in the sexes all the way down to the molecular level, so that drug developers have a solid base on which to test these differences in humans. The responsibility for obtaining this evidence falls to basic, preclinical, and clinical researchers of all kinds, communicating and working together. A participant pointed out that beyond the costs of development, a company must also bear the costs of educating physicians and providers about the use of the product. Nakamura added that over the past few years, there has been discus- sion that the business model for CNS disorder medication development is basically failing, and that many pharmaceutical firms are leaving this thera- peutic area. Studying sex differences, to the extent that they are a compli- cating factor, adds costs when developing a profitable drug. On the other hand, there is a limited business case for developing reasonable molecules that are inexpensive. Perhaps the NIH could help share the risk and lever- age the strengths of federally funded research and industry by performing proof-of-concept studies while providing industry with the opportunity to develop distributable drugs. A participant said that in recent years, charities such as the Bill and Melinda Gates Foundation have funded the development of compounds that are either not patentable, or are for a rare disease in countries where people cannot pay for products. Perhaps this alternative funding approach should be considered for sex differences. Lee noted that many companies are now involved in various consor- tiums, such as the Foundation for NIH’s Biomarker Consortium, where money is pooled and risk is shared. Lee also said that the blockbuster busi- ness model is really in the past. Moving forward, there is definitely consid- eration of personalized medicine, but the investment has to be justified. Another industry participant concurred that industry is moving away

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 SEX DIFFERENCES IN TRANSLATIONAL RESEARCH from big blockbuster drugs and is much more comfortable with smaller markets. In personalized healthcare, it is implicit that not only is the target population restricted, but the efficacy of the drug is potentially increased and thus has a higher chance. A product may be approved for the 20 per- cent of the people who have a particular mutation or amplification of a gene, with payers willing to reimburse the manufacturer for it because it works. Another participant wondered if a sex difference is something that a drug developer would simply rather not know because that information prevents them from marketing the product to everyone, potentially cutting the marketing in half. An industry participant responded that he was not aware of many drugs in development that were so much more efficacious for one sex than the other that they merited pursuing a separate, sex-based efficacy claim. However, safety problems detected in animals can often be restricted to one sex, or the therapeutic index may be different. The company is then faced with the difficult decision of whether to continue development of the drug for one sex, or stop and try to switch to another molecule in the family (assuming it is not a mechanism-based toxicology or adverse event). Risk/ benefit also needs to be considered. If toxicology in one sex is bad, should the drug still be developed for the opposite sex? If the other sex takes it inadvertently or doctors do not read the label or prescribe it off-label for the other sex anyway, that is a significant risk. Encouraging Basic Research into Sex Differences Many participants asserted that if basic researchers were somehow required to study both males and females, the amount of information avail- able about sex differences could be rapidly expanded, but such a mandate would be too expensive, and there would be a huge pushback from the research community. Other, more practical options could be to require study sections to rate grants on their comparison of sexes, or to put greater power in the hands of program officers, asking them to commit a certain amount of their funding to basic research grants that consider sex differ- ences. There could be a study section, or similar alternative mechanism, for vetting which sex differences in human conditions and diseases should be funded. A participant added that, as a previous recipient of NIH funding, he believed that forcing researchers to include both sexes in animal research was a bit unpalatable, but that it would be reasonable to ask them to justify why they use one sex or another, or both.

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 SEX DIFFERENCES AND IMPLICATIONS Interdisciplinary Collaboration As the panel presentations demonstrated, there are commonalities and shared factors across different clinical conditions with respect to sex differ- ences. Given limited funds, participants suggested that one way to have a greater impact is to target funding toward those commonalities. One way is to encourage interdisciplinary cooperation through Requests for Applica- tions. This would not need to be a large multicenter effort, but could be two researchers from different disciplines who are working in collaboration. A participant noted that the NIH interdisciplinary programs are intended to do this.