TEAM SCIENCE AT A PROGRAMMATIC LEVEL. Sundeep Khosla, M.D. Mayo Clinic College of Medicine

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1 TEAM SCIENCE AT A PROGRAMMATIC LEVEL Sundeep Khosla, M.D. Mayo Clinic College of Medicine

2 APPROACH TO TRANSLATIONAL RESEARCH IN OSTEOPOROSIS Epidemiological Studies Clinical Investigation Mouse and Cellular Models Translation into Practice

3 FRACTURES IN MEN Incidence/1, person-yr Hip Vertebrae Colles Men Women Age group, yr Cooper & Melton, Trends Endocrinol Metab 8:225, 1992

4 LIFETIME RISK OF FRACTURE AT AGE 5 YEARS (%) White women White men Hip fracture Vertebral fractue Forearm fracture Any of the Annual healthcare costs ~ $17B Cummings & Melton, Lancet 359:1761, 22

5 ESTROGEN VERSUS ANDROGEN EFFECTS ON THE SKELETON TRADITIONAL VIEW Oophorectomy/menopause in women loss of E BR/ BF Rapid bone loss Therefore, E regulates bone metabolism in women

6 EFFECT OF ORCHIDECTOMY ON BMD IN MEN 1.4 BMD, gm/cm Stepan et al. JCEM 69:523, Time after Orchidectomy, yrs

7 ESTROGEN VERSUS ANDROGEN EFFECTS ON THE SKELETON TRADITIONAL VIEW Oophorectomy/menopause in women loss of E BR/ BF Rapid bone loss Therefore, E regulates bone metabolism in women Orchidectomy in men loss of T BR/ BF Rapid bone loss Therefore, T regulates bone metabolism in men

8 ROLE OF ESTROGEN IN THE MALE SKELETON Evidence from experiments of nature An estrogen receptor (ER) mutant male (Smith et al. NEJM 331:156, 1994) and two aromatase deficient males (Morishima et al. JCEM 8:3689, 1995; Carani Et al. NEJM 337:91, 1997) were described All three individuals had unfused epiphyses, high rates of bone resorption (and formation), and osteopenia

9 INSIGHTS FROM THE ERα AND AROMATASE DEFICIENT MALES E is required for epiphyseal closure Presence of E is necessary for the acquisition of bone mass during puberty in boys

10 ISSUES LEFT UNRESOLVED BY THE ERα AND AROMATASE DEFICIENT MALES What if any, is the role of E in regulating bone remodeling in adult men with mature skeletons? What is the role of E or T deficiency in mediating age-related bone loss in men

11 APPROACH TO TRANSLATIONAL RESEARCH IN OSTEOPOROSIS Epidemiological Studies Clinical Investigation Mouse and Cellular Models Translation into Practice

12 DISSECTING RELATIVE CONTRIBUTIONS OF E VS T TO BONE TURNOVER IN MEN Visit Entry Baseline Final Weeks Leuprolide Leuprolide Letrozole T + E Patch Falahati-Nini et al. JCI 16: , 2 A: No Patch B: E Patch C: T Patch D: T + E Patch T Patch

13 IN VIVO EFFECTS OF SEX STEROIDS ON BONE RESORPTION MARKERS 4 3 ** *** *** P <.1 ** P <.5 * P <.5 Dpd % change 2 1 NTx -1 Group A Group B (-T, -E) (-T, +E) Group C (+T, -E) Group D (+T, +E)

14 IN VIVO EFFECTS OF SEX STEROIDS ON BONE RESORPTION MARKERS 4 3 ** *** *** P <.1 ** P <.5 * P <.5 Dpd % change 2 1 * NTx -1 Group A Group B (-T, -E) (-T, +E) Group C (+T, -E) Group D (+T, +E)

15 IN VIVO EFFECTS OF SEX STEROIDS ON BONE RESORPTION MARKERS % change ** *** * * ** *** P <.1 ** P <.5 * P <.5 Dpd ANOVA E effect: P =.5 T effect: P =.232 NTx -1 Group A Group B (-T, -E) (-T, +E) Group C (+T, -E) Group D (+T, +E) ANOVA E effect: P =.2 T effect: P =.85

16 IN VIVO EFFECTS OF SEX STEROIDS ON SERUM OSTEOCALCIN LEVELS Serum Osteocalcin, % change *** *** P <.1 Group A (-T, -E) Group B (-T, +E) Group C (+T, -E) Group D (+T, +E)

17 IN VIVO EFFECTS OF SEX STEROIDS ON SERUM OSTEOCALCIN LEVELS Serum Osteocalcin, % change *** *** P <.1 Group A (-T, -E) Group B (-T, +E) Group C (+T, -E) Group D (+T, +E)

18 IN VIVO EFFECTS OF SEX STEROIDS ON SERUM OSTEOCALCIN LEVELS Serum Osteocalcin, % change ANOVA *** E effect: P =.2; T effect: P =.13 *** P <.1 Group A (-T, -E) Group B (-T, +E) Group C (+T, -E) Group D (+T, +E)

19 E VS T EFFECTS ON BONE TURNOVER IN MEN: SUMMARY In men, E is the dominant sex steroid regulating bone resorption, although T (in the absence of aromatization to E) may make a smaller contribution (< 3%) Both E and T contribute to the maintenance of bone formation

20 E DEFICIENCY IS THE PRIMARY MEDIATOR OF BONE LOSS IN HYPOGONADAL MEN Healthy men (age 2-5 yrs) given GnRH and aromatase blocker for 16 weeks Treated with 5 doses of T (, 1.25 g, 2.5 g, 5 g, 1 g) All subjects had suppressed E 2 levels ( pg/ml) T levels: 43, 234, 378, 497, and 985 ng/dl Regardless of the T level, in the setting of low E 2 levels, bone loss (total, cortical, trabecular density, cortical area and thickness) was identical in all the T groups Yu et al. ASBMR, 212 (#1199)

21 RALOXIFENE IN MEN Study Design 5 elderly men, mean age 69 years Randomized to placebo or raloxifene, 6 mg/d, for 6 months Baseline and 6 month assessment of bone turnover markers Doran et al. JBMR 16: , 21

22 RALOXIFENE IN MEN Changes in PTH and bone turnover markers Placebo Raloxifene P-value PTH, pmol/l +.2 ± ± BSAP, U/L -1 ± 1 ±.1 Urine NTx, nmol/d -17 ± 12 3 ± 11.19

23 RALOXIFENE IN MEN Change in Urine NTx Excretion as a Function of Baseline E 2 Levels Urinary NTx Excretion, nmol /24 hr Raloxifene R =.57 P < Baseline Serum Estradiol, pg/ml Placebo R = -.15 P = Baseline Serum Estradiol, pg/ml

24 RALOXIFENE IN MEN Change in Urine NTx Excretion as a Function of Baseline E 2 Levels Urinary NTx Excretion, nmol /24 hr Raloxifene R =.57 P <.1 26 pg/ml Baseline Serum Estradiol, pg/ml Placebo R = -.15 P = Baseline Serum Estradiol, pg/ml

25 APPROACH TO TRANSLATIONAL RESEARCH IN OSTEOPOROSIS Epidemiological Studies Clinical Investigation Mouse and Cellular Models Translation into Practice

26 RELATIONSHIP OF E 2 VS. T LEVELS TO FRACTURE RISK IN OLDER MEN Serum sex steroids analyzed by GC-MS at baseline in older men (n = 2639, mean age 75 years) Fractures occurring after baseline were validated (average follow up of 3.3 years) Mellström et al. JBMR 23:1552, 28

27 RELATIONSHIP OF E 2 VS. T LEVELS TO FRACTURE RISK IN OLDER MEN (Cont d) Age-adjusted HR for risk of first fracture Low E 2 : 16 pg/ml Low T: 336 ng/dl (Lowest quartiles for each) I: nl E 2, nl T II: low E 2, nl T III: nl E 2, low T IV: low E 2, low T

28 RELATIONSHIP OF E 2 VS. T LEVELS TO FRACTURE RISK IN OLDER MEN (Cont d) Poisson regression model 12 Incidence/1 person-years E2, pg/ml

29 BIOAVAILABLE VS SHBG-BOUND SEX STEROIDS NON-BIOAVAILABLE BIOAVAILABLE T T E 2 E 1 Free (1-3%) ~5% SHBG (~9 kd) E 2 E T 2 E 1 E 1 ALBUMIN (~66 kd) ~5%

30 SERUM TOTAL TESTOSTERONE AND ESTRADIOL LEVELS AS A FUNCTION OF AGE IN ROCHESTER, MN MEN AND WOMEN Total T, ng/dl Total E 2, pg/ml Pre Post Pre Post Age, yrs Age, yrs Khosla et al. JCEM 83:2266, 1998

31 SERUM BIOAVAILABLE TESTOSTERONE AND ESTRADIOL LEVELS AS A FUNCTION OF AGE IN ROCHESTER, MN MEN AND WOMEN 2 3 Bioavailable T, ng/dl Bioavailable E 2, pg/ml Pre Post Pre Post Age, yrs Age, yrs Khosla et al. JCEM 83:2266, 1998

32 CHANGES IN SERUM SHBG IN MEN 6 SHBG, nmol/l 4 2 < Age, yrs Khosla et al. JCEM 83:2266,

33 SUMMARY: AGE-RELATED CHANGES IN SEX STEROIDS Abrupt decrease in total and bioavailable estrogen levels in women at the time of menopause More gradual decreases in serum bioavailable testosterone and estrogen levels in men starting at age 5-6 years Marked increase in men in serum SHBG levels starting at age 5-6 years

34 APPROACH TO TRANSLATIONAL RESEARCH IN OSTEOPOROSIS Epidemiological Studies Clinical Investigation Mouse and Cellular Models Translation into Practice

35 IN VIVO EFFECTS OF SEX STEROIDS ON BONE RESORPTION MARKERS % change ** *** * * ** *** P <.1 ** P <.5 * P <.5 Dpd NTx -1 Group A Group B (-T, -E) (-T, +E) Group C (+T, -E) Group D (+T, +E)

36 IN VIVO EFFECTS OF SEX STEROIDS ON SERUM OSTEOCALCIN LEVELS Serum Osteocalcin, % change *** *** P <.1 Group A (-T, -E) Group B (-T, +E) Group C (+T, -E) Group D (+T, +E)

37 DIFFERENTIAL EFFECTS OF E AND T ON BONE RESORPTION MARKERS IN YOUNG MEN Percent Change Percent Change Urinary Deoxypyridinoline Serum Osteocalcin Serum N-telopeptide Time after GnRH, weeks Time after GnRH, weeks Leder et al. JCEM 88: 24, 23 Percent Change Percent Change Serum PINP

38 DIFFERENTIAL EFFECTS OF E AND T ON BONE RESORPTION MARKERS IN YOUNG MEN Percent Change Percent Change Leder et al. JCEM 88: 24, Urinary Deoxypyridinoline Phase I. Uncoupling Serum Osteocalcin Percent Change Percent Change -1 Serum N-telopeptide Serum PINP Time after GnRH, weeks Time after GnRH, weeks Phase I. Uncoupling

39 DIFFERENTIAL EFFECTS OF E AND T ON BONE RESORPTION MARKERS IN YOUNG MEN Percent Change Percent Change Leder et al. JCEM 88: 24, Urinary Deoxypyridinoline Phase II. Re-coupling Serum Osteocalcin Percent Change Percent Change -1 Serum N-telopeptide Serum PINP Time after GnRH, weeks Time after GnRH, weeks Phase II. Re-coupling

40 ACUTE CHANGES IN BONE FORMATION AND OC # s FOLLOWING OVX IN RATS BFR/BS, µm 3 /µm 2 per day (X1-2 ) Sham OVX ** *P<.2, **P<.2 vs Sham Pre Days following OVX * OCs/BS (%) *P<.5 SHAM * OVX Lean et al. J Endocrinol 142:119, 1994

41 OSTEOCLAST OSTEOBLAST REGULATION OF OSTEOBLAST OSTEOCLAST FORMATION/FUNCTION Stimulatory Factors Inhibitory Factors OC PRECURSORS Differentiation and activation ACTIVE OC OC APOPTOSIS OPG IL-6 IL-7 PGE 2 GM-CSF M-CSF RANKL RANKL Osteoclast-derived TGFβ coupling factors TGFβ OSTEOPROGENITOR/ OSTEOBLASTS

42 BONE MARROW-BONE INTERFACE

43 BONE REMODELING COMPARTMENT Hauge et al. JBMR 16:1575, 21

44 SCHEMATIC OF THE BONE REMODELING COMPARTMENT Khosla, Westendorf, Oursler JCI 118:421, 28

45 POTENTIAL MECHANISMS FOR COUPLING BONE FORMATION TO BONE RESORPTION Cell-cell contact Factors released from the bone matrix Osteoclast-derived secreted factors

46 BIDIRECTIONAL EPHRIN SIGNALING BETWEEN OSTEOCLASTS AND OSTEOBLASTS Mundy & Elefteriou Cell 126:441, 26; Zhao et al. Cell Metab 4:111, 26

47 ROLE OF TGFβ1 IN COUPLING BONE RESORPTION TO BONE FORMATION Iqbal & Zaidi Nat Med 15:729, 29; Tang et al. Nat Med 15:757, 29

48 SCHEMATIC OF THE BONE REMODELING COMPARTMENT

49 OSTEOCLAST CONDITIONED MEDIA STIMULATES MINERALIZATION ALIZARIN RED Osteoclast Precursor CM Mature Osteoclast CM Alizarin red units OC Precursor Conditioned Media Mature OC Conditioned Media * Pederson et al. PNAS 15:2764, 28

50 MARROW-DERIVED OSTEOCLAST COUPLING FACTOR EXPRESSION Pederson et al. PNAS 15:2764, 28

51 COUPLING FACTOR HYPOTHESIS BMP6 Wnts S1P SCLEROSTIN

52 APPROACH TO TRANSLATIONAL RESEARCH IN OSTEOPOROSIS Epidemiological Studies Clinical Investigation Mouse and Cellular Models Translation into Practice

53 CLINICAL IMPLICATIONS Use of serum estradiol levels in the evaluation of male osteoporosis

54 POTENTIAL CLINICAL APPROACH Male with low T (< 3 ng/ml), osteoporosis/increased fracture risk E 2 normal (> 16 pg/ml by mass spec): standard pharmacological therapy (e.g., bisphosphonate) E 2 low (< 16 pg/ml): Consider trial of T therapy and monitor BMD

55 CHANGES IN SERUM E 2 FOLLOWING TRANSDERMAL T REPLACEMENT 5 E 2, pg/ml Months Wang et al. JCEM 89:285, 24

56 CLINICAL IMPLICATIONS Use of serum estradiol levels in the evaluation of male osteoporosis Use of selective estrogen receptor modulators in male osteoporosis

57 RALOXIFENE IN MEN WITH PROSTATE CANCER ON GnRH Smith et al. JCEM 89:3841, 24 Percent change Lumbar spine P=.7 Percent change Total hip P<.1 Raloxifene n = 19 Percent change Trochanter P< Month Percent change Femoral neck P= Month No raloxifene n =22

58 EFFECTS OF THE SERM, TOREMIFINE, ON VERTEBRAL FRACTURES IN MEN WITH PROSTATE CANCER 1284 men with prostate cancer on ADT randomized to receive 8 mg toremifine or placebo for up to 24 months 5% reduction noted in vertebral fractures in the toremifene treated group (P =.5) Smith et al. J Urol 184:1316, 21

59 CLINICAL IMPLICATIONS Use of serum estradiol levels in the evaluation of male osteoporosis Use of selective estrogen receptor modulators in male osteoporosis Cautionary note regarding possible efficacy of nonaromatizable selective androgen receptor modulators in preventing bone loss in men

60 CLINICAL IMPLICATIONS Use of serum estradiol levels in the evaluation of male osteoporosis Use of selective estrogen receptor modulators in male osteoporosis Cautionary note regarding possible efficacy of nonaromatizable selective androgen receptor modulators in preventing bone loss in men Based on osteoclast-derived osteoblast coupling factors, potential development of novel bone formation-stimulating agents

61 ACKNOWLEDGEMENTS B. Lawrence Riggs L. Joseph Melton III Shreyasee Amin Merry Jo Oursler Jennifer Westendorf Matthew Drake David Monroe Terry Therneau Elizabeth Atkinson Sara Achenbach Richard Robb/Jon Camp Louise McCready Amanda Tweed Margaret Holets Kelley Hoey Jim Peterson Dan Fraser Matt Roforth Ulrike Mödder Farhan Syed Patrick Doran Ali-Falahati Nini Kristy Nicks K. Chokalingam

62 RESEARCH SUPPORT MAYO CTSA MAYO FOUNDATION