5  -Reductase

1. OH 5-Reductase OH o H Testosterone 5 -Dihydrotestosterone AR o ARAs ARAs Nucleus Cytoplasm ARAs ARAs AR AR Pol II complex PSA ARE ARE TATA Androgens and Androgen Receptor Mechanisms, Functions, and Clinical Applications Chawnshang Chang Ph.D. George Whipple Professor University of Rochester Medical Center

2. Conventional Anti-Androgens and Their Functions

3. Potential Anti-androgens based on the Current Concept of AR-Activation Pathway

4. Strategy of Discovering New Anti-Androgens Using cell-based assays to measure gene products that are regulated by the androgen-mediated AR-activation pathway. Looking into Natural Products, especially Chinese herbs, for new anti-androgens. Thus, to increase the chances of finding novel anti-androgens with new antagonistic mechanisms that are different from the conventional anti-androgens.

5. ASCJ Compounds A group of NEW chemical compounds (ASCJ), derivatives of a natural product isolated from a Chinese Herb, was discovered to possess high anti-androgenic activity.

6. The Unique Characteristics of ASCJ Compounds 1. ASCJ inhibits AR activity via a mechanism that is entirely different from any conventional anti-androgens, i.e., theyenhance degradation of AR. 2. ASCJ suppresses activity of bothwild type and mutant AR regardless in the presence or absence of androgen. 2. ASCJ, unlike some conventional anti-androgens, does not possess “residual androgenic activity”. In contrast, ASCJ suppresses residual androgenic activity induced by conventional anti-androgens.

7. ASCJ Specifically Reduced AR Expression LNCaP Cell Line

8. ASCJ Enhances Degradation of AR (in the presence of Cyclohexamide) V J E V J E V J E V J E LNCaP cells were cultured in medium with ASCJ, control, and vitamin E for 20 h. Subsequently, cyclohexamide (a protein synthesis inhibitor) was added to the cell cultures. The cells were harvested, at the designated times, and AR was detected by Western blot.

9. Potential Indications for ASCJ Compounds • Topical and Oral drugs for acne and baldness. • Oral or Parenteral drugs for prostate cancer and benign prostate hyperplasia (BPH). • Drugs for other androgen or AR-related disorders.

10. ASCJ Compound as Topical Drug for Acne ?

11. Hair Follicle and Sebaceous Glands

12. Androgen acts on the skin's sebaceous glands to stimulate sebum (oil) secretion; when the opening of follicle was blocked, a microcomedone is formed. Blackhead A blackhead occurs when the trapped sebum and bacteria open to the surface and turn black due to melanin, the skin's pigment. Whitehead When the trapped sebum and bacteria stay below the skin surface, a whitehead is formed. Drawings credit: www..Acne.org

14. Photo Credit: W.F. Bergfeld MD, FACP Cleveland Clinic

15. Photo Credit: W.F. Bergfeld MD, FACP Cleveland Clinic

16. Photo Credit: W.F. Bergfeld MD, FACP Cleveland Clinic

17. Conventional Anti-Acne Drugs • Microcomedones: Retina, Accutante, benzoyl peroxide, azelaic acid (unplug pores, but cause skin burning, peeling, scaling and allergic reactions). • Bacteria and Inflammation: Antibiotics, sodium sulfacetamide (Antibiotics resistant bacteria). • Oral Contraceptive: Birth control pill, Ortho Tri-Cyclen **None of the conventional topical drugs are targeted at blocking androgen stimulation (the cause of acne); and none of the conventional anti-androgens are effective as topical drug for acne.

18. A Novel Topical Drug for Acne ? ASCJ degrades AR => Inhibits androgen-induced sebum secretion => Acnesubside ?

19. Animal Model (Fuzzy Rats) for Studying Sebaceous Glands Castrated Male Rat (5 weeks later) Female Fuzzy Rat Male Fuzzy Rat Fuzzy rat model: F. Ye et al., Skin Pharmacol. 10:288-297 (1997)

20. ASCJ9 CONTROL Week 0 -1 ASCJ induces sebaceous glands regression in male rats. Week 3 Week 4 Week 5

22. After treated with ASCJ cream, for 5 wks, the number and size of glands (duct and lobe) were significantly reduced.

24. ASCJ cream tested in human volunteers (Taiwan) Age Sex Compound* Treatment Effective** Recovery time*** 17 F J15 twice a day ++ 1 week 15 M J15 as often as needed +++ 1 week 15 M J15 as often as needed +++ 1 week 38 F J15 as often as needed ++ 2 weeks 52 F J15 twice a day ++ 2 weeks 42 F J15 twice a day ++ 2 weeks 18 F J9 4 times +++ 1 week 24 F J9 twice a day +++ 1 week 17 M J9 once a day +++ 2 weeks * ASCJ15 in 1 mM; ASCJ9 in 2.5 mM ** Pimples usually subside within 5-7 days of treatment. *** Recover time: acne disappear, (n>45, with >90% effective rate). No skin allergy, peeling, redness or other adversary effects were reported.

25. Anti-Acne Activity of ASCJ in Human Volunteer Before treatment with ASCJ cream One month after treatment

26. A Novel Topical Drug for Acne ? ASCJ degrades AR => Sebaceous glands reduction => Acne subside Yes !

27. A Potential Topical New Drug for Baldness ASCJ degrades AR => Blocks androgen stimulation on follicular cells => Inhibits hair loss?

28. Androgenetic Alopecia (Baldness) Male pattern baldness, a genetic inherited hair disorder, has been a part of the human race for as long as historical records. It affects between 50% and 80% of Caucasian men. Boldness occurred in 30% of men in their thirties, 40% in their forties, and so on until 80% of men are affected when 80 or more years old. Despite its standard name of “male pattern baldness” androgenetic alopecia is also the most common form of hair loss in women.

29. Androgen reaches Follicular cells via circulation.

30. Androgenetic Alopecia, A Genetic Inherited Hair Growth Disorders

31. FDA-Approved Drugs for Baldness Finasteride (Propecia):oral, (5a-reductase inhibitor) Cause soft tender breast, impotence, less sexual desire in some men, difficulty in achieving an erection. Minoxidil (Rogaine): topical,(a vessel dilator not an anti-androgen) Cause scaling, itching, burnings scalp, swelling face. When enter the blood stream it can cause: blurred vision, chest pain, decrease in sexual ability or desire, irregular heartbeat, headache, swelling of face, and rapid weight gain. Some womenmay experience hair growth on the forehead, upper lip, cheeks, lower arms and/or lower legs. **No conventional anti-androgens are effective when used as topical drug for baldness!

32. An Androgen-Induced “Hair Growth Suppression” in Mice, An Animal Model Testosterone applied to the back of mice suppresses hair growth Mouse model used: H.Uno et al., J. Cutaneous, Aging & Cosm Derm 1:193 (1990)

33. ASCJ overcomes testosterone-induced hair growth suppression (2 right mice) Testosterone suppresses hair growth (2 left mice)

34. A New Topical Drug for Baldness ? ASCJ degrades AR => Blocking androgen stimulation => Preventing hair loss and stimulating new hair growth? Very Likely!

35. Hope Sprouts Eternal ASCJ Newsweek, Feb. 9, 2004, by Karen Springen

36. Other Potential Indications for ASCJ Compound • Wound Healing (Topical): Androgens are know to involved in delay of wound healing, especially in diabetic patients and in aging men. • Spinal and Bulbar Muscular Atrophy (Kennedy’s Disease): This neuronal disorder is known to cause by mutant AR proteins that aggregated in neuron cells and caused muscular atrophy.

37. Androgen Inhibits cutaneous wound healing Background 1. Androgen receptors are detectable in keratinocyte, inflammatory cells and fibroblast in wounded area, suggesting that androgens might be involved in the regulation of inflammation and repair process. 2.Mice received castration or flutamide treatment their cutaneous wound healing accelerated andaccompanied by an attenuated inflammatory response, i.e., reduced macrophage invasion, and increased matrix collagen deposition. 3.AR might be involved in following mechanisms: — Androgens possess both pro- and anti-inflammatory actions to modulate the expression of various cytokines, eg. IL-1, IL-10, and TNF. — Androgens might influence the angiogenic response that follows cutaneous injury. — Androgens might be important regulators of matrix protein deposition during wound healing.

38. Day 4 WT KO Fig. 1The activity of wound healing. (A) Wound pictures comparison of the WT and ARKO mice. (B) Quantitative comparison of the wound areas of WT and ARKO mice. A B

39. A B C WT 40X 200X D E Red arrow: PMN Green arrow: lymphocyte KO 40X 200X Fig. 2Histology comparison of Day 6 wounds with H&E Stain. Compare WT and ARKO wounds, the wound size (the length of arrows) of ARKO mice (A) is smaller than that in WT mice (D). Besides, the cell infiltration in WT wound (B) is more obvious than in ARKO wound (E). Under higher magnification (C), these infiltrating cells includes Polymorphonuclear cells (PMN, red arrow), lymphocytes (green arrows).

40. A B WT 40X 200X C D KO 40X 200X Fig. 3 Trichrome Stain for collagen deposition in Day 6 wound. Compare to WT mice, the ARKO mice has more collagen deposition in the wound area. Blue color: collagen fiber Red arrowsindicate the collagen deposition in wound area

41. Control Wound mWT mWT mKO mKO fWT fWT A 1 1 2 2 1 1 2 2 1 1 AR -Actin C B Fig. 4(A) RT-PCR for AR gene expression in Day 6 wound tissue. The AR expression increase in male wild type and female wild type wounds, but not in the male ARKO wounds. (B,C) Immunohistochemistry of AR in Day 1 wound. It shows AR positive staining (brown color) in epithelial cells(black arrow), hair follicles (red arrow), and fibroblasts (blue arrow). AR positive staining is also in the infiltrating macrophage (green arrow) but not in lymphocyte (purple arrow).

42. Control Wound A mWT mWT mKO mKO fWT fWT 1 1 2 2 1 1 2 2 1 1 TGF iNOS TLR-4 -Actin B Fig. 5 RT-PCR analysis for inflammatory factors that might involved in the wound healing process. (A) mRNA extracted from day 6 wound tissues. (B) Real-time RT-PCR analysis of iNOS gene expression in wound tissue from day 0 to day 3

43. Summary: • 1.Accelerating wound healing process in ARKO mice compare to WT mice. • 2.The infiltration in ARKO wound is less server than in WT wound. • 3.Collagen deposition in ARKO wound is more than in WT wound. • 4.AR expression was detected in epithelial cells, hair follicles, fibroblast, and infiltrating macrophage, but not detected in infiltrating lymphocytes. Besides, AR expression was increased in wound tissues compare to normal skin. • 5.iNOS gene expression in wound tissue is increase in WT mice, but not in the ARKO wounds.

44. ASCJ Fasten Wound Healing in Mice Control Cream ASCJ9 Cream Mouse #1 Mouse #2 Mouse #3 Mouse #4 Day 0 Day 5 Day 10

45. ASCJ Compounds Toxicity Test Compound Surv/total Mean day weight Comments to death change ________________________________________________________________ Etoposide 1/4 8 d +2 g Hunching & ruffled furs JC9* 4/4 >28 d +4 g Normal appearance; (selected for drug develop) (small residual comp. left in abdominal cavity); normal internal organs. JC15* 4/4 >28 d + 4 g Hunching & ruffled furs; male sex organs fused DMOS/Corn oil 4/4 >28 d + 4 g Normal appearance and (vehicle control)normal internal organs. *Animal received 100 mg/kg daily injection (ip) for 14 days, and observed for another 14days before the experiment was terminated. Note: Mice in separate experiments received single JC9 injection at 200 mg/kg (3) and 500 mg/kg (2) their appearance remained normal for up to 10 days ; the other two mice received 1 injection of JC9 at 1 g/kg injection, one mouse died (in 3 hr.) and the other one survived 6 days when experiment was terminated.

46. Pharmacokinetics of ASCJ9 5 mM of JC9 is detectable, in plasma, at 75 min after i.v. injection (16 mg/kg body weight)

47. 12w 20w 25w 32w 38w Mating test Pair 2 Pair 3 Jc-9 vehicle Plugs 0 0 Pregnancy 0 0 Litters 0 0 Mating test Jc-9 vehicle Plugs 2 0 Pregnancy 2 0 Litters 2 0

48. ASCJ Compounds, Ideal Drug Candidates • Unique Anti-Androgen that inhibits androgen activity via enhancing AR degradation and does not affect endogenous androgen. • Potent efficacy applied either topically or systemically. • Low toxicity, and safe to use systemically. • Relatively high bioavailability in vivo. • One group of compounds for Multiple Indications.

49. Acknowlegments Shuyuah Yeh Yi-Fen Lee Miyamoto h Karen Wolf Hui-Kuan Lin Chao Xie Shih-Rong Shyr (Stone) Loretta Collins All of the members in Dr. Chang’s Lab

50. CHAWNSHANG CHANG : GEORGE WHIPPLE PROFESSOR. George Whipple Lab Members: 4 Junior Faculty 12 PostDrs 20 PhD Students University of Rochester New York, USA