Inhibition of Testicular Retinoic Acid Biosynthesis for Male Contraception. John K. Amory MD, MPH University of Washington October 30th, 2011

Transcription

1 Inhibition of Testicular Retinoic Acid Biosynthesis for Male Contraception John K. Amory MD, MPH University of Washington October 30th, 2011

2 Bisdichloroacetyldiamines (BDADs) Shown to reversibly inhibit spermatogenesis in 50 years ago Development abandoned because they were found to cause a disulfiram reaction when alcohol was consumed Shown to suppress spermatogenesis in many species including rodents, cats, dogs, monkeys and man!

3 The effect of a BDAD on spermatogenesis in a representative human subject over 27 weeks of treatment Heller CG, Moore DJ, Paulsen CA. Suppression of spermatogenesis and chronic toxicity in men by a new series of Bis (dichloroacetyl) Diamines. Toxicol Appl Pharmacol :1-11.

4 Disulfiram Reactions Disulfiram prevents metabolism of alcohol to acetic acid via inhibition of the liver isozyme of aldehyde dehydrogenase (ALDH2) Disulfiram used for aversion therapy of alcoholism S ALDH2 NAD + NADH NAD + NADH O H 3 C CH 2 OH H 3 C C H Ethanol Alcohol Acetaldehyde Aldehyde Dehydrogenase Dehydrogenase N S S N S Disulfiram H 3 C O C OH Acetic Acid

5 An aldehyde dehydrogenase is necessary for retinoic acid biosynthesis in spermatogonia Type A Spermatogonia Differentiation (A A1 transition) STRA8 Retinoic Acid-RAR Retinol-RBP from Sertoli cells Retinoic acid Retinaldehyde ALDH1A2 Slide courtesy of Michael Griswold STRA6 Retinol Hypothesis: BDADs exert their contraceptive effect by inhibiting spermatogonial retinoic acid biosynthesis

6 ALDH1A2 is Testes Specific --Target enzyme is the testicular isozyme of aldehyde dehydrogenase (ALDH1A2), which is uniquely expressed in the testes --In the mouse, expression of Aldh1a2 occurs prior to the onset of spermatogenesis Hsu et al. Biochemica Biophysica Acta 2000;

7 ALDH1A2 is a retinal dehydrogenase, which converts retinal to retinoic acid Paik et al (unpublished observations)

8 The BDAD WIN18,446 inhibits ALDH1A2 Paik et al (unpublished observations)

9 The BDAD WIN 18,446 inhibits Stra8 expression by retinol but not by retinoic acid in primary cultures of murine testicular gonocytes * p<0.05 ** p<0.001 Hogarth et al. Biol Reprod :

10 In vivo Rabbit Studies Goal: to assess impact of WIN 18,446 (200 mg/kg) on spermatogenesis and fertility over 16 weeks of treatment Followed animals for changes in testosterone, liver and kidney function and blood counts, testicular histology, and testicular retinoic acid Allowed animals to recover post-treatment

11 Rabbit Ejaculate Collection

12 Ejaculated Sperm Concentrations with BDAD treatment in Rabbits 500 Treatment Sperm Concentration (Millions/ml) * Hemi-orchiectomy * * * * * * * * * * Time (weeks) *p<0.05 c.f. baseline * Amory et al. J Andrology :

13 Testicular Histology (100x) Control Rabbit Testis With normal spermatogenesis Rabbit Testis after treatment with the BDAD WIN 18,446 (200 mg/kg orally, daily x 16 weeks) Amory et al. J Andrology :

14 Testicular Histology (400x) Control Rabbit Testis with normal spermatogenesis Rabbit Testis after treatment with the BDAD WIN 18,446 (200 mg/kg orally, daily x 16 weeks)

15 Serum Testosterone/Lab tests 3 Serum Testosterone Before During and After Treatment with Win 18,446 (200 mg/kg) orally x 16 weeks Treatment Period No changes in liver, Kidney function or Blood counts 1.5 Serum Testosterone (ng/ml) 1 No changes in sexual behavior Time (weeks) Testicular volume decreased during treatment

16 Testicular and Epididymal Weights 4 Organ Weight during treatment with the BDAD WIN 18,446 at 200mg/kg p.o. daily (n=4) Testis Epididymis Organ Weight (grams) * * 1 * Control 4 weeks 8 weeks 16 weeks

17 Testicular Retinoic Acid Decrease in testicular retinoic acid occurs before drops in ejaculated sperm counts Tissue Retinoic Acid 10 (pmol/gram) 5 * * ** Residual testicular retinoic acid during treatment may represent retinoic acid synthesized in testicular interstitium by ALDH1A1, or contamination with blood. 0 Baseline 4 Weeks 8 Weeks 16 Weeks p<0.05 compared with baseline * * p<0.01 compared with baseline

18 Fertility 0/4 animals able to impregnate a female at end of treatment All animals fertile after recovered sperm counts (mean embryo count ~7)

19 Summary The BDAD WIN 18,446 inhibits ALDH1A2 enzyme activity in vitro In cultures of mouse testicular cells, WIN 18,446 inhibited expression of Stra8 by retinol, but not by retinoic acid In vivo, WIN18,446 significantly reduced tissue retinoic acid concentrations and abrogated spermatogenesis and fertility. Testosterone remains normal throughout treatment. No evidence of systemic toxicity

20 Conclusion WIN 18,446 reversibly suppresses spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1A2 Retinol-RBP from Sertoli cells or serum Novel, specific inhibitors of ALDH1A2, may have utility as male contraceptives Type A Spermatogonia STRA6 Spermatogonial Differentiation STRA8 Retinoic Acid-RAR Retinoic acid Retinaldehyde Retinol Azoospermia BDADs ALDH1a2

21 Contraceptive Hypothesis Sadly, numerous alterations of the BDADs failed to result in compounds with specific inhibitory activity for ALDH1A2 vs. ALDH2, Alternative pharmacophores and a better understanding of the molecular pharmacology will be required to develop novel, specific inhibitors of ALDH1A2.

22 ALDH1A2-structure/function ALDH1a2 is a 220kD tetramer (4 x 55 kd) with a cytosolic localization Enzymatic activity is NAD+ dependent Crystal structure of murine ALDH1a2 (at 2.7A resolution) Lamb & Newcomer, Biochemistry (1999) 38:

23 ALDH1A2 Crystallization Have crystallized ALDH1A2 to 2.9 Ångstroms resolution Attempting to improve resolution with truncation & substitution strategies to better characterize active site and substrate interactions Such information may help direct rational design of novel inhibitors The structure of a human ALDH1A2. Only one subunit of the tetrameric protein is shown Stenkamp et al., (unpublished Observations)

24 Novel ALDH1a2 Pharmacophores Identified by High Throughput Screening Inhibitors of Interest

25 Future Work Characterize high-throughput screen hits Use computational modeling with the solved crystal structure to optimize leads Identify compounds with acceptable pharmacokinetic characteristics Test impact of compounds on spermatogenesis and fertility in mice

26 Thank You University of Washington William Bremner MD, PhD Alex Goldstein PhD Charles Muller PhD Jisun Paik PhD Nina Isoherranen PhD Ronald Stenkamp PhD Tim Martins PhD Washington State University Mike Griswold PhD Chris Small MS Cathryn Hogarth PhD NIH Diana Blithe (U01HD060408)