In Mice Treated with L-39 Over 28 Days Was 1.25-Fold

British Journal of Cancer (2000) 83(1), 74–82 © 2000 Cancer Research Campaign doi: 10.1054/ bjoc.2000.1136, available online at http://www.idealibrary.com on Anti-tumour effects and pharmacokinetic profile of 17-(5′-isoxazolyl)androsta-4,16-dien-3-one (L-39) in mice: an inhibitor of androgen synthesis IP Nnane*, BJ Long, Y-Z Ling†, DN Grigoryev and AM Brodie Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA Summary 17-(5′-Isoxazolyl)androsta-4,16-dien-3-one (L-39), a novel androstene derivative, was synthesized and evaluated in vitro and in vivo. L-39 showed potent and non-competitive inhibition of human testicular microsomal 17α-hydroxylase/C17,20-lyase with an IC50 value of 59 nM and Ki of 22 nM. L-39 also showed potent and competitive inhibition of 5α-reductase in human prostatic microsomes with IC50 and Ki values of 33 and 28 nM respectively. L-39 (5 µM) has also been shown to manifest anti-androgenic activity in cultures of human prostate cancer cell lines (LNCaP) by preventing the labelled synthetic androgen R1881 (5 nM) from binding to the androgen receptors. Androgen- dependent human próstate cancer xenografts (PC-82) were grown in nude mice and the effects of L-39 (50 mg kg–1 day–1) on tumour growth and prostate-specific antigen (PSA) levels were determined after 28 days. L-39 significantly (P < 0.01) diminished tumour growth and wet weights to a similar extent as castration or flutamide treatment. L-39 also significantly (P < 0.01) reduced serum PSA levels by more than 80% in the mice bearing human prostate cancer xenografts. Pharmacokinetic studies were also conducted in male Balb/c mice. After subcutaneous administration of a single bolus dose, L-39 was rapidly absorbed into the systemic circulation. Peak plasma levels occurred at 0.75 h and then declined with a t1/2 of 1.51 h. The bioavailability of L-39 after subcutaneous administration was 28.5%. These results demonstrate that L-39 is a potent inhibitor of androgen synthesis and is effective in reducing the growth of human prostate cancer xenografts in nude mice. Although improvements in the bioavailability are necessary, L-39 is a potential lead compound with this profile as an inhibitor of prostate cancer growth. © 2000 Cancer Research Campaign Keywords: 17α-hydroxylase/C17,20-lyase; 5α-reductase; androgens; prostate cancer; pharmacokinetics Prostate cancer is second only to lung cancer as the leading cause role than T in the organogenesis and homeostasis of the prostate of death and is the most prevalent cancer amongst men in the USA (Wilson, 1996). Current endocrine therapies such as orchidectomy and Europe and accounts for about a third of all cancers diagnosed and luteinizing hormone releasing hormone (LHRH) agonists each year in men. In the UK, the incidence of prostate cancer has result in reduced androgen production by the testis and are useful almost doubled over the last 30 years. Similarly, in 1998, it was in the early stages of prostate cancer. However, these treatment estimated that more than 334 500 cases were diagnosed and more options fail to alter androgen production by the adrenals that may than 41 800 men died of the disease in the USA (Dijkman and contribute androgen precursors to the prostate. The addition of Debruyne, 1996). The prostate gland is under the influence of flutamide, an anti-androgen to inhibit the action of androgens on androgens and often becomes enlarged in older men. It has clearly the prostate, is only partially effective as the disease frequently been demonstrated that prostate cancers are androgen-dependent progresses due to mutations in the androgen receptor, which can and that withdrawal of androgen supply produced favourable utilize flutamide as an agonist. It would appear that total androgen response in patients with adenocarcinoma of the prostate blockade can be therapeutically more effective than conventional (Saunders, 1963; Rosenberg and Eschenbach, 1990). Localized androgen ablation therapy (Labrie et al, 1983). However, more prostate cancer is curable and although the metastatic form of the effective therapeutic strategies for combating prostate cancer are disease is difficult to manage, endocrine therapy does reduce the needed. This may be achieved through dual inhibition of 17α- death rate from the disease and is first-line treatment for all hydroxylase/C17,20-lyase and 5α-reductase (Li et al, 1992; Klus patients. In the testis and adrenals, 17α-hydroxylase/C17,20-lyase et al, 1996). Several derivatives of oestrogens, progestins and converts the C21 steroid precursors to the corresponding C19 andro- androgens which diminish androgen levels by inhibiting key gens. Testosterone (T) is further converted to the more potent enzymes in the androgen synthesis cascade have been described androgen dihydrotestosterone (DHT) by 5α-reductase in the (Ayub and Levell 1987; Angelastro et al, 1989; Nakajin et al, prostate (Bruchovsky and Wilson, 1968). Both T and DHT stimu- 1989; Jarman et al, 1990; Njar and Brodie, 1999). Currently, late prostatic growth, although DHT plays a much more important ketoconazole, an active imidazole fungicide, is the only 17α- hydroxylase/C17,20-lyase inhibitor that is used clinically to reduce Received 9 November 1999 testosterone biosynthesis in the treatment of patients with Revised 4 February 2000 *Present address: Department of Pharmaceutical Sciences, Temple University Accepted 10 February 2000 School of Pharmacy, Philadelphia, PA 19140, USA. †Present address: School of Pharmaceutical Sciences, Beijing Medical University, Correspondence to: AM Brodie China. 74 Effects of L-39 in vitro and in vivo 75 N (Rahway, NJ, USA) and ketoconazole was purchased from Sigma \ Chemical Company (St Louis, MO, USA). [21-3H]-17α-hydroxy- O pregnenolone (13.61 µCi µmol–1) was prepared in our laboratory as previously described (Njar et al, 1998). [1,2,6,7-3H]- Testosterone (96.5 Ci mmol–1) and [4-14C]-dihydrotestosterone (56.5 mCi mmol–1) were obtained from Dupont (Boston, MA, USA). Silica gel thin layer chromatographic (TLC) plates (20 × 20 cm, w/uv 254, 500 microns, GF) were obtained from Analtech (Newark, DE, USA). The active [125I]-T coated-tube radioimmunoassay (RIA) kits and the [125I]-DHT coated tube RIA kits for quantitative measurement of T and DHT, respectively, were purchased from DSL Inc. (Webster, TX, USA). All O other reagents were purchased from Sigma Chemical Company Figure 1 Structure of 17-(5′-isoxazolyl)androsta-4,16-dien-3-one (L-39) (St Louis, MO, USA). Preparation of microsomes advanced prostate cancer (Small et al, 1997). However, ketoconazole is not very potent and it inhibits several other steroidogenic Human testes and prostate tissue (from patients with benign enzymes and has a number of significant side-effects (Ayub and prostatic hyperplasia, BPH) were obtained from Dr James Mohler, Levell, 1987; Trachtenberg, 1984). 5α-Reductase occurs in two Director, Urologic Oncology, University of North Carolina at isoforms namely, type I and type II. The type II isoenzyme is the Chapel Hill and stored at –70°C prior to use. Testicular and predominant form in the human prostate. Finasteride, which was prostatic microsomes were prepared as described previously (19). recently approved for the treatment of benign prostatic hyper- Briefly, human testis or prostate was washed with saline (0.9%), trophy (BPH), is a more potent inhibitor of the type II than the type blotted dry and weighed. The tissue was minced and homogenized I isoform (Cunningham and Hirshkowitz, 1995). However, finas- in a blender with two volumes of sucrose (250 mM). The teride is only effective against benign prostatic hypertrophy (BPH) homogenates were added to 50-ml plastic centrifuge tubes and in patients with minimal disease and although the compound centrifuged at 10 000 g for 30 min. The resulting supernatant was reduced DHT levels, it also increased serum T levels (Peters and centrifuged at 109 000 g for 1 h using an ultra-centrifuge. The Sorkin, 1993). microsomal pellet was covered with 2 ml of phosphate buffer We have previously reported the synthesis and testing of several (0.1 M) and stored at –70°C until required for assay. The microsomal protein content was determined by the Lowry method steroidal inhibitors of 17α-hydroxylase/C17,20-lyase and 5α-reductase (Li et al, 1995, 1996; Ling et al, 1997; Njar et al, 1998). These (Lowry et al, 1951). compounds were demonstrated to be effective dual inhibitors of human testicular 17α-hydroxylase/C -lyase and prostatic 17,20 17α-hydroxylase/C17,20-lyase activity 5α-reductase in vitro (Nnane et al, 1998, 1999; Grigoryev et al, 1999). In animal studies, the compounds diminished the levels of The measurement of the activity of the human 17α-hydroxylase/ circulating T and DHT in male rat tissues. In LNCaP cell cultures, C17,20-lyase in testicular microsomes, in the absence and presence several of our novel steroidal compounds including 17-(5′-isoxa- of inhibitors was performed as described previously (Li et al, zolyl)androsta-4,16-dien-3-one (L-39; Figure 1) inhibited cellular 1995). Briefly, the 17α-hydroxylase/C17,20-lyase activity was 3 proliferation and were effective at slowing LNCaP human determined by measuring the release of [ H]-acetic acid during the 3 prostatic cells grown in male severe combined immunodeficient conversion of [21- H]-17α-hydroxypregnenolone to dehydroepi- (SCID) mice as tumours. L-39 (5 µM) has also been shown to androsterone. The incubations were carried out in a total volume manifest anti-androgenic activity in cultures of human prostate of 1.01 ml. Sample tubes were supplied with 10 µl of propylene 3 cancer cell lines (LNCaP) by preventing the labelled synthetic glycol, 300 000 dpm of [21- H]-17α-hydroxypregnenolone (13.61 –1 androgen R1881 (5 nM) from binding to the androgen receptors µCi µmol ) and the indicated inhibitors.

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