ANDROGEN SIGNALING AXIS AS DRUG TARGET IN CASTRATION-RESISTANT PROSTATE CANCER: Preclinical development of novel treatments and models

Abstract: Prostate cancer is a major global challenge due to the increasing number of aging population and frequency of diagnosis. Already in 1941, Huggins and Hodges showed that metastatic prostate cancer is responsive to androgens. This important observation has led to new therapies, androgen-deprivation therapy being the standard of care for men with metastatic disease. During the past decade new treatments have been primarily targeted to the androgen signaling axis either by 1) inhibition of androgen production in the adrenal glands and in the cancerous tissue, or 2) blocking the binding of androgens to androgen receptor (AR) in cells and inhibiting AR nuclear translocation. Despite this remarkable progress, the disease is still incurable and there is urgent need for better, more effective treatment strategies. This study is a part of the nonclinical development and characterization of novel therapies for castration-resistant prostate cancer (CRPC). Darolutamide (ODM-201), currently being in phase III studies, is a structurally different, novel, oral, high-affinity second-generation AR antagonist, which showed a minimal blood-brain barrier penetration in our nonclinical models, and therefore it may have a better safety profile compared to other second-generation antiandrogens. Based on our in vitro studies, darolutamide is a very potent AR antagonist and also showed antagonism in all studied AR mutations, including the AR(F877L) mutation where the clinical reference compound, enzalutamide, showed agonism. Another investigational compound, ODM-204, is a non-steroidal, orally active inhibitor of CYP17A1 enzyme and AR antagonist. We showed that the both compounds had significant tumor growth inhibition in the CRPC xenograft models. In addition, ODM-204 demonstrated potent inhibition in CYP17A1 enzyme both in vitro in cell based assay and isolated microsomes, and in vivo in rodent and monkey efficacy models. In the nonclinical pharmacokinetic studies both compounds showed favorable profiles, therefore supporting further studies in CRPC patients. It is well known that in CRPC overexpressed AR allows multiple ligands to work as an agonist, including several steroids secreted form adrenal glands. As mice adrenal glands, in contrast to human, have not been shown to synthetize androgens, the relevance of mice xenograft models in studying CRPC has been questioned. As part of this study, we showed that mouse adrenals produce significant amounts of steroids that contribute to the ARdependent growth of CRPC xenograft tumor model. This study therefore provides novel data from nonclinical development of new drug candidates for CRPC, and translational research tools and information for further drug development.Tiivistelmä: Eturauhassyöpä on maailmanlaajuinen ongelma johtuen väestön ikääntymisestä ja lisääntyvistä diagnooseista. Jo 1941 Huggins ja Hodges osoittivat, etäpesäkkeisen eturauhassyövän olevan riippuvainen mieshormoneista eli androgeeneista. Tämä merkittävä havainto on johtanut lukuisien uusien hoitojen kehittämiseen ja androgeenien tuotannon esto on ollut levinneen taudin keskeinen hoito jo pitkään. Viimeisen vuosikymmenen aikana on hoidot ovat kehittyneet merkittävästi. Ensisijaisesti ne ovat keskittyneet 1) androgeenien tuotannon estämiseen lisämunuaisissa ja syöpäkudoksessa, tai 2) androgeenien sitoutumisen estämiseen androgeenireseptoreihin ja rajoittamaan reseptorien kulkeutumista tumaan. Uusista lääkkeistä huolimatta, sairaus on yhä parantumaton ja uusia tehokkaampia hoitomuotoja tarvitaan. Tämä tutkimus on osa uusien hoitojen prekliinistä kehitystä kastraatioresistenttiin eturauhassyöpään. Jo Faasi III kliinisiin tutkimuksiin edennyt darolutamide (ODM-201) on uudenlainen, suun kautta otettava korkea-affiniteettinen toisen polven antiandrogeeni, joka ei prekliinisissä malleissa ole läpäissyt veri-aivoestettä ja siksi se saattaa olla muita toisen polven antiandrogeenejä turvallisempi. In vitro tulostemme perusteella darolutamide on tehokas AR-antagonisti kaikissa tutkimissamme AR mutaatioissa, myös AR(F877L) mutaatiossa, jossa kliininen vertailuyhdiste entsalutamidi osoitti agonismia. ODM-204 on uusi kokeellinen, ei-steroidaalinen, oraalinen lääkeaine, joka estää CYP17A1-entsyymiä ja toimii AR-antagonistina. Tutkimuksissamme molemmilla yhdisteillä havaittiin merkittävää tuumorin kasvun estoa kastraatioresistentin eturauhassyövän eläinmalleissa. Lisäksi ODM- 204 osoitti tehokasta CYP17A1-entsyymin toiminnan estoa sekä in vitro solu-mallissa, että eristetyissä mikrosomifraktioissa, ja in vivo tehomalleissa jyrsijöissä ja apinassa. Yhdisteiden lupaavat prekliiniset farmakokineettiset tulokset tukivat jatkotutkimusten tekemistä kastraatioresistenttiä eturauhassyöpää sairastavilla potilailla. Kastraatioresistentissä eturauhassyövässä AR:n määrä kasvaa ja se voi aktivoitua useiden erilaisten yhdisteiden, mukaan lukien lisämunuaisessa tuotettujen steroidihormonien avulla. Ksenograftimallien soveltuvuus kyseisen syövän tutkimukseen onkin kyseenalaistettu, koska toisin kuin ihmisellä, hiiren lisämunuaisen ei ole osoitettu tuottavan androgeeneja. Kuitenkin osana tutkimustamme osoitimme hiiren lisämunuaisen tuottavan merkittäviä määriä steroidihormoneja, jotka edistävät eläinmallissa syövän kasvua. Tämä tutkimus tarjoaa uutta tietoa kastraatioresistentistä eturauhassyövästä ja sen hoitoon kehitettävistä lääkkeistä, sekä uusia prekliinisiä työkaluja lääkekehityksen tueksi

High intratumoral dihydrotestosterone is associated with antiandrogen resistance in VCaP prostate cancer xenografts in castrated mice

Antiandrogen treatment resistance is a major clinical concern in castration-resistant prostate cancer (CRPC) treatment. Using xenografts of VCaP cells we showed that growth of antiandrogen resistant CRPC tumors were characterized by a higher intratumor dihydrotestosterone (DHT) concentration than that of treatment responsive tumors. Furthermore, the slow tumor growth after adrenalectomy was associated with a low intratumor DHT concentration. Reactivation of androgen signaling in enzalutamide-resistant tumors was further shown by the expression of several androgen-dependent genes. The data indicate that intratumor DHT concentration and expression of several androgen-dependent genes in CRPC lesions is an indication of enzalutamide treatment resistance and an indication of the need for further androgen blockade. The presence of an androgen synthesis, independent of CYP17A1 activity, has been shown to exist in prostate cancer cells, and thus, novel androgen synthesis inhibitors are needed for the treatment of enzalutamide-resistant CRPC tumors that do not respond to abiraterone.Peer reviewe

Optimized design and analysis of preclinical intervention studies in vivo

Recent reports have called into question the reproducibility, validity and translatability of the preclinical animal studies due to limitations in their experimental design and statistical analysis. To this end, we implemented a matching-based modelling approach for optimal intervention group allocation, randomization and power calculations, which takes full account of the complex animal characteristics at baseline prior to interventions. In prostate cancer xenograft studies, the method effectively normalized the confounding baseline variability, and resulted in animal allocations which were supported by RNA-seq profiling of the individual tumours. The matching information increased the statistical power to detect true treatment effects at smaller sample sizes in two castration-resistant prostate cancer models, thereby leading to saving of both animal lives and research costs. The novel modelling approach and its open-source and web-based software implementations enable the researchers to conduct adequately-powered and fully-blinded preclinical intervention studies, with the aim to accelerate the discovery of new therapeutic interventions.Peer reviewe

Discovery and development of ODM-204: A Novel nonsteroidal compound for the treatment of castration-resistant prostate cancer by blocking the androgen receptor and inhibiting CYP17A1

We report the discovery of a novel nonsteroidal dual-action compound, ODM-204, that holds promise for treating patients with castration-resistant prostate cancer (CRPC), an advanced form of prostate cancer characterised by high androgen receptor (AR) expression and persistent activation of the AR signaling axis by residual tissue androgens. For ODM-204, has a dual mechanism of action. The compound is anticipated to efficiently dampen androgenic stimuli in the body by inhibiting CYP17A1, the prerequisite enzyme for the formation of dihydrotestosterone (DHT) and testosterone (T), and by blocking AR with high affinity and specificity. In our study, ODM-204 inhibited the proliferation of androgen-dependent VCaP and LNCaP cells in vitro and reduced significantly tumour growth in a murine VCaP xenograft model in vivo. Intriguingly, after a single oral dose of 10-30 mg/kg, ODM-204 dose-dependently inhibited adrenal and testicular steroid production in sexually mature male cynomolgus monkeys. Similar results were obtained in human chorionic gonadotropin-treated male rats. In rats, leuprolide acetate-mediated (LHRH agonist) suppression of the circulating testosterone levels and decrease in weights of androgen-sensitive organs was significantly and dose-dependently potentiated by the co-administration of ODM-204. ODM-204 was well tolerated in both rodents and primates. Based on our data, ODM-204 could provide an effective therapeutic option for men with CRPC.</p

Adrenals Contribute to Growth of Castration-Resistant VCaP Prostate Cancer Xenografts

The role of adrenal androgens as drivers for castration-resistant prostate cancer (CRPC) growth in humans is generally accepted; however, the value of preclinical mouse models of CRPC is debatable, because mouse adrenals do not produce steroids activating the androgen receptor. In this study, we confirmed the expression of enzymes essential for de novo synthesis of androgens in mouse adrenals, with high intratissue concentration of progesterone (P-4) and moderate levels of androgens, such as androstenedione, testosterone, and dihydrotestosterone, in the adrenal glands of both intact and orchectomized (ORX) mice. ORX alone had no effect on serum P-4 concentration, whereas orchectomized and adrenalectomized (ORX + ADX) resulted in a significant decrease in serum P-4 and in a further reduction in the Low levels of serum androgens (androstenedione, testosterone, and dihydrotestosterone), measured by mass spectrometry. In line with this, the serum prostate-specific antigen and growth of VCaP xenografts in mice after ORX + ADX were markedly reduced compared with ORX alone, and the growth difference was not abolished by a glucocorticoid treatment. Moreover, ORX + ADX altered the androgen-dependent gene expression in the tumors, similar to that recently shown for the enzalutamide treatment. These data indicate that in contrast to the current view, and similar to humans, mouse adrenals synthesize significant amounts of steroids that contribute to the androgen receptor dependent growth of CRPC.Peer reviewe

ODM-204, a Novel Dual Inhibitor of CYP17A1 and Androgen Receptor: Early Results from Phase I Dose Escalation in Men with Castration-resistant Prostate Cancer

Background: Most prostate cancer patients develop castration-resistant prostate cancer (CRPC) after androgen deprivation therapy treatment. CRPC growth is mediated mostly by androgen receptor signalling driven by primary androgens synthesised largely by the CYP17A1 enzyme. Objective: To evaluate the safety profile and dose-limiting toxicities of ODM-204. Design, setting, and participants: In this open, uncontrolled, nonrandomised, multi-centre, tolerability and pharmacokinetic first-in-man phase I dose escalation study, patients with metastatic CRPC were randomised to receive ODM-204 in sequential cohorts of five dose levels (ie, 50, 100, 200, 300, and 500 mg twice daily) concomitantly with prednisone. Intervention: ODM-204, a novel, orally administered, investigational, nonsteroidal dual inhibitor of CYP17A1 and androgen receptor. Outcome measurements and statistical analysis: ODM-204 plasma concentrations, serum testosterone, and prostate-specific antigen (PSA) levels were evaluated and imaging of lesions was performed. Results and limitations: Of the 23 patients enrolled into the study, 60.9% experienced mild adverse effects considered to be related to the study treatment, which were fatigue, increased/decreased appetite, nausea, asthenia, diarrhoea, and weight decrease. ODM-204 area under the curve (AUC(0-12)) values increased dose dependently until the 300 mg dose. The AUC was lower on day 8 after repeated dosing compared with day 1 from the 200 mg dose upwards. Decreases in testosterone levels were seen with ODM-204 treatment confirming androgen deprivation. Of the patients, 13% also demonstrated a >50% decrease in PSA at week 12 and continued ODM-204 treatment for over a year. Conclusions: ODM-204 was well tolerated up to the highest evaluated dose. There were decreases in both testosterone and PSA levels, suggesting preliminary antitumour activity in the treatment of CRPC. The pharmacokinetic properties of the molecule, however, prevent further development. Patient summary: This study looked at the safety of ODM-204, a novel dual inhibitor of CYP17A1 and the androgen receptor, in castration-resistant prostate cancer patients. ODM-204 treatment was found to be well tolerated, and it also reduced both serum testosterone and prostate-specific antigen levels, but the properties of the molecule prevent further development. (C) 2018 European Association of Urology. Published by Elsevier B.V.Peer reviewe

Antiandrogens Reduce Intratumoral Androgen Concentrations and Induce Androgen Receptor Expression in Castration-Resistant Prostate Cancer xenografts

The development of castration-resistant prostate cancer (CRPC) is associated with the activation of intratumoral androgen biosynthesis and an increase in androgen receptor (AR) expression. We recently demonstrated that, similarly to the clinical CRPC, orthotopically grown castration-resistant VCaP (CR-VCaP) xenografts express high levels of AR and retain intratumoral androgen concentrations similar to tumors grown in intact mice. Herein, we show that antiandrogen treatment (enzalutamide or ARN-509) significantly reduced (10-fold, P <0.01) intratumoral testosterone and dihydrotestosterone concentrations in the CR-VCaP tumors, indicating that the reduction in intratumoral androgens is a novel mechanism by which antiandrogens mediate their effects in CRPC. Antiandrogen treatment also altered the expression of multiple enzymes potentially involved in steroid metabolism. Identical to clinical CRPC, the expression levels of the full-length AR (twofold, P <0.05) and the AR splice variants 1 (threefold, P <0.05) and 7 (threefold, P <0.01) were further increased in the antiandrogen-treated tumors. Nonsignificant effects were observed in the expression of certain classic androgen-regulated genes, such as TMPRSS2 and KLK3, despite the low levels of testosterone and dihydrotestosterone. However, other genes recently identified to be highly sensitive to androgen-regulated AR action, such as NOV and ST6GalNAc1, were markedly altered, which indicated reduced androgen action. Taken together, the data indicate that, besides blocking AR, antiandrogens modify androgen signaling in CR-VCaP xenografts at multiple levels.Peer reviewe