Pekka Kallio

Scientific Basis of a Matrix Metalloproteinase‐8 Specific Chair‐side Test for Monitoring Periodontal and Peri‐implant Health and Disease

ABSTRACT: Matrix metalloproteinases (MMPs), especially collagenase‐2 (MMP‐8), are key mediators of irreversible tissue destruction associated with periodontitis and peri‐implantitis. MMP‐8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri‐im‐plant sulcular fluid (PISF) from progressing periodontitis and peri‐implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112‐131 [1994]; Teronen et al. J. Dent. Res. 76: 1529‐1537 [1997]). We have developed monoclonal antibodies to MMP‐8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504‐31509 [1997]) that can be used in a chair‐side dipstick test to monitor the course and treatment of periodontitis and peri‐implantitis. Monoclonal and polyclonal antibody tests for MMP‐8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont.Res. 22: 386‐393 [1988]) in periodontal and peri‐implant health and disease. In future a chair‐side functional and/or immunological MMP‐test can be useful to diagnose and monitor periodontal and peri‐implant disease and health.

Androgen Receptor and Mechanism of Androgen Action

Androgen receptor is the intracellular protein that mediates biological actions of physiological androgens (testosterone and 5 alpha-dihydrotestosterone). Androgen receptor belongs to a large family of ligand-dependent proteins whose function is to modulate expression of genes and gene networks in a cell- and tissue-specific manner. The present overview describes the structurally important domains of the receptor protein, and discusses several aspects in the structure-function relationship, using naturally occurring receptor mutants in androgen insensitivity patients or experimental animals as examples. In addition, characteristics of androgen receptor expressed in a heterologous system are described, and their potential usefulness in specific molecular studies discussed.

Interaction of Androgen Receptors with Androgen Response Element in Intact Cells ROLES OF AMINO- AND CARBOXYL-TERMINAL REGIONS AND THE LIGAND

Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). Native rat and human ARs interfered with pCMV-ARE2-CAT expression in a hormone-dependent fashion. Low steroid-independent interference seemed to occur because of the ligand binding domain (LBD), which was transcriptionally inhibitory also in a heterologous context. AR devoid of LBD (rARΔ641–902) decreased pCMV-ARE2-CAT activity by 50%. The rARΔ46–408 mutant devoid of the NH2-terminal transcription activation region exhibited ligand-dependent promoter interference of a similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro. Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized agonist-dependent promoter interference, whereas cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial agonists/antagonists. Collectively, interaction of ARs with ARE in intact cells does not require the presence of the COOH-terminal or NH2-terminal domain and/or their interaction. In the context of native AR, however, the androgen-induced conformational change in LBD is mandatory for generation of a transcriptionally competent receptor that binds to DNA in intact cells.

Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies.

Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR, and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.

Genetic regulation of androgen action

The androgen receptor (AR) belongs to the superfamily of nuclear receptors that employ complex genetic mechanisms to guide the development and physiological functions of different target tissues. Upon interaction with its cognate hormone, AR activates or represses gene transcription through association with specific DNA elements and/or proteins. This review summarizes briefly our current view of androgen action, with a special emphasis on genetic factors that may modulate the response.

ODM-201, a new generation androgen receptor inhibitor for castration-resistant prostate cancer: Preclinical and phase I data.

65^ Background: Castration resistant prostate cancer (CRPC) is characterized by persistent, high level androgen receptor (AR) expression and resistance to conventional AR inhibitors. ODM-201 is a novel AR inhibitor with unique pharmacologic properties that has shown promising results in preclinical and clinical studies. METHODS AR binding affinity of ODM-201 to wild type AR was determined in cytosolic lysates obtained from ventral prostates of castrated rats using a competition binding assay. Additionally, effects of ODM-201 on the growth of castration resistant VCaP tumors was evaluated. Tumors were established by subcutaneous injection of VCaP prostate cancer cells into male nude mice. After initial tumor growth, mice were castrated. ODM-201 (50 mg/kg QD or BID orally) was initiated upon tumor regrowth. Since the risk of seizures has been reported for some second generation AR inhibitors, ODM-201 concentrations in mouse brain homogenates were studied after repeated oral administration to assess the risk. The clinical effects of ODM-201 (100, 200, 300, 500, 700, 900 mg BID) was examined in a Phase I/II dose-escalation trial in patients (N=24) with progressive metastatic CRPC. RESULTS ODM-201 binds to wild type AR with superior affinity compared to enzalutamide (Ki of 9 and 39 nM, respectively). In the VCaP CRPC model, ODM-201 significantly (p<0.001 compared to castrated control), and more efficiently than enzalutamide, inhibited tumor growth. After oral administration, tissue/plasma ratio of ODM-201 in mouse brain homogenates was negligible in all studied doses. In the Phase I/II clinical study, ODM-201 (100 to 700 mg results available) was well tolerated, and most commonly reported adverse events were asthenia, diarrhea and nausea. A PSA response (defined as ≥ 50% decrease) was obtained in 13/15 patients evaluable at 12 weeks with all patients achieving partial response or stable disease by RECIST/PCWG2 criteria. CONCLUSIONS ODM-201 is a new generation AR inhibitor with superior preclinical efficacy compared to enzalutamide and bicalutamide, it does not enter the brain in preclinical studies, and it shows very promising activity and no significant toxicity in patients with CRPC. CLINICAL TRIAL INFORMATION NCT01317641.

Androgen Receptor in Transcriptional Regulation

Male sex steroids (androgens) sculpture a male body in a number of ways, such as development of male sex organs; growth of facial, body, and pubic hair; enlargement of vocal cords (deepening of the voice); loss of hair at temples; production of sperm; development of muscle strength; growth of prostate, and development of masculine behavior. Each of these events is regulated by tissue-specific mechanisms through the androgen receptor that activates and/or represses gene networks to elicit its distinct physiological actions. The principal events leading to hyperplastic and/or hypertrophic responses to androgens are, however, believed to be similar, if not identical, in reproductive and nonreproductive tissues (Berger and Watson 1989). Recent studies in a number of laboratories have delineated the ways by which androgen receptor interacts with specific DNA elements — located within and/or around responsive genes — to increase the rate of gene transcription. Much less information is available, however, about mechanisms by which androgens bring about gene repression. In order to elicit defined biological (androgenic) responses, the receptor protein ought to communicate not only with DNA motifs, but also with other transcription factors or coactivators. Finally, practically nothing is known about the role of androgen receptor protein in the stabilization of specific mRNA species.

Abstract LB-113: Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models

Background: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine-based inhibitors including JQ1, I-BET762, and OTX015. Phase I clinical trials have now been initiated with this agent based on its potent anti-tumor activity in various in vitro and in vivo models of hematologic malignancies and solid tumors. In view of the recent publications implicating a role for BET protein BRD4 in the suppression of PD-L1 expression, an immune checkpoint ligand for PD-1, we sought to evaluate ODM-207 for its effect on immune-mediated anti-tumor efficacy in pre-clinical models. Methods and Results: Mouse splenocytes were stimulated with anti-CD3 and anti-CD28 in the presence or absence of ODM-207 for four days and changes in immune cell population were analyzed by FACS. Results revealed an increase in the level of activated cytotoxic CD8+ T cells as indicated by increased intracellular IFNγ and granzyme B with ODM-207 treatment. After confirming the lack of direct anti-proliferative activity on the mouse colon carcinoma cell line CT26, in vivo evaluation of ODM-207 was carried out in the syngeneic CT26 subcutaneous tumor model established in BALB/c mice. Daily oral administration of ODM-207 at 30 mg/kg was well tolerated in this model and resulted in a statistically significant inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ODM-207 was comparable to that with a commercially available anti-mouse PD1 antibody. Studies to characterize the immune changes in the tumor and anti-tumor activity of ODM-207 in combination with an anti-mouse PD1 antibody are currently underway and the results will be presented. Conclusions: In summary, these studies demonstrate the anti-tumor activity of BET inhibitor in a syngeneic model of colon carcinoma in the absence of a direct anti-proliferative activity on tumor cells. Observed tumor growth inhibition correlated with the in vitro activation of cytotoxic CD8+ T cells supporting the immune-mediated effect leading to tumor growth inhibition. In view of the remarkable success with the immune-based therapeutic approaches, these findings are relevant in devising appropriate strategies for the continued clinical development of ODM-207. Citation Format: Pratima Deshpande, Ravi Krishna Babu, Prashant Yallappa Vadnal, Mahaboobi Jaleel, Murali Ramachandra, Chandrasekhar Abbineni, Susanta Samajdar, Anu-Maarit Moilanen, Pekka Kallio. Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2017-LB-113