A Multifunctional LNA Oligonucleotide-Based Strategy Blocks AR Expression and Transactivation Activity in PCa Cells.

The androgen receptor (AR) plays a critical role in the development of prostate cancer (PCa) through the activation of androgen-induced cellular proliferation genes. Thus, blocking AR-mediated transcriptional activation is expected to inhibit the growth and spread of PCa. Using tailor-made splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we successfully redirected splicing of the AR precursor (pre-)mRNA and destabilized the transcripts via the introduction of premature stop codons. Furthermore, the SSOs simultaneously favored production of the AR45 mRNA in lieu of the full-length AR. AR45 is an AR isoform that can attenuate the activity of both full-length and oncogenic forms of AR by binding to their common N-terminal domain (NTD), thereby blocking their transactivation potential. A large screen was subsequently used to identify individual SSOs that could best perform this dual function. The selected SSOs powerfully silence AR expression and modulate the expression of AR-responsive cellular genes. This bi-functional strategy that uses a single therapeutic molecule can be the basis for novel PCa treatments. It might also be customized to other types of therapies that require the silencing of one gene and the simultaneous expression of a different isoform.

Oligonucleotides to the (Gene) Rescue: FDA Approvals 2017-2019.

Four decades have passed since oligonucleotides were first used to manipulate gene expression. There were few FDA approvals prior to 2016, mostly of drugs that eventually exhibited poor performance in the market. The aura of their younger siRNA relatives had also faded during the past 15 years. However, several FDA approvals have occurred in the past 4 years, restoring hope that a new era has dawned in oligonucleotide/siRNA clinical therapeutics. Here, we review the field of oligonucleotide therapeutics and provide an update on FDA approvals of oligonucleotides from 2017 until the second quarter of 2019. We take into consideration the ethical issues looming over the still somewhat limited efficacy of these molecules, the toxicity of treatment, and the exorbitant cost of these therapeutic agents, which limits accessibility for many.

Ammonium and arsenic trioxide are potent facilitators of oligonucleotide function when delivered by gymnosis.

Oligonucleotide (ON) concentrations employed for therapeutic applications vary widely, but in general are high enough to raise significant concerns for off target effects and cellular toxicity. However, lowering ON concentrations reduces the chances of a therapeutic response, since typically relatively small amounts of ON are taken up by targeted cells in tissue culture. It is therefore imperative to identify new strategies to improve the concentration dependence of ON function. In this work, we have identified ammonium ion (NH4+) as a non-toxic potent enhancer of ON activity in the nucleus and cytoplasm following delivery by gymnosis. NH4+ is a metabolite that has been extensively employed as diuretic, expectorant, for the treatment of renal calculi and in a variety of other diseases. Enhancement of function can be found in attached and suspension cells, including in difficult-to-transfect Jurkat T and CEM T cells. We have also demonstrated that NH4+ can synergistically interact with arsenic trioxide (arsenite) to further promote ON function without producing any apparent increased cellular toxicity. These small, inexpensive, widely distributed molecules could be useful not only in laboratory experiments but potentially in therapeutic ON-based combinatorial strategy for clinical applications.

Randomized phase 2 therapeutic equivalence study of abiraterone acetate fine particle formulation vs. originator abiraterone acetate in patients with metastatic castration-resistant prostate cancer: The STAAR study.

BACKGROUND: This multicenter, randomized, open-label, active-controlled study evaluated therapeutic equivalence, steady-state pharmacokinetics, and safety of a novel abiraterone acetate fine particle formulation (AAFP) 500mg plus methylprednisolone vs. the originator AA (OAA) 1000mg plus prednisone in men with metastatic castrate-resistant prostate cancer (mCRPC). The primary endpoint was a comparison of average of serum testosterone levels on treatment days 9 and 10 between groups. METHODS: Men with progressive mCRPC, receiving gonadotropin-releasing hormone agonist or antagonist therapy, and with a serum testosterone level of <50ng/dl were randomized 1:1 to either AAFP 500mg daily plus 4mg methylprednisolone orally twice daily (BID), or OAA 1000mg daily plus 5mg prednisone BID for 84 days. Serum testosterone, serum prostate-specific antigen (PSA), steady-state (trough) abiraterone pharmacokinetics, and safety were evaluated. RESULTS: Fifty-three patients were enrolled (n = 24, AAFP; n = 29, OAA). Mean age was 75.1 years and 54.7% had Gleason>7. Over 90% of patients in each group achieved absolute testosterone levels of ≤1ng/dl during the study. The averaged absolute testosterone levels ≤0.1ng/dl were achieved in 25% of AAFP-treated patients and 17% of OAA-treated patients. A PSA-50 response was observed in>65% of patients in both groups on days 28, 56, and 84 (P = NS, all timepoints). Days 9 and 10 averaged rounded-up least squares (LS) mean (SE) serum testosterone levels were comparable (1.05ng/dl [0.04], AAFP; 1.02ng/dl [0.03], OAA; P = 0.4703 for LS mean difference). The geometric mean ratio between groups was 1.021 (90% CI: 0.965-1.081); the 90% CI fell within 80.0% to 125.0% equivalence limits. The LS mean differences in abiraterone trough plasma concentrations were not statistically significant at any visit. Adverse event frequency was comparable between arms (75.0%, AAFP; 82.8%, OAA). Musculoskeletal events were more common among OAA-treated patients (37.9% vs. 12.5%). CONCLUSION: Therapeutic equivalence between AAFP 500mg daily and OAA 1000mg daily based on serum testosterone levels was confirmed in mCRPC patients. Both agents led to similar PSA-50 response rates. Abiraterone trough levels were similar between treatments. No new safety concerns were observed.

Impact of an alternative steroid on the relative bioavailability and bioequivalence of a novel versus the originator formulation of abiraterone acetate.

PURPOSE: The originator abiraterone acetate (OAA) formulation is used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). This study evaluated the bioavailability and bioequivalence of a novel formulation, abiraterone acetate fine particle (AAFP), versus OAA on a steady-state background of steroids. METHODS: Thirty-seven healthy male subjects were randomized in a crossover design to receive methylprednisolone (4 mg twice daily) or prednisone (5 mg twice daily) for 12 days in Period 1. On Day 11 of Period 1, subjects given methylprednisolone received a single dose of AAFP 500 mg, and subjects given prednisone received a single dose of OAA 1000 mg under fasted conditions. After a 2-week steroid washout period, subjects received the alternate treatments in Period 2. RESULTS: There were no statistical differences regarding area under the curve (AUC) and maximum concentration (C max) between AAFP and OAA. The bioavailability of abiraterone from AAFP versus OAA by geometric mean ratio was AUC0-∞, 95.9% (90% confidence interval [CI] 86.0-106.9); AUC0-t , 99.2% (88.7-110.9); and C max, 116.8% (102.2-133.4). The coefficient of variation (CV) was smaller for AAFP versus OAA (AUC0-∞, CV 44.23 vs. 55.61%; AUC0-t , 45.17 vs. 58.16%; C max, 54.55 vs. 65.65%, respectively). Both treatments were safe and well tolerated. CONCLUSIONS: AAFP plus methylprednisolone provided abiraterone exposure that was comparable to OAA plus prednisone with respect to C max and AUC. Less drug exposure variability was observed with AAFP compared with OAA. Reduced pharmacokinetic variability may positively influence clinical outcomes and warrants further study in mCRPC patients.

FDA-Approved Oligonucleotide Therapies in 2017.

Oligonucleotides (oligos) have been under clinical development for approximately the past 30 years, beginning with antisense oligonucleotides (ASOs) and apatmers and followed about 15 years ago by siRNAs. During that lengthy period of time, numerous clinical trials have been performed and thousands of trial participants accrued onto studies. Of all the molecules evaluated as of January 2017, the regulatory authorities assessed that six provided clear clinical benefit in rigorously controlled trials. The story of these six is given in this review.

Protein Kinase C-α is a Critical Protein for Antisense Oligonucleotide-mediated Silencing in Mammalian Cells.

We have identified the existence of a productive, PKC-α-dependent endocytotic silencing pathway that leads gymnotically-delivered locked nucleic acid (LNA)-gapmer phosphorothioate antisense oligonucleotides (ASOs) into late endosomes. By blocking the maturation of early endosomes to late endosomes, silencing the expression of PKC-α results in the potent reduction of ASO silencing ability in the cell. We have also demonstrated that silencing of gene expression in the cytoplasm is vitiated when PKC-α expression is reduced. Restoring PKC-α expression via a reconstitution experiment reinstates the ability of ASOs to silence. These results advance our understanding of intracellular ASO trafficking and activity following gymnotic delivery, and further demonstrate the existence of two distinct silencing pathways in mammalian cells, one in the cytoplasmic and the other in the nuclear compartment.

Antisense oligonucleotides in cancer.

PURPOSE OF REVIEW: Over the past several dozen years, regardless of the substantial effort directed toward developing rational oligonucleotide strategies to silence gene expression, antisense oligonucleotide-based cancer therapy has not been successful. This review focuses on the most likely reasons for this lack of success, and on the barriers that still need to be overcome to make a clinical cancer treatment reality out of the promise of antisense therapy. RECENT FINDINGS: Considerable progress has been made in the design and delivery of nucleic acid fragments. Chemical modifications have considerably improved oligonucleotide absorption, distribution and metabolism while at the same time reducing toxicity. Nevertheless, the delivery and the cellular uptake of these molecules are still not adequate to provide the desired therapeutic outcome. Recent therapeutic interventional phase III trials of antisense oligodeoxyribonucleotides for a cancer indication will be discussed, in addition to those studies that markedly improve the scientific understanding of the properties of these molecules. SUMMARY: We still do not have a marketed antisense oligonucleotide for a cancer indication. This is because critical aspects of the cellular, tumor pharmacology and delivery properties of these agents are still not well understood.

Targeting angiopoietin-2 signaling in cancer therapy.

INTRODUCTION: Over the past decade, several anti-angiogenic strategies have been devised to target a wide spectrum of malignancies. The most widely utilized approach involves abrogation of vascular endothelial growth factor receptor signaling through either consumption of ligand (i.e., with the monoclonal antibody bevacizumab) or through direct inhibition of the receptor tyrosine kinase domain (i.e., with small molecules such as sunitinib or sorafenib). While these agents do appear to delay cancer progression in the clinic, they are not curative approaches. AREAS COVERED: A novel anti-angiogenic strategy involves inhibition of signaling along the Ang/Tie-2 axis, a pathway critical for mediating endothelial and perivascular cell interactions. While several agents (i.e., AMG-386 and regorafenib) have reached late stages of clinical development, others (i.e., ARRY614 and CEP-11981) are in their relative infancy. Herein, we will outline the clinical trajectory of wide spectrum Ang/Tie-2 inhibitors, with attention to data evaluating combinations with cytotoxic therapy or other targeted agents. EXPERT OPINION: Provided that these approaches to not drastically augment toxicity, they may represent the ideal path for further development of this class of agents.

Enzalutamide for the treatment of prostate cancer.

INTRODUCTION: The FDA approval of docetaxel for metastatic castration-resistant prostate cancer (mCRPC) in 2005 marked a major milestone, as it was the first approved agent for this disease that demonstrated a survival advantage in Phase III assessment of this disease. Since 2009, several other agents have been approved by FDA, including sipuleucel-T, abiraterone, cabazitaxel and enzalutamide . Enzalutamide, a potent antiandrogen that blocks nuclear translocation of the androgen receptor (AR), is the most recently approved of these agents. AREAS COVERED: The clinical development of enzalutamide is discussed, with attention given as to how this agent will most appropriately be used among a growing list of agents for mCRPC. A MEDLINE search was conducted to identify all relevant published datasets pertaining to the drug. In addition, relevant ASCO and ESMO abstracts were searched. EXPERT OPINION: The current role and sequencing of enzalutamide may change drastically based on studies such as PREVAIL (a Phase III pre-chemotherapy assessment of enzalutamide) and planned studies to assess relevant combinations (i.e., enzalutamide with abiraterone). Outside of clinical efficacy, issues such as drug cost may ultimately dictate our utilization of agents such as enzalutamide for mCPRC. Although the development of biomarkers to guide therapy for mCRPC is ideal, there are inherent challenges in establishing biomarker-driven treatment.

Preoperative therapy for localized prostate cancer: a comprehensive overview.

At the 2012 American Society of Clinical Oncology (ASCO) Annual Meeting, two studies of preoperative systemic therapy for localized prostate cancer garnered significant attention. In the first, investigators evaluated various permutations of conventional hormonal therapies prior to prostatectomy, with detailed biomarker studies focused on tissue androgens. In the second, investigators assessed the novel CYP17 lyase inhibitor abiraterone prior to prostatectomy. Both studies provide a wealth of biological information, but the question remains - will preoperative systemic therapy ultimately be incorporated into clinical algorithms for prostate cancer? Herein, the existing literature for both preoperative hormonal and chemotherapeutic approaches is reviewed. We performed a MEDLINE search of published prospective and retrospective clinical studies assessing preoperative systemic therapy for prostate cancer from 1982 onwards, revealing a total of 75 publications meeting these criteria. Of these, 55 possessed a number of patients (i.e., greater than 10) deemed worth of the current analysis. Beyond outlining these datasets, we discuss the relevance of clinical and pathologic endpoints in assessing preoperative therapy.

Paclitaxel-based high-dose chemotherapy with autologous stem cell rescue for relapsed germ cell tumor: clinical outcome and quality of life in long-term survivors.

BACKGROUND: High-dose chemotherapy (HDCT) is a viable and potentially curative approach for patients with relapsed or refractory germ cell tumors (GCTs). However, no comparative data exist to define the optimal chemotherapeutic strategy, and little is known about the quality of life (QOL) of long-term survivors. Herein we attempt to characterize the QOL in long-term survivors who received high-dose paclitaxel, etoposide, carboplatin, and ifosfamide (TECTIC). PATIENTS AND METHODS: Details of the TECTIC regimen and clinical outcomes for the initial 33 patients have been reported. In the present study, we report the clinical data for 15 additional patients. Using the European Organization for the Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-Core 30 (QLQ-C30) and the Functional Assessment of Cancer Therapy-Taxane (FACT-T) questionnaires, we surveyed all patients who survived at least 4 years after HDCT. RESULTS: Forty-eight patients were enrolled and 46 patients received protocol therapy. For all 48 patients, the median progression-free survival (PFS) and overall survival (OS) were 11.8 months (range, 5.8-not reached) and 21.7 months (range, 12.7-not reached), respectively. Seventeen patients were progression free at a median of 123.2 months (51.6-170.2 months), and 6 patients remain alive after progression with a median OS of 68.8 months (47.6-147.1 months). Of the 23 surviving patients, 18 were accessible and consented to telephone interviews. Compared with historical cohorts, survivors had a higher global health scale score (87.04 vs. 75.62; P = .02) but a lower physical functioning score (68.89 vs. 92.66; P = .0001) by the QLQ-C30 scale. CONCLUSIONS: HDCT with the TECTIC regimen produces durable remissions in patients with relapsed or refractory GCTs with acceptable QOL in long-term survivors.

Antisense 2'-Deoxy, 2'-Fluroarabino Nucleic Acids (2'F-ANAs) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids.

Gymnosis is the process of the delivery of antisense oligodeoxynucleotides to cells, in the absence of any carriers or conjugation, that produces sequence-specific gene silencing. While gymnosis was originally demonstrated using locked nucleic acid (LNA) gapmers, 2'-deoxy-2'fluroarabinonucleic acid (2'F-ANA) phosphorothioate gapmer oligonucleotides (oligos) when targeted to the Bcl-2 and androgen receptor (AR) mRNAs in multiple cell lines in tissue culture, are approximately as effective at silencing of Bcl-2 expression as the iso-sequential LNA congeners. In LNCaP prostate cancer cells, gymnotic silencing of the AR by a 2'F-ANA phosphorothioate gapmer oligo led to downstream silencing of cellular prostate-specific antigen (PSA) expression even in the presence of the androgenic steroid R1881 (metribolone), which stabilizes cytoplasmic levels of the AR. Furthermore, gymnotic silencing occurs in the absence of serum, and silencing by both LNA and 2'F-ANA oligos is augmented in serum-free (SF) media in some cell lines when they are treated with oleic acid and a variety of ω-6 polyunsaturated fatty acids (ω-6 PUFAs), but not by an aliphatic (palmitic) fatty acid. These results significantly expand our understanding of and ability to successfully manipulate the cellular delivery of single-stranded DNA molecules in vitro.Molecular Therapy - Nucleic Acids (2012) 1, e43; doi:10.1038/mtna.2012.35; advance online publication 18 September 2012.

Direct regulation of androgen receptor activity by potent CYP17 inhibitors in prostate cancer cells.

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.

Therapeutic oligonucleotides: the road not taken.

Antisense oligonucleotide therapeutics have been in development for almost 25 years without a single U.S. Food and Drug Administration-approved product in cancer. The reasons for this absence stem, in part, from a deep lack of understanding about how to deliver these molecules to cancer cells in vivo.

LDH correlation with survival in advanced melanoma from two large, randomised trials (Oblimersen GM301 and EORTC 18951).

PURPOSE: In a randomised study (GM301; dacarbazine with/without oblimersen), patients with advanced melanoma were stratified based on performance status, metastatic site and lactate dehydrogenase (LDH). Progression-free survival and response and durable response rates showed a highly significant difference favouring dacarbazine-oblimersen and a nearly significant survival difference. All efficacy parameters significantly favoured dacarbazine-oblimersen in patients with normal baseline LDH [1.1xupper limit of normal (ULN)]. Each stratification factor was assessed for an interaction with treatment on survival and an interaction was detected only for LDH. EXPERIMENTAL DESIGN: Baseline LDH values in Study GM301 treatment groups were combined and analysed using cutoffs above and below 1xULN. Baseline LDH in EORTC study 18951 (dacarbazine, cisplatin, interferon-alfa-2b with/without interleukin-2 in advanced melanoma) was independently analysed using the same approach. In Study GM301, the relation between treatment effect and LDH, treatment effect and tumour size, LDH and tumour size and LDH and disease site were determined. RESULTS: In Study GM301 (N=760) and Study 18951 (N=325), LDH was within the upper range of normal for a large number of patients. This was not exhibited in the general population, suggesting such values may be elevated rather than normal in melanoma. A highly ordered and monotonic relationship was apparent between LDH and survival: survival worsened as LDH became more elevated, even when LDH remained within normal range. LDH and tumour size were poorly correlated; elevated LDH was not associated with any one disease site. LDH was highly predictive of oblimersen effect. CONCLUSION: In designing studies, LDH should be considered, regardless of tumour size or disease site.