RX-3117 (fluorocyclopentenyl cytosine): a novel specific antimetabolite for selective cancer treatment

ABSTRACT

Introduction: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer.

Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule.

Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types.

Trial registration: ClinicalTrials.gov identifier: NCT02030067.

Trial registration: ClinicalTrials.gov identifier: NCT03189914.

KEYWORDS:

• Cytidine analogs
• fluorocyclopentenyl cytosine
• uridine-kinase 2
• DNA methyltransferase

1. Introduction

Nucleoside analogs are a class of antitumoural drugs commonly used to treat cancer (Figure 1). Their cytotoxic activity is due to their incorporation in RNA or DNA, inhibition of their synthesis and cell proliferation [1]. In particular, gemcitabine (Gemzar, 2ʹ,2ʹ-difluoro-2ʹ-deoxycytidine) is commonly used as single agent or with nab-paclitaxel for the treatment of pancreatic cancer patients and in combination with cisplatin for lung and bladder cancer and is also registered for ovarian cancer and head-neck cancer [2]; Cytarabine (cytosine arabinoside; ara-C) is part of the treatment of pediatric and adult leukemia [3]; 5-Azacytidine (aza-CR; Vidaza; azacitidine) and 5-aza-2ʹdeoxycytidine (aza-CdR; Decitabine, Dacogen) are widely used for hematological malignancies, such as myelodysplastic syndrome [4,5]. However, one common issue for these drugs is resistance [6]. This led to the development of many analogs. Cyclopentenylcytosine (CEPC) is a cytidine analog with a modification in the sugar and was evaluated for efficacy in several tumors, including neuroblastoma, but failed because of neurotoxicity [7]. Another cytidine analog with a modification in the side chain of the base is ethynyl-cytidine (ETC; TAS-106) which had a unique mechanism of action, acting predominantly on RNA polymerase I, II and III [8,9]. This drug failed because of uncontrollable neutropenia, neurotoxicity and lack of efficacy. Most recently RX-3117 (Fluorocyclopentenylcytosine) was developed (Box 1). It was synthesized starting from D-ribose using a Wittig reaction, Swern oxidation, stereoselective Grignard reaction, ring-closing metathesis, oxidative rearrangement and electrophilic fluorination [10,11]. It is a novel promising cytidine-analog with a modification on the ribose molecule consisting of a carbon-fluorine bond instead of oxygen and a double bond. It presents interesting features both in bioavailability and pharmacodynamics/efficacy [12,13]. It has an excellent bioavailability compared to other nucleoside analogs, including gemcitabine, and a strong effect on a wide panel of cancer cell lines and xenograft models, including gemcitabine-resistant ones [12–14].

Figure 1. Structural formulas of RX-3117 and the related nucleoside analogs.

cyclopentenylcytosine (CPEC), ethynylcytidine (ETC; TAS106), azacytidine (azaCR), aza-2ʹ-deoxycytidine (aza-CdR), gemcitabine and the normal nucleoside cytidine.

2. Overview of the market

Pancreatic cancer, more specifically pancreatic ductal adenocarcinoma (PDAC) is one of the most dismal cancers and is the seventh leading cause of cancer death globally [15,16]. Currently more than 330,000 patients are diagnosed worldwide [15] and 55,440 patients are diagnosed every year in the US, where it is the 4th cause of death and expected to become the 2nd cause of death in 2030, since the majority of patients will be seen in the age group of 65 to 75 years, especially those who have a history of heavily smoking, obesity and alcohol consumption [16]. The 5-year survival for patients with advanced or metastatic disease is less than 5%, with overall just 8.5% (period 2008–2014) [16]. It is expected that the market will be over 1 billion US$ in 2018. Current treatment options are limited since the disease is usually diagnosed in an advanced stage (III-IV). Only a small percentage of the patients (12–15%) can be treated with a surgery with a curative intent. Surgery may be curative only for patients with stage I/II disease. For patients eligible to undergo complete tumor resection, with macroscopically completely removed tumors the 5-year overall survival was 10.4% for observation alone and 20.7% for gemcitabine only adjuvant treated patients [17]. Most stage III/IV patients are treated with gemcitabine in combination with Abraxane® (nab-paclitaxel), or with FOLFIRINOX, while patients with poor PFS may receive single-agent gemcitabine. Overall median survival for patients with advanced cancer even with treatment is about 10 months. FOLFIRINOX (5-fluorouracil-leucovorin combined with oxaliplatin and irinotecan), is usually given to patients with a good performance status, since it is a relatively toxic regimen [18]. In a retrospective study in several community hospitals patients with a good performance received FOLFIRINOX, while gemcitabine/Abraxane was usually given to patients with a poor PFS; when very poor, gemcitabine was given to a minority as an alternative single agent. Therefore, more effective, less toxic treatments are desperately needed.

The prevalence of urothelial bladder cancer (aUBC) is approximately 2.5 million patients worldwide with about 420,000 new patients diagnosed every year [16]. Risk factors include smoking and exposure to certain chemicals. The majority of patients (70%) are diagnosed with early-stage superficial bladder cancer that can be treated with transurethral resection (TURBT) followed by intravesical Bacillus Calmette–Guerin (BCG) or mitomycin. Only about 10% of the patients are diagnosed with advanced tumors that have penetrated into the bladder wall muscle and spread regionally or to distant sites but prognosis in these patients is poor with a 5-year survival at only 15%. These late-stage patients are usually treated with chemotherapy (with or without radiation). Gemcitabine in combination with cisplatin or carboplatin is the standard of care. More recently, PD-1 inhibitors have been approved in PD-L1 expressing tumors, but it is not limited to these tumors only (e.g. also for patients whose cancer cannot be treated with platinum chemotherapy) for second-line use in patients who develop resistance to gemcitabine-based chemotherapy and about 30% of the patients will have an objective response. The majority of patients do not respond to PD-1 inhibitors and there is a need for additional effective and tolerated treatment options. The bladder cancer market is currently estimated at $2B globally and expected to increase to $8B in the next 10 years due to aging populations and inclusion of PD-1 inhibitors.

3. Mechanism of action

3.1. Transport and activation

Peters et al. characterized the main aspects of the metabolism of RX-3117 and its putative role in anticancer activity, taking in consideration various common pathways which drugs can take to be activated to its cytotoxic intracellular metabolites [12]. A postulated scheme of its metabolism and main targets is presented in Figure 2. However, for some steps in this scheme proof is lacking albeit proof for alternatives is not present either.

Figure 2. Postulated metabolism and mechanism of action of RX-3117.

Uptake of RX-3117 is mediated by hENT1, although a role for other transporters cannot be excluded. RX-3117 is a poor substrate for CDA, but an excellent substrate for UCK2. The monophosphate is subsequently being phosphorylated by either UMPK or CMPK (or both) and thereafter by NDPK. RX-3117 is incorporated into RNA and DNA, but it is not clear in which form the nucleotide (RX-3117-TP or dRX-3117-TP) is incorporated, since inhibition of RR did not decrease its incorporation. Molecular modeling revealed that in both forms RX-3117 can bind to the DNMT1-DNA complex (insert), inhibiting its function. DNMT1 can be translocated to the cytosol, and the binding of RX-3117 to DNMT1 enables ubiquitination leading to breakdown. Phosphorylated forms of RX-3117 can be broken down by a nucleotidase (e.g. NT5C3), while the triphosphate might be a substrate for either MTH1, DCTPP1 or SAMHD1.

One of the main steps for a drug to exert its cytotoxicity, as for other nucleoside analogs, is the uptake into the cell. This is mediated by the human equilibrative nucleoside transporter (hENT) and the human concentrative nucleoside transporter (hCNT) family [19]. An inhibitor of ENT1, dipyridamole, reduced sensitivity against RX-3117, indicating that hENT1 was responsible for uptake of the drug (similarly to gemcitabine), possibly excluding other transporters [12]. A cell line deficient in hENT1 and lacking other transporters was also resistant to RX-3117 (Figure 3).

Figure 3. Deficiency of hENT1 as resistance mechanism for RX-3117.

CEM CCRF cells deficient in hENT1 were exposed to RX-3117 for 72 h, and the sensitivity was determined using an MTT assay as described earlier [12]. This cell line is completely dependent on hENT1 for uptake of nucleosides and nucleoside analogs.

According to the similarity to other cytidine-analogs, two enzymes were tested, known to be involved in cytidine and cytidine analog metabolism: UCK (Uridine-cytidine kinase in both forms UCK1 and UCK2), is responsible for the activation of Aza-CR [20], and dCK (deoxycytidine kinase) for gemcitabine and aza-CdR [21]. To analyze the contribution of these enzymes to RX-3117 metabolism, protection studies were performed with (deoxy)nucleosides, uridine, cytidine, and deoxycytidine. Deoxycytidine did not protect any of the analyzed cell lines against RX-3117, in contrast to uridine and cytidine, which were able to protect cells from RX-3117 effects in a dose-dependent manner [12]. This demonstrated that dCK was not involved in activation of RX-3117, since deoxycytidine would have reverted the sensitivity, as was found earlier for gemcitabine [22]. However, uridine and cytidine protected cells from RX-3117.

The activation enzyme UCK exists in two forms, UCK1 and UCK2 [23]. Using UCK-siRNAs transfections, UCK2-siRNA could completely downregulate UCK2 at the mRNA and protein-level, protecting cells from the RX-3117 cytotoxicity [24]. On the contrary, UCK1-siRNA was ineffective in protecting cells against RX-3117. Moreover, expression of UCK2 (activity, protein, and mRNA) correlated with cellular sensitivity to RX-3117. As a positive control ethynylcytidine was also investigated, known to be a specific substrate for UCK2 [8]. UCK1 downregulation with siRNA did not affect the sensitivity to both ethynylcytidine and RX-3117. Interestingly, UCK2 expression is low in human normal tissues, in comparison with cancer cells which are sensitive to RX-3117 [24]. Subsequent metabolism of RX-3117 to its diphosphate (RX-3117-DP) and triphosphate (RX-3117-TP) is possibly mediated by uridine monophosphate kinase (UMPK) and cytidine monophosphate kinase (CMPK) and Nucleoside diphosphate kinase (NDPK), respectively, but no definite proof has been obtained for involvement of these enzymes. However, the formation of these nucleotides has been demonstrated by the use of radioactive RX-3117 [12] and by the use of a sensitive LC-MS-MS [25]. Accumulation of these ribonucleotides appeared to be correlated to the sensitivity of RX-3117 in several cancer cell lines [12]. No proof has been obtained that RX-3117-DP is reduced to its deoxynucleotide derivative dRX-3117-DP by ribonucleotide reductase and subsequently to dRX-3117-TP. Analysis of the cellular nucleotide content after incubation of several non-small cell lung cancer (NSCLC) cell lines, did not show any trace of the formation of deoxyribonucleotides, despite the use of a sensitive LC-MS-MS assay. Moreover, the deoxy form of RX-3117, 2ʹ-deoxy-fluoropentenylcytosine (RX-3128), did not exhibit any cytotoxicity to cancer cells (Table 1). Furthermore, inhibition of ribonucleotide reductase by a specific inhibitor triapine did not reduce the incorporation of radiolabelled RX-3117 into DNA (unpublished data). These data question the nature of the derivative of RX-3117 incorporated into DNA, and it is hypothesized that RX-3117 has not to be converted to its deoxyribonucleotide in order to be incorporated into DNA.

Table 1. Cytotoxicity of RX-3117 derivatives in human cancer cell lines.

Cell lines	RX-3117		Deaminated RX-3117	Deoxy RX-3117	Deoxy & deaminated RX-3117	RX-3117 diastereomer
	IC50 (µM)	Growth at 1 µM	Growth at 1 µM	Growth at 20 µM	Growth at 20 µM	Growth at 25 µM
HCT-116	0.39	1.0%		79.4||%	81.9%	97.0%
MDA-MB-231	0.18	1.4%		100%	71.1%	100%
PANC-1	0.62	3.2%		71.4%	89.1%	100%
Caki-1	0.84	6.4%		100%	100%
MCF7	0.34	16.4%	96.3%
A549	0.34	2.8%	100%
MKN45	0.50	3.8%	100%
U251	0.83	10.6	92.1%

Values for RX-3117 are given as μM. For all derivatives of RX-3117 no IC50 values (concentration that leads to 50% growth inhibition) could be reached and growth at a high concentration (1, 20 or 25 μM) is given. 100% means no growth inhibition.

3.2. Degradation enzymes

CDA (cytidine deaminase) is a degradation enzyme, which is involved in the degradation step of many cytidine analog drugs, such as gemcitabine and ara-C [26,27]. However, using different approaches, it was demonstrated enzymatically that RX-3117 is a very poor substrate for CDA, while tetrahydrouridine (THU; an inhibitor of CDA) did not affect cellular sensitivity to RX-3117, whereas it had a strong effect on the sensitivity of cells to other drugs, such as gemcitabine, aza-CR and aza-CdR [12].

Next to CDA, the nucleotides formed from RX-3117 can be degraded. Several enzymes have been described to be involved in nucleotide breakdown [28]. A clear role for various nucleotidases and phosphatases has been described for breakdown of normal and analog nucleotides. Most likely the cytosolic NT5C3 is responsible for the breakdown of the monophosphate to RX-3117. For the breakdown of deoxyribonucleotides several enzymes have been described, such as nudix hydrolase 1 (NUT1/MTH1), deoxycytidine triphosphatase (DCTPP1) and SAM and HD domain-containing protein 1 (SAMHD1) [28] but a potential role in breakdown of RX-3117 nucleotides has not yet been demonstrated.

3.3. Degradation of RNA and DNA

RX-3117 was reported to be incorporated into both DNA and RNA to a comparable extent, but the exact nature of the incorporated metabolite was not identified. Moreover, RX-3117 showed a concentration-dependent inhibition of DNA/RNA synthesis, with cell-line specificity, but without a relation with cell-line sensitivity to the drug [12]. DNA synthesis inhibition was found at lower concentrations of RX-3117, in comparison to RNA inhibition. In some cell lines even 100 µM RX-3117 did not inhibit RNA synthesis. Additionally, the drug did not seem to affect RNA integrity after 48 h of RX-3117 exposure. According to this evidence, RX-3117 mechanism of action was compared with aza-CR, which can be incorporated both to RNA and DNA, but exerts its cytotoxic effect due to its incorporation in the DNA, not affecting RNA integrity as well [12]. The incorporation of RX-3117 into DNA led to DNA damage as found with positive staining for the DNA damage marker gamma H2AX, accumulation of cells in the S-phase, causing apoptosis as shown by increased annexin-5 staining [29] (Figure 4).

Figure 4. DNA damage and induction of apoptosis in A2780 ovarian cancer cells and A549 and SW1573 NSCLC cells.

(a) Quantification of γ H2A.X and DNMT1 by Western Blot of nuclear extracts of A2780 cells; H3 is the loading control for the nuclear fraction: (b) Quantification of Annexin V in RX-3117 treated (5 µM for A549 and 10 µM for SW1573 cells for 4 days); (c) Positive staining for γ H2A.X as a marker for DNA damage in A549 cells after 4 days of exposure.

3.4. Effect on DNA methyltransferase

An interesting aspect of the mechanism of action of RX-3117 is the inhibition of DNA methyltransferase 1 (DNMT1) [12,14], but not DNMT3a. Inhibition of DNMT1 has been demonstrated in different cancer cell lines and was related to their sensitivity to the drug. DNMT1 is crucial for maintenance methylation [30]. Different RX-3117 concentrations were needed in various cell lines to reach the same level of DNMT1 suppression. Interestingly, this effect was predominantly noticed at the protein level, since the effect on mRNA levels was less [31]. In a comparison with aza-CdR and aza-CR as reference hypomethylation agents, it was demonstrated that the decrease in DNMT1 levels exerts a functional effect as well. Methylation usually leads to suppression of the promoter of a number of genes; hypomethylation will result in the activation of these genes, as has been demonstrated for genes such as O-methylguanine DNA methyltransferase and tumor suppressor genes [32]. The proton-coupled folate transporter not only showed an increased protein and gene expression, but also an increased transport activity, similar to aza-CR and aza-CdR. Moreover, treatment with RX-3117 led to a decrease in overall methylation of the DNA.

The mechanism of DNMT1 downregulation seems to be related to an increased protein breakdown. Normally DNMT1 acts on the DNA during the so-called base flipping for methyl transfer [30]. Treatment of the NSCLC cell line A549 and the pancreatic cancer cell line SUIT-028 with RX-3117 demonstrated trapping of DNMT1 to DNA. Moreover, treatment with RX-3117 led to an increased ubiquitination of DNMT1, which translocated to the cytosol, resulting in increased degradation, which could be inhibited by the specific proteasome inhibitor bortezomib. It was concluded that RX-3117 treatment would still allow initiation of the methyl transfer but cannot proceed [32].

RX-3117 has also been evaluated for its radiosensitizing potential. Firstly, using a clonogenic assay, it was demonstrated that pre-incubation of cells with RX-3117 led to a radiosensitizing effect in the majority of the analyzed cell lines [29]. Moreover, RX-3117 had a significant effect on radiation induced-DNA damage with a delayed DNA repair in irradiated cells, after RX-3117 treatment. RX-3117 and radiation were also used to investigate the cell cycle distribution. RX-3117 and irradiation led to accumulation of cells in G2 and S phase [29]. RX-3117 might be an effective radiosensitizing agent for NSCLC.

4. Effect on in vitro and in vivo models of cancer

Both in vitro and in vivo studies have been performed to characterize the antitumor effect of RX-3117 in comparison with other drugs belonging to the same family. Initial studies in tumor cell lines showed a promising activity pattern in many types of cancer with IC50 in the low µM, particularly in breast (0.18 µM IC50), lung (0.25 µM IC50), and colon (0.28 µM IC50) cancer cell lines [14]. This pattern was verified in a panel of 59 cell lines, with different pathological origins [12]. Altogether more than 100 different human cancer cell lines were tested. Interestingly, RX-3117 had no effect on melanomas. In addition to that, many cancer cells resistant to other (deoxy-) cytidine analogues were sensitive to RX-3117, which for instance showed a similar efficacy pattern in A549, SW1573, CEM and their gemcitabine-resistant cell lines.

Interestingly, it was shown that RX-3117 had a time-dependent sensitivity to RX-3117, with a higher efficacy in cells at a long exposure [12].

Yang et al. [13] initially tested the efficacy of RX-3117 in nine xenograft models, showing potent activity of the drug in many types of cancer, including gemcitabine-resistant xenografts. Altogether the drug was effective against 17 human colon tumors, human renal cancer, and human pancreatic cancer xenograft models, comparable or better than other analogs or other effective drugs such as paclitaxel.

In a recent study, combination treatments of RX-3117 with Abraxane or anti-PD1 immunotherapy were tested in colorectal and pancreatic xenograft models [33]. Combination treatment with Abraxane + RX-3117 showed a better tumor growth inhibition, compared to RX-3117 alone, while the patient from whom the xenograft was obtained, showed resistance to Abraxane alone. Moreover, RX-3117 + anti-PD1 combination also showed promising activity, with a higher level of survival compared to anti-PD1 treatment alone.

5. Clinical trials

5.1. Phase 0 study

RX-3117 was investigated for its pharmacokinetics and bioavailability in order to clarify its plasma profile and its best administration method. In particular, oral and intravenous administrations have been compared. A limited cohort of patients with solid tumors was treated with a 50 mg or 100 mg single RX-3117 oral dose or a 20 mg single intravenous dose [34]. First results indicated a different plasma profile between these two administration methods. The plasma profile of the orally administered drug had a T_max of 2.16 and 2.49 h and an average C_max of 1.18 µM (303.3 ng/mL) and 1.21 µM (311.43 ng/mL) for 50 mg and 100 mg dose, respectively. On the contrary, intravenously administered drug led to a C_max of 4.44 µM (1143.63 ng/mL) and a T_max of 0.25 h. From these studies, using the plasma profile of the 20 mg i.v. dose, the oral bioavailability of RX-3117 was calculated to be 55.7% and 33.4%, respectively, and appeared to be safe and well tolerated in patients [34].

5.2. Phase I and II studies and pharmacokinetics

RX-3117 has been evaluated for its safety and is being evaluated for efficacy in several phase I and phase II clinical trials. Here we summarize the preliminary outcomes of ongoing studies of RX-3117 in pancreatic and bladder cancer patients.

First, Phase I studies have been performed to evaluate pharmacokinetics (PK) and safety and to determine the MTD (Maximum tolerated dose) of oral administration, in a group of patients with relapsed/refractory solid tumors [35]. RX-3117 was administered orally 3 times a week, for three weeks with one week off for each 4-week cycle, in a dose escalation schedule from 30 to 2000 mg. The drug showed a linear PK with a plateau at 1500 mg, with peak plasma concentrations varying from 32 to 1858 ng/mL (0.12–7.22 µM) after the 1st dose and 99–1703 ng/mL (0.34–6.62 μM) after the 7th dose. The measured AUC was 164–20,544 h ng/mL (0.64 µmol r/L – 79 µmol h/L) after the 1st dose and 702–20,919 h ng/mL (2.73 µmol h/L – 81 µmol h/L) after the 7th dose [36]. At several doses, early antitumor activity was found in pancreas, colorectal and mesothelioma cancers with some cases of tumor burden reductions.

Additionally, two alternative more frequent administration schedules were compared: the first with 500–700 mg RX-3117 orally administered 5 times a week, for 3 weeks with 1-week-off; the second one with 500 mg of RX-3117 orally administered 7 times a week, for 3 weeks, with 1-week off, in 4-week cycles. For the 5 times a week administration schedule, the maximal tolerated dose (MTD) was 700 mg, with a C_max of 3.84 µM (989 ng/mL) and the AUC 8663 h.ng/mL. In the Phase Ib clinical trial, 12 patients experienced stable disease for up to 276 days and 3 patients showed evidence of a decrease of tumor burden. The 700 mg dose appears to be safe and well tolerated with predictable pharmacokinetics and selected for further Phase II clinical trials, in line with the better pharmacokinetic properties [35].

Next, RX-3117 has been evaluated as a single agent in two phase IIa clinical trials which were also intended to extend analyses of safety and tolerability. For both pancreas and aUC (advanced urothelial cancer) Phase II trials, patients received a dose of 700 mg of RX-3117 orally once-a-day for 5 consecutive days with 2 days off per week for 3 weeks with 1 week off in each 4-week cycle, although some patients with aUC were treated for 4 straight weeks in the 28 days cycle. The most recent data are on 45 patients with refractory or relapsed metastatic pancreatic cancer (of which 93% progressed on gemcitabine therapy and 42 patients were evaluable for final efficacy analysis). In this study, 13 patients had an increased progression-free survival for 2 months or more and 5 patients had disease stabilization for more than 4 months. One patient had a partial response. In this clinical trial, the reported adverse effects were mild to moderate anemia (19%), mild to moderate fatigue (15%), mild to moderate diarrhoea (11%), severe anemia (9%) (Clinical trial number NCT02030067; evaluation not yet according to RECIST criteria) [36]. RX-3117 with Abraxane (nab-paclitaxel) is being investigated in a 4-week cycle in a Phase 1b/Phase 2a clinical trial of RX-3117 (700 mg orally for 5 consecutive days/2 days off) with nab-paclitaxel (125 mg/m² i.v. weekly) for 3 weeks with one week off in patients with metastatic pancreatic cancer [37]. Preliminary updated data (Clinical Trial number NCT03189914; Jan 2019 [38]) revealed a disease control rate of 92% (22/24 patients) at 8 weeks, including a CR after 6 cycles of therapy, 8 PR and 13 SD [38]. PK analyses indicated that RX-3117 and nab-paclitaxel did not interfere with each other, with comparable Cmax and AUC for RX-3117 as in the single-agent study. Toxicity evaluation is preliminary and not yet complete (Grade 3–4 neutropenia 29%, anemia 11%, diarrhea 8%). Enrollment will continue until 40 evaluable patients have been identified. The study will continue until all patients have completed their last on-study visit.

The Phase IIa clinical trial for advanced urothelial cancer (aUC) showed similar encouraging results as for pancreatic cancer. Up to now, 35 patients have been treated who were refractory to either gemcitabine (31 patients) or immunotherapy (27 patients), and 31 patients were evaluable for efficacy analysis [39]. Remarkably, seven patients experienced disease stabilization for more than 2 months and 19% for more than 4 months, with one of them even more than 310 days; one patient with one CR is continuing treatment after 14 months; 5 patients had a SD for more than 4 months, with one patient with an SD at 10 months and one at 6 months. For four patients a tumor reduction was found ranging from 13.9% to 20%. The reported preliminary adverse effects were G1/2 diarrhea (14%), fatigue (9%), nausea (9%), vomiting (9%), G1/G2 anemia (7%), and G3 thrombocytopenia (7%). The study continues to enroll patients with aUC in Stage 2 (NCT02030067) [40].

Rexahn has received Orphan Drug designation for RX-3117 for pancreatic cancer from the FDA and from the EMA.

6. Potential biomarkers for RX-3117

6.1. Transporters and target enzymes

6.1.1. Transporters

From the initial mechanism of action studies, several potential markers were identified in various model systems and characterized for their expression in human normal and tumor tissues. These potential biomarkers are either related to its metabolism or its identified targets. RX-3117 is transported into the cell by the hENT1. Since inhibition of hENT results in a decreased sensitivity and since a deficiency of hENT even in complete resistance, it can be postulated that at least a certain expression of hENT is required for cells to be sensitive to RX-3117. For gemcitabine, it has been demonstrated that sensitivity of PDAC is related to hENT1 expression, either measured by PCR [40] or by immunohistochemistry [41]. Patients with a higher ENT1 expression had a longer survival. Care has to be taken that a proper antibody is being used for immunostaining since the original antibody developed by Cass et al. clearly showed a relation with efficacy of gemcitabine [41] and patients with a higher expression had a longer survival. However, the antibody SP120 also stained positive in cells with a deficient hENT expression [42]; this antibody was used in a pivotal prospective study comparing gemcitabine and the prodrug CO1.01 (CP-4126) which can bypass the hENT transporter and shows sensitivity in transport inhibited cells [43]. In this clinical study, no difference in survival of gemcitabine-treated patients with low and high positive staining with SP120 was found [44] so that it was incorrectly concluded that hENT was not related to gemcitabine’s efficacy. Using another proper antibody, a relation was found for high and low expression of hENT, which was validated with PCR, and showed a large difference in hENT in xenografts and patient samples.

Figure 5. Expression of UCK2 in several human xenografts and tumors from patients with a different histological origin; liver metastasis of colon cancer, small cell lung cancer (H69) and pancreatic cancer..

6.1.2. Uridine-cytidine kinase

Another potential biomarker is the expression of UCK2. This enzyme is abundantly expressed in tumors, but not in normal tissues, except for placenta [24]. Since UCK1, which is abundant in normal tissues, cannot activate RX-3117, this gives a very interesting potential selective activation of RX-3117 in tumors. Expression of UCK2 has been demonstrated to be high in many tumor tissues, as shown using PCR and specific antibodies (Figure 5) [45]. It plays a role in intrinsic resistance to RX-3117, although in cells with acquired resistance no deficiency of UCK2 was found (unpublished results). Therefore, UCK2 has the potential to be used to select patients with a high expression of this enzyme, which has been found in several lung cancer xenografts and samples from human colon cancer, pancreatic cancer and bladder cancer [24,46].

6.1.3. Degradation enzymes

Several enzymes are postulated to play a role in RX-3117 activation (e.g. UMPK, CMPK, NDPK) or in the degradation of RX-3117 nucleotides (nucleotidases, MTH1, SAMHD1, DCTPP1) [28] but initial data did not show a relation with resistance in a cell line panel in which acquired resistance to RX-3117 was induced [47] (Sarkisjan et al., unpublished). However, similar to UCK2 this does not exclude that intrinsic resistance might be related to drug sensitivity and should be investigated more in detail.

6.2. DNA methyltransferases

Another potential biomarker is DNMT1. DNMT1 expression can be downregulated by RX-3117, which affects DNA methylation. DNMT1 shows a large variation in expression in various cancer cell lines, human xenografts and human tumor tissues [48]. This can easily be measured using either PCR or immunohistochemistry, both in tumor tissue and normal cells such as white blood cells. Moreover, since RX-3117 treatment leads to a decreased DNA methylation, this feature can be evaluated in patients as well, either by measurement of global DNA methylation in surrogate tissue or by measurement of the expression of target genes, which are regulated by promoter methylation, such as the above-mentioned tumor suppressor genes. Since it will be inconvenient to monitor methylation in tumors, alternative sources should be investigated, e.g. by using surrogate tissues or by analysis of liquid biopsy specimens, such as circulating tumor cells (CTCs) or circulating free DNA (cfDNA). Sensitive techniques such as pyrosequencing might be used for this purpose.

7. Conclusion

RX-3117 is a novel cytidine analog with several interesting features that make it distinct from currently used antimetabolites. Its anticancer activity is dependent on the accumulation of RX-3117 nucleotides; the first activation step is catalyzed by the tumor-specific enzyme UCK2, and its triphosphate is incorporated into RNA and DNA. However, its mechanism of action also includes inhibition of DNMT1. RX-3117 is not catabolized by CDA, which explains its excellent oral bioavailability. The drug is well tolerated and oral administration is used in ongoing Phase II studies where promising anticancer activity has been found.

8. Expert opinion

According to the promising data from in vitro and in vivo studies, and ongoing Phase I/II studies, RX-3117 appears to be a safe and well-tolerated drug. It has various promising features which make it different from other successful cytidine analogs such as gemcitabine and ara-C or analogs that failed such as cyclopentenylcytosine and ethynylcytidine. These features include, (1) a selective activation in tumors by UCK2, an enzyme not present in normal tissues; (2) an excellent oral bioavailability; and (3) a potent antitumor activity against a variety of human xenografts, including novel PDX and gemcitabine-resistant tumors. These aspects have all been covered in several patents, not only USA and European but also various other countries. There are promising clinical data from the initial Phase I and Phase II studies on advanced bladder and pancreatic cancer, such as the feasibility of several combinations including combinations with targeted cytotoxic chemotherapy, tyrosine kinase targeted drugs and novel checkpoint inhibitors. The mechanism of action studies revealed the potential of many promising biomarkers including the tumor specific UCK2, as well as hENT1 and DNMT1. However, the exact mechanism of action for this drug still remains to be elucidated, since a potential mechanism (or mechanisms) of acquired resistance has (have) not yet been elucidated either, indicating that the drug has several novel attractive features [48]. Based on these favourable properties the drug will be an improvement compared to current therapies for PDAC such as gemcitabine-based combinations and the toxic FOLFIRINOX. In advanced bladder cancer the toxic MVAC therapy has already been replaced by gemcitabine-cisplatin, but RX-3117 (alone or in combination) is likely to be a favourable alternative. Because of its ease of administration (oral) and excellent safety profile, it is likely to be prescribed. This is already indicated by the rapid progress of ongoing clinical studies. However, to achieve this aim, the current clinical studies need to retain the current favorable results. Proper Phase III studies (focusing on similar patient populations and using a proper active Phase II administration schedule) in comparison with gemcitabine-based therapies need to be performed for registration, focusing on at least equivalence, which may be a feasible outcome in about 5 years.

Considering the excellent in vitro and in vivo data on other tumor types, such as non-small cell lung cancer, this disease may be another potential tumor type. Current immunotherapy studies may be an important hurdle in the recruitment and therefore development. However, the lack of anticancer activity of immunotherapy in various tumor types may be an opportunity for RX-3117. Therefore, clinical trials should not only evaluate biomarkers such as UCK2, transporters and the target DNMT1, but should also include the analysis of secreted cytokines and chemokines. A future opportunity might then be a combination with immune checkpoint inhibitors. Such combinations, when positive, will likely replace current therapy modalities.

Box 1. Drug summary

• Drug name: 5-fluorocyclopentenyl cytosine (RX-3117);

• Molecular formula: C₁₀H₁₂FN₃O₄; Molecular weight: 257.221 g/mol

• Isomeric SMILE: C1=CN(C(=O) N=C1N) [C@H]2[C@@H] ([C@@H](C(=C2F)CO)O)O

• IUPAC: 4-amino-1-[(1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl) cyclopent-2-en-1-yl] pyrimidin-2-one

• The drug is covered by several patents US, European and several other countries, regarding its potential therapeutic use (US7405214, submitted 2005, granted 2008), process of preparation (US2017158662 and US533958: submitted 2015 and 2016; granted 2016); methods of use (US9782410, submitted 2016)

• Phase: the drug is being evaluated in phase 2 for advanced bladder cancer (ClinicalTrials.gov Identifier: NCT02030067) and pancreatic cancer (ClinicalTrials.gov Identifier: NCT03189914)

• Indication: when active in the first Phase 2, various potential indications (pancreatic ductal adenocarcinoma, advanced urothelial cancer, and non-small cell lung cancer) may receive the drug 1^st line.

• Pharmacology: >50% oral bioavailability; Tmax was reached within 2–3 h with Cmax in the high micromolar range; linear PK with no drug accumulation.

• The drug is taken up by the equilibrative nucleoside transporters and initially activated by UCK2.

• The drug is incorporated into DNA, causing DNA damage, but also inhibition of DNA methyltransferase I.

  

Clinical trials in bladder and pancreatic cancer are ongoing.

Declaration of interest

GJ Peters has received research funding and conference reimbursement from Rexahn Pharmaceuticals (USA), research funding from Eli Lilly & Co (Netherlands) and Taiho Pharmaceutical (Japan) and consultancy fees from Taiho Pharmaceutical (Japan), Clear Creek Bio (USA) and Ellipses Pharma Ltd, UK. E Benaim, C Heaton, DJ Kim, YB Lee, C Peterson and J Poore are employees of Rexahn and own stock in the company. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

One peer reviewer is engaged in clinical trials that are assessing RX-3117. Peer reviewers on this manuscript have no other relevant financial or other relationships to disclose.

Acknowledgements

We appreciate the contributions of Julie Poore, Callie Heaton, Christine Peterson and Ely Benaim, all from Rexahn Pharmaceuticals, Inc, Rockville, MD, USA

Additional information

Funding

This paper was not funded.