Potent Inhibitor Of Topoisomerase Research Articles

In silico Study of Novel Tryptanthrin-Based Topoisomerase Inhibitors

Background: Over the past ten years, a remarkable number of changes have occurred in the field of cancer drug research. Most anticancer drugs from the first generation work by breaking down DNA, preventing its production, interfering with cell division processes, or attaching to microtubules. The potential use of tryptanthrin as well as its analogues is well documented for anticancer properties. Objective: To design a novel hybrid of tryptanthrin analogs with expected anticancer activity. Method: By changing the C-6 carbonyl position of the tryptanthrin molecule, a set of 72 derivatives of substituted-6-benzylidine-6H-indolo[2,1-b] quinazoline-12-one was developed. These ligands were screened in silico using Schrodinger Glide extra precision docking against DNA topoisomerase using doxorubicin and teniposide as references to identify their potential anticancer properties. Further, these ligands were subjected to an in silico ADMET study to identify their drug likeliness. Results: Combined results of molecular docking and in silico ADMET study suggest that out of the total 72 ligands, 6 ligands RC 51, RC 29, RC 42, RC 3, RC 54, and RC 63 were showing very better binding affinity than the natural ligand adenylyl-imidodiphosphate and the two standard reference drugs- doxorubicin and teniposide. Conclusion: Our computational approach was successful in identifying ligands that are potentially potent topoisomerase inhibitors. These can be tested further using in vitro and in vivo analysis.

Identification of a novel 10-hydroxyevodiamine prodrug as a potent topoisomerase inhibitor with improved aqueous solubility for treatment of hepatocellular carcinoma

Natural product evodiamine (Evo) and its synthetic derivatives represent an attractive dual Topo 1/2 inhibitors with broad-spectrum antitumor efficacy. However, the clinical applications of these compounds have been impeded by their poor aqueous solubility. Herein, a series of water-soluble 10-substituted-N(14)-phenylevodiamine derivatives were designed and synthesized. The most potent compound 45 featuring a quaternary ammonium salt fragment achieved robust aqueous solubility and nanomolar potency against a panel of human hepatoma cell lines Huh7, HepG2, SK-Hep-1, SMMC-7721, and SMMC-7721/DOX (doxorubicin-resistant cell). Further studies revealed that 45 could inhibit Topo 1 and Topo 2, induce apoptosis, arrest the cell cycle at the G2/M stage and inhibit the migration and invasion. Compound 45 exhibited potent antitumor activity (TGI = 51.1 %, 10 mg/kg) in the Huh7 xenograft model with acceptable safety profile. In addition, a 21-day long-term dose toxicity study confirmed that the maximum tolerated dose of compound 45 was 20 mg/kg. Overall, this study presented a promising Evo-derived candidate for the treatment of hepatocellular carcinoma.

Dose optimization and exposure-response analyses to support optimal dose of ABBV-400, a novel C-met–targeting antibody drug conjugate, in patients with metastatic colorectal cancer (mCRC).

3030 Background: ABBV-400 is an antibody drug conjugate (ADC) consisting of a c-Met targeting antibody conjugated to a potent inhibitor of topoisomerase 1 (Top1) payload. ABBV-400 has shown encouraging activity and demonstrated tolerability of dosages up to 3.0 mg/kg Q3W as monotherapy in patients with advanced solid tumors in an ongoing phase 1 study (1). Dosage optimization is a critical aspect of oncology drug development and FDA Project Optimus has focused on reformulating the dosage selection paradigm for oncology products. The phase 1 study included a dose optimization phase in mCRC patients in which 3 doses were evaluated (1.6, 2.4 and 3.0 mg/kg Q3W) to determine the optimal dose for further development. Herein, exposure-response analyses are reported to determine optimal monotherapy dose in mCRC patients. Methods: Data across a range of doses (1.6 – 6.0 mg/kg Q3W) from the phase 1 study (NCT05029882) were used. Population pharmacokinetics (PK) and exposure-response (E-R) analyses were conducted to characterize ABBV-400 conjugate and unconjugated payload PK and the relationship between exposures and efficacy (objective response rate [ORR]) and safety (Grade (Gr) ≥ 3 neutropenia, anemia and thrombocytopenia) endpoints. Relative dose intensity (RDI) analysis was performed. Results: ABBV-400 conjugate and payload PK were adequately described by a combined multi-analyte population PK model with first order kinetics (n=204). ABBV-400 conjugate average concentration was a better predictor of response and showed that higher exposure correlated with higher probability of response (n=122 CRC, ORR, p < 0.05) and safety events (n=204, Gr ≥ 3 neutropenia, anemia and thrombocytopenia, p < 0.001). Dose intensity analyses showed greater number of dose reductions for 3 mg/kg Q3W with effective (actual) dose received of 2.6 mg/kg Q3W in mCRC patients. E-R analyses for safety and efficacy and dose intensity analyses in mCRC 3L+ patients indicate that both 2.4 and 3.0 mg/kg doses provided meaningful efficacy (ORR); while the 3.0 mg/kg dose may provide higher response rates, the 2.4 mg/kg Q3W dosing regimen provides an optimal balance of safety and efficacy compared to 3.0 mg/kg Q3W. Conclusions: Population PK, E-R for efficacy and safety and RDI analyses and totality of clinical data supported the selection of 2.4 mg/kg Q3W as optimal ABBV-400 monotherapy dose for further study in mCRC patients. 1. Sharma M, et al. ASCO 2023. Abstract 3015. Clinical trial information: NCT05029882 .

Interaction of Camptothecin Anticancer Drugs with Ribosomal Proteins L15 and L11: A Molecular Docking Study.

The antitumor drug topotecan (TPT) is a potent inhibitor of topoisomerase I, triggering DNA breaks lethal for proliferating cancer cells. The mechanism is common to camptothecins SN38 (the active metabolite of irinotecan) and belotecan (BLT). Recently, TPT was shown to bind the ribosomal protein L15, inducing an antitumor immune activation independent of topoisomerase I. We have modeled the interaction of four camptothecins with RPL15 derived from the 80S human ribosome. Two potential drug-binding sites were identified at Ile135 and Phe129. SN38 can form robust RPL15 complexes at both sites, whereas BLT essentially gave stable complexes with site Ile135. The empirical energy of interaction (ΔE) for SN38 binding to RPL15 is similar to that determined for TPT binding to the topoisomerase I-DNA complex. Molecular models with the ribosomal protein L11 sensitive to topoisomerase inhibitors show that SN38 can form a robust complex at a single site (Cys25), much more stable than those with TPT and BLT. The main camptothecin structural elements implicated in the ribosomal protein interaction are the lactone moiety, the aromatic system and the 10-hydroxyl group. The study provides guidance to the design of modulators of ribosomal proteins L11 and L15, both considered anticancer targets.

Antibody-drug conjugates targeting TROP-2: Clinical development in metastatic breast cancer

Antibody drug conjugates (ADCs) combine the potent cytotoxicity of chemotherapy with the antigen -specific targeted approach of antibodies into one single molecule. Trophoblast cell surface antigen 2 (TROP-2) is a transmembrane glycoprotein involved in calcium signal transduction and is expressed in multiple tumor types. TROP-2 expression is higher in HER2-negative breast tumors (HR+/HR-) and is associated with worse survival. Sacituzumab govitecan (SG) is a first-in-class TROP-2-directed ADC with an anti-TROP-2 antibody conjugated to SN-38, a topoisomerase inhibitor via a hydrolysable linker. This hydrolysable linker permits intracellular and extracellular release of the membrane permeable payload enabling the “bystander effect” contributing to the efficacy of this agent. There was significant improvement in progression free survival (PFS) and overall survival (OS) with SG versus chemotherapy in pretreated metastatic triple negative breast cancer (TNBC), resulting in regulatory approval. Common adverse events (AE) reported were neutropenia and diarrhea. SG also demonstrated clinical activity versus chemotherapy in a phase III trial of HR+/HER2-metastatic breast cancer (MBC) and is under evaluation in first-line metastatic and early stage TNBC as well. Datopotamab deruxtecan (Dato-DXd) is a TROP-2 ADC that differs from SG in that it has a cleavable tetrapeptide linker and a more potent topoisomerase inhibitor payload. This construct is highly stable in circulation with a longer half-life than SG, and undergoes cleavage in presence of intracellular lysosomal proteases. Dato-DXd demonstrated preliminary efficacy in unselected metastatic TNBC, with common AEs of low-grade nausea and stomatitis. Dato-DXd is being investigated in phase III studies in metastatic TNBC and HR+/HER2- MBC. These novel TROP-2 ADCs have the potential to deliver enhanced efficacy with reduced toxicity in MBC and possibly in early stage breast cancer (EBC).

A Phase Ib Study of the Simmitecan Single Agent and in Combination With 5-Fluorouracil/Leucovorin or Thalidomide in Patients With Advanced Solid Tumor.

Background: Simmitecan is a potent inhibitor of topoisomerase I with anti-tumor activity. This phase Ib trial was conducted to investigate the safety and anti-tumor effect of simmitecan alone or in combination with other drugs. Methods: Eligible patients with advanced solid tumor had no further standard treatment options. Patients were allocated to receive simmitecan alone, simmitecan in combination with 5-fluorouracil (5-FU)/leucovorin (LV), or simmitecan in combination with thalidomide, 14 days a cycle, until disease progression or unacceptable toxicity occurred. Results: A total of 41 patients were enrolled, with a median age of 55 (range 29–69) years. Among them, 13 patients received simmitecan monotherapy, 10 received simmitecan + 5-FU/LV, and 18 received simmitecan + thalidomide. No dose-limiting toxicity occurred. Overall, the most common grade 3/4 adverse event (AE) was neutropenia (46.2, 70.0, and 88.9%, respectively, in simmitecan, simmitecan + 5-FU/LV, and simmitecan + thalidomide cohorts), and treatment-related severe AEs included anemia and febrile neutropenia (7.7% each in simmitecan cohort), diarrhea (10% in simmitecan +5-FU/LV cohort), and febrile neutropenia (5.6% in simmitecan + thalidomide cohort). The majority of patients (24/41, 58.3%) had progressed on prior irinotecan; nevertheless, partial response was achieved in one colorectal cancer patients treated with simmitecan + thalidomide. The disease control rates of simmitecan, simmitecan + 5-FU/LV, and simmitecan + thalidomide cohorts were 46.2, 80.0, and 61.1%, respectively. Conclusion: This study demonstrated a manageable safety profile of simmitecan as a single agent or as part of a combination therapy. There have not been any safety concerns with simmitecan in combination when compared to simmitecan alone. Simmitecan + 5-FU/LV regimen seemed to have a better efficacy. Nonetheless, the efficacy of this regimen needs to be further explored in the subsequent study.

Abstract 6249: CBX-12: A low pH targeting alphalex™-exatecan conjugate for the treatment of solid tumors

Abstract Topoisomerase inhibitors are potent DNA damaging agents with great potential as anti-cancer drugs for a wide range of solid tumors. However, dose-limiting toxicities such as myelosuppression and gastric toxicity have prevented them from reaching their full clinical potential. Targeting topoisomerase inhibitors with antibodies (i.e. antibody-drug conjugates; ADCs) may enhance the therapeutic window of these agents, but this approach typically limits applicability to a small subset of patients with tumors expressing the target antigen. We recently developed the alphalexTM tumor-targeting platform to overcome the limitations of ADC-based therapeutic strategies. Rather than targeting a specific antigen, alphalexTM consists of a unique variant of pH-Low Insertion Peptide, (pHLIP®; references 1-3) which targets the low pH environment of the tumor, a universal feature characteristic of all tumors due to the Warburg effect. These alphalexTM conjugates form an alpha helix only in low pH conditions, allowing for insertion of the peptide within the cancer cell membrane, delivery of C-terminal warheads across the membrane, and subsequent intracellular release of the agent via glutathione reduction of the linker, thereby allowing for tumor-specific intracellular delivery in an antigen-independent manner. We report the discovery and development of CBX-12, an alphalexTM conjugate of the potent topoisomerase inhibitor, exatecan. CBX-12 provides additional proof of mechanism to the alphalexTM platform by displaying remarkable tumor-targeting properties in preclinical models. CBX-12 displays enhanced stability in plasma in vivo, undergoing only 0.003% warhead release over 30 hours in circulation and demonstrating exquisite selectivity for tumor over normal tissues in mouse tumor models. Notably, CBX-12 allows for efficient delivery of exatecan into tumors due to a highly optimized cleavable linker, allowing CBX-12 to display extraordinary efficacy in a HER2-negative tumor model in an antigen-independent manner. At 10 mg/kg, CBX-12 treatment almost completely suppressed growth of human colorectal tumors in mice, with complete sparing of bone marrow. In contrast, in animals dosed with the equimolar free exatecan (1.15 mg/kg) there was substantial tumor growth accompanied by neutropenia and weight loss. This superior profile of CBX-12 allow us to greatly enhance efficacy relative to dosing equimolar amounts of unconjugated exatecan, which causes significant, dose-limiting bone marrow toxicity. We have demonstrated that CBX-12 is both safe and has potent anti-tumor activity in preclinical models, and we plan to rapidly move forward with the clinical development of CBX-12 as our lead candidate.

Design and Synthesis of Arylnaphthalene Lignan Lactone Derivatives as Potent Topoisomerase Inhibitors.

Arylnaphthalene lignan lactones are a class of natural products containing the phenyl-naphthyl skeleton. Some arylnaphthalene lignan lactones have been used in clinical practice as antitumor agents, due to their cytotoxicity and inhibitory activities against DNA topoisomerase I (Topo I) and topoisomerase II (Topo II). This study presents the design and synthesis of arylnaphthalene lignan lactones derivatives. The inhibitory activities against Topo I and Topo IIα and antitumor activities of these compounds were assayed. A series of arylnaphthalene lignan lactones derivatives have been designed and synthesized, using the Diels-Alder reaction and Suzuki reaction as the key steps. Their antiproliferation activities were evaluated by sulforhodamine B assay on human breast cancer MDAMB-231, MDA-MB-435 and human cervical cancer HeLa cells. DNA relaxation assays were employed to examine the inhibitory activity of compounds 1-22 on Topo I and Topo IIα in vitro. Flow cytometry analysis was performed to study the drug effects on cell cycle progressions. Seven compounds exhibited the modest anti-proliferation activity with IC50 values between 1.36 and 20 μM. Compounds 3, 19 and 22 showed potent inhibitory activities with IC50 values less than 1 μM. DNA relaxation assay revealed that compound 22 showed potent inhibitory activity against Topo IIα in vitro. Compound 22 also induced DNA breaks in MDA-MB-435 cells evidenced by comet tails and the accumulation of γ-H2AX foci. The ability of 22 in inducing DNA breaks mediated by Topo IIα resulted in G2/M phase arrest and apoptosis. This work indicates that arylnaphthalene lignan lactones derivatives represent a novel type of Topo IIα inhibitory scaffold for developing new antitumor chemotherapeutic agents.

A phase Ib study of simmitecan (LP) single-agent and in combination with 5-fluorouracil/leucovorin (5-FU/LV) or thalidomide (T) in patients with advanced solid tumor.

3523 Background: Simmitecan (LP), a novel 9-substituted lipophilic camptothecin derivative, is a potent inhibitor of topoisomerase I with anti-tumor activity single agent or in combination with other anti-tumor agents, i.e. thalidomide or anti-PD-1 antibody in xenograft models. In this phase Ib study (NCT02870036), we evaluate the safety and anti-tumor effects of LP as monotherapy or in combination therapies in patients (pts) with advanced solid tumors. Methods: This open-label, multi-cohort phase Ib study was conducted at 3 academic centers in China. Eligible pts had advanced cancer without standard treatment options. Prior irinotecan treated failure pts was eligible. In the single agent study, enrolled pts was allocated to received intravenous(iv) 50, 80, or 120 mg/m2 of LP Q2W to determine the safety of LP. After single agent study finished, enrolled pts allocated in LP+5-FU/LV cohorts received 50, 65, or 80 mg/m2 of LP then LV 400 mg/m2 and 5-FU bolus at 400 mg/m2 followed by 5-FU continuous iv of 2400 mg/m2 Q2W; pts in LP+T cohorts received LP 65 mg/m2 Q2W + thalidomide 50mg QD or thalidomide 100mg QD, or LP 80 mg/m2 Q2W + thalidomide 50mg QD. Treatment repeated in 28-day cycles until disease progression or unacceptable toxicity. Results: Between October 2016 to February 2019, 41 pts, median age of 55.1 (range 29-69) years, were enrolled, 13 in LP monotherapy, 10 in LP + 5-FU/LV and 18 in LP + T. Over all, no dose limited toxicity (DLT) occurred. The most common (≥20%) grade 3/4 AE among three regimens was neutropenia (44%, 70% and 89% respectively in LP, LP+5FU/LV and LP+T),and treatment related SAEs were similar, i.e. anemia and febrile neutropenia (11.1% each) in LP, diarrhea (10%) in LP+5-FU/LV, febrile neutropenia (5.6%) in LP+T. Majority of enrolled pts (24/41, 59%) had progressed on prior irinotecan, nevertheless, partial response (PR) was observed in 1 colorectal cancer pt treated with LP 80 mg/m2 + thalidomide 50mg. Disease control rate (DCR, SD≥12 weeks or CR or PR) were 60%, 80% and 61% and median progress free survival were 2.71, 4.17 and 2.76 months respectively in LP, LP + 5-FU/LV and LP+T cohorts. Conclusions: This study showed the safety profiles are manageable, either LP monotherapy or combination therapies, no DLT or safety concern was elicited in combination compared to single agent, and the LP + 5-FU/LV regimen showed higher DCR. A phase II study in pts with metastatic NEC is on-going to explore safety and efficacy of LP + 5-FU/LV regimen combined with toripalimab, an anti-PD-1 antibody. Clinical trial information: 02870036 .

Measurement of NLG207 (formerly CRLX101) nanoparticle-bound and released camptothecin in human plasma

Camptothecin (CPT), a potent inhibitor of topoisomerase I and HIF-1α, failed to demonstrate utility as an anti-cancer agent in early clinical trial investigations, primarily due to limited clinical activity and significant toxicity attributable to unfavorable physicochemical properties (e.g. low plasma solubility, pH-labile lactone ring). NLG207 (formerly CRLX101), a nanoparticle-drug conjugate (NDC) of CPT designed to optimize plasma pharmacokinetics and facilitate drug delivery to tumors, is included as part of combination treatment in two Phase II clinical trials ongoing at the National Cancer Institute (NCT02769962 and NCT03531827). To better understand the potential for drug-drug interactions and to correlate drug exposure to clinical outcomes and pharmacodynamic biomarkers, a robust analytical method was developed to measure CPT in human plasma. Two sample processing methods were developed to quantify both NDC-bound CPT and free CPT, primarily via alteration of pH conditions. A solid-phase extraction recovered >79 % of CPT prior to quantitative analysis by ultra HPLC-MS/MS. Dynamic calibration ranges of 10 to 10,000 ng/mL and 1 to 1000 ng/mL for total and free CPT, respectively were utilized to capture clinical ranges. NLG207 NDCs demonstrated significant rates of CPT release in human plasma at room temperature after 2 h but were shown to be stable at 4 °C for 24 h and through 4 freeze/thaw cycles. This assay was used to quantitate CPT plasma concentrations in clinical samples to confirm clinical utility following NLG207 treatment in subjects with advanced prostate cancer.

Three-Component Approach to Pyridine-Stabilized Ketenimines for the Synthesis of Diverse Heterocycles.

Ketenimines are versatile synthetic intermediates capable of performing novel transformations in organic synthesis. They are normally generated in situ due to their inherent instability and high level of reactivity. Herein, we report pyridine-stabilized ketenimine zwitterionic salts, which are prepared through click chemistry from readily accessible alkynes and sulfonyl azides. To demonstrate their synonymous reactivity to ketenimines, these salts have been utilized in a cascade sequence to access highly functionalized quinolines including the core structures of an antiprotozoal agent and the potent topoisomerase inhibitor Tas-103.

A phase I study of the oral administration of irinotecan in combination with the potent P-glycoprotein (P-gp) inhibitor HM30181A.

3032 Background: Irinotecan is a prodrug of the potent topoisomerase inhibitor SN-38. In animals, oral administration of irinotecan with the selective minimally absorbed P-gp inhibitor HM30181A increased the bioavailability of irinotecan. Oral administration of irinotecan may also increase the conversion to SN-38. Objectives: To determine the MTD and DLT of orally administered irinotecan in combination with HM30181A 15 mg on day 1 of a 21-day cycle. Additional objectives include determining the recommended phase 2 dose and the PK of irinotecan and SN-38. Methods: This was a phase 1 dose escalation study enrolling cohorts of 3-6 patients with advanced malignancies. Patients had Hb ≥9 gm/dL, ANC ≥ 1.5x109/L, platelets ≥ 100x109/L, adequate hepatic and renal function, ECOG 0-1 and were not homozygous for UGT1A1*28. Patients were administered HM30181A 15 mg and oral irinotecan 20, 40, 80, 120, 160, 200, 240, 280 and 320mg/m2. Results: Thirty male and female patients, mean age 60.9 (range 33-78) were enrolled into this ongoing study. The most common cancers were ovarian (6), colorectal (4), breast (4), endometrial (3), and pancreatic (3). The median number of cycles administered was 3 (range 1-9). Treatment-related Grade 3-4 AEs were experienced by 12 (40%) subjects. The most common were nausea 7 (23%), vomiting 6 (20%) and abdominal pain 3 (10%). Treatment-related SAEs were experienced by 6 (20%) patients (nausea or vomiting in 4 subjects). DLTs occurred in 2 patients at the 320 mg/m2 dose level (neutropenia and C. Difficile diarrhea) and additional patients are being enrolled at the 280mg/m2 dose level to define the MTD. Acute cholinergic diarrhea has not been observed. The best response was stable disease in 9/21 evaluable patients. PK at the three highest dose levels is summarized below. Conclusions: Oral administration of HM30181A in combination with irinotecan tablets results in pharmacologically active concentrations of SN-38. Confirmation of the MTD when dosed on a 21-day cycle is ongoing. Phase 2 studies are being planned. Clinical trial information: NTC02250157. [Table: see text]

Design and Synthesis of 3,4-diarylpyrrole Analogues as Potent Topoisomerase Inhibitors.

The natural products containing a common 3,4-diarylpyrrole skeleton have attracted considerable attention due to their unique structures and multiplex biological activities. In our previous study, lycogarubin C was synthesized and showed cytotoxicity against MDAMB- 231, A549, PC3 and HeLa cell lines and topoisomerase II inhibitory activities. We present the design, synthesis and antitumor studies of 3,4-diarylprrole derivatives. Their antitumor activities and inhibitory activities against Topo I and Topo IIα of these compounds were assayed. A series of 3,4-diarylpyrrole analogues have been designed and synthesized. Their antiproliferation activities were evaluated by sulforhodamine B assay on human breast cancer MDAMB- 231, MDA-MB-435 and human cervical cancer HeLa cells. Four compounds showed modest inhibitory activities against the growth of the three cell lines with IC50 below 50 μM. DNA relaxation assay revealed that compound 19o showed potent inhibitory activity against Topo IIα in vitro. 19o also induced DNA breaks in MDA-MB-435 cells evidenced by comet tails and the accumulation of γ-H2AX foci. The ability of 19o in inducing DNA breaks mediated by Topo IIα resulted in G2/M phase arrest and apoptosis. This work indicates that 3,4-diarylpyrrole derivatives represent a novel type of Topo IIα inhibitory scaffold for developing new antitumor chemotherapeutic agents.

A randomized phase II trial of CRLX101 in combination with bevacizumab versus standard of care in patients with advanced renal cell carcinoma

BackgroundNanoparticle-drug conjugates enhance drug delivery to tumors. Gradual payload release inside cancer cells augments antitumor activity while reducing toxicity. CRLX101 is a novel nanoparticle–drug conjugate containing camptothecin, a potent inhibitor of topoisomerase I and the hypoxia-inducible factors 1α and 2α. In a phase Ib/2 trial, CRLX101+bevacizumab was well tolerated with encouraging activity in metastatic renal cell carcinoma (mRCC). We conducted a randomized phase II trial comparing CRLX101+bevacizumab versus standard of care (SOC) in refractory mRCC. Patients and methodsPatients with mRCC and 2–3 prior lines of therapy were randomized 1:1 to CRLX101+bevacizumab versus SOC, defined as investigator’s choice of any approved regimen not previously received. The primary end point was progression-free survival (PFS) by blinded independent radiological review in patients with clear cell mRCC. Secondary end points included overall survival, objective response rate and safety. ResultsIn total, 111 patients were randomized and received≥1 dose of drug (CRLX101+bevacizumab, 55; SOC, 56). Within the SOC arm, patients received single-agent bevacizumab (19), axitinib (18), everolimus (7), pazopanib (4), sorafenib (4), sunitinib (2), or temsirolimus (2). In the clear cell population, the median PFS on the CRLX101+bevacizumab and SOC arms was 3.7months (95% confidence interval, 2.0–4.3) and 3.9months (95% confidence interval 2.2–5.4), respectively (stratified log-rank P=0.831). The objective response rate by IRR was 5% with CRLX101+bevacizumab versus 14% with SOC (Mantel–Haenszel test, P=0.836). Consistent with previous studies, the CRLX101+bevacizumab combination was generally well tolerated, and no new safety signal was identified. ConclusionsDespite promising efficacy data on the earlier phase Ib/2 trial of mRCC, this randomized trial did not demonstrate improvement in PFS for the CRLX101+bevacizumab combination when compared with approved agents in patients with heavily pretreated clear cell mRCC. Further development in this disease is not planned. Clinical trial identificationNCT02187302 (NIH).

Pro-Angiogenic Effects of Low Dose Ethoxidine in a Murine Model of Ischemic Hindlimb: Correlation between Ethoxidine Levels and Increased Activation of the Nitric Oxide Pathway.

Ethoxidine, a benzo[c]phenanthridine derivative, has been identified as a potent inhibitor of topoisomerase I in cancer cell lines. Our group has reported paradoxical properties of ethoxidine in cellular processes leading to angiogenesis on endothelial cells. Because low concentration ethoxidine is able to favor angiogenesis, the present study aimed to investigate the ability of 10−9 M ethoxidine to modulate neovascularization in a model of mouse hindlimb ischemia. After inducing unilateral hindlimb ischemia, mice were treated for 21 days with glucose 5% or with ethoxidine, to reach plasma concentrations equivalent to 10–9 M. Laser Doppler analysis showed that recovery of blood flow was 1.5 fold higher in ethoxidine-treated mice in comparison with control mice. Furthermore, CD31 staining and angiographic studies confirmed an increase of vascular density in ethoxidine-treated mice. This ethoxidine-induced recovery was associated with an increase of NO production through an enhancement of eNOS phosphorylation on its activator site in skeletal muscle from ischemic hindlimb. Moreover, real-time RT-PCR and western blots have highlighted that ethoxidine has pro-angiogenic properties by inducing a significant enhancement in vegf transcripts and VEGF expression, respectively. These findings suggest that ethoxidine could contribute to favor neovascularization after an ischemic injury by promoting the NO pathway and VEGF expression.

In vitro Investigation of Metabolic Profiling of a Potent Topoisomerase Inhibitors Fluorescein Hydrazones (FLHs) in RLMs by LC-MS/MS.

Metabolic profiling of newly reported two topoisomerase inhibitors namely fluorescein hydrazones (FLHs) were studied in rat liver microsomes (RLMs) and the data were acquired in a liquid chromatography (LC) ion trap mass spectrometry. (E)-3',6'-dihydroxy-2-((2-nitrobenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one (1) was bio-transformed into two mono-hydroxylated, one double hydroxylated and a tetra hydroxylated metabolites, on the other hand, (Z)-3',6'-dihydroxy-2-((2-oxoindolin-3-ylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one (2) was bio-transformed into eight possible metabolites by cleavage reduction, hydroxylation, cleavage reduction together with hydroxylated and azo-reduction in RLMs in presence of NADPH-generating system. No metabolites were generated in the absence of either NADPH or microsomes for the compounds (1 and 2), indicating a likely involvement of CYP450 enzymes and the cofactor NADPH in the metabolisms. The structures of those metabolite were elucidated by MS/MS fragmentation study.

In-silico Designing of Novel Camptothecin Analogues as Potent Inhibitors of Topoisomerase I: A Molecular Docking, QSAR, and ADME-T Study

Cancer is the second largest cause of deaths across the globe. The current treatments available for cancer include surgical removal of the infected tissue, radiotherapy, chemotherapy and combinatorial therapeutic approach. Most of the commercially available anti-cancer drugs and their unique modes of action are decoded by the cancer cells, making them less effective. Hence, there is an increasing demand for newer class of drugs with better pharmacological properties and fewer side effects. Camptothecin (CPT), the quinoline alkaloid isolated from Camptotheca acuminata is a wellknown Topoisomerase I inhibitor. It has a proven anti-cancer activity against various cell lines. However, its poor pharmacological properties like low solubility and a narrow therapeutic index calls for better therapeutic alternatives. In this study, eight novel analogues of camptothecin were rationally designed based on the structure-activity relationship of the substituent groups. The designed analogues were further analyzed in-silico for their drug-likeness, quantitative structure-activity relationship (QSAR) and ADME-T properties. All the novel analogues were docked with topoisomerase I using Autodock 4.2 and the docking results were validated with the help of an online docking server, Patchdock. In this study, we have found that the therapeutic profile of some of the designed analogues was comparable to that of Topotecan, the FDA approved drug and better than the parent molecule Camptothecin. Keywords: ADME-T, cancer, camptothecin, topoisomerase I, in-silico designing, molecular docking, QSAR.

Synthesis, characterization, and evaluation of mPEG-SN38 and mPEG-PLA-SN38 micelles for cancer therapy.

7-Ethyl-10-hydroxy camptothecin (SN38) is a potent topoisomerase inhibitor and a metabolite of irinotecan. Its clinical development has been hampered by its poor solubility. To address this problem, methoxy poly(ethylene glycol)-2000 (mPEG2K)–SN38 and mPEG2K–poly(lactide) (PLA1.5K)–SN38 conjugates were prepared and then dispersed into an aqueous medium to form micelles. Physicochemical characteristics of SN38–polymer conjugate micelles, for example, micelle diameter, zeta potential, morphology, and drug content, were then evaluated. The results showed that the mean diameters of mPEG2K–SN38 and mPEG2K–PLA1.5K–SN38 micelles were ~130 and 20 nm, respectively. These two micelles had similar drug contents. mPEG2K–PLA1.5K–SN38 micelles were more homogeneous than mPEG2K–SN38 micelles. Moreover, in vitro drug release behavior of the micelles was studied by high performance liquid chromatography. SN38 release from mPEG2K–SN38 micelles was much faster than from mPEG2K–PLA1.5K–SN38 micelles. In vitro cytotoxicity, cellular uptake, and apoptosis assays of the SN38–polymer conjugate micelles were carried out on BEL-7402 human liver cancer cells. In vivo biodistribution and antitumor tumor efficacy studies were carried out in a nude mouse xenograft model derived from BEL-7402 cells. The results showed that mPEG2K–PLA1.5K–SN38 micelles were significantly more effective than mPEG2K–SN38 micelles in tumor inhibition, and the inhibitory effect of mPEG2K–PLA1.5K–SN38 micelles on tumor growth was significantly greater than that of mPEG2K–SN38 micelles (1,042 vs 1,837 mm) at 30 days. In conclusion, mPEG–PLA–SN38 is a promising anticancer agent that warrants further investigation.

Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone.

Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycone), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable production of the etoposide aglycone in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.

20. Carboxylesterase-Secreting Neural Stem Cells Increase Efficacy of Irinotecan in Orthotopic Glioma Models: Translation Toward the Clinic

Human neural stem cells (NSCs) are serving as vehicles for targeted delivery of cytosine deaminase/5-flucytosine enzyme/prodrug therapy to recurrent glioma patients in first in human Phase 1 clinical trials (IND 14041). The initial safety/feasibility clinical study demonstrated safety, non-tumorigenicity, non-immunogenicity with 1 round of treatment, and proof of concept for localized conversion of 5-Flucytosine to the active chemotherapeutic agent, 5-Fluorouracil. We have now developed a second generation NSC brain tumor therapy, engineering NSCs to secrete a modified human carboxylesterase (hCE1m6) that converts irinotecan (IRN) to the potent topoisomerase inhibitor SN-38. Biodistribution, efficacy, and safety/toxicity studies have been completed and an Investigational New Drug application has been filed with the Food and Drug Administration (IND 16265).Early studies on the CE-expressing human NSCs was described in Metz, et al., Stem Cells Transl Med. 2013 Dec;2(12):983), demonstrating tumor tropism in orthotopic human glioma models and tumor localized conversion of IRN to SN-38 in a time-dependent manner. The current preclinical studies show in vivo biodistribution of varying doses of intracranially delivered CE-NSCs and optimization of a clinically relevant adult IRN treatment regimen based on pharmacokinetic and long term survival studies. These studies demonstrated a statistically significant increase in long-term survival distributions with the addition of CE-NSCs + IRN as compared to IRN alone (see figure). These data, together with our preclinical safety/toxicity data, support clinical translation of this schema to phase I clinical trials for patients with recurrent glioma. View Large Image | Download PowerPoint Slide