Topotecan Concentrations Research Articles

Quantification of Unencapsulated Drug in Target Tissues Demonstrates Pharmacological Properties and Therapeutic Effects of Liposomal Topotecan (FF-10850)

PurposeQuantifying unencapsulated drug concentrations in tissues is crucial for understanding the mechanisms underlying the efficacy and safety of liposomal drugs; however, the methodology for this has not been fully established. Herein, we aimed to investigate the enhanced therapeutic potential of a pegylated liposomal formulation of topotecan (FF-10850) by analyzing the concentrations of the unencapsulated drug in target tissues, to guide the improvement of its dosing regimen.MethodsWe developed a method for measuring unencapsulated topotecan concentrations in tumor and bone marrow interstitial fluid (BM-ISF) and applied this method to pharmacokinetic assessments. The ratios of the area under the concentration–time curves (AUCs) between tumor and BM-ISF were calculated for total and unencapsulated topotecan. DNA damage and antitumor effects of FF-10850 or non-liposomal topotecan (TPT) were evaluated in an ES-2 mice xenograft model.ResultsFF-10850 exhibited a much larger AUC ratio between tumor and BM-ISF for unencapsulated topotecan (2.96), but not for total topotecan (0.752), than TPT (0.833). FF-10850 promoted milder DNA damage in the bone marrow than TPT; however, FF-10850 and TPT elicited comparable DNA damage in the tumor. These findings highlight the greater tumor exposure to unencapsulated topotecan and lower bone marrow exposure to FF-10850 than TPT. The dosing regimen was successfully improved based on the kinetics of unencapsulated topotecan and DNA damage.ConclusionsTissue pharmacokinetics of unencapsulated topotecan elucidated the favorable pharmacological properties of FF-10850. Evaluation of tissue exposure to an unencapsulated drug with appropriate pharmacodynamic markers can be valuable in optimizing liposomal drugs and dosing regimens.

New Dual Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 and 2 Based on Deoxycholic Acid: Design, Synthesis, Cytotoxicity, and Molecular Modeling.

Deoxycholic acid derivatives containing various heterocyclic functional groups at C-3 on the steroid scaffold were designed and synthesized as promising dual tyrosyl-DNA phosphodiesterase 1 and 2 (TDP1 and TDP2) inhibitors, which are potential targets to potentiate topoisomerase poison antitumor therapy. The methyl esters of DCA derivatives with benzothiazole or benzimidazole moieties at C-3 demonstrated promising inhibitory activity in vitro against TDP1 with IC50 values in the submicromolar range. Furthermore, methyl esters 4d-e, as well as their acid counterparts 3d-e, inhibited the phosphodiesterase activity of both TDP1 and TDP2. The combinations of compounds 3d-e and 4d-e with low-toxic concentrations of antitumor drugs topotecan and etoposide showed significantly greater cytotoxicity than the compounds alone. The docking of the derivatives into the binding sites of TDP1 and TDP2 predicted plausible binding modes of the DCA derivatives.

Abstract 4204: CDK4/6 inhibition mitigates chemotherapy-induced immunosuppression and enhances the efficacy of chemo- immunotherapy in small cell lung cancer models

Abstract Small cell lung cancer (SCLC) is an aggressive form of lung cancer with a notably low survival rate. Chemotherapy and more recently its combination with immunotherapy are the standard of care for extensive stage SCLC. Although this combinatorial treatment provides clinical benefit, it can be hampered by the effect of chemotherapy on inducing immunosuppression. Cyclin dependent kinases 4/6 (CDK4/6) phosphorylate retinoblastoma (Rb) protein and promote proliferation of Rb-intact hematopoietic stem progenitor cells (HSPCs) by allowing cells to progress through S phase. Rb loss is a signature mutation in SCLC tumors, making these tumor cells unresponsive to CDK4/6 inhibition. Therefore, we hypothesized that CDK4/6 inhibitors could be utilized to block the proliferation of Rb-intact HSPCs and protected them from chemotherapy without altering the effect of chemotherapy on Rb-deficient SCLC cells. We treated Rb-deficient SCLC cancer cells and Rb-intact NK and T cells with increasing concentrations of cisplatin, etoposide and topotecan with or without the CDK4/6 inhibitor G1T28. Cell proliferation, apoptosis and cell cycle were analyzed using CellTiter-Glo, Annexin V staining, and PI-based cell cycle analysis. In vitro, CDK4/6 inhibition did not alter the impact of chemotherapy on Rb-deficient SCLC cell viability, whereas with Rb-intact NK and T cells, CDK4/6 inhibitor treatment induced cell cycle arrest and prevented apoptosis induced by chemotherapeutic agents. In the TKOTmG Rb-deficient mouse model of SCLC, CDK4/6 inhibition improved the efficacy of cisplatin plus etoposide. In vivo studies using the KP-1 syngeneic mouse models showed that treatment with a CDK4/6 inhibitor improved the efficacy of chemotherapy and immunotherapy individually or chemo-immunotherapy combinations by reducing the tumor growth rate and prolonging animal survival. CDK4/6i treatment prior to chemotherapy preserved white blood cell counts in comparison to chemotherapy alone. Tissue and blood immune marker analysis showed that CDK4/6 inhibitor treatment increased both the circulating and tumor-resident NK cells. Collectively, we demonstrated that CDK4/6 inhibition does not antagonize the therapeutic impact of chemotherapy on Rb-deficient SCLC cells but enhances the efficacy of chemotherapy and immunotherapy in KP-1 syngeneic SCLC mouse model. CDK4/6i also at least partially abrogated the chemotherapy-induced reductions in subpopulatons of immune cells. CDK4/6 inhibition may be considered as a therapeutic strategy for mitigating chemotherapy-induced immunosuppression and enhance antitumor immunity in SCLC patients. Citation Format: Thiruvengadam Arumugam, Irene G. Muñoz, Alissa Poteete, Fahoa Zhang, Monique Nilsson, John S. Yi, Jessica A. Sorrentino, Andrew P. Beelen, John V. Heymach. CDK4/6 inhibition mitigates chemotherapy-induced immunosuppression and enhances the efficacy of chemo- immunotherapy in small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4204.

The Role of Cryotherapy in Vitreous Concentrations of Topotecan Delivered by Episcleral Hydrogel Implant.

Transscleral diffusion delivery of chemotherapy is a promising way to reach the vitreal seeds of retinoblastoma, the most common intraocular malignancy in childhood. In this in vivo study, the delivery of topotecan via lens-shaped, bi-layered hydrogel implants was combined with transconjunctival cryotherapy to assess whether cryotherapy leads to higher concentrations of topotecan in the vitreous. The study included 18 New Zealand albino rabbits; nine rabbits received a topotecan-loaded implant episclerally and another nine rabbits received transconjunctival cryotherapy superotemporally 2 weeks before implant administration. Median vitreous total topotecan exposures (area under the curve, AUC) were 455 ng·h/mL for the cryotherapy group and 281 ng·h/mL for the non-cryotherapy group, and were significantly higher in the cryotherapy group, similar to maximum levels. Median plasma AUC were 50 ng·h/mL and 34 ng·h/mL for the cryotherapy and non-cryotherapy groups, respectively, with no statistically significant differences between them. In both groups, AUC values in the vitreous were significantly higher than in plasma, with plasma exposure at only approximately 11–12% of the level of vitreous exposure. The results confirmed the important role of the choroidal vessels in the pharmacokinetics of topotecan during transscleral administration and showed a positive effect of cryotherapy on intravitreal penetration, resulting in a significantly higher total exposure in the vitreous.

Development and Validation of an HPLC Method for Analysis of Topotecan in Human Cerebrospinal Fluid and Its Application in Elimination Evaluation of Topotecan after Intraventricular Injection.

Simple SummaryIntrathecal administration of anticancer drugs is an effective strategy to treat meningeal leukemia and lymphoma. Topotecan is an anticancer drug, and its anticancer effect is expected via intrathecal administration. In this study, we developed a method to measure the concentration of topotecan in the cerebrospinal fluid (CSF) for clinical monitoring of personalized medicine. By controlling the unique structural feature of topotecan, a pH-dependent lactone ring-opening/closing reversible reaction, we established a simple high-performance liquid chromatography (HPLC) method to measure the total topotecan concentration in the CSF and confirmed that this method can be applied to clinical samples. In the present situation, wherein the concentrations were monitored, the disappearance of topotecan in the CSF was delayed, and signs of adverse effects were confirmed when a high concentration of topotecan was observed.Intrathecal administration of anticancer drugs is an effective dosage strategy, but the elimination of intraventricular drugs is not uniform in all patients. For safety, a system to evaluate local pharmacokinetics in the ventricles after administration is desired. In this study, we developed a simple and reproducible method to measure topotecan concentration in the cerebrospinal fluid (CSF) and confirmed its clinical applicability. High-performance liquid chromatography (HPLC) analysis was performed using a C18 column to measure the total topotecan concentration in the CSF. Clinical CSF samples were obtained from a 1-year old child with poor CSF absorption and stagnation. The patient received topotecan via an intraventricular subcutaneous reservoir. The HPLC method complied with the validation criteria. The lower limit of quantitation of this method was 0.04 µM. Using the developed method, we could determine the difference in topotecan CSF concentrations at 24 and 48 h after administration. The patient’s topotecan elimination rate was extremely low, and signs of adverse effects were observed at high CSF concentration of topotecan. The developed method could detect the delay in topotecan elimination after intrathecal injection. The findings of this study are valuable for the development of personalized treatments for the intrathecal administration of anticancer drugs.

Three different mutations in the DNA topoisomerase 1B in Leishmania infantum contribute to resistance to antitumor drug topotecan

BackgroundThe evolution of drug resistance is one of the biggest challenges in leishmaniasis and has prompted the need for new antileishmanial drugs. Repurposing of approved drugs is a faster and very attractive strategy that is gaining supporters worldwide. Different anticancer topoisomerase 1B (TOP1B) inhibitors have shown strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs. However, cancer cells and Leishmania share the ability to become rapidly resistant. The aim of this study was to complete a whole-genome exploration of the effects caused by exposure to topotecan in order to highlight the potential mechanisms deployed by Leishmania to favor its survival in the presence of a TOP1B inhibitor.MethodsWe used a combination of stepwise drug resistance selection, whole-genome sequencing, functional validation, and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the action of TOP1B inhibitor topotecan.ResultsWe demonstrated that L. infantum is capable of becoming resistant to high concentrations of topotecan without impaired growth ability. No gene deletions or amplifications were identified from the next-generation sequencing data in any of the three resistant lines, ruling out the overexpression of efflux pumps as the preferred mechanism of topotecan resistance. We identified three different mutations in the large subunit of the leishmanial TOP1B (Top1BF187Y, Top1BG191A, and Top1BW232R). Overexpression of these mutated alleles in the wild-type background led to high levels of resistance to topotecan. Computational molecular dynamics simulations, in both covalent and non-covalent complexes, showed that these mutations have an effect on the arrangement of the catalytic pentad and on the interaction of these residues with surrounding amino acids and DNA. This altered architecture of the binding pocket results in decreased persistence of topotecan in the ternary complex.ConclusionsThis work helps elucidate the previously unclear potential mechanisms of topotecan resistance in Leishmania by mutations in the large subunit of TOP1B and provides a valuable clue for the design of improved inhibitors to combat resistance in both leishmaniasis and cancer. Our data highlights the importance of including drug resistance evaluation in drug discovery cascades.Graphical abstract

The effect of iodine-131 beta-particles in combination with A-966492 and Topotecan on radio-sensitization of glioblastoma: An in-vitro study

Glioblastoma tumors are resistant to radiotherapy, and the need for drugs to induce radio-sensitization in tumor cells has always been a challenge. Besides, radiotherapy using targeted radionuclide would be effective even for resistant tumors. It has been shown topoisomerase I and poly (ADP-ribose) polymerase (PARP) enzymes have critical roles in the repair process of DNA injury in cells. Therefore, the inhibition of the activity of these enzymes can halt this process and result in the accumulation of damaged DNA in cells and the induction of cell death in tumors. In the present research, the impact of beta-particles of iodine-131 in combination with Topotecan (TPT), as the inhibitor of topoisomerase I, and A-966492, as the inhibitor of the PARP enzyme on the possible increase of radio-sensitivity of glioblastoma cells was assessed.For this purpose, a human glioblastoma cell line, U87MG, was cultured in flasks coated with Poly-Hema to achieve 300 μm-diameter spheroids. Then, nontoxic concentrations of A-966492 and TPT were applied in the cell culture media. The viability of the cells treated with iodine131 in combination with A-966492 and TPT was determined by the clonogenic assay. The expression rate of gamma-H2AX, as a biomarker of DNA double-strand breaks, was analyzed using immunofluorescence microscopy to unravel the effect of TPT, A-966492 (1 μM), and radiation on the cell death induction.The combination of each TPT or A-966492 with radiation resulted in the increased rate of cell death, and the ratios of sensitizer enhancement at 50% survival (SER50) were elevated by 1.45 and 1.25, respectively. Chemo- and radio-sensitization were promoted when iodine-131 was combined with A-966492 and TPT, with the SER50 of 1.68. Also, the expression of γ-H2AX was significantly increased in cells treated with A-966492 and TPT combined with radiation.The results demonstrated that iodine-131, in combination with A-966492 and TPT, had marked effects on radio-sensitizing and can be used as a targeted radionuclide for targeting radiotherapy in combination with topoisomerase I and PARP inhibitors to enhance radiotherapy in clinics.

Controlling the release rate of topotecan from PLGA spheres and increasing its cytotoxicity towards glioblastoma cells by co-loading with calcium chloride

It has been suggested that local administration of topotecan (TT) could increase its efficacy in the treatment of glioblastoma. In this context, a PLGA implant model in the form of spheres with a porous core and stiff surface, loaded with TT and CaCl2 was developed. An array of formulations differing from each other by the type of PLGA used, the integrity of the surface, the concentrations of TT and CaCl2 added during the preparation, and the volume of water in the PLGA mix, was prepared, screened and explored by computerized multifactorial analysis. This analysis enabled the simultaneous identification of the most influential experimental factors on the experimental responses, which were pre-determined as the efficiency of TT loading and the TT % cumulative release at 14 days. The multifactorial analysis also revealed how the interactions among the experimental factors affect the performance of the various formulations. Thus, TT concentration and its factorial interaction with the concentration of CaCl2 added during the spheres’ preparation were identified as most prominent on the loading efficiency, while the surface integrity (intact or punctured) and CaCl2 amount in the spheres were identified as most prominent on the TT % cumulative release from the spheres. TT was found to be cytotoxic towards glioblastoma U87 MG cells, an activity which was enhanced, synergistically, in the presence of CaCl2 (the relative viability was reduced from 36 to 28% with combination indices of 1.0, 0.37, 0.13 and 0.06 for EC50, EC75, EC90 and EC95, respectively). Interestingly, dividing the TT dose into 3 equal portions, replenished daily to the incubation medium, increased TT cytotoxicity. The relative viability was then reduced from 35 to 7% and in the presence of CaCl2 – from 28 to 1.9%, suggesting that a local, slow input of TT could be effective in the treatment of glioblastoma by an adjacent TT implant. The increased effect of CaCl2 on cytotoxicity was also observed when it was co-loaded into the TT spheres. In that case, the cells’ viability was reduced from 72 to 27%. It is suggested that the PLGA spheres could be used for tunable local delivery of TT in post-resection adjuvant therapy of glioblastoma.

DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells

SummaryNeuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 μM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. Network analysis revealed that topotecan upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Results of DNA damage assay, cell cycle, and quantitative analyses of membrane cholesterol supported the validity of these resistance factors and their applicability to all neuroblastoma cells. Our results provide a model for high dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.

Abstract 328: DNA damage inhibitors with topotecan in a complex spheroid model in PDM and established cell lines

Abstract Many solid tumor treatment regimens include combination chemotherapy. Over thirty-five human tumor cell lines, some recently developed and some well-established, were grown as complex spheroids, tumor cells mixed with HUVEC and MSC, to better model human solid tumors. The complex spheroids were established for 3 days, then the drug(s) and/or investigational agent(s) were added. After 7 days, the experiment was terminated with CellTiterGlo 3D. Cell viability was determined relative to a vehicle treated control and IC50s were calculated from concentration response curves. The response to topotecan was determined over a concentration range achievable in patients alone and in combination with nedisertib (M3814) or VX984, DNA PK inhibitors, M6620 or BAY-1895344, ATR inhibitors, MK-1775, a Wee-1 inhibitor, AZD-1390, an ATM inhibitor or olaparib or talazoparib, PARP inhibitors. The clinical Cmax of topotecan is 0.15 uM and the topotecan concentration range tested was 0.001 to 0.3 uM. Topotecan IC50's spanned more than 2 logs from 0.003 uM to >>0.3 uM. The ATRi inhibitors were tested over the concentration range from 0.01 to 10 uM. The M6620 IC50's covered 2 logs from 0.015 to >>1 uM and the BAY-189-5344 IC50 range was similar. Combinations of topotecan and M6620 produced greater than additive cytotoxicity in Rh28 and Rh30 rhabdomyosarcoma and 328373-195-R-J1 squamous cell carcinoma with >1 log increase in cell killing. BAY-1895344 in combination with topotecan was greater than additive in the COR L88 SCLC line and the 287954-098-R-J1 Ewing sarcoma line. The combination of topotecan and M6620 resulted in approximately additive cell killing in 929823-356-R-J2 squamous carcinoma, and NCI-H196 and NCI-H1618 SCLC with 4-6-fold increase in cell killing. Topotecan along with the ATM inhibitor AZD-1390, produced greater than additive cell killing in the 287954-098-R-J1 Ewing sarcoma line at the lower concentrations of topotecan (0.003 and 0.01 uM) tested. Primarily additive effects of the ATM inhibitor and topotecan were seen in most other cell lines tested. The combination of topotecan and the Wee-1 inhibitor MK1775 was tested in complex spheroids of the same cell lines. Only in the NCI-H196 SCLC line did topotecan and MK1775 produce greater than additive cell killing. There was a 1.5- to 4-fold increase in cell killing with topotecan and MK1775 in the RH28 and Rh30 rhabdomyosarcomas, 929823-356-R-J2 and 328373-195-R-J1 squamous cell carcinomas and the NCI-H1618 SCLC which was additive to sub-additive. The combination of topotecan with the DNA PK inhibitor M3814 produced little or no increase in cell killing (1.2- to 2-fold) compared with topotecan alone in complex spheroids. These findings may provide guidance regarding selection of agent combinations worthy of further investigation. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Beverly A. Teicher, Austin Doyle, John Wright, Charles Kunos, Thomas Silvers, Rene Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Nathan Coussens, Joel Morris, James Doroshow. DNA damage inhibitors with topotecan in a complex spheroid model in PDM and established cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 328.

Abstract 4098: Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance

Abstract Background: Although enzalutamide (ENZ) is an effective treatment for prostate cancer (PC), development of ENZ resistance is inevitable. One mechanism for resistance is upregulation of ARV7, a splice variant of the full-length androgen receptor (FLAR) that lacks the androgen binding and hinge region domains. Nuclear translocation of FLAR requires the hinge region for coupling to tubulin or importin. ARV7 gains nuclear entry independent of the canonical pathway. FLAR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α) under normoxic conditions in the presence of dihydrotestosterone (DHT). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, enabling nuclear localization and transcriptional activity of both transcription factors. We reported that topotecan (TPT) blocks HIF1α mRNA translation by targeting topoisomerase I (TOPO1) resident on HIF1α mRNA. We determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. We also evaluated if TOPO1 resided on FLAR/ARV7 mRNA, providing an additional target for TPT. Methods: 22Rv1 ENZ resistant PC cells that express FLAR and ARV7 protein were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of DHT, ENZ, and TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the XTT viability assay. Results: Topo-I RNA IP demonstrated its linkage to HIF-1α, FLAR and ARV7 mRNA. Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots under normoxic conditions in the presence of DHT and under hypoxic conditions. TPT and ENZ treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α, FLAR and ARV7 nuclear localization. TPT had an IC50 of 20 nM in normoxia and 91 nM in hypoxia. ENZ had an IC50 of 24.4 uM in normoxia and 388.7 uM in hypoxia. Dose-response curves for TPT / ENZ combinations at concentrations achieved clinically demonstrated synergistic activity, with combination index values of 0.54 under normoxic and 0.19 under hypoxic conditions. Conclusions: Our data indicate that Topo I resides on HIF-1α, FLAR and ARV7 mRNA, providing a potential target for TPT-mediated inhibition of their translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support exploration of TPT in combination with ENZ for treatment of PC. Citation Format: John P. Fruehauf, Diana C. Gomez, Leslie Spitalny, Jai-Hyun Kim. Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4098.

Validation of an effective implantable pump-infusion system for chronic convection-enhanced delivery of intracerebral topotecan in a large animal model.

Intracerebral convection-enhanced delivery (CED) has been limited to short durations due to a reliance on externalized catheters. Preclinical studies investigating topotecan (TPT) CED for glioma have suggested that prolonged infusion improves survival. Internalized pump-catheter systems may facilitate chronic infusion. The authors describe the safety and utility of long-term TPT CED in a porcine model and correlation of drug distribution through coinfusion of gadolinium. Fully internalized CED pump-catheter systems were implanted in 12 pigs. Infusion algorithms featuring variable infusion schedules, flow rates, and concentrations of a mixture of TPT and gadolinium were characterized over increasing intervals from 4 to 32 days. Therapy distribution was measured using gadolinium signal on MRI as a surrogate. A 9-point neurobehavioral scale (NBS) was used to identify side effects. All animals tolerated infusion without serious adverse events. The average NBS score was 8.99. The average maximum volume of distribution (Vdmax) in chronically infused animals was 11.30 mL and represented 32.73% of the ipsilateral cerebral hemispheric volume. Vdmax was achieved early during infusions and remained relatively stable despite a slight decline as the infusion reached steady state. Novel tissue TPT concentrations measured by liquid chromatography mass spectroscopy correlated with gadolinium signal intensity on MRI (p = 0.0078). Prolonged TPT-gadolinium CED via an internalized system is safe and well tolerated and can achieve a large Vdmax, as well as maintain a stable Vd for up to 32 days. Gadolinium provides an identifiable surrogate for measuring drug distribution. Extended CED is potentially a broadly applicable and safe therapeutic option in select patients.

Ultrasound-Triggered Spatiotemporal Delivery of Topotecan and Curcumin as Combination Therapy for Cancer.

Chemotherapy is limited by the low availability of drug at the tumor site and drug resistance by the tumor. In this report we describe a combination therapy for codelivery of two anticancer drugs with spatiotemporal control by ultrasound pulses. We developed curcumin and topotecan-coencapsulated nanoconjugates Cur_Tpt_NC.MB to have an ultrasound contrast property. MDA MB 231 and B16F10 cells were incubated with Cur_Tpt_NC.MB and exposed to ultrasound. Ultrasound exposure reduced the IC50 concentration of topotecan and curcumin significantly (P < 0.05) compared with free drug. Antitumor efficacy study of the Cur_Tpt_NC.MB in B16F10 melanoma tumor-bearing C57BL/6 mice showed that ultrasound exposure of right tumor reduced growth by 3.5 times compared with the unexposed left tumor of same mice and 14.8 times compared with a group treated with a physical mixture of drugs. These results suggest that the nanotherapeutic system we developed induces site-specific inhibition of tumor growth at a high rate and has the potential to be used as a therapeutic regimen for spatiotemporal delivery of dual drugs for treatment of cancer.

PHARMACOLOGIC TOPOISOMERASE-I INHIBITION CAUSES DNA DAMAGE AND MORTALITY IN ACTIVATED CD4+ T CELLS

Topoisomerase-I is required for DNA replication. It acts by preventing torsional stress caused by DNA winding during replication fork progression. Topoisomerase-I inhibitors are widely used in many cancer therapies, in light of their anti-proliferative activity. However, their use as chemotherapeutics is associated with significant toxicity due to the off-target effects on healthy cells. We analyzed the dose-time-toxicity profile of a clinically employed topoisomerase-I inhibitor, i.e. topotecan, on primary CD4+T cells. This cell type was chosen to model a typical in-vivo interaction, due to the wide use of topotecan in the treatment of T-cell lymphomas. Our results show that a clinically achievable concentration of topotecan can induce toxic effects in healthy CD4+ T cells as early as 7 hours of the in vitro treatment. Toxicity of the drug was markedly increased by prolonging the post-treatment follow-up, but not by increasing concentrations, suggesting that clinical doses of topotecan can induce cell death and DNA damage in non-cancerous activated CD4+ T lymphocytes.

Synchronized Ratiometric Codelivery of Metformin and Topotecan through Engineered Nanocarrier Facilitates In Vivo Synergistic Precision Levels at Tumor Site.

The combination of metabolic modulators with chemotherapy holds vast promise for effective inhibition of tumor progression and invasion. Herein, a ratiometric codelivery platform is developed for metformin (MET), a known metabolic modulator and topotecan (TPT), a chemotherapeutic drug, by engineering lipid bilayer-camouflaged mesoporous silica nanoparticles (LB-MSNs). In an attempt to deliver and maintain high tumor site concentrations of MET and TPT, a novel ion pairing-assisted loading procedure is developed using pamoic acid (PA) as an in situ trapping agent. PA, a hydrophobic counterion, increases the hydrophobicity of MET and TPT and facilitates MSNs with exceptionally high payload capacity (>40 and 32 wt%, respectively) and controlled release profile. Further, the synergy between MET and TPT determined by a modeling approach helps to afford synchronized delivery of both the drugs. Coloaded MET and TPT LB-MSNs present synergistic cytotoxicity against MDA-MB-231/4T1 cells and effectively promote apoptosis via mitochondrial membrane depolarization and cell cycle arrest. Extended pharmacokinetic profiles in preclinical models with fourfold to sevenfold longer circulation half-life and 7.5-100 times higher tumor site concentrations correspond to a significant increase in pharmacodynamic efficacy. Taken together, the developed codelivery approach effectively addresses the challenges in the chemotherapeutic efficacy of MET and TPT collectively.

The combination of A-966492 and Topotecan for effective radiosensitization on glioblastoma spheroids

Radiotherapy is one of the modalities in the treatment of glioblastoma patients, but glioma tumors are resistant to radiation and also chemotherapy drugs. Thus, researchers are investigating drugs which have radiosensitization capabilities in order to improve radiotherapy. PARP enzymes and topoisomerase I enzymes have a critical role in repairing DNA damage in tumor cells. Thus, inhibiting activity of these enzymes helps stop DNA damage repair and increase DSB lethal damages. In the current study, we investigated the combination of TPT as a topoisomerase I inhibitor, and A-966492 as a novel PARP inhibitor for further radiosensitization. U87MG cells (a human glioblastoma cell line) were cultured in Poly-Hema coated flasks to reach 300 μm-diameter spheroids. Treatments were accomplished by using non-toxic concentrations of A-966492 and Topotecan. The surviving fraction of treated cells was determined by clonogenic assay after treatment with drugs and 6 MV X-ray. The γ-H2AX expression was measured by an immunofluorescence staining method to examine the influence of A-966492, TPT and radiation on the induction of double stranded DNA breaks. Treatments using the A-966492 drug were conducted in concentration of 1 μM. Combining A-966492 and TPT with radiation yielded enhanced cell killing, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER50) 1.39 and 1.16 respectively. Radio- and chemo-sensitization was further enhanced when A-966492 was combined with both X-ray and TPT, with SER50 of 1.53. Also γ-H2AX expression was higher in the group treated with a combination of drugs and radiation.A-966492 is an effective PARP inhibitor and has significant radio-sensitivity on U87MG spheroids. By accumulating cells in the S phase and by inhibiting the DNA damage repair, TPT enhanced radio-sensitivity. A-966492 combined with TPT as a topoisomerase I inhibitor had additive radio-sensitizing effects. As a result, applying PARP and topoisomerase I inhibitors can be a suitable strategy for improving radiotherapy in clinics.

Abstract 1192: Regorafenib (Stivarga®) reverses BCRP-mediated multidrug resistance in vitro and in vivo

Abstract Overexpression of breast cancer resistance protein (BCRP) has been shown to produce multidrug resistance (MDR) in various kinds of cancers, such as breast, colon, lung and ovarian cancers. Regorafenib, an oral multi-kinase inhibitor, was found to have inhibitory effects on BCRP-mediated MDR both in vitro and in vivo. Regorafenib significantly sensitized BCRP-overexpressing cancerous cells to BCRP substrates by increasing their intracellular accumulation. There are no significant changes in the expression level or the subcellular distribution of BCRP in the cells exposed to regorafenib. Our mechanism studies revealed that regorafenib inhibited the ATP-driven efflux function of BCRP. Our induced-fit docking and molecular dynamics simulations suggested the existence of strong and stable interactions between regorafenib and BCRP protein. Animal study revealed that the combination of regorafenib and topotecan resulted in great inhibitory effects on the growth of S1-M1-80 xenograft tumors. Regorafenib significantly increased the intratumoral concentration of topotecan but had no significant effects on topotecan plasma concentration. In conclusion, our study indicated that regorafenib could be useful in combating multidrug resistance in cancer treatments. Citation Format: Yun-Kai Zhang, Yi-Jun Wang, Guan-Nan Zhang, Wen-Ji Zhang, Meng-Ning Wei, Zhi Shi, Zhe-Sheng Chen. Regorafenib (Stivarga®) reverses BCRP-mediated multidrug resistance in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1192. doi:10.1158/1538-7445.AM2017-1192

In vitro assay for measuring real time topotecan release from liposomes: release kinetics and cellular internalization.

Topotecan is a drug that is under investigation for the treatment of neuroblastoma and has been encapsulated into liposomes to improve its therapeutic efficacy. However, liposomal formulations still need to be optimized for drug retention and new techniques to measure drug release are required to better understand this process. Here, a novel in vitro method based on fluorescence de-quenching and an automated microscopy imaging platform were developed for monitoring, in real time, the release of topotecan from a liposomal formulation. Drug release from liposomes was monitored for up to 15h under different conditions including topotecan concentrations, fetal bovine serum amounts (0-20%), and temperatures (25 and 37°C). A cell-based assay was used to assess liposome association with cells in culture and to quantify amounts of topotecan internalized into cells after release from liposomes. Our results show that the liposomal topotecan concentration had an influence on drug release kinetics: there was a reduction in release rate as a function of increasing concentration. Our data also show that topotecan release from the liposomal formulation was dependent on serum concentration where faster release was observed at higher serum concentrations, and on temperature where faster release was found at 37°C. This real-time liposomal drug release assay allows for better understanding of the factors important in governing release of topotecan. The assay will be essential towards designing liposomal formulations of topotecan (and potentially of other camptothecin derivatives such as irinotecan) with optimized retention times and better therapeutic efficacy for testing in the clinic.

Abstract A37: Blockade of ARV7:HIF1α; heterodimers after toptoecan reverses enzalutamide resistance in 22Rv1 cells

Abstract Background: Although enzalutamide (ENZ) is initially effective for the treatment of castration-resistant prostate cancer (CRPC), development of ENZ resistance is inevitable. One mechanism for resistance is the emergence of ARV7, an alternative splicing variant of the full length androgen receptor (FL AR) which lacks the androgen binding and hinge region domains. Nuclear translocation of FL AR utilizes the hinge region for coupling to tubulin or importin. ARV7 protein appears to gain nuclear entry independent of the canonical pathway. FL AR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, facilitating its nuclear localization and transcriptional activity. We previously found that TPT blocks HIF1α translation by targeting topoisomerase I resident on HIF1α mRNA. We therefor determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. Methods: 22Rv1 ENZ resistant prostate cancer cells express comparable levels of full length AR and truncated ARV7 protein. 22Rv1 cells were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the Luminescent cell viability assay. Results: Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots. Compared with control cells, TPT treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α accompanied by decreased ARV7 nuclear localization. Topo-I immunoprecipitation confirmed its linkage to HIF-1α mRNA. Surprisingly, we found that Topo-I RNA-IP pulled down AR mRNA as well. Dose-response curves for the TPT / ENZ combination demonstrated synergistic activity, with combination index values of 0.6. Under hypoxic conditions, TPT had an IC50 of 120 nM. pretreatment of 22Rv1 cells with a minimally toxic dose of 15 nM TPT reduced the ENZ IC50 from 93 nM to 60 nM. Conclusions: Our data indicate that Topo I residing on HIF-1α mRNA provided a druggable target for TPT-mediated inhibition of HIF1α mRNA translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support further exploration of TPT modulation of ENZ resistance in the clinic. Citation Format: John P. Fruehauf, Nathan Farrokh, Sarmen Sarkissian, Jai-Hyun Kim. Blockade of ARV7:HIF1α; heterodimers after toptoecan reverses enzalutamide resistance in 22Rv1 cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A37.

Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1−10 μM) or TOPO (0.1 nM−1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.