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Purpose: 7-t-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a novel third generation camptothecin selected for development based on the blood stability of its pharmacologically active lactone form and its high potency in preclinical models. Here, we report the initial phase I experience with i.v. AR-67 in adults with refractory solid tumors.Experimental Design and Methods: AR-67 was infused over 1 hour daily five times, every 21 days, using an accelerated titration trial design. Plasma was collected on the 1st and 4th day of cycle 1 to determine pharmacokinetic parameters.Results: Twenty-six patients were treated at nine dosage levels (1.2-12.4 mg/m2/d). Dose-limiting toxicities were observed in five patients and consisted of grade 4 febrile neutropenia, grade 3 fatigue, and grade 4 thrombocytopenia. Common toxicities included leukopenia (23%), thrombocytopenia (15.4%), fatigue (15.4%), neutropenia (11.5%), and anemia (11.5%). No diarrhea was observed. The maximum tolerated dosage was 7.5 mg/m2/d. The lactone form was the predominant species in plasma (>87% of area under the plasma concentration-time curve) at all dosages. No drug accumulation was observed on day 4. Clearance was constant with increasing dosage and hematologic toxicities correlated with exposure (P < 0.001). A prolonged partial response was observed in one subject with non–small cell lung cancer. Stable disease was noted in patients with small cell lung cancer, non–small cell lung cancer, and duodenal cancer.Conclusions: AR-67 is a novel, blood-stable camptothecin with a predictable toxicity profile and linear pharmacokinetics. The recommended phase II dosage is 7.5 mg/m2/d five times every 21 days. Clin Cancer Res; 16(2); 673–80

Supplementary Data from A Phase I Study of 7-t-Butyldimethylsilyl-10-Hydroxycamptothecin in Adult Patients with Refractory or Metastatic Solid Malignancies

Supplementary Figure 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]

Supplementary Table 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

In this paper, we review the management of neonatal opioid withdrawal syndrome (NOWS) and clinical pharmacology of primary treatment agents in NOWS, including morphine, methadone, buprenorphine, clonidine, and phenobarbital. Pharmacologic treatment strategies in NOWS have been mostly empirical, and heterogeneity in dosing regimens adds to the difficulty of extrapolating study results to broader patient populations. As population pharmacokinetics (PKs) of pharmacologic agents in NOWS become more well‐defined and knowledge of patient‐specific factors affecting treatment outcomes continue to accumulate, PK/pharmacodynamic modeling and simulation will be powerful tools to aid the design of optimal dosing regimens at the patient level. Although there is an increasing number of clinical trials on the comparative efficacy of treatment agents in NOWS, here, we also draw attention to the importance of optimizing the dosing regimen, which can be arguably equally important at identifying the optimal treatment agent.

Background: Intravenous irinotecan hydrochloride (IRN-IV) is approved for the treatment of adult colorectal cancer. IRN-IV is also used off-label to treat a range of adult and pediatric tumors including recurrent Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, hepatoblastoma, Wilms tumor, gynecologic cancers, lung cancer and medulloblastoma. Previously, a regimen of IRN-IV administered as a 60-min i.v. infusion daily for 5 days in combination with other agents such as temozolomide has been recommended use in treating children with solid tumors (Blaney. ClinCanRes, 2001). Protracted administration schedule of intravenous irinotecan is inconvenient for patients, so oral regimens utilizing IRN-IV have been developed (Wagner. ClinSarcRes, 2015). Unfortunately, the palatability of the intravenous preparation is poor, leading to reduced compliance especially in younger pediatric patients. Development of an advanced formulation to improve tolerability and patient compliance is an important unmet need. VAL-413 is a novel formulation developed to improve palatability of oral irinotecan. Methods: Eligibility: Up to 20 patients ≥ 1 year of age or ≤ 30 years of age with recurrent pediatric solid tumors and adequate bone marrow, renal and liver function, for whom irinotecan therapy is a treatment option will be enrolled. Trial Design: Two different dose levels of VAL-413, 90mg/m2/day or 110mg/m2/day will be studied in combination with fixed-dose temozolomide using a standard 3 + 3 phase I design. In the event the starting dose of 90 mg/m2/day is not tolerable due to toxicity, a lower dose of 75 mg/m2/day may be implemented. Treatment: During the first cycle of treatment, each patient will receive 4 daily doses of VAL-413 and one daily dose of the intravenous preparation of irinotecan taken orally (IRN-IVPO). During all subsequent cycles, only VAL-413 will be given with temozolomide in 5-day courses administered every 21 days, as tolerated. Outcome Measures: Toxicity is assessed by NCI CCTCAEv5; tumor response is assessed by RECIST 1.1. A palatability survey instrument will assess palatability of VAL-413 vs. IRN-IVPO; comparative intrapatient pharmacokinetics of irinotecan and its metabolites will be assessed. This trial is ongoing (CT.gov: NCT04337177), with no DLT observed to date. Citation Format: Jeffrey Bacha, Dennis Brown, Sarath Kanekal, Neil Sankar, Lorena Lopez, Noymi Yam, Lars Wagner, James Geller, Markos Leggas, Patrick Thompson, Javier Osterheld. A pilot clinical study of orotecan (oral irinotecan HCl, VAL-413) in patients with recurrent pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT135.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32Etreatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector functionin vitro. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

Herein we describe the ability of the permissive glycosyltransferase (GT) OleD Loki to convert a diverse set of > 15 histone deacetylase (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters. Representative glycosyl esters were subsequently evaluated in assays for cancer cell line cytotoxicity, chemical and enzymatic stability, and axolotl embryo tail regeneration. Computational substrate docking models were predictive of enzyme-catalyzed turnover and suggest certain HDACis may form unproductive, potentially inhibitory, complexes with GTs.

Ewing sarcoma is the second most common bone cancer seen in children and adolescents. Previous reports have demonstrated that the main driver of malignancy in this disease is an aberrant transcription factor that is expressed by gene fusions between the EWSR1 gene and an ETS family transcription factor such as FLI1 or ERG. Here we review recent preclinical and clinical advances in drug development for the treatment of Ewing sarcoma. We also discuss the rationale for promising combination therapies that have been considered in the interest of developing treatments for Ewing sarcoma. The main driver of malignancy in this disease is EWS-FLI1. There have been valiant efforts to develop targeted therapies targeting EWS-FLI1, epigenetic factors, factors that affect transcription and the repurposing of previously approved drugs has also been of interest. The underlying mechanisms of how EWS-FLI1 contributes to malignancy in Ewing sarcoma have been extensively studied. Through these observations, EWS-FLI1 targeting and inhibition of aberrant transcription of downstream targets has been proposed as a potential pharmacologic treatment. Improved understanding of how newly developed compounds affect this disease, keeping associated toxicities in mind have led to structure activity relationship studies that have demonstrated improved efficacy and toxicity when treating at the preclinical level. Although there have been many challenges translating these promising results in the clinic, there are further studies ongoing to improve these efforts.

AR-67 is a lipophilic camptothecin analog currently under clinical investigation using a Cremophor EL based formulation. However, as potential toxicity limitations exist in the clinical use of Cremophor, an alternative cyclodextrin (SBE-β-CD) based formulation has been proposed. Pharmacokinetic (PK) studies were conducted in mice and the SBE-β-CD based formulation was compared with the Cremophor EL formulation. PK studies were conducted following intravenous or oral administration of AR-67 in either Cremophor or SBE-β-CD formulation in mice. Noncompartmental analysis was used to determine the plasma and tissue drug distribution. A non-linear mixed effects (population) PK model was developed to fit both the oral and intravenous data and to estimate key PK parameters. The effect of formulation was explored as a covariate in the PK model. AR-67 in the SBE-β-CD formulation had similar plasma PK and biodistribution to that in the Cremophor EL formulation. The proposed two-compartment model described the plasma PK of AR-67 in both formulations adequately. AR-67 in the SBE-β-CD formulation exhibited dose linearity following both oral and intravenous administration. Our studies indicate that SBE-β-CD is a viable alternative to Cremophor EL as a pharmaceutical excipient for formulating AR-67.

Background One of the most prominent opioid analgesics in the United States is the high potency agonist fentanyl. It is used in the treatment of acute and chronic pain and as an anesthetic adjuvant. When used inappropriately, however, ingestion of just a few milligrams of fentanyl or other synthetic opioid can cause opioid-induced respiratory depression (OIRD), often leading to death. Currently, the treatment of choice for OIRD is the opioid receptor antagonist naloxone. Recent reports, however, suggest that higher doses or repeated dosing of naloxone (due to recurrence of respiratory depression) may be required to reverse fully fentanyl-induced respiratory depression, rendering this treatment inadequate. To combat this synthetic opioid overdose crisis, this research aims at identifying a novel opioid reversal agent with enhanced efficacy towards fentanyl and other synthetic opioids. Methods A series of naltrexone analogues were characterized for their ability to antagonize the effects of fentanyl in vitro utilizing a modified forskolin-induced cAMP accumulation assay. Lead analogue 29 was chosen to undergo further PK studies, followed by in vivo pharmacological analysis to determine its ability to antagonize opioid-induced antinociception in the hot plate assay. Results A series of potent MOR antagonists were identified, including the highly potent analogue 29 (IC50 = 2.06 nM). Follow-up PK studies revealed 29 to possess near 100% bioavailability following IP administration. Brain concentrations of 29 surpassed plasma concentrations, with an apparent terminal half-life of ~ 80 min in mice. In the hot plate assay, 29 dose-dependently (0.01–0.1 mg/kg; IP) and fully antagonized the antinociception induced by oxycodone (5.6 mg/kg; IP). Furthermore, the dose of 29 that is fully effective in preventing oxycodone-induced antinociception (0.1 mg/kg) was ineffective against locomotor deficits caused by the KOR agonist U50,488. Conclusions Methods have been developed that have utility to identify enhanced rescue agents for the treatment of OIRD. Analogue 29, possessing potent MOR antagonist activity in vitro and in vivo, provides a promising lead in our search for an enhanced synthetic opioid rescue agent.

Intravenous irinotecan hydrochloride (IRN-IV) is approved for the treatment of adult colorectal cancer. IRN-IV is also widely used off-label for a range of adult and pediatric solid tumors including recurrent Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, hepatoblastoma, Wilms tumor, gynecologic cancers, lung cancer and medulloblastoma. Recently, commercially available intravenous irinotecan has been administered orally (IRN-IVPO) in pediatric patients to improve convenience, reduce in-clinic time and overall cost of treatment. Unfortunately, the palatability of the intravenous preparation is poor, leading to reduced compliance especially in younger pediatric patients. Development of an advanced formulation to improve tolerability and patient compliance is an important unmet need. VAL-413 is a novel formulation developed to improve palatability of oral irinotecan. We will report on an ongoing Phase 1-2 first-in-human clinical trial of VAL-413, designed to test the hypothesis that VAL-413 improves palatability without significant pharmacokinetic differences from IRN-IVPO using intra-patient comparison of pharmacokinetic profiles. The evaluation of two dose levels will help to formally define a recommended dose for advanced clinical studies. Trial Design: Patients ≥ 1 year of age or ≤ 30 years of age with recurrent pediatric solid tumors and adequate bone marrow, renal and liver function, for whom irinotecan therapy is a treatment option, are eligible. Up to 20 eligible patients will receive VAL-413 90mg/m2/day or 110mg/m2/day using a standard 3 + 3 design. During the first cycle of treatment, each patient will receive four daily doses of VAL-413 and one daily dose of IRN-IVPO, together with five (5) days of concurrent temozolomide. Pharmacokinetic assessments will follow both VAL-413 and IRN-IVPO administration using a limited sampling strategy. During all subsequent cycles, only VAL-413 will be given with temozolomide in 5-day courses administered every 21 days. Up to 17 cycles of treatment may be administered on this study. Outcome Measures: Toxicity is assessed by NCI CCTCAEv5; tumor response is assessed by RECIST 1.1. A palatability survey instrument will assess palatability of VAL-413 vs. IRN-IVPO; and comparative intrapatient pharmacokinetics of irinotecan and its metabolites will be assessed in all patients. Citation Format: James Geller, Patrick Thompson, Javier Oesterheld, Dennis M. Brown, Jeffrey Bacha, Sarath Kanekal, Lorena Lopez, Markos Leggas, Neil Sankar, Noymi Yam, Lars M. Wagner. A pilot clinical study of VAL-413 (Orotecan®, oral irinotecan HCl) in patients with recurrent pediatric solid tumors [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 CT256.

Background: Recent studies demonstrated that the aberrant oncogenic activity of ETS transcription factor fusions such as EWS-FLI1 and EWS-ERG in Ewing sarcoma can be targeted with the natural product mithramycin (MTM), which is known to have poor pharmacology. We hypothesized that the pharmacological properties of MTM can be ameliorated by rational semisynthetic modifications that improve its pharmacokinetic (PK) liabilities and expand its efficacy toxicity window. Further, we reasoned that the MTM core of each analog can facilitate DNA docking while the substitutions may interact uniquely with oncogenic ETS transcriptional complexes in different cancers. Here we present the pharmacologic properties of two analogs and their unique transcriptome profiles in TC32 Ewing sarcoma and VCaP prostate cancer cells, both of which depend on the aberrant oncogenic activity of an ETS gene fusion. Methods: MTM analogs were generated by semisynthetic conjugation to the 3-side chain of MTM. Cytotoxicity (GI50) was assessed using the resazurin assay following 72 hr incubation with test compounds (0.3nM-10uM). PK properties were assessed in athymic nu/nu mice after a bolus tail-vein injection and concentrations were assessed in plasma using LC/MS/MS methods. The maximum tolerated dose (MTD) was estimated based on weight loss and blood hematology. In vivo efficacy was assessed in subcutaneous xenografts established, which were dosed SIDx5 at the respective MTD or fractions of the MTD. Transcriptomic profiles were assessed in cells exposed to their respective 72 hr GI50 for 24 hr and in livers of mice that were dosed SIDx5. Results: Here we demonstrate that select modifications displayed unique cytotoxicity and efficacy Ewing sarcoma (EWS-FLI1) and VCaP prostate (TMPRSS2-ERG) cancer cell lines. Analogues with bulky amino acid substitutions in the 3-side chain of MTM had significantly improved PK in mice, as compared to MTM. Select analogs displayed selective efficacy in xenograft models of Ewing sarcoma at doses ranging as low as 1/3 of the MTD. Significantly, analogues of MTM displayed significantly different transcriptomic profiles in each cell line, which suggests that these molecules have unique properties despite sharing a core DNA-binding structure with MTM. Conclusions: MTM analogues with bulky hydrophobic amino acid substitutions on the 3-side chain showed the greatest improvement in PK properties and efficacy. Presumably PK are affected due to increased protein binding and an increase in the pKa, which leads to a lower ionized fraction and decreased uptake in the liver by organic anion transporters. This increase in plasma exposure may lead to increased drug partition in the tumor, as compared to MTM, and, therefore, improved efficacy. Transcriptomic data suggest that the analogs have unique transcriptional effects and therefore unlikely to manifest the same toxicity profile as MTM. Citation Format: Markos Leggas, Ambika Dudhate, Jurgen Rohr. Selective targeting of aberrant ETS transcription factors using uniquely modified mithramycin analogs [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 1851.

TPS10063 Background: Intravenous irinotecan hydrochloride (IRN-IV) is approved for the treatment of adult colorectal cancer. IRN-IV is also widely used off-label for a range of adult and pediatric solid tumors including recurrent Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, hepatoblastoma, Wilms tumor, gynecologic cancers, lung cancer and medulloblastoma. Previously, a regimen of IRN-IV administered as a 60-min i.v. infusion daily for 5 days, every 21 days has been recommended use in treating children with solid tumors (Blaney. ClinCanRes, 2001 ). Protracted administration schedule of intravenous irinotecan is inconvenient for patients, so oral regimens utilizing IRN-IV have been developed (Wagner. ClinSarcRes, 2015 ). Unfortunately, the palatability of the intravenous preparation is poor, leading to reduced compliance especially in younger pediatric patients. Development of an advanced formulation to improve tolerability and patient compliance is an important unmet clinical need. VAL-413 is a novel formulation developed to improve palatability of oral irinotecan. Methods: Eligibility: Up to 20 patients ≥ 1 year and ≤ 30 years of age with recurrent pediatric solid tumors and adequate bone marrow, renal and liver function, for whom irinotecan therapy is a treatment option will be enrolled. Trial Design: Two different dose levels of VAL-413, 90mg/m2/day or 100mg/m2/day will be studied in combination with a fixed-dose of temozolomide using a standard 3 + 3 phase I design. In the event that the starting dose of 90 mg/m2/day is not tolerable due to toxicity, a lower dose of 75 mg/m2/day may be implemented. Treatment: During the first cycle of treatment, each patient will receive 4 daily doses of VAL-413 and one daily dose of the intravenous preparation of irinotecan taken orally (IRN-IVPO). During all subsequent cycles, only VAL-413 will be given with temozolomide in 5-day courses administered every 21 days, as tolerated. Outcome Measures: Toxicity is assessed by NCI CCTCAEv5; tumor response is assessed by RECIST 1.1. A palatability survey instrument will assess palatability of VAL-413 vs. IRN-IVPO; and comparative intrapatient pharmacokinetics of irinotecan and its metabolites will be assessed in all patients. This trial is ongoing (CT.gov: NCT04337177). Enrolment of the first dose level is ongoing, with no DLT observed to date. Clinical trial information: NCT04337177.

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2′-oxime (MTM(ox)) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM(ox) analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2′-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTM(ox)32E (a Phe–Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTM(ox)32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is

Introduction The purpose of this study was to evaluate the efficacy and tolerability of carfilzomib plus irinotecan (C/I) in patients with relapsed small-cell lung cancer (SCLC). Patients and Methods Patients with SCLC who progressed after 1 platinum-containing regimen for recurrent or metastatic disease were eligible. Patients were stratified as: sensitive (SS) (progressive disease > 90 days after chemotherapy) or refractory (RS) (progressive disease 30 to 90 days after chemotherapy) and received up to 6 cycles of C/I; imaging was performed every 2 cycles. The primary endpoint was 6-month overall survival (OS). Results All 62 patients enrolled were evaluable for efficacy and adverse events. 6-month OS was 59% in the platinum SS and 54% in the platinum RS. The overall response rate was 21.6% (2.7% complete response, 18.9% partial response) in SS (n = 37) and 12.5% (all partial response) in RS (n = 25). The disease control rate was 68% (SS) and 56% (RS). Progression-free survival and OS were 3.6 months (95% confidence interval [CI], 2.6-4.6 months) and 6.9 months (95% CI, 4.3-12.3 months) in SS, and 3.3 months (95% CI, 1.8-3.9 months) and 6.8 months (95% CI, 4.1-11 months) in RS. Twenty-nine (47%) patients experienced ≥ grade 3 adverse events; 8 (12.9%) subjects had grade 4 toxicities. Three treatment-related deaths occurred: myocardial infarction (possible), lung infection (possible), and sepsis (probable). Conclusion In patients with relapsed SCLC, C/I was effective in the treatment of SS and RS. With 4.8% grade 5 toxicity, C/I is a viable option for relapsed patients with SCLC with performance status 0 to 1, particularly in platinum-resistant patients, or subjects who cannot receive immunotherapy.

Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of

BACKGROUND: AR-67 is a novel camptothecin analogue at early stages of drug development. The phase 1 clinical trial in cancer patients with solid tumors was completed and a population pharmacokinetic model (POP PK) was developed to facilitate further development of this investigational agent. METHODS: Pharmacokinetic data collected in the phase 1 clinical trial were utilized for the development of a population POP PK by implementing the non-linear mixed effects approach. Patient characteristics at study entry were evaluated as covariates in the model. Subjects (N=26) were treated at nine dosage levels (1.2–12.4 mg/m(2)/day) on a daily × 5 schedule. Hematological toxicity data were modeled against exposure metrics. RESULTS: A two-compartment POP PK model best described the disposition of AR-67 by fitting a total of 328 PK observations from 25 subjects. Following covariate model selection, age remained as a significant covariate on central volume. The final model provided a good fit for the concentration versus time data and PK parameters were estimated with good precision. Clearance, inter-compartmental clearance, central volume and peripheral volume were estimated to be 32.2 L/h, 28.6 L/h, 6.83 L and 25.0 L, respectively. Finally, exposure-pharmacodynamic analysis using E(max) models showed that plasma drug concentration versus time profiles are better predictors of AR-67-related hematologic toxicity were better predictors of leukopenia and thrombocytopenia, as compared to total dose. CONCLUSIONS: A POP PK model was developed to characterize AR-67 pharmacokinetics and identified age as a significant covariate. Exposure PK metrics C(max) and AUC were shown to predict hematological toxicity. Further efforts to identify clinically relevant determinants of AR-67 disposition and effects in a larger patient population are warranted.

Purpose Cohort studies report associations between statin use and improved survival in patients with cancer. We used pharmacoepidemiologic methods to evaluate the survival of patients with cancer who received statins alone or in ostensibly synergistic drug combinations. Materials and Methods Patients with cancer who were diagnosed from 2010 to 2013 were identified in a large health care claims database. The rate of all-cause death up to 1 year after diagnosis was compared by Cox proportional hazard regression. Sensitivity analyses included age stratification, statin type and intensity, and comparison with or without bisphosphonates and dipyridamole. Results Among 312,907 identified patients with cancer, treatment groups included statin users (n = 65,440), nonstatin users who received medications that block cholesterol absorption (n = 9,289), and nonusers (n = 226,007). Statin use before diagnosis was associated with improved overall survival compared with no treatment (hazard ratio [HR], 0.85; 95% CI, 0.80 to 0.91) and specifically in patients with leukemia, lung, or renal cancers. Nonstatin users had increased overall survival compared with no treatment (HR, 0.73; 95% CI, 0.62 to 0.85); when stratified, this difference held true only for pancreatic cancer and leukemia. No differences were observed between statin and nonstatin groups. Bisphosphonate use alone had no effect (n = 4,528), but patients who used both statins and bisphosphonates (n = 4,090) had increased survival compared with no treatment (HR, 0.60; 95% CI, 0.45 to 0.81). The effect of the combination of dipyridamole and statin use (n = 651) was not significant compared with no treatment. Conclusion This study suggests that the combination of statins with drugs that affect isoprenylation, such as bisphosphonates, improves survival in patients with cancer. Consideration of pathway-specific pharmacology allows for hypotheses testing with the pharmacoepidemiologic approach. Prospective evaluation of these findings warrants clinical investigation and preclinical mechanistic studies.

T-cell-based immunotherapy revolutionized the treatment of immune suppressive cancers. Blocking T cell inhibitory receptors such as programmed death 1 (PD1) or their ligands (like PD-L1) enhances antitumor T cell responses. However, many cancers employ multiple methods of immune suppression, including the production of potent regulatory cytokines like Interleukin-10 (IL-10). B cell Chronic Lymphocytic Leukemia (CLL) is one such B cell malignancy that elicits widespread immune dysfunction including numerous functional deficits in T cells. Human and mouse CLL cells (Eμ-Tcl1) produce IL-10 and PD-L1, which dampen antitumor immunity. Since immune checkpoint blockade experienced limited success in CLL, we investigated IL-10 suppression as a novel combination therapy. Exogenous and CLL-derived IL-10 decreased human and mouse T cell functionality in vitro. Further, CLL-derived IL-10 can be blocked with antibodies or can be reduced with small molecule inhibitors to improve CD8+ T cell functionality in vivo. CLL IL-10 production depends on the transcription factor Sp1. Hence, we utilized a novel analogue of the Sp1 inhibitor mithramycin to reduce CLL IL-10 secretion. IL-10 inhibition improved responses to anti-PD-L1 therapy, with increased survival and decreased CLL burden compared to anti-PD-L1 alone. This method improved CD8+ T cell proliferation, effector-memory cell frequency, and interferon-γ production. CD8+ T cells were also more abundant, with fewer exhausted T cells even at very advanced stages of disease. Current cancer therapies do not target IL-10 and our studies provide evidence for targeting IL-10 to increase the efficacy of T cell immunotherapies in human CLL, and possibly other cancers with T cell suppression.

Tumors of the brain and of the central nervous system (CNS) are the most common solid tumors in children. Intravenous irinotecan hydrochloride (IRN-IV) is approved for the treatment of adult colorectal cancer. IRN-IV is also used off-label in a wide range of treatment regimens for recurrent adult and pediatric tumors including glioblastoma, pediatric glioma and medulloblastoma. Recently, commercially available IRN-IV has been administered orally (IRN-IVPO) in pediatric patients to reduce intravenous administration-related side effects, improve convenience and reduce clinic time and costs. Unfortunately, the poor palatability of this preparation leads to poor compliance, especially in pediatric patients. VAL-413 is novel formulation developed to improve tolerability of oral irinotecan. The current study (NCT04337177) evaluates the safety and pharmacokinetics of VAL-413 administered with temozolomide for treatment of recurrent pediatric solid tumors. METHODS: Up to 20 patients ≥ 1 to ≤ 30 years of age with recurrent pediatric solid tumors will be enrolled. The primary objective is to establish the recommended phase II dose of VAL-413 when given in combination with temozolomide. Secondary objectives include characterization of the pharmacokinetics of VAL-413 vs. IRN-IVPO, evaluating palatability of VAL-413, assessing the toxicity profile of VAL-413 in combination with temozolomide, and assessment of tumor response. Toxicity is evaluated based on CTCAEv5 and tumor response based on RECISTv1.1. An update on the progress of this trial will be presented.

Introduction: Chromosomal translocations of ETS family transcription factors (TF) are found in Ewing sarcoma, prostate cancer, and leukemia and lead to expression of aberrant ETS transcription factors driving tumorigenesis. Mithramycin (MTM) inhibits EWS-FLI1, the most common ETS-related TFs in Ewing sarcoma through interference at its DNA binding sites on promoters. However, MTM has a narrow therapeutic window marked by severe liver and hematologic toxicities and poor pharmacokinetic (PK) properties, as demonstrated recently in a pediatric clinical trial with Ewing sarcoma patients. Here, we sought to develop analogues with specificity toward ETS family TFs and improved pharmacologic properties. Methods: Guided by crystal structures of MTM/DNA/ETS, we used semisynthetic approaches to prepare a library of analogs by substitution on the 3-side chain of MTM-SA and MTM (analogs). To select analogs with specific activity toward cells with ETS-related chromosomal translocations, analog cytotoxicity was determined in TC32 cells before and after EWS-FLI1 silencing. To confirm selectivity, analogs were tested in a panel of Ewing sarcoma cell lines (n=8) expressing EWS-ETS translocations and compared to a panel of non-Ewing cell lines (n=9) that do not express these translocations. CETSA, FRET, and luciferase reporter assays were used to evaluate interactions with EWS-FLI1 and SP1 proteins and gene promoter regions they bind. Inhibition of select transcriptionally regulated genes was determined by qRT-PCR and immunoblotting. Select analogs were tested in vivo to assess PK in mice and primates, identify the maximum tolerated dose (MTD), and determine dose-dependent efficacy in mouse xenograft models. Results: MTM analogs had higher GI50 in silenced EWS-FLI1 cells and > 10-fold lower GI50 in ETS-dependent cell lines vs. non-ETS dependent cancer cells, as compared to ~3-fold for MTM. Target engagement was demonstrated in dose-dependent manner using a luciferase reporter TC32 cell line under control of the NR0B1 promoter, which is regulated by EWS-FLI1, and CETSA experiments showed increased physical interaction of the analogs with EWS-FLI1 as compared to MTM. Treatment with MTM and analogs produced comparable dose-dependent displacement of an ETS DNA binding domain from GGAA (microsatellite-like) DNA fragments as demonstrated by FRET. Exposure to analogs led to concentration-dependent downregulation of EWS-FLI1 and Sp1 regulated genes. In vivo studies showed that MTM analogs attained higher exposure at their respective MTD and had lower clearance in mice and primates. Unlike MTM, analogs reversed tumor growth and increased survival at the MTD and 2/3 of MTD. Conclusion: We demonstrated that MTM analogs are selectively more cytotoxic in tumor cell lines dependent on ETS family TFs and have vastly improved PK properties as confirmed in a primate model. Given their effectiveness at 2/3 of the MTD, they are expected to have an improved therapeutic window. Citation Format: Markos Leggas, Joseph Eckenrode, Yang Liu, Abhisek Mandal, Reiya Hayden, Oleg Tsodikov, Jon Thorson, Jurgen Rorh. Novel mithramycin analogues with improved pharmacologic profile and efficacy in ETS transcription factor-driven tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A41.

Summary Peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3) are two major antioxidant proteins that play a critical role in maintaining redox homeostasis for tumor progression. Here, we identify the prototypical pyranonaphthoquinone natural product frenolicin B (FB) as a selective inhibitor of Prx1 and Grx3 through covalent modification of active-site cysteines. FB-targeted inhibition of Prx1 and Grx3 results in a decrease in cellular glutathione levels, an increase of reactive oxygen species (ROS), and concomitant inhibition of cancer cell growth, largely by activating the peroxisome-bound tuberous sclerosis complex to inhibit mTORC1/4E-BP1 signaling axis. FB structure-activity relationship studies reveal a positive correlation between inhibition of 4E-BP1 phosphorylation, ROS-mediated cancer cell cytotoxicity, and suppression of tumor growth in vivo. These findings establish FB as the most potent Prx1/Grx3 inhibitor reported to date and also notably highlight 4E-BP1 phosphorylation status as a potential predictive marker in response to ROS-based therapies in cancer.

Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure-activity relationships of the gilvocarcin group of antitumor antibiotics due to a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary O-methyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2'-OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important hydrogen bond donor for the interaction with histone H3, and converting 3'-OH into an OCH3 group improved the bioactivity. Bis-methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two hydrogen bond donors in the sugar are necessary for optimal binding.

Tipifarnib, a farnesyltransferase inhibitor (FTI), was initially designed to disrupt RAS farnesylation and membrane localization necessary for RAS function. However, alternative geranylgeranylation has been postulated as an escape mechanism by which RAS bypasses the effect of FTI treatment. In this study, we demonstrate that simvastatin, an HMG-CoA reductase inhibitor, augments the cytotoxic effect of tipifarnib by blocking the alternative geranylgeranylation of RAS. Notably, this effect was accompanied by disruption of RAS membrane localization and ERK downregulation. In addition, the apoptotic effect of this combination was associated with downregulation of the antiapoptotic Mcl-1 protein and activation of the caspase cascade. These findings demonstrate that combining tipifarnib and simvastatin was successful in targeting RAS/ERK signaling and inducing apoptosis in leukemia cells. Both simvastatin and tipifarnib were used at clinically achievable doses, which make the combination promising for future clinical studies.

Although preclinical studies on camptothecin antitumor effect have demonstrated the superiority of low-dose protracted dosing, these findings were not replicated in the clinic. 7-t-butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a camptothecin analogue currently under investigation in early phase clinical trials. To maximize the therapeutic potential of AR-67, we sought to identify factors that affect response to treatment. After determining the maximum tolerated dose using neutropenia as a toxicity endpoint, xenografts received AR-67 under varying dosing schedules and were monitored for survival. On the last treatment day, tumor tissue was collected and topoisomerase 1 (Top1), γH2AX, caspase 3 and PARP protein content was evaluated. AR-67 plasma and tumor pharmacokinetics were also studied in mice and cancer patients who were administered AR-67 as a 1-h intravenous infusion on days 1, 4, 8, 12 and 15 every 21 days. Low-dose protracted dosing schedules increased animal survival compared to less frequent, but higher-dose courses and the expression of Top1 and γH2AX were schedule dependent. Fatigue and neutropenia were the dose-limiting toxicities identified in patients receiving AR-67. Finally, elimination of AR-67 from the tumor site was slower in both xenografts and tumor of a patient enrolled in the pilot clinical trial. We demonstrated that low-dose protracted dosing schedules of AR-67 are therapeutically effective and Top1 reflects the biological activity of AR-67 in xenografts. Moreover, the tumor pharmacokinetics as well as the efficacy and safety of AR-67 given intermittently to cancer patients warrant further investigation.

Human genome sequencing revealed that only ~1.5% of the DNA sequence coded for proteins. More and more evidence has uncovered that a substantial part of the 98.5% so-called “junk” DNAs actually code for noncoding RNAs. Two milestones, chemical drugs and protein drugs, have already appeared in the history of drug development, and it is expected that the third milestone in drug development will be RNA drugs or drugs that target RNA. This review focuses on the development of RNA therapeutics for potential cancer treatment by applying RNA nanotechnology. A therapeutic RNA nanoparticle is unique in that its scaffold, ligand, and therapeutic component can all be composed of RNA. The special physicochemical properties lend to the delivery of siRNA, miRNA, ribozymes, or riboswitches; imaging using fluogenenic RNA; and targeting using RNA aptamers. With recent advances in solving the chemical, enzymatic, and thermodynamic stability issues, RNA nanoparticles have been found to be advantageous for in vivo applications due to their uniform nano-scale size, precise stoichiometry, polyvalent nature, low immunogenicity, low toxicity, and target specificity. In vivo animal studies have revealed that RNA nanoparticles can specifically target tumors with favorable pharmacokinetic and pharmacodynamic parameters without unwanted accumulation in normal organs. This review summarizes the key studies that have led to the detailed understanding of RNA nanoparticle formation as well as chemical and thermodynamic stability issue. The methods for RNA nanoparticle construction, and the current challenges in the clinical application of RNA nanotechnology, such as endosome trapping and production costs, are also discussed.

B-cell Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world, accounting for nearly one third of all leukemia cases. In CLL abnormal B-cells accumulate in the blood and lymphoid organs leading to serious immune dysfunction. This immune suppression is in part due to CLL-produced mediators that downregulate T-cell responses, such as the regulatory cytokine Interleukin-10 (IL-10). We previously found that eliminating T-cell IL-10 signaling enhanced their ability to control CLL growth in vivo. Therefore, we investigated the potential for IL-10 blockade to enhance the anti-tumor activity of CD8+ T-cells. In our studies we use human CLL cells as well as the Eμ-Tcl1 mouse model of CLL, in which the oncogene Tcl1 is expressed under the immunoglobulin VH promoter and µ-enhancer. IL-10 production by CLL cells depends on the transcription factor Sp1, and we found that the Sp1 inhibitor mithramycin (MTM) suppresses CLL IL-10 production. However, MTM is not well tolerated in vivo, so we synthesized novel, less toxic analogues of MTM to test for IL-10 suppression. One of these MTM analogues similarly suppresses mouse and human CLL IL-10 with little to no effect on effector T-cell cytokines and viability. Therefore, we treated mice with this analogue in the adoptive transfer model of Eμ-Tcl1, and later combined this with anti-PD-L1 checkpoint blockade to determine its effects on anti-tumor immunity. Here we show that this MTM analogue enhances the efficacy of anti-CLL T-cells in vivo by suppressing CLL IL-10 production, allowing for increased CD8+ T-cell proliferation, effector memory cell prevalence, and CD8+ interferon-γ (IFN-γ) production. Treatment slowed the growth of Eμ-TCL1 CLL cells in the spleen and blood and reduced the spread of CLL to the bone marrow. Furthermore, suppressing IL-10 in this manner improved responses to anti-PD-L1 treatment, decreasing the burden of CLL cells and the functionality of CD8+ T-cells in comparison to anti-PD-L1 alone. The overall number and frequency of CD8+ T-cells was higher in double treated mice, with more IFN-γ+ CD8+ cells, more effector memory cells, and fewer exhausted T-cells. This paradigm shifting approach is novel as current therapies for CLL do not target IL-10 and it may increase the efficacy of T-cell-based immunotherapies in human CLL. T-cell-based immunotherapies have experienced limited success in trials with CLL, and since there is no cure for this disease, our approach may provide a new avenue for combination therapies. Moreover, IL-10 blockade could be applicable to other B-cell malignancies and even solid tumors where T-cell suppression plays a significant role. Disclosures Hildebrandt: Axim Biotechnologies: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Sangamo: Equity Ownership; Novartis: Equity Ownership; Axim Biotechnologies: Equity Ownership; Juno Therapeutics: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Novartis: Equity Ownership; Insys Therapeutics: Equity Ownership; Abbvie: Equity Ownership; GW Pharmaceuticals: Equity Ownership; Cardinal Health: Equity Ownership; Immunomedics: Equity Ownership; Endocyte: Equity Ownership; Clovis Oncology: Equity Ownership; Cellectis: Equity Ownership; Aetna: Equity Ownership; CVS Health: Equity Ownership; Celgene: Equity Ownership; Bluebird Bio: Equity Ownership; Bristol-Myers-Squibb: Equity Ownership; crispr therapeutics: Equity Ownership; IDEXX laboratories: Equity Ownership; Johnson & Johnson: Equity Ownership; Pfizer: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Procter & Gamble: Equity Ownership; Vertex: Equity Ownership; Bayer: Equity Ownership; Scotts-Miracle: Equity Ownership; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Research Funding; Pharmacyclics: Research Funding; Astellas: Other: Travel.

Introduction: Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcription factors drive tumorigenesis. The transcription factors EWS-FLI1 and EWS-ERG are common translocations in Ewing sarcoma and bind DNA at GGAA repeats leading to expression of genes that drive tumor growth. Pharmacologic inhibition of EWS-FLI1 with mithramycin (MTM) was shown to inhibit expression of downstream genes and tumor growth in mice. But despite this specific inhibitory activity, MTM has a narrow therapeutic window with hematologic and hepatic toxicity attributed to displacement of the ubiquitously acting Sp1 transcription factor. Thus, a synthetic effort was initiated to develop MTM analogues with reduced toxicity and increased specificity for ETS binding sites. Structural studies informed the design of MTM analogues that may stabilize transcriptional complexes leading to the disruption of transcriptional activity and DNA damage. In vitro cytotoxicity assays demonstrated that MTM analogues have significantly higher cytotoxicity in EWS-ETS expressing cell lines. Here we present mechanistic evidence for the differences in biochemical activity among MTM and its novel analogues. Methods: Qualitative interactions between drug-DNA-protein were assessed and optimized by electrophoretic mobility shift assays (EMSA). Time-resolved fluorescence energy transfer (TR-FRET) assays were used to quantitatively determine ERG displacement from DNA in the presence of MTM and analogues. Expression of proteins indicating DNA damage (c-PARP, γ-H2AX) and phosphorylation at the C-terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines. Results: Using TR-FRET, we observed that MTM displaced DNA bound ERG more potently and in a concentration dependent manner as compared to MTM analogues. As compared to MTM, treatment with MTM analogues resulted in higher expression of DNA damage markers, γ-H2AX and c-PARP, specifically in cell lines containing EWS-ETS translocations in a concentration dependent manner. Conclusion: These studies provide insights regarding differences among MTM and analogues in DNA binding and interactions with DNA associated proteins in the presence and absence of EWS-ETS expression. Our results suggest that MTM analogues may bind and stabilize transcriptional complexes. These differences will provide the basis for structure activity relationships and for the development of analogues with decreased in vivo toxicity. Future work will incorporate co-immunoprecipitation studies to determine if physical protein interactions are being disrupted by MTM and analogues and cellular thermal shift assays to directly probe drug interactions with EWS-ETS proteins and with RNAPII. Note: This abstract was not presented at the meeting. Citation Format: Reiya C. Hayden, Caixia Hou, Prithiba Mitra, Abhisek Mandal, Jurgen Rohr, Jon Thorson, Oleg Tsodikov, Markos Leggas. Mithramycin analogues disrupt ETS transcription factor DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2954.

A high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of morphine, morphine's major metabolites morphine-3-glucuronide and morphine-6-glucuronide, and clonidine, to support the pharmacokinetic analysis of an ongoing double-blinded randomized clinical trial that compares the use of morphine and clonidine in infants diagnosed with neonatal abstinence syndrome. Plasma samples were processed by solid-phase extraction and separated on an Inertsil ODS-3 (4 μm) column using an 0.1% formic acid in water-0.1% formic acid in methanol gradient. Detection of the analytes was conducted in the positive multiple reaction monitoring mode. The range of quantitation was 1-1000 ng/mL for morphine, morphine-3-glucuronide and morphine-6-glucuronide, and 0.25-100 ng/mL for clonidine. Intra-day and inter-day accuracy and precision were ≤15% for all analytes across the quantitation range. Extraction recovery rates were ≥94% for morphine, ≥90% for M3G, ≥87% for M6G and ≥ 79% for clonidine. Matrix effect ranged from 85-94% for clonidine to 101-106% for M3G. The method fulfilled all predetermined acceptance criteria and required only 100 μL of starting plasma volume. Furthermore, it was successfully applied to 30 clinical trial plasma samples.

Paclitaxel (PTX) nanocrystals (200 nm) were produced by crystallization from a solution. Antitumor efficacy and toxicity were examined through a survival study in a human HT-29 colon cancer xenograft murine model. The antitumor activity of the nanocrystal treatments was comparable with that by the conventional solubilization formulation (Taxol®), but yielded less toxicity as indicated by the result of a survival study. Tritium-labeled PTX nanocrystals were further produced with a near infrared (NIR) fluorescent dye physically integrated in the crystal lattice. Biodistribution and tumor accumulation of the tritium-labeled PTX nanocrystals were determined immediately after intravenous administration and up to 48 h by scintillation counting. Whole-body optical imaging of animals was concurrently carried out; fluorescent intensities were also measured from excised tumors and major organs of euthanized animals. It was found that drug accumulation in the tumor was less than 1% of 20mg/kg intravenous dose. Qualitatively correlation was identified between the biodistribution determined by using tritium-labeled particles and that using optical imaging, but quantitative divergence existed. The divergent results suggest possible ways to improve the design of hybrid nanocrystals for cancer therapy and diagnosis. The study also raises questions of the general role of the enhanced permeability and retention (EPR) effect in tumor targeting and the effectiveness of bioimaging, specifically for theranostics, in tracking drug distribution and pharmacokinetics.

Simvastatin (SIM) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor widely used in hyperlipidemia therapy. SIM has recently been studied for its anticancer activity at doses higher than those used for the hyperlipidemia therapy. This prompted us to study the pharmacokinetics of high-dose SIM in cancer patients. For this purpose, an LCâMS/MS method was developed to measure SIM and its acid form (SIMA) in plasma and peripheral blood mononuclear cells (PBMCs) obtained from patients. Chromatographic analyte separation was carried out on a reverse-phase column using 75:25 (% v/v) acetonitrile:ammonium acetate (0.1Â M, pH 5.0) mobile phase. Detection was performed on a triple quadrupole mass spectrometer, equipped with a turbo ion spray source and operated in positive ionization mode. The assay was linear over a range 2.5â500Â ng/mL for SIM and 5â500Â ng/mL for SIMA in plasma and 2.5â250Â ng/mL for SIM and 5â250Â ng/mL for SIMA in cell lysate. Recovery was >58% for SIM and >75% for SIMA in both plasma and cell lysate. SIM and SIMA were stable in plasma, cell lysate and the reconstitution solution. This method was successfully applied for the determination of SIM and SIMA in plasma and PBMCs samples collected in the pharmacokinetic study of high-dose SIM in cancer patients. Keywords: Simvastatin, LCâMS/MS, Human plasma, Cell lysate, Pharmacokinetic, High-dose

Four new benzamides, pyramidamycins A-D (2–5) along with the new natural 3-hydroxyquinoline-2-carboxamide (6) were isolated from the crude extract of Streptomyces sp. DGC1. Additionally, five other known compounds namely 2-aminobenzamide (anthranilamide) (1), 4′,7-dihydroxyisoflavanone (7), 2′-deoxy-thymidine, 2′-deoxy-uridine and adenosine were also isolated and identified. The structures of the new compounds 2–6 were elucidated by 1D and 2D NMR studies along with HRMS analyses. The isolated compounds 1–6 contained the same amide side chain. The isolated compounds 1–7 were biologically evaluated in comparison with landomycin A against a prostate cancer cell line (PC3) and non small cell lung cancer cell line (H460) for 48 hrs and against several bacterialstrains. Pyramidamycin C (4) was the most active compound against both PC3 and H460 cell lines (GI50 = 2.473 μM and GI50 = 7.339 μM, respectively). Benzamides (1–3) demonstrated inhibitory activity against Kocuria rosea B-1106 (a diameter halo of 13±2 mm for 1; 10±2 mm for 2 and 3). Compound 6 was slightly active against both Escherichia coli DH5α and Micrococcus luteus NRRL B-2618 (diameter halos 8±2 mm and 9±2 mm, respectively). Taxonomically, the amplified 500 bp 16S rRNA fragment of the Streptomyces sp. DGC1 had 99% identity (BLAST search) to the 16S rRNA gene of Streptomyces atrovirens strain NRRL B-16357.

(-)Pantoprazole [(-)PAN] accumulated in rat milk stereoselectively, and this accumulation was attributed to rat Abcg2 (rAbcg2). In contrast, flux experiments at 25 μM showed that (+)pantoprazole [(+)PAN] was preferentially transported by rAbcg2. The purpose of the current study was to comprehensively evaluate the transport of PAN isomers in empty-Madin-Darby canine kidney II (MDCKII) and MDCKII cells expressing the human/rat (ABCG2/rAbcg2) isoforms at concentrations ranging from 3 to 200 μM. The apical-to-basolateral and basolateral-to-apical directional flux and the asymmetry efflux ratios were virtually identical for both isomers in empty (mock transfected)-MDCKII monolayers but were concentration dependent for both isomers in ABCG2 (human/rat)-MDCKII. Kinetic analysis using predicted cellular concentrations showed that (-)PAN had an 8-fold lower K(M) compared with (+)PAN for both rAbcg2 (0.25 versus 1.85 μM) and ABCG2 (0.6 versus 5.32 μM). (+)PAN had a 3-fold higher T(Max) compared with the (-)PAN for both rAbcg2 (7.86 versus 2.49 nmol/h · cm(2)) and ABCG2 (10.2 versus 3.29 nmol/h · cm(2)). Effective ABCG2 surface-area permeability of (-)PAN was 9920 and 5480 (μl/h)/cm(2) for rAbcg2 and ABCG2, respectively, compared with the (+)PAN isomer (4250 and 1920 μl/h · cm(2), respectively). These results indicate a stereoselective interaction of PAN with similar kinetic parameters for both human and rat ABCG2. (-)PAN is a better substrate than (+)PAN for ABCG2/rAbcg2 and provide a rationale for the preferential accumulation of (-)PAN into rat milk.

Camptothecin analogues are anticancer drugs effective when dosed in protracted schedules. Such treatment is best suited for oral formulations. AR-67 is a novel lipophilic analogue with potent efficacy in preclinical models. Here we assessed factors that may influence its oral bioavailability in rats. Plasma pharmacokinetic (PK) studies were conducted following administration of AR-67 lactone or carboxylate doses alone or after pre-dosing with inhibitors of the efflux transporters P-gp and Bcrp. A population PK model that simultaneously fitted to oral and intravenous data was used to estimate the bioavailability (F) and clearance of AR-67. An inverse Gaussian function was used as the oral input into the model and provided the best fits. Covariate analysis showed that the bioavailability of the lactone, but not its clearance, was dose dependent. Consistent with this observation, the bioavailability of AR-67 increased when animals were pretreated orally with GF120918 or Zosuquidar. Absorption of AR-67 is likely affected by solubility of its lactone form and interaction with efflux pumps in the gut. AR-67 appears to be absorbed as the lactone form, most likely due to gastric pH favoring its formation and predominance. F increased at higher doses suggesting saturation of efflux mechanisms.