Durvalumab: a newly approved checkpoint inhibitor for the treatment of urothelial carcinoma.

Until a recent introduction to checkpoint inhibitors, there were limited second-line chemotherapy options for urothelial carcinoma (UC) patients with disease progression after first-line, platinum-based treatment. Outcomes for patients with advanced disease over the past 30 years have highlighted a need for new and better therapy. In response to evolving interest, durvalumab (MEDI4736) was introduced as a potential treatment for advanced stages of UC. Durvalumab is a selective, high-affinity, human IgG1 kappa monoclonal antibody engineered with a triple mutation to reduce toxicity. This checkpoint inhibitor has shown promise in advanced UC and is currently the topic of much discussion in the cancer research community. This review article will explore the details surrounding durvalumab, while also giving a brief overview of additional immunotherapeutic agents utilized for UC.

In vivo electron paramagnetic resonance imaging of differential tumor targeting using cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.

PURPOSE: Electron paramagnetic resonance spectroscopy promises quantitative images of important physiologic markers of animal tumors and normal tissues, such as pO(2), pH, and thiol redox status. These parameters of tissue function are conveniently reported by tailored nitroxides. For defining tumor physiology, it is vital that nitroxides are selectively localized in tumors relative to normal tissue. Furthermore, these paramagnetic species should be specifically taken up by cells of the tumor, thereby reporting on both the site of tumor formation and the physiological status of the tissue. This study investigates the tumor localization of the novel nitroxide, cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidin-yloxyl 3 relative to the corresponding di-acid 4. METHODS: We obtained images of nitroxide 3 infused intravenously into C3H mice bearing 0.5-cm(3) FSa fibrosarcoma on the leg, and compared these with images of similar tumors infused with nitroxide 4. RESULTS: The ratio of spectral intensity from within the tumor-bearing region to that of normal tissue was higher in the mice injected with 3 relative to 4. CONCLUSION: This establishes the possibility of tumor imaging with a nitroxide with intracellular distribution and provides the basis for EPR images of animal models to investigate the relationship between crucial aspects of tumor microenvironment and malignancy and its response to therapy.

Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1.

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5' untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model.

Phase I study of UCN-01 and perifosine in patients with relapsed and refractory acute leukemias and high-risk myelodysplastic syndrome.

BACKGROUND: The PI3K-Akt pathway is frequently activated in acute leukemias and represents an important therapeutic target. UCN-01 and perifosine are known to inhibit Akt activation. METHODS: The primary objective of this phase I study was to determine the maximum tolerated dose (MTD) of UCN-01 given in combination with perifosine in patients with advanced acute leukemias and myelodysplastic syndrome. Secondary objectives included safety, pharmacokinetics, pharmacodynamics, and efficacy. Perifosine 150 mg every 6 h was given orally on day 1 followed by 100 mg once a day continuously in 28-day cycles. UCN-01 was given intravenously over 3 h on day 4 at three dose levels (DL1=40 mg/m(2); DL2=65 mg/m(2); DL3=90 mg/m(2)). RESULTS: Thirteen patients were treated (DL1, n=6; DL2, n=4; DL3, n=3) according to a traditional "3+3" design. Two patients at the DL3 experienced dose-limiting toxicity including grade 3-4 pericardial effusion, hypotension, hyperglycemia, hyperkalemia, constitutional symptoms and grade 5 pneumonitis. Other frequent toxicities were grade 1-2 nausea, diarrhea, vomiting, fatigue and hyperglycemia. The MTD was determined to be UCN-01 65 mg/m(2) with perifosine 100 mg a day. No appreciable direct Akt inhibition could be demonstrated in patients' mononuclear cells using Western blot, however, reduced phosphorylation of the downstream target ribosomal protein S6 in leukemic blasts was noted by intracellular flow cytometry. No objective responses were observed on this study. CONCLUSION: UCN-01 and perifosine can be safely administered, but this regimen lacked clinical efficacy. This approach may have failed because of insufficient Akt inhibition in vivo.

Identification and characterization of the major alternative promoter regulating Bcrp1/Abcg2 expression in the mouse intestine.

Mouse models are often used to predict drug absorption in humans. Mouse Bcrp1 protein exhibits sequence and functional homology with human BCRP protein. Additionally, BCRP/Bcrp1 expression is regulated by alternative promoter usage in humans and mice; however, the precise intestine-specific alternative promoter utilized in either species is yet to be determined. Therefore we sought to identify and characterize the mouse intestinal Bcrp1 promoter. Using real-time quantitative RT-PCR and 5' RACE PCR we first established the predominance of a single Bcrp1 first exon (E1b) in the Bcrp1 mRNA isolated throughout the mouse intestine. Simultaneously using 5' RACE PCR we identified E1C as the predominant BCRP 5' UTR expressed in the human intestine. Next we established functional activity for the murine promoter upstream of E1b using reporter assays. Subsequently using deletion-construct analysis we found the core promoter region to span -231 to -42bps from the transcriptional start site of E1b. We then predicted a cAMP response element (CRE) as a transcription factor binding site unique only to the E1b promoter region, using in silico methods. We finally established functional interaction of phospho-CREB (p-CREB) protein with the CRE on the E1b promoter using both functional assays and chromatin immunoprecipitation assays. In conclusion, mouse intestinal Bcrp1 expression is regulated by a single alternative promoter upstream of E1b, the predominant Bcrp1 mRNA isoform expressed in the mouse intestine. Furthermore, Bcrp1 E1b mRNA expression is regulated by binding of p-CREB to its cis site on the mouse E1b promoter region.

Comparison of pharmacokinetic and pharmacodynamic profiles of aspirin following oral gavage and diet dosing in rats.

Aspirin is one of the oldest drugs and has been purported to have multiple beneficial effects, including prevention of cardiovascular disease and cancer, in addition to its original indication for treatment of inflammation, fever and pain. In cancer chemoprevention studies using animal models, two methods of aspirin administration have been employed: oral gavage and diet. The untested assumption was that exposure and the resultant pharmacological effects are similar for these two administration methods when dosing is normalized on the basis of mg/kg body weight/day. This study examined and compared time-dependent plasma and colon mucosal concentrations of aspirin metabolite salicylate (aspirin concentrations were below level of quantification), plasma thromboxane B(2) concentrations, and colon mucosal prostaglandin E(2) concentration following these two different dosing paradigms in rats. Diet dosing yielded relatively constant plasma and colon salicylate concentration vs. time profiles. On the other hand, oral gavage dosing led to a rapid peak followed by a fast decline in salicylate concentration in both plasma and colon. Nevertheless, the exposure as measured by the area under plasma or colon concentration-time curve of salicylate was linearly related to dose irrespective of the dosing method. Linear relationships were also observed between colon and plasma salicylate areas under the curve and between colon prostaglandin E(2) and plasma thromboxane B(2) areas under the curve. Therefore, more easily accessible plasma salicylate and thromboxane B(2) concentrations were representative of the salicylate exposure and prostaglandin E(2) pharmacodynamic biomarker in the target colon, respectively.

Phase I and pharmacokinetic study of 7-hydroxystaurosporine and carboplatin in advanced solid tumors.

PURPOSE: Based on preclinical data showing synergy between 7-hydroxystaurosporine (UCN-01) and platinum agents, a phase I trial of carboplatin with UCN-01 administered as a 3 h infusion in patients with advanced solid tumors was done. The primary goals of this trial were to evaluate the tolerability of this combination and the pharmacokinetics of UCN-01 when administered over 3 h and to compare the tolerability and pharmacokinetics with previously described schedules. PATIENTS AND METHODS: Patients with advanced solid tumors, good performance status, normal organ function, and no potentially curative therapy were eligible for the trial. Carboplatin was escalated from an area under the curve (AUC) of 3 to an AUC of 5. UCN-01 was escalated from 50 to 90 mg/m(2). RESULTS: Twenty-three patients with advanced solid tumors (20 with prior platinum treatment) received a total of 60 cycles of therapy. Full doses of both agents (carboplatin AUC 5, UCN-01 90 mg/m(2) in cycle 1, 45 mg/m(2) in subsequent cycles) could be administered. The major toxicity noted was hypotension, which could be abrogated with the use of saline prehydration and posthydration. No responses were seen; however, seven patients were able to receive more than two courses of therapy. Of note, two of three patients with refractory, progressive small cell lung cancer were able to receive six cycles of therapy without evidence of progression. One patient experienced resolution of paraneoplastic syndrome of inappropriate antidiuretic hormone. The pharmacokinetic variables C(max) and t(1/2) of the 3 h infusion were essentially identical to those previously observed when UCN-01 was administered over 72 h. The average t(1/2) for cycle 1 was 506 +/- 301 h, and the mean C(max) for all dose levels was >30 micromol/L. The mean AUC over the dosing interval for each dose level ranged from approximately 6,000 to 9,000 micromol/L h. Thus, the AUC of UCN-01 after the 3 h infusion was lower than was observed after a 72 h infusion. CONCLUSION: The regimen of carboplatin and UCN-01 (administered as a 3 h infusion) was well tolerated. Further development of this combination, particularly in small cell lung cancer, is warranted.

Randomized phase II trial of matrix metalloproteinase inhibitor COL-3 in AIDS-related Kaposi's sarcoma: an AIDS Malignancy Consortium Study.

PURPOSE: Matrix metalloproteinases (MMPs) are involved in tumor metastasis and are overexpressed in Kaposi's sarcoma (KS) cells. In a phase I trial of the MMP inhibitor COL-3 in patients with AIDS-related KS, the drug was well tolerated, KS regression was observed, and MMP-2 levels decreased significantly in responders compared with nonresponders. The aim of this trial was to extend these initial observations. PATIENTS AND METHODS: This was a randomized, parallel-group, phase II study. COL-3 was administered orally once daily at one of two doses (group A received 50 mg and group B received 100 mg) to patients with AIDS-related KS. Antiretroviral therapy was permitted but not required. Serial tumor assessments and plasma levels of MMPs were obtained. Study end points were progressive KS and recurrent dose-limiting toxicity. RESULTS: Seventy-five patients received COL-3: 37 in group A and 38 in group B. Fifty-seven patients (76%) had received prior KS therapy. Thirty-three patients (44%) had more than 50 KS lesions. The response rate in group A was 41%, which was significantly greater than the prespecified target rate of 20% (95% CI, 25% to 58%; P = .003); the response rate of group B was 29% (P = not significant). There were significant declines in MMP-2 and MMP-9 plasma levels from baseline to minimum value with treatment (MMP-2, P < .001; MMP-9, P = .001). The most common adverse events were photosensitivity and rash. CONCLUSION: COL-3, when administered as 50 mg/d, is both active and well tolerated in the treatment of AIDS-related KS. COL-3 is a promising agent for the treatment of this opportunistic neoplasm of AIDS.

Phase I and pharmacokinetic study of flavopiridol followed by 1-beta-D-arabinofuranosylcytosine and mitoxantrone in relapsed and refractory adult acute leukemias.

PURPOSE: The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-beta-D-arabinofuranosylcytosine (ara-C) and mitoxantrone. EXPERIMENTAL DESIGN: Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels. RESULTS: Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with > or =50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%). CONCLUSIONS: Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d x 3 days in combination with cytotoxic and biological agents for acute leukemias.

A phase I and pharmacologic study of idarubicin, cytarabine, etoposide, and the multidrug resistance protein (MDR1/Pgp) inhibitor PSC-833 in patients with refractory leukemia.

This study was conducted to define the maximum tolerated dose (MTD), dose limiting toxicity (DLT), and pharmacokinetics of idarubicin when administered with and without the P-glycoprotein inhibitor PSC-833 in combination with cytarabine, and etoposide. Fifteen patients with relapsed and refractory acute leukemia were enrolled and received cytarabine as a 7-day continuous infusion, with etoposide and idarubicin administered for any three consecutive days during the cytarabine infusion. Two hours prior to the second dose of idarubicin, PSC-833 administration was initiated. The pharmacokinetics of idarubicin alone and with PSC-833 was assessed at three idarubicin dose levels (6, 8 and 10 mg/m(2)). The MTD of idarubicin in this combination was 8 mg/(m(2) day) with a DLT of oral mucositis. The complete remission rate (on an intent-to-treat basis) for this regimen was 33%, with a median duration of 6 months. The clearance of idarubicin was 140 +/- 200 and 181 +/- 94.3 l/h for idarubicin alone and with PSC-833, respectively. The volume of distribution of the central compartment was 423 +/- 443 and 337 +/- 394 l for idarubicin alone and in combination with PSC-833, respectively. This combination including PSC-833 was well tolerated. Although a pharmacokinetic interaction might have been expected, PSC-833 did not significantly alter the disposition of idarubicin.

Liquid chromatography method for the quantitation of the breast cancer resistance protein ABCG2 inhibitor fumitremorgin C and its chemical analogues in mouse plasma and tissues.

Fumitremorgin C (FTC) was recently discovered to be a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2). FTC was shown to reverse multidrug resistance mediated by BCRP and to increase the cytotoxicity of several anticancer agents in vitro. To support in vivo studies a reverse phase HPLC method with ultraviolet detection was developed to quantitate FTC in mouse plasma and tissues. Further, assay method validation was performed for the determination of FTC in mouse plasma. Plasma standard curves ranged from 0.03 to 30 microg/ml, while the various tissue assay ranges differed to some extent. The sample preparation consisted of acetonitrile precipitation with separation accomplished with a C18 Novapak column and a C18 pre-column utilizing an isocratic mobile phase of ammonium acetate and acetonitrile. UV detection was set at 225 nm for FTC and at 312 nm for roquefortine, the internal standard. The retention times were approximately 9.5 min for FTC and 13.0 min for roquefortine. The recoveries for FTC and roquefortine from plasma were 90.8+/-5.8% and 111.6+/-13.6, respectively. The reported assay can be used for future study of BCRP resistance in vivo in different biological matrices. Further, we found that a more potent analogue of FTC, Ko143, was able to be extracted and detected, with a maximal UV absorbance at 320 nm under the conditions reported.

Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis.

To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.

Clinical pharmacology of flavopiridol following a 72-hour continuous infusion.

BACKGROUND: Flavopiridol, a novel flavone derivative, inhibits cyclin-dependent kinase-1. We initiated a Phase I trial in patients with refractory solid tumors to determine the maximum tolerated dose and characterize the adverse effect profile. OBJECTIVE: To characterize the clinical pharmacology of flavopiridol. METHODS: Serial plasma samples were collected and analyzed by HPLC using electrochemical detection. The pharmacokinetics were analyzed by noncompartmental analysis. Enterohepatic recirculation was studied by analyzing fecal samples, with an attempt to correlate cholecystokinin and post-infusional peak concentrations. The plasma protein binding was studied using equilibrium dialysis. RESULTS: Seventy-six patients were treated with flavopiridol at 13 dose levels for a total of 504 cycles of treatment. The average steady-state concentration was 26.5 and 253 nM at 4 and 122.5 mg/m2, respectively. The clearance ranged from 49.9 to 2943 mL/min, with nonlinearity at doses >50 mg/m2/d. A post-infusional increase in plasma flavopiridol concentrations was noted in a subset of patients and generally occurred between 3 and 24 hours after the end of infusion. Flavopiridol was found in fecal matter, suggesting enterohepatic recirculation. There was nonsaturable plasma protein binding of flavopiridol (fu = 6%). CONCLUSIONS: The dose-limiting toxicity for the Phase I trial of flavopiridol was secretory diarrhea. We failed to identify a clear relationship between dose or concentration and diarrhea. At 50 and 78 mg/m2/d, the mean steady-state plasma concentrations were 278 and 390 nM. These concentrations were well above those noted for in vitro antiproliferative activity. Nonlinear elimination was observed at doses above 50 mg/m2/d, and postinfusional peaks appear to be related to enterohepatic recirculation.