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Purpose: Camptothecins contain a lactone ring that is necessary for antitumor activity, and hydrolysis of the lactone ring yields an inactive carboxylate species. Human serum albumin (HSA) and alpha-1 acid glycoprotein (AGP) are clinically significant plasma proteins thought to have important roles in camptothecin lactone stability. Herein, we examined the effect(s) of HSA and AGP on the lactone stability of Karenitecin, a novel, highly lipophilic camptothecin analog, currently at the phase 3 clinical testing stage., Methods: An AGP-immobilized protein column was used to develop HPLC methods to evaluate the effect(s) of physiologically relevant HSA and AGP concentrations on the lactone/carboxylate ratio and hydrolysis kinetics of Karenitecin, camptothecin (CPT), and topotecan (TPT)., Results: Physiologically relevant concentrations of HSA and AGP substantially slowed Karenitecin lactone hydrolysis. AGP was notably more effective at protecting the Karenitecin lactone from hydrolysis than HSA was in promoting hydrolysis. Additionally, AGP reversed the hydrolysis of partially hydrolyzed Karenitecin lactone. In contrast, HSA and AGP had minimal effects on hydrolysis of the TPT lactone, while the AGP/HSA solutions dramatically accelerated hydrolysis of the CPT lactone., Conclusion: AGP strongly enhances the lactone stability of Karenitecin. Since Karenitecin is highly protein-bound in human plasma and exhibits greater lactone stability, relative to other camptothecins, in patient plasma samples, this newly identified role of AGP in promoting lactone stability may have important implications for the design of more effective anticancer agents within the Karentecin™ and camptothecin classes.

Reversing abnormal gene silencing in cancer cells due to DNA hypermethylation of promoter CpG islands may offer new cancer prevention or therapeutic approaches. Moreover, such approaches may be broadly applicable to enhance the efficacy of radiotherapy, chemotherapy, or immunotherapy. Here, we demonstrate the powerful utility of a novel gene reporter system to permit studies of the dynamics, mechanisms, and translational relevance of candidate therapies of this type in human colon cancer cells. The reporter system is based on in situ modification of the endogenous locus of the tumor-suppressor gene SFRP1, a pivotal regulator of the Wnt pathway that is silenced by DNA hypermethylation in many colon cancers. The modified SFRP1-GFP reporter allele used remained basally silent, like the unaltered allele, and it was activated only by drug treatments that derepress gene silencing by reversing DNA hypermethylation. We used the established DNA methyltransferase inhibitor (DNMTi) 5-aza-deoxycitidine (DAC) to show how this system can be used to address key questions in the clinical development of epigenetic cancer therapies. First, we defined conditions for which clinically relevant dosing could induce sustained induction of RNA and protein. Second, we found that, in vivo, a more prolonged drug exposure than anticipated was essential to derepress gene silencing in significant cell numbers, and this has implications for generating effective anticancer responses in patients with hematopoietic or solid tumors. Finally, we discovered how histone deacetylase inhibitors (HDACi) alone, when administered to cells actively replicating DNA, can robustly reexpress the silenced gene with no change in promoter methylation status. Taken together, our findings offer a new tool and insights for devising optimal clinical experiments to evaluate DNMTi and HDACi, alone or in combination, and with other cancer treatments, as agents for the epigenetic management and prevention of cancer., (©2014 American Association for Cancer Research.)

Purpose: We conducted a phase I trial of BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate, Tavocept™), a novel chemoprotective and antitumor enhancing agent administered in combination with paclitaxel and cisplatin. The primary aim was to determine a safe and potentially efficacious BNP7787 dose for preventing and mitigating paclitaxel- and cisplatin-induced toxicities and to evaluate for preliminary evidence of efficacy of treatment., Patients and Methods: Twenty-two patients with stage IIIB/IV non-small cell lung cancer (NSCLC) received BNP7787 alone 1 week before co-administration of BNP7787 with paclitaxel followed by cisplatin. Twenty-one patients were treated with BNP7787 in escalating doses of 4.1-41.0 g/m² concurrently with paclitaxel 175 mg/m² and cisplatin 75 mg/m² every 3 weeks., Results: The appropriate dose was determined to be 18.4 g/m² of BNP7787 although no dose-limiting toxicity was observed up to 41.0 g/m². Mild intravenous site discomfort, thirst, and nausea were the most common toxicities. One patient developed grade 2 skin rash, which was severe enough to preclude further study treatment. The AUC(0-inf) of the metabolite mesna was approximately 6.3% of the AUC(0-inf) of BNP7787. Co-administration of paclitaxel and cisplatin did not appear to influence the pharmacokinetics of BNP7787 and mesna. The overall response rate was encouraging; 43% including 11 patients with prior chemotherapy., Conclusions: The recommended dose for phase II/III studies is 18.4 mg/m² of BNP7787 in combination with paclitaxel and cisplatin. Further studies are warranted to assess whether BNP7787 prevents and mitigates common and serious paclitaxel- and cisplatin-related side effects and enhances the efficacy of paclitaxel and cisplatin in advanced NSCLC patients.

Karenitecin is a highly lipophilic camptothecin analogue with a lactone ring that is relatively resistant to inactivating hydrolysis under physiologic conditions. This phase I clinical trial was conducted to determine the maximum tolerated dose (MTD) of karenitecin in adults with recurrent malignant glioma (MG), to describe the effects of enzyme-inducing antiseizure drugs (EIASDs) on its pharmacokinetics, and to obtain preliminary evidence of activity. Karenitecin was administered intravenously over 60 min daily for 5 consecutive days every 3 weeks to adults with recurrent MG who had no more than one prior chemotherapy regimen. The continual reassessment method was used to escalate doses, beginning at 1.0 mg/m(2)/day, in patients stratified by EIASD use. Treatment was continued until disease progression or treatment-related dose-limiting toxicity (DLT). Plasma pharmacokinetics was determined for the first daily dose of karenitecin. Thirty-two patients (median age, 52 years; median KPS score, 90) were accrued. Seventy-eight percent had glioblastoma, and 22% had anaplastic glioma. DLT was reversible neutropenia or thrombocytopenia. The MTD was 2.0 mg/m(2) in daggerEIASD patients and 1.5 mg/m(2) in -EIASD patients. The mean (+/-SD) total body clearance of karenitecin was 15.9 +/- 9.6 liters/h/m(2) in daggerEIASD patients and 10.2 +/- 3.5 liters/h/m(2) in -EIASD patients (p = 0.02). No objective responses were observed in 11 patients treated at or above the MTD. The total body clearance of karenitecin is significantly enhanced by the concurrent administration of EIASDs. This schedule of karenitecin, a novel lipophilic camptothecin analogue, has little activity in recurrent MG.

Purpose: A phase II trial of the novel camptothecin karenitecin (BNP1350) was conducted to determine its efficacy and tolerability in patients with metastatic melanoma. Patients were biopsied to determine topoisomerase expression at baseline and response to therapy., Patients and Methods: Eligible patients had metastatic melanoma with up to three prior chemotherapy and/or any number of immunotherapy regimens. Treatment consisted of an i.v. infusion of 1 mg/m(2) karenitecin daily for 5 days with cycles repeated every 3 weeks. Fine-needle aspiration biopsies were done before treatment and on day 3 to determine topoisomerase expression from patients' tumors., Results: Forty-three patients were evaluable for response and toxicity. Most patients (72%) had stage M1C disease and were previously exposed to chemotherapy (56%). The investigational agent was well tolerated with limited gastrointestinal side effects or fatigue. The major toxicity seen was reversible noncumulative myelosuppression. One patient had a complete response after 11 months of therapy. No partial responses were seen, but 33% of the patients had disease stabilization lasting > or =3 months. Topoisomerase I, IIalpha, and IIbeta expression and localization were determined in a subset of patients. Topoisomerase I expression was highest, followed by topoisomerase IIbeta and topoisomerase IIalpha., Conclusion: Karenitecin was a well-tolerated investigational agent in this phase II study; side effects were generally mild and mostly hematologic. Karenitecin has significant activity in metastatic melanoma. Melanoma metastases express high levels of topoisomerase I. We did not observe any compensatory increase in topoisomerase II upon treatment with karenitecin.

Purpose: BNP1350 (7-[(2-trimethylsilyl)ethyl]-20(S)-camptothecin, karenitecin), a highly lipophilic camptothecin, a high percentage of which is maintained in the active lactone form under physiologic conditions, has recently entered clinical trials in adults and children. BNP1350 has shown significant preclinical antitumor activity against a wide variety of adult and pediatric tumor cell lines. This study was undertaken to define the pharmacokinetics of BNP1350 in both plasma and cerebrospinal fluid (CSF) in a nonhuman primate model., Methods: Four nonhuman primates with indwelling Ommaya reservoirs received BNP1350, 0.1 mg/kg i.v, administered as a 60-min infusion. Frequent plasma and CSF samples were obtained for quantitation of BNP1350 concentrations using reverse-phase high-pressure liquid chromatography (HPLC)., Results: Disappearance of the lactone form from the plasma was biexponential with a mean distribution half-life of 57.5 min (CV +/-33%) and an elimination half-life of 457 min (CV +/-24%). The volume of distribution for the central compartment was 1.36 l/kg (CV +/-27%) and clearance from the central compartment was 10.6 ml/kg per minute (CV +/-28%). The peripheral compartment volume of distribution was 1.96 l/kg (CV +/-8.4%). Peak CSF lactone concentration, which occurred at 12 to 25 min after the end of the infusion, was 0.33 n M (CV +/-71%)., Conclusions: The ratio of the CSF AUC to the plasma AUC was less than 5% (range 0.4% to 3.0%), similar to other highly protein-bound topoisomerase inhibitors such as 9-aminocamptothecin and SN-38 (the active metabolite of irinotecan).

Purpose: To determine the maximum tolerated dose (MTD) of gamma-methylene-10-deazaaminopterin (MDAM), a unique antifolate structurally similar to methotrexate (MTX), in the treatment of patients with solid tumors and to characterize toxicity and pharmacokinetic profiles of MDAM administered intravenously for five consecutive days repeated every 21 days., Methods: A group of 18 patients with treatment-refractory colorectal cancer (CRC) were given MDAM at increasing dose levels from 80 to 300 mg/m2 per day intravenously for 5 days every 3 weeks., Results: A total of 18 patients were entered into the study. Grade 2 or less nausea, vomiting, diarrhea, anorexia and fatigue were observed at doses > or =160 mg/m2 per day. Both patients enrolled at 300 mg/m2 per day experienced grade 3 stomatitis and one patient had grade 4 granulocytopenia. At 270 mg/m2 per day, grade 3 stomatitis (n=2), thrombocytopenia (n=1) and hyperbilirubinemia (n=1) were observed. All toxicities were relatively brief in duration and reversible. Leucovorin rescue was not required. Of 17 evaluable patients, no complete or partial responses were observed, and 3 patients demonstrated stable disease. Pharmacokinetic analyses were performed in 16 of the 18 patients receiving MDAM at doses of 80, 160, 240, 270 and 300 mg/m2. Normalized clearance of MDAM was approximately 1.5 times that reported for MTX (125 vs 80 ml/min per m2) in adults., Conclusion: MDAM is a novel antifolate with potential pharmacokinetic and safety advantages over MTX. Based on the results of this phase I study, stomatitis emerged as the dose-limiting toxicity and the recommended starting dose for phase II trials using this schedule and route of administration is 240 mg/m2 per day.

Background: BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate) was evaluated in a phase I clinical trial with paclitaxel and cisplatin to assess the safety and potential efficacy for preventing or reducing cisplatin- and paclitaxel-induced toxicities. During this trial the effects of BNP7787 administration on the total concentrations (oxidized plus free) of cysteine, homocysteine and GSH in plasma, free and total GSH in WBC and rate of urinary excretion of cysteine were studied. The pharmacokinetics of ultrafilterable (free, non-protein bound) platinum were also determined after cisplatin (75 mg/m(2)) treatment which followed paclitaxel (175 mg/m(2)) and BNP7787 (8.2 to 27.6 g/m(2))., Methods: Plasma thiols were measured by HPLC with fluorescence detection and platinum was measured by atomic absorption spectrophotometry., Results: BNP7787 administration produced a significant depletion of all plasma thiols in all the patients studied. Differences were noted in the kinetics of BNP7787-induced depletion of cysteine and other thiols. A significant depletion of cysteine occurred with a time lag of about 2 h after the end of BNP7787 infusion, while a reversible depletion of GSH and homocysteine occurred immediately following the start of BNP7787 infusion, with the plasma thiol/disulfide nadir corresponding to the end of infusion. The mean half-life of cysteine depletion following BNP7787 administration was 2.2 h, significantly longer than for homocysteine (0.23 h), or GSH (0.18 h; P<0.05 for both). A several-fold increase in the urinary excretion of cysteine occurred following BNP7787 administration in all patients. The BNP7787-induced thiol/disulfide depletion in plasma was not affected by cisplatin administration ( P>0.05). BNP7787 administration had no effect on the ultrafilterable platinum pharmacokinetics. The 2-h lag in the depletion of cysteine, the most abundant thiol in plasma, suggests that the process may be related to the formation of free mesna from BNP7787 and that increased levels of mesna are not in circulation until after 2 h after BNP7787 administration. No effect of BNP7787 was seen on the GSH concentration in WBC, possibly reflecting the inability of these cells to take up BNP7787., Conclusion: The results suggest that BNP7787 has the potential to enhance cisplatin antitumor activity by depleting the reactive thiols in plasma.

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2 +/- 0.5 min and 8.0 +/- 0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at -70 degrees C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.

Purpose: Camptothecins have emerged as an important new class of antitumor drugs. Camptothecin derivatives such as CPT-11 and topotecan are commercially available and approved for the treatment of colorectal (CPT-11) and ovarian and small-cell lung cancer (topotecan). This study was designed to test the efficacy of karenitecin, a novel highly lipophilic camptothecin derivative, against a panel of human tumor xenografts derived from adult and pediatric central nervous system malignancies growing in athymic nude mice., Methods: Xenografts evaluated were derived from childhood high-grade gliomas (D-212 MG, D-456 MG), adult high-grade gliomas (D-54 MG, D-245 MG), medulloblastomas (D-341 MED, D-487 MED), and ependymomas (D-528 EP, D-612 EP), as well as sublines with demonstrated resistance to procarbazine (D-245 MG (PR)) and busulfan (D-456 (BR)). In replicate experiments, karenitecin was given at 1.0 mg/kg per dose via intraperitoneal injection for a period of 10 consecutive days, which is the dosage lethal to 10% of treated animals., Results: Karenitecin produced statistically significant (P < or = 0.001) growth delays in all subcutaneous xenografts tested, including the sublines resistant to procarbazine and busulfan. Growth delays ranged from 12.1 days in D-456 MG (BR) to 90+ days in D-212 MG and D-341 MED. Karenitecin also produced statistically significant (P < or = 0.001) increases in survival of animals bearing D-341 MED intracranial xenografts (69% increase) and those bearing D-456 MG xenografts (62% increase)., Conclusion: These preclinical studies confirm that karenitecin is active against human central nervous system xenografts and should undergo clinical evaluation in patients with malignant central nervous system tumors.

In a patient with advanced cancer, neurosurgery relieved severe pain that had been uncontrolled by very high doses of opioids. On reduction of opioid dosage he had visual and auditory hallucinations and showed deterioration of consciousness progressing to coma. Naloxone worsened his mental state whereas morphine restored it to normal. This syndrome may be related to opioid withdrawal at receptors other than the mu receptors that have been linked to analgesia and drug abuse.