Stephen T. Ingalls

Development of β-Lapachone Prodrugs for Therapy Against Human Cancer Cells with Elevated NAD(P)H:Quinone Oxidoreductase 1 Levels

β-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces β-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to β-lapachone. Thus, β-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non–small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of β-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to β-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to β-lapachone. Once converted, β-lapachone derivatives caused NQO1-dependent, μ-calpain-mediated cell death in human cancer cells identical to that caused by β-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect β-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of β-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to β-lapachone. The use of β-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

Quantification of carnitine and acylcarnitines in biological matrices by HPLC electrospray ionization-mass spectrometry.

BACKGROUND Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished. METHODS Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine. RESULTS Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided. CONCLUSIONS This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.

Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue.

A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: (1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1M potassium phosphate, pH 6.7, and (2) purification using 2-(2-pyridyl)ethyl-functionalized silica gel. Recoveries determined by adding radiolabeled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-, or arachidonyl-coenzyme A to powdered rat liver varied 93-104% for tissue extraction and 83-90% for solid-phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters differing widely in chain length and saturation.

Method for isolation of non-esterified fatty acids and several other classes of plasma lipids by column chromatography on silica gel

A method is described for isolation from human plasma of non-esterified fatty acids, cholesteryl esters, triglycerides, cholesterol and diglycerides, monoglycerides, and some phospholipids by extraction and silica gel column chromatography. All of these lipid classes except diglycerides and cholesterol were separated cleanly in seven elution steps. Diglycerides and cholesterol were isolated together. Recovery of model compounds which represent the most significant classes of plasma lipids during the column chromatographic step was nearly complete. The overall recovery of added heptadecanoic acid from plasma specimens was 81% after both sample isolation steps. The overall recovery of added synthetic pentadecanoic acid and heptadecanoic acid ester lipid homologues from plasma was 80-91% after both sample preparation steps. About 6 h are required for extraction and isolation in duplicate of these lipid classes from twenty plasma specimens. Alternatively, non-esterified fatty acids can be isolated from twenty plasma specimens in duplicate within 4 h by a variation of the full procedure.

Phase I Clinical and Pharmacokinetic Study of Rebeccamycin Analog NSC 655649 Given Daily for Five Consecutive Days

PURPOSE Rebeccamycin analog (NSC 655649) is active against a variety of both solid and nonsolid tumor cell lines. We performed a phase I trial to determine the maximum-tolerated dose (MTD) of rebeccamycin analog when given on a daily x 5 schedule repeated every 3 weeks, characterize the toxicity profile using this schedule, observe patients for antitumor response, and determine the pharmacokinetics of the agent and pharmacodynamic interactions. PATIENTS AND METHODS Thirty assessable patients received a total of 153 cycles according to the following dose escalation schema: 60, 80, 106, 141, and 188 mg/m(2)/d x 5 days. RESULTS Grade 2 phlebitis occurred in all patients before the use of central venous access, placed at dose level 4 and higher. Dose-limiting toxicity (DLT), grade 4 neutropenia, occurred at 188 mg/m(2)/d x 5 days in both previously treated and chemotherapy-naive patients. Pharmacokinetic analysis revealed a three-compartmental model of drug elimination and a long terminal half-life (154 +/- 55 hours). The percentage drop in absolute neutrophil count correlates with the area under the curve infinity. The presence of a second peak during the elimination phase as well as a high concentration of NSC 655649 in biliary fluid compared with the corresponding plasma measurement (one patient) is suggestive of enterohepatic circulation. Two partial responses, two minor responses, and six prolonged (> 6 months) cases of stable disease were observed. Of these, three patients with gallbladder cancer and one patient with cholangiocarcinoma experienced either a minor response or a significant period of freedom from progression. CONCLUSION The recommended phase II dose for NSC 665649 on a daily x 5 every 3 weeks schedule is 141 and 165 mg/m(2)/d for patients with prior and no prior therapy, respectively, with DLT being neutropenia. During this phase I trial, encouraging antitumor activity was been observed.

A Phase I and Pharmacodynamic Study of Fludarabine, Carboplatin, and Topotecan in Patients With Relapsed, Refractory, or High-Risk Acute Leukemia

Purpose: A novel regimen designed to maximize antileukemia activity of carboplatin through inhibiting repair of platinum-DNA adducts was conducted in poor prognosis, acute leukemia patients. Experimental Design: Patients received fludarabine (10 to 15 mg/m2 × 5 days), carboplatin (area under the curve 10 to 12 by continuous infusion over 5 days), followed by escalated doses of topotecan infused over 72 hours (fludarabine, carboplatin, topotecan regimen). Twenty-eight patients had acute myelogenous leukemia (7 untreated secondary acute myelogenous leukemia, 11 in first relapse, and 10 in second relapse or refractory), 1 patient had refractory/relapsed acute lymphoblastic leukemia, and 2 patients had untreated chronic myelogenous leukemia blast crisis. Six patients had failed an autologous stem cell transplant. Patients ranged from 19 to 76 (median 54) years. Measurement of platinum-DNA adducts were done in serial bone marrow specimens. Results: Fifteen of 31 patients achieved bone marrow aplasia. Clinical responses included 2 complete response, 4 complete response with persistent thrombocytopenia, and 2 partial response. Prolonged myelosuppression was observed with median time to blood neutrophils ≥200/μl of 28 (0 to 43) days and time to platelets ≥20,000/μl (untransfused) of 40 (24 to 120) days. Grade 3 or greater infections occurred in all of the patients, and there were 2 infection-related deaths. The nonhematologic toxicity profile was acceptable. Five patients subsequently received allografts without early transplant-related mortality. Maximum tolerated dose of fludarabine, carboplatin, topotecan regimen was fludarabine 15 mg/m2 × 5, carboplatin area under the curve 12, and topotecan 2.55 mg/m2 over 72 hours. An increase in bone marrow, platinum-DNA adduct formation between the end of carboplatin infusion and 48 hours after the infusion correlated with bone marrow response. Conclusions: Fludarabine, carboplatin, topotecan regimen is a promising treatment based on potential pharmacodynamic interactions, which merits additional study in poor prognosis, acute leukemia patients.

Validated Method for the Quantification of Free and Total Carnitine, Butyrobetaine, and Acylcarnitines in Biological Samples

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.

Determination of plasma non-esterified fatty acids and triglyceride fatty acids by gas chromatography of their methyl esters after isolation by column chromatography on silica gel

Non-esterified fatty acids (NEFA) and triglycerides were isolated from human plasma by column chromatography on silica gel. Eight principal fatty acids of each of these lipid classes were determined by gas chromatography of their methyl ester derivatives and quantified relative to multipoint standard curves. Within-day relative standard deviations for plasma non-esterified fatty acid and triglyceride fatty acid determinations were 2.4 and 3.2%, respectively. Day-to-day relative standard deviations for plasma non-esterified fatty acid and triglyceride fatty acid determinations were 1.4 and 1.1%, respectively. The total plasma concentration and the relative proportions of the eight non-esterified fatty acids determined by this method were significantly different from results obtained according to two generally accepted methods for direct plasma non-esterified fatty acid determination without a specific isolation step. These comparisons suggested that considerable fatty acid ester lipid hydrolysis occurred during these direct determination procedures, and that this hydrolysis resulted in 3-fold overestimation of plasma NEFA content by those methods. Measured levels of arachidonic acid are substantially overestimated by these direct determination methods in which non-esterified fatty acids are not isolated before derivatization.

Management of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone-induced methemoglobinemia.

The anticancer agent 3-aminopyridine-2-carboxaldehyde thiosemicarbazone is a ribonucleotide reductase inhibitor. It inactivates ribonucleotide reductase by disrupting an iron-stabilized radical in ribonucleotide reductases small subunits, M2 and M2b (p53R2). Unfortunately, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone also alters iron II (Fe(2+)) in hemoglobin. This creates Fe(3+) methemoglobin that does not deliver oxygen. Fe(2+) in hemoglobin normally auto-oxidizes to inactive Fe(3+) methemoglobin at a rate of nearly 3% per day and this is counterbalanced by a reductase system that normally limits methemoglobin concentrations to less than 1% of hemoglobin. This balance may be perturbed by symptomatic toxicity levels during 3-aminopyridine-2-carboxaldehyde thiosemicarbazone therapy. Indications of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone sequelae attributable to methemoglobinemia include resting dyspnea, headaches and altered cognition. Management of methemoglobinemia includes supplemental oxygen, ascorbate and, most importantly, intravenously administered methylene blue as a therapeutic antidote.

Pharmacokinetics of O6-benzylguanine (NSC637037) and its metabolite, 8-oxo-O6-benzylguanine.

O6‐Benzylguanine and its metabolite, 8‐oxo‐O6‐benzylguanine, are equally potent inhibitors of the DNA repair enzyme, O6‐alkylguanine‐DNA alkyltransferase. Pharmacokinetic values are derived from cancer patients participating in a phase I trial (10 or 20 mg/m2 of O6‐benzylguanine in a single bolus dose or 10 to 120 mg/m2 as a 60‐min constant infusion). A two‐compartment model fits the plasma concentration versus time profile of O6‐benzylguanine. O6‐Benzylguanine is eliminated rapidly from the plasma compartment in humans (t1/2α and t1/2β are 2 ± 2 min and 26 ± 15 min [mean ± SD, n = 7], respectively), and its plasma clearance (513 ± 148 mL/min/m2) is not dose dependent. Metabolite kinetics are evaluated using both a novel approach describing the relationship between O6‐benzylguanine and 8‐oxo‐O6‐benzylguanine and classical metabolite kinetics methods. With increasing doses of O6‐benzylguanine, the plasma clearance of 8‐oxo‐O6‐benzylguanine decreases, prolonging elimination of the metabolite. This effect is not altered by coadministration of BCNU. The urinary excretion of drug and metabolites is minimal.