Pharmacokinetic and metabolism studies using uniformly stable isotope labeled proteins with HPLC/CRIMS detection.

We present a novel method for performing pharmacokinetic and metabolism studies on macromolecules that offers advantages over the existing techniques of radiolabeling, immunoassay or bioassays. Our strategy uses macromolecules with stable isotopes uniformly distributed throughout the structure. The stable isotope enrichment is detected using high performance liquid chromatography combined with chemical reaction interface mass spectrometry (HPLC/CRIMS). HPLC/CRIMS is a technique where analytes are first eluted from an HPLC column and then dissociated in a microwave reaction chamber. The dissociated analytes are oxidized using SO2 and the resulting small molecules are detected by the mass spectrometer. The stable-isotope labeled analyte is distinguished from the matrix carbon by monitoring the enrichment of 13CO2. In order to demonstrate the feasibility of performing pharmacokinetic and metabolism studies using this technique, uniformly 13C, 15N-labeled rat growth hormone was administered intravenously to rats and blood samples were collected. Raw plasma samples were analysed by HPLC/CRIMS. Growth hormone was detectable for 1 h following administration. The absolute amounts detected ranged from a high of 66 pmol to a low of 825 fmol in a 20 microL plasma sample. The data were modeled using PCNONLIN and were consistent with a one compartment model. The calculated half-life was 7.7 +/- 0.7 min, with a clearance of 4.5 +/- 0.3 mL min(-1), values consistent with literature reports for growth hormone in rats. No circulating growth hormone metabolites were detected in the plasma. This paper demonstrates a novel technique for performing pharmacokinetic studies of proteins. The uniform labeling strategy also presents a viable comprehensive method for obtaining metabolism data on macromolecules.

Isotopic differences in human growth hormone preparations.

The 13C/12C isotope ratios have been measured for human pituitary growth hormone and three commercial growth hormone products in an attempt to differentiate endogenous versus exogenous origin. This might be a strategy to detect doping, as has recently been recognized for testosterone. While all preparations are statistically different from each other, we find that only Humatrope from Lilly has a carbon isotope ratio that is markedly different from those of human growth hormone or Genentech's Nutropin and Protropin. The low renal clearance of growth hormone reduces the applicability of this concept.

Quantitation of human plasma levels of the anticancer agent carboxyamidotriazole by high-performance liquid chromatography.

The predominant cause of death of cancer patients is growth and metastasis of their tumors. By targeting signal transduction pathways as sites of therapeutic intervention, we have identified a novel anticancer drug carboxyamidotriazole (CAI). A straight-forward and reliable method of detection and quantitation of human CAI plasma levels using solid-phase organic extraction followed by isocratic reversed-phase chromatography is now reported. This assay detected CAI over the concentration range 0.04-10.0 micrograms/ml, which brackets the range shown to be physiologically and biochemically effective. Linearity was demonstrated by linear regression analysis of calibration curves (r2 = 0.999). Equivalence of recovery of extracted versus non-extracted CAI over a broad concentration range was demonstrated (r2 = 0.998, coefficients of variability < 10%). The method was applied to quantitate CAI plasma levels from patients now entered on the Phase I clinical trial underway at the National Cancer Institute.

Chloroquinoxaline sulfonamide: a sulfanilamide antitumor agent entering clinical trials.

Chloroquinoxaline sulfonamide (CQS) has been developed to the clinical trial stage based on its activity in the Human Tumor Colony Forming Assay (HTCFA). In the HTCFA, CQS demonstrated inhibition of colony formation against breast, lung, melanoma and ovarian carcinomas. The mechanism of action of CQS is unknown. It does not appear to inhibit folate metabolism as does the structurally similar sulfaquinoxaline. Preclinical toxicology studies in dogs and rats have shown that CQS is toxic to lymphoid organs, bone marrow, gastrointestinal tract, pancreas, CNS, adrenal glands and testes. Toxicity was generally reversible with the exception of testicular atrophy in dogs and rats which occurred late and was not reversible within the study time frame. The pharmacokinetic data indicate that CQS binds to serum proteins in a dose and species specific manner. Terminal half-lives appear to vary between species from 60 hours in mice, 15 hours in rats, and 45-132 hours in dogs. Preliminary data indicate a longer terminal half-life in humans. Two phase I trials are ongoing using a 60 min infusion schedule once every 28 days. The starting dose for each trial was 18 mg/m2.