Philip L. Stetson

Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.

The paraoxonase gene family contains at least three members: PON1, PON2, andPON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g.lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.

Improved regional selectivity of hepatic arterial mitomycin by starch microspheres

Biodegradable starch microspheres, 40 μm in diameter, were administered through hepatic arterial catheters in 16 subjects with primary and metastatic liver tumors. These microspheres temporarily obstruct blood flow at the precapillary arteriole (microcirculation) level. Our study was undertaken to determine whether such occlusion would enhance hepatic deposition of, and thereby decrease systemic exposure to, simultaneously administered hepatic arterial mitomycin C (mito). When mito (10 mg/m2 over 1 min) was given with 90 × 106 microspheres (10 subjects), there was a 17% to 70% reduction in systemic mito exposure. When mito (10 mg/m2 over 1 min) was given with 36 × 106 microspheres (six subjects), there was a 15% to 60% reduction in systemic exposure, which may correlate with dose‐dependent shunting (8% to 29%) through the liver to the lung (and hence to the systemic circulation), attributed to the starch microspheres. No life‐threatening myelosuppression was noted; hepatic toxicity consisted of transient pain and elevation of liver enzymes.

THE DEPENDENCE OF HALOGENATED PYRIMIDINE INCORPORATION AND RADIOSENSITIZATION ON THE DURATION OF DRUG EXPOSURE

The influence of the duration of exposure to the halogenated pyrimidines iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) on incorporation into DNA and the resulting radiosensitization was studied in cultured human colon cancer cells. Cells were incubated with either 10 microM BrdUrd or IdUrd for periods up to 7 days. They were also assessed for up to 4 days after removal of drug from the medium. Replacement of thymidine by fraudulent bases was measured using a sensitive gas chromatographic, mass spectrometric (GC/MS) assay. Incorporation of BrdUrd and IdUrd plateaued at 35% and 30%, respectively, after 4 days of exposure. Prolonging the time of exposure to 7 days increased cytotoxicity without affecting either incorporation or radiosensitization. Incorporation remained constant for 1-2 days after removal of drug from the medium. Radiosensitization was linearly related to incorporation throughout the range of conditions assessed. These data suggest that it may be possible to develop a predictive assay for radiosensitization based on measurements of halogenated pyrimidine incorporation in a tumor biopsy specimen. They also suggest that a clinical approach based on repeated short exposures to halogenated pyrimidines may present certain advantages over the current practice of prolonged continuous exposure. A Phase I/II trial using IdUrd and external beam irradiation for the treatment of patients with poor prognosis soft tissue sarcomas has been initiated based on this concept.

Steady‐state pharmacokinetics of delavirdine in HIV‐positive patients: Effect on erythromycin breath test

The steady‐state kinetics of delavirdine and desisopropyldelavirdine were evaluated in human immunodeficiency virus‐positive patients after escalating oral doses and after repeated oral administrations at the same dose level.

Intra-arterial bromodeoxyuridine radiosensitization of malignant gliomas

In the 1950s it was first observed that mammalian cells exposed to the halogenated deoxyuridines were more sensitive to ultraviolet light and radiation than untreated cells. This prompted early clinical trials with bromodeoxyuridine (BUdR) which showed mixed results. More recently, several Phase I studies, while establishing the feasibility of continuous intravenous (IV) infusion of BUdR, have reported significant dose limiting skin and bone marrow toxicities and have questioned the optimal method of BUdR delivery. To exploit the high mitotic activity of malignant gliomas relative to surrounding normal brain tissue, we have developed a permanently implantable infusion pump system for safe, continuous intraarterial (IA) internal carotid BUdR delivery. Since July 1985, 23 patients with malignant brain tumors (18 grade 4, 5 grade 3) have been treated in a Phase I clinical trial using IA BUdR (400-600 mg/m2/day X 8 1/2 weeks) and focal external beam radiotherapy (59.4 Gy at 1.8 Gy/day in 6 1/2 weeks). Following initial biopsy/surgery the infusion pump system was implanted; BUdR infusion began 2 weeks prior to and continued throughout the 6 1/2 week course of radiotherapy. There have been no vascular complications. Side-effects in all patients have included varying degrees of anorexia, fatigue, ipsilateral forehead dermatitis, blepharitis, and conjunctivitis. Myelosuppression requiring dose reduction occurred in one patient. An overall Kaplan-Meier estimated median survival of 20 months has been achieved. As in larger controlled series, histologic grade and age are prognostically significant. We have shown in a Phase I study that IA BUdR radiosensitization is safe, tolerable, may lead to improved survival, and appears to be an efficacious primary treatment of malignant gliomas.

Constant intraperitoneal 5-fluorouracil infusion through a totally implanted system.

Five‐day continuous intraperitoneal (ip) infusions of 5‐fluorouracil (FU) were injected into five patients as part of a phase 1 clinical pharmacology study. They received 20 courses through a totally implanted catheter/injection port system. Six courses are evaluable for kinetic parameters and all courses are evaluable for toxicity. In each course a 2 to 3 log FU concentration differential in favor of the peritoneal cavity was achieved and maintained. Steady‐state venous plasma FU concentrations averaged 0.34 µM, whereas steady‐state ip FU concentrations averaged 697 µM. Mean total body clearance (TBC) in these patients was 18.4 l/min and mean permeability‐area (PA) product for diffusion from the peritoneum was 13.7 ml/min. Mean TBC of 20 l/min with ip FU infusion was observed in one patient who also received a 24‐hr IV FU infusion for comparison. The TBC during the later infusion was 5.9 l/min. In this patient, calculations indicate 75% extraction of drug during the passage from the peritoneum to the systemic circulation, presumably representing in large part hepatic extraction of FU taken into the portal venous circulation. Ip constant infusion and bolus kinetics were compared in one patient. TBC for the ip bolus was 14.3 l/min, which was approximately half of the TBC of 29.5 l/min determined during the 5‐day constant ip infusion. Thus constant ip infusion of FU (1 gm/day) can provide an improved regional advantage over bolus ip FU because of an increased TBC. Toxicity was acceptable in all courses. Dose limiting toxicity was regional, namely moderate chemical peritonitis seen in two of the five patients on repeated courses. There was no myelosuppression, alopecia, nausea, or vomiting. There were no infectious complications. The only patient with measurable disease had an objective response in hepatic metastases from gastric cancer. The implanted device was well tolerated and facilitated peritoneal fluid sampling.

The nitroglycerin polymer gel matrix system: a new method for administering nitroglycerin evaluated with plasma nitroglycerin levels.

A new topical dosage form of nitroglycerin has been developed in which nitroglycerin and its vehicle are incorporated in a polymer gel matrix of fixed dimension. Venous plasma nitroglycerin concentrations were measured for 24 h after application of nitroglycerin polymer gel systems measuring 10 cm and containing 2% nitroglycerin (wt/wt) to 10 normal male volunteers. Five of these subjects were subsequently tested with 20-cm2 gel systems of similar composition. With the 10-cm2 nitroglycerin polymer gel system, venous plasma nitroglycerin concentrations were 0.83 ± 0.26 ng/ml at 4 h and 0.89 ± 0.23 ng/ml at 24 h. For the 20-cm2 system, venous plasma nitroglycerin concentrations were 1.83 ± 0.55 ng/ml at 8 h and 1.81 ± 0.13 ng/ml at 24 h. The only adverse effect noted was headache, which affected all the subjects tested with the 20-cm2 appliance. The plasma nitroglycerin concentrations obtained with the polymer gel system appear to be in a clinically useful range: thus this new form of topical nitroglycerin should be of benefit to patients in whom sustained plasma nitroglycerin concentrations are desirable, e.g., those patients with chronic congestive heart failure and angina on awakening.

Gas chromatography-mass spectrometric characteristics and assay of tele-methylhistamine☆

To date, te/e-methylhistamine, the product of enzymatic histamine methylation, has not been measured by a suitable method. Tefe- andpros-methylhistamine both react quantitatively with trifluoroacetic and heptafluorobutyric anhydrides at room temperature under anhydrous conditions to yield either mono- or bis-derivatives in the absence or presence of pyridine, respectively. The extraction of tissue homogenates, de~vati~tion, and analysis by gas chromatography-mass fragmentography permits the detection of I rig/ml tele-methylhistamine, using pros-methylhistamine as the internal standard. Rat brain contains te/emethylhistamine in a concentration similar to that of histamine (42 rig/g). The content of this amine in tissue has been compared with that obtained by other methods.

An assay of deoxyadenosine and adenosine in human plasma by HPLC

Abstract We have developed a method for the simultaneous measurement of deoxyadenosine and adenosine in human plasma. A barium hydroxidezinc sulfate extraction procedure circumvented acid precipitation and preserved the deoxyadenosine. Deoxyadenosine and adenosine were separated using reverse-phase high-pressure liquid chromatography and their identities were confirmed by the enzymatic peak shift technique. A radioactive internal standard was used to measure extraction efficiency and to prove completeness of enzyme treatment for the peak shift technique. This assay is sensitive to 100 n m of either nucleoside in plasma and can be completed within 1 hr. This technique can be used to quantitate plasma deoxyadenosine and adenosine in patients with congenital adenosine deaminase deficiency or in patients being treated with ADA inhibitors.

Analysis of bromodeoxyuridine incorporation into DNA: comparison of gas chromatographic/mass spectrometric, CsCl gradient sedimentation, and specific radioactivity methods.

A sensitive new method for the quantitation of 5-bromodeoxyuridine (BrdUrd) incorporated into DNA by GC/MS analysis of enzymatically released Thy and bromouracil (BrUra) is presented. The hydrolysis procedure was characterized and found to give uniform results when sample size was 1-10 micrograms DNA and incubation time for DNA digestion was between 40 min and 16 h. Samples of DNA containing 3H-labeled BrdUrd were analyzed in parallel by the GC/MS technique and by specific radioactivity and buoyant density measurements, in order to compare the three methods. The GC/MS procedure gave values for percentage replacement of Thy by BrUra which were higher than those obtained by specific activity and lower than those obtained by buoyant density. This GC/MS method can detect 1% replacement in a 1-microgram DNA sample, equivalent to approximately 10(5) cells or 0.1 mg tissue, and will permit sensitive and quantitative analysis of the presence of this chemotherapeutic agent/radiosensitizer in cellular DNA from biopsy samples of normal or tumor tissue.