Charles J. Decedue

A colorimetric assay of pancreatic lipase: rapid detection of lipase and colipase separated by gel filtration

A rapid assay for pancreatic lipase (E.C., glycerol-ester hydrolase 3.1.1.3) is described. The assay is based on the color change of a pH indicator as butyric acid is released from the substrate tributyrin. A mixture made with tributyrin and the water soluble components of the assay is ideally suited for use as a rapid test as, for example, when assaying chromatography fractions. Quantitative data can be obtained by measuring the disappearance of absorbance at 557 nm versus a blank reaction. The assay has been used in the rapid preparation of colipase-free lipase and colipase.

A synaptic membrane glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein: isolation and immunochemical characterization

Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamate/N-methyl-D-aspartate (NMDA) receptors, were used previously to clone a cDNA for a glycine-, glutamate-, and thienylcyclohexylpirperidine (TCP)-binding protein, pGlyBP (Kumar et al., Biochem. Biophys. Res. Commun. 216, 390-398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relatively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55-60 kDa protein was purified from rat brain synaptic membranes by chromatographic separation through matrices derivatized with 5,7-di-chlorokynurenic acid (5,7-DCK) followed by chromatography on a matrix derivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [3H]glycine, NMDA- and glutamate-sensitive L-[3H]glutamate, and MK-801-sensitive [3H]TCP binding sites. The purified protein bound [3H]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (KD = 280 nM) and low affinity (KD = 30 microM) glycine binding sites. Glycine binding was inhibited by D-serine and R-(+)-3-amino-1-hydroxypyrrolidin-2-one(R-(+)-HA-966). The KD values for high and low affinity sites of glycine binding as well as those for the inhibition by R-(+)-HA-966 were very similar to the KDs for glycine binding to the expressed pGlyBP. Both L-glutamate and glycine activated [3H]TCP binding to the isolated proteins, but with relatively low affinity. The anti-43 kDa antibodies reacted strongly with the 55-60 kDa protein. Based on these results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cloned pGlyBP.

Response of HDL Cholesterol, Apoprotein A-I, and LCAT to Exercise Withdrawal

The effect of short-term exercise withdrawal on plasma lipoproteins, apoprotein A-I (Apo A-I), and lecithin:cholesterol acyltransferase (LCAT) was studied in moderately trained lifestyle exercisers. Eight endurance-trained men, age 18-45 years (means = 29 years), withdrew from aerobic activity for 6 weeks, while an age and fitness-matched control group (n = 9) maintained normal exercise habits. A baseline period that included two blood samplings preceded the detraining intervention. Plasma total cholesterol (TCHOL), HDL cholesterol (HDL-C) and triglyceride (TG) levels were determined weekly. Other blood variables (HDL2-C, HDL3-C, Apo A-I, and LCAT), % fat, and aerobic capacity (VO2max) were measured pre-, mid-, and post-experiment. A two-way repeated measures analysis of variance (ANOVA) indicated that the 6-week exercise withdrawal period failed to elicit significant mean changes in any blood variable, % fat, or VO2max. Therefore, a short-term layoff from aerobic activity by moderately trained, chronic exercisers generally does not adversely affect the blood lipoprotein profile or aerobic capacity.

Phase I dose escalation safety study of nanoparticulate paclitaxel (CTI 52010) in normal dogs

Background Paclitaxel is highly effective in the treatment of many cancers in humans, but cannot be routinely used in dogs as currently formulated due to the exquisite sensitivity of this species to surfactant-solubilizing agents. CTI 52010 is a formulation of nanoparticulate paclitaxel consisting of drug and normal saline. Our objectives were to determine the maximally tolerated dose, dose-limiting toxicities, and pharmacokinetics of CTI 52010 administered intravenously to normal dogs. Methods Three normal adult hound dogs were evaluated by physical examination, complete blood count, chemistry profile, and urinalysis. Dogs were treated with staggered escalating dosages of CTI 52010 with a 28-day washout. All dogs were treated with a starting dosage of 40 mg/m2, and subsequent dosages were escalated at 50% (dog 1), 100% (dog 2), or 200% (dog 3) with each cycle, to a maximum of 240 mg/m2. Dogs were monitored by daily physical assessment and weekly laboratory evaluation. Standard criteria were used to grade adverse events. Plasma was collected at regular intervals to determine pharmacokinetics. Dogs were euthanized humanely, and necropsy was performed one week after the last treatment. Results The dose-limiting toxicity was grade 4 neutropenia and the maximum tolerated dosage was 120 mg/m2. Grade 1–2 gastrointestinal toxicity was noted at higher dosages. Upon post mortem evaluation, no evidence of organ (liver, kidney, spleen) toxicity was noted. Conclusion CTI 52010 was well tolerated when administered intravenously to normal dogs. A starting dosage for a Phase I/II trial in tumor-bearing dogs is 80 mg/m2.

Immobilization of ascorbic acid oxidase

Ascorbic acid has long been recognized as an important vitamin [1], and has been shown to exist in high concentrations in the central nervous system of small animals and man. The regional distribution of AA in rat [2] and human [3] brain has been reported. There appear to be homeostatic mechanisms for controlling AA levels, as well as an active uptake mechanism [4]. This, in addition to the many newly reported effects of AA on neuronal tissue, of which just a few are listed, suggests an important role(s) for this molecule in the functioning of mammalian brain [5-7]. Ascorbic acid oxidase (EC 1.10.3.3, AAO) has become a useful tool in the manipulation of [AA] in neuronal tissue preparations [8,9] for the purpose of gaining information on AA directly, as well as removing it as an interference in the electrochemical determination of catecholamine neurotransmitters, serotonin, and their key metabolites. With the intention of obtaining a form of this extremely useful enzyme suitable for use in flowing stream analyses (for example, direct electrochemical analysis of neuronal tissue perfusates), we have immobilized AAO onto Sepharose 4B activated by cyanogen bromide (CNBr). In [10] the enzyme was immobilized in an enzyme electrode configuration; however, this method is unsuitable for the type of flowing stream applications inten-

Molecular weight differences among potato peroxidases

Abstract The isoenzyme of potato peroxidase A5 has MW 105 000; C3—94 000; C4 and C5—56 500 C6—48 500. The isoenzymes retain activity on SDS-gels thereby allowing direct measurement of monomeric MW, even in crude extracts. One of the isoperoxidases showed anomalous behaviour on SDS-electrophoresis.

Evaluation of intravenous and subcutaneous administration of a novel, excipient-free, nanoparticulate formulation of paclitaxel in dogs with spontaneously occurring neoplasia

Carriers used to solubilize taxane chemotherapy drugs cause severe hypersensitivity. Nanoparticle formulations can provide improved dissolution and bioavailability of taxanes. Thus, a nanoparticulate, excipient-free formulation of paclitaxel (CTI52010) was evaluated in tumour-bearing dogs with intravenous and subcutaneous delivery. Tumour-bearing dogs were treated with intravenous CTI52010 using a modified rapid dose escalation scheme. Subcutaneous administration was then planned for a small cohort of dogs for comparison. For both groups, serial blood samples were collected after first dosing for pharmacokinetic analysis by LCMSMS. Tumour response was measured using RECIST criteria. Toxicity was recorded using VCOG-CTCAEv1.1. Fifteen dogs were treated with intravenous delivery at increasing dosages (80-136 mg/m2 ), with one objective response in the urethral component of a prostatic carcinoma (probable transitional cell carcinoma) and four dogs with durable stable disease (two carcinomas, two sarcomas). Pharmacokinetic data indicate a rapid initial clearing of the drug from serum followed by an extended elimination half-life, similar to normal dogs and suggesting reticuloendothelial clearance. Parameters and toxicity were highly variable and a maximally tolerated dosage could not be reliably confirmed. Three dogs were treated with subcutaneous delivery and no drug was detected in circulation, resulting in termination of the study. This novel formulation of paclitaxel is well tolerated in dogs and no unique toxicity or hypersensitivity was noted. The preliminary responses suggest biologic activity. The lack of systemic absorption after subcutaneous administration suggests a possible role for intratumoural anticancer therapy.