Screening method for 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid in urine using hollow fiber membrane solvent microextraction with in-tube derivatization.

An efficient and inexpensive screening technique for the simultaneous clean-up, extraction, and derivatization of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from urine has been developed. Using the principles of solvent microextraction in the form of a 20-microl volume of solvent placed inside a permeable hydrophobic polypropylene hollow fiber membrane, the analyte of interest is preconcentrated inside this tubing as the bulk sample solution is stirred for a given extraction time. The pH of the sample is raised by adding buffer after which the charged moiety is extracted as an ion pair with tetramethylammonium hydrogen sulfate. Using a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and octane as the extraction solvent allows the GC-unstable carboxylic acid metabolite to be derivatized during the extraction without prior sample clean-up steps such as filtration of the urine. After an 8-min extraction, a 6-microl portion is drawn up with a syringe and directly injected into a gas chromatograph for separation and analysis. Samples as low as 10 ng ml(-1) were analyzed successfully and the limit of detection was estimated at 1.0 ng ml(-1) with relative standard deviations lower than 10% in the final protocol.

Exogenous ochronosis.

Ochronosis is an uncommon condition characterized by yellow-brown pigment deposits in the dermis. It occurs in exogenous and endogenous forms. We report a case of exogenous ochronosis in a Hispanic woman and discuss the different forms of this condition. We treated the lesions with Q-switched ruby laser.

Novel agents effective against solid tumors: the diarylsulfonylureas. Synthesis, activities, and analysis of quantitative structure-activity relationships.

A series of diarylsulfonylureas with exceptionally broad-spectrum activity against syngeneic rodent solid tumors in vivo is described. Their discovery resulted from a program dedicated to in vivo screening for novel oncolytics in solid tumor models, rather than traditional ascites leukemia models. The structures, oral efficacy, side-effect profile, and mechanism of action of these sulfonylureas appear to be distinct from previously known classes of oncolytics. An extensive series of analogues was prepared to probe structure-activity relationships (SAR), with particular focus on the substituent patterns of each aryl domain. Quantitative analysis of these substituent SARs, using the method of cluster significance analysis, showed the lipophilicity of the substituents to be the dominant determinant of activity. One compound from the series, LY186641 (104, sulofenur), has progressed to Phase I clinical trials as an antitumor drug.

Effect of host age upon interleukin-2-mediated anti-tumor responses in a murine fibrosarcoma model.

The age-associated decline in immune function may be an important factor in both the pathogenesis of neoplastic diseases and the response to immunopharmacological therapies. With the increased efforts to develop immunotherapy with such agents as interferon and interleukin-2 (IL-2), the question of the effect of host age upon response is of practical importance. Phase I and phase II clinical trials of IL-2 have included primarily young patients, and toxicity and efficacy have not been reported with specific reference to host age. In this study, we examined young and old mice with regard to in vitro natural killer and lymphokine-activated killer (LAK) cell functions. We also assessed the effects of exogenously administered recombinant human IL-2 in tumor-bearing mice of various ages. We found that natural killer cell function was demonstrably lower in old mice but that LAK cell function was comparable (young versus old). Furthermore, IL-2 treatment was successful in increasing survival time in old mice, similar to results in young mice. Our observations allow the prediction that immune senescence per se does not preclude successful anti-neoplastic treatment with IL-2.

Immunologic enhancement of B16 melanoma growth.

In certain experimental tumor models, tumor growth is less pronounced in immune deficient animals. Characteristically, tumors such as the murine B16 melanoma and Lewis lung carcinoma (3LL) are weakly antigenic. We proposed that with such tumors that are weakly antigenic, growth is enhanced by T-cell factors. Young mice were inoculated with irradiated B16 cells in complete Freunds adjuvant (CFA) on three occasions, each separated by 2 weeks. Specific antibody (IgG) to B16 membrane antigens was detected by an enzyme-linked immunosorbent assay (ELISA) after the first injection, and it continued to rise for 6 weeks. B16 growth was compared in 20 mice that had received irradiated B16 in CFA or CFA alone by the same schedule previously. Despite the previous sensitization, the rates of tumor appearance and growth were similar. In an additional experiment involving 23 mice that had received B16 immunization, the period of time in which a palpable tumor developed after the injection of viable B16 cells did not correlate with anti-B16 antibody level. It appeared that detectable antibody to B16 antigens was of little consequence. To explain why B16 primary growth and metastases were reduced in immune deficient hosts, we proposed that lymphocytes might enhance tumor growth. To demonstrate this, splenic lymphocytes from tumor-bearing (B16 or 3LL) or control mice were injected with B16 cells into young, immune competent hosts. Tumors (B16) developed earlier and growth was more rapid in mice that received spleen cells from tumor-bearing (B16) mice. Subsequent cell depletion experiments to determine the mediator of tumor enhancement implicated a T-cell fraction that was neither of T-helper nor T-suppressor cell type phenotypically. Immune deficiency states that are associated with dysfunction of those cells that account for tumor enhancement might explain the reduced tumor aggressiveness that is observed frequently in these conditions.