A high-performance liquid chromatography-tandem mass spectrometry method for the clinical combination study of carboplatin and anti-tumor agent eribulin mesylate (E7389) in human plasma.

An LC/MS/MS method was developed to quantify carboplatin and eribulin mesylate (E7389) in human plasma and urine. For carboplatin, sample clean-up by protein precipitation and supernatant injection into a Waters Spherisorb((R)) S5 SCX column was used. Liquid-phase extraction and reverse-phase chromatography on a Polaris C18 column were used for eribulin. Quantitation involved LC/MS/MS with positive electrospray ionization. Accuracy, precision, linearity, range, specificity, recovery and stability were also evaluated. Both compounds were stable in human plasma (>or=80 days at -80 degrees C), at room temperature (>or=4h), following three freeze-thaw cycles and in 50/50 methanol/H(2)O (<4 degrees C for >or=252 days).

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.

Mass spectral analyses of labile DOTA-NHS and heterogeneity determination of DOTA or DM1 conjugated anti-PSMA antibody for prostate cancer therapy.

Prostate specific membrane antigen (PSMA) is a well-characterized glycoprotein overexpressed on the surface of prostate cancer cells. The novel radiopharmaceutical 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) radiolabeled with Yttrium (90Y) or Indium (111In) conjugated with anti-PSMA genetically engineered humanized monoclonal antibody (huJ591) has been investigated to target prostate cancer cells. The immunoconjugate of huJ591 with the analog of the cytotoxic drug maytansine, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) has also been developed at Millennium Pharmaceuticals. Activation of the DOTA molecule, resulting in 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS), allows conjugation with the anti-PSMA antibody through lysine residues in the antibody. The objectives of the study were to characterize the unstable chemical properties of DOTA-NHS before bioconjugation with huJ591, evaluate the binding profiles of DOTA to huJ591, and calculate trace metal elements (which may disturb 90Y or 111In labeling efficacy to the DOTA-huJ591 conjugate). A novel LC/MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry) quantitation method was developed to monitor the stability of DOTA-NHS in solid form and its bioconjugation chemistry reactions. Meanwhile, metal analysis was quantified by Inductively Coupled Plasma Mass Spectrometry (ICP/MS) to estimate the amounts of trace metals in DOTA-NHS and ensure radiolabeling efficiency of the conjugate at the radiopharmacy. MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry) was used to identify levels of DOTA or DM1conjugation in DOTA-huJ591 and DM1-huJ591 conjugates, respectively.