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Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide–amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy. © 2001 Wiley-Liss, Inc.

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.

5-Ethyl-5-(4-hydroxy-3-methoxyphenyl) barbituric acid was identified as a new, minor metabolite of phenobarbital in man. The identity of this O-methylcatechol metabolite was confirmed by an unequivocal chemical synthesis, and by GC—MS studies. Mephobarbital and the 1,3-dimethyl, 1-ethyl, and 1,3-diethyl analogues of phenobarbital yielded the corresponding N-alkylated O-methylcatechol metabolites, all of which were confirmed by synthesis. The N-alkyl barbiturates each gave additionally at least one O-methylcatechol metabolite in which N-dealkylation had occurred. These metabolites accounted for ∼1–5% of the orally administered dose in man.

2-Methoxyestradiol (2ME2, Panzem®) is an endogenous metabolite of estradiol with antiproliferative, proapoptotic, and antiangiogenic activity. Some of these activities are mediated through the downregulation of HIF1-α. Panzem® capsules have been evaluated in several oncology clinical trials, including multiple myeloma where some patients have been on therapy for more than 4 years with stable disease. A new formulation of 2ME2 with enhanced absorption, Panzem® NCD, is currently in Phase 1 and 2 clinical studies in oncology. In anticipation of combination studies with Panzem® NCD, we evaluated the effectiveness of 2ME2 alone or with the proteosome inhibitor, Velcade® (bortezomib), in the RPMI-8226 myeloma xenograft tumor model. Preliminary in vitro studies indicated significantly increased antiproliferative activity when 2ME2 (1.5μM) was combined with Velcade® (5nM) compared to Velcade® alone (p<0.05 ANOVA). Additionally, 2ME2 in combination with Velcade® also resulted in decreased nuclear NFκB in response to TNFα, as compared to either agent alone. Subsequent to these studies, female CB17 SCID mice were injected subcutaneously with 1 x 106RPMI-8226 tumor cells. Starting 10 days after tumor cell inoculation when mean tumor volume was approximately 100–200 mm3, cohorts of mice (n=8/group) began treatment with (a) vehicle alone ad libitumin the drinking water; (b) 2ME2 300 mg/kg ad libitumin the drinking water; (c) Velcade® 1.5 mg/kg i.p., biw; or (d) 2ME2 (300 mg/kg) in combination with Velcade® (1.5 mg/kg). On study day 27, mean tumor volume in vehicle-treated, control mice was approximately 3400 mm3. Under these experimental conditions, treatment with either 2ME2 alone or Velcade® alone generated a treatment/control (T/C) value of 0.66 and 0.64, respectively. In comparison, 2ME2 in combination with Velcade® resulted in a T/C value of 0.35. Velcade® is a well recognized and commonly used antineoplastic agent in multiple myeloma. The degradation of HIF1-α is accomplished through the proteosome. These studies demonstrate that a combination strategy using Velcade® and 2ME2 can enhance the antitumor effectiveness of either agent alone in the treatment of a xenograft model of human multiple myeloma.