Lack of methemoglobinemia with flutamide.

OBJECTIVE: To determine whether the nonsteroidal antiandrogenic drug flutamide is a clinically relevant inducer of methemoglobinemia in patients with prostatic cancer. METHODS: Fifty consecutive outpatients with prostatic cancer stage D2 entered the study (age 71.1 +/- 7.3 y). Five patients were lost to follow-up; the remaining 45 patients received the recommended oral dose of flutamide 250 mg three times daily. Total hemoglobin (Hb) and methemoglobin (Met-Hb) concentrations were measured on varying days using an ultraviolet/visible-spectrophotometric method with an intra- and interday variability < 8%. In 12 patients, Met-Hb was analyzed before initiating flutamide therapy and after therapy was begun. RESULTS: On average, 2.6 venous blood samples per patient were analyzed with a mean Met-Hb concentration of 1.9% of total Hb. Mean concentrations of > or = 3% were detected in only six patients (13%). The data from 12 patients evaluated before and after initiating flutamide therapy were without significantly different changes. During the study period, no clinical signs of methemoglobinemia were reported or observed. CONCLUSIONS: This study found no clinically relevant increase of Met-Hb concentrations in elderly patients with prostatic cancer during chronic treatment with flutamide. However, clinicians should be aware of the very rare possibility of flutamide-induced methemoglobinemia.

Purification of estramustine-binding protein and production of monoclonal antibodies to its different components.

Estramustine-binding protein (EMBP) is a heterodimeric 46-kDa glycoprotein that is secreted from the prostate. Upon reductive cleavage it decomposes into two closely related components, C1 and C2, and the shared glycosylated peptide C3. EMBP binds estramustine and estromustine, the active metabolites of estramustine phosphate (Estracyt), which is a drug with antimitotic properties used in the treatment of prostatic carcinoma. In the present study, a two-step procedure (i.e., anion-exchange and Con A-Sepharose chromatography) is described for the isolation of EMBP in high yield from rat prostate tissue. Mouse monoclonal antibodies (mAbs) were produced using the major DEAE-Sepharose fraction of EMBP as an immunogen. Eleven mAbs were selected by screening in a solid-phase ELISA. One displayed high-affinity binding with soluble EMBP (Ka approximately 3 x 10(10) M-1) and crossreacted with a human prostate tumor extract in a radioimmunoassay. The epitopes defined by these mAbs were analyzed by Western immunoblotting. All constituents of EMBP, except component C1, were identified by at least one antibody. Nine visualized either one or both of the two EMBP subunits under denaturing conditions, two of which retained their reactivity after reduction of disulfide bridges. One epitope was exposed to its mAb only when the antigen was in its reduced state. The immunoreactivity was eliminated by protease treatment, whereas deglycosylation with glycopeptidase F had a minimal effect. EMBP has been detected in tissues other than the prostate as well as in prostate neoplastic specimens and in several other human malignancies. Hence, these mAbs will be a useful tool in the characterization of EMBP in different tissues and in evaluating existing and in defining new indications for Estracyt therapy.

Biological activity of prostate-specific antigen isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution.

Human prostate-specific antigen (PSA), a 33 kDa kallikrein-like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate-buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS-PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS-stable complexes with the two major extracellular protease inhibitors in blood, alpha 1-antichymotrypsin (alpha 1-ACH) and alpha 2-macroglobulin (alpha 2-M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors alpha 1-ACH and alpha 2-M.

Monoclonal antibody to the prostate specific antigen.

Somatic cell hybrids were made from mouse myeloma cells and spleen cells derived from BALB/c mice immunized with homogenized epithelial fractions of BPH. The screening by immunoperoxidase staining on human prostate and non-prostate tissue resulted in one monoclonal antibody identifying a prostate specific antigen. Upon SDS-PAGE and Western blot this antigen exhibited a single band at the position of 34 kDa molecular weight. The immunoreactivity of the prostate antigen was found to be localized exclusively in the epithelial lining of ducts and secretions of normal prostate, BPH and prostate cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule and inhibited the binding of Anti-PSA antibody to PSA by about 80 to 90% in the RIA.

Effect of cyproterone acetate in comparison to flutamide on the ventral prostate of adult male castrated Copenhagen-Fisher rats and on Dunning R-3327 H tumors.

In this investigation the effect of CPA was tested in comparison to FL after the procedure of double blinding on the ventral prostate of 70 adult male castrated Copenhagen-Fisher rats and on the Dunning R-3327 H tumor. Total androgen blockade by castration plus CPA or by castration plus FL induced significant decrease in prostate weight compared to the androgen deprivation by castration alone. No significant difference between CPA and FL was observed. Furthermore it was impossible to exaggerate this effect with higher doses of CPA of FL. The Dunning R-3327 H tumor did not become palpable 60 days after inoculation of the tumor cells indicating that androgen deprivation by total androgen blockade by castration plus CPA or plus FL did not exhibit any proliferative activity on the hormone-sensitive Dunning R-3327 H tumor cells.

Monoclonal antibody that defines the prostate specific antigen.

A monoclonal antibody to a protein with a single band in the 30-kD region was obtained from fusion of Balb/C mouse spleen cells immunized with epithelial fractions of BPH, with the myeloma cell line P3 x 63 Ag8 6.5.3 by standard procedures. This antigen was characterized in immunobinding studies with various cellular and target antigens and in immunoperoxidase staining.

The effect of short term treatment with cyproterone acetate or flutamide on the metabolism of 5 alpha-dihydrotestosterone in human testicular tissue.

Testicular tissue obtained from ten patients orchiectomized for prostatic cancer was incubated with [3H]5 alpha-dihydrotestosterone (DHT) in order to study the metabolic transformation into 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Throughout 5 days before surgery four subjects were treated with cyproterone acetate (CA). To three patients flutamide (F) was administered for the same period of time. Three subjects remained untreated. Compared to the control group the administration of CA decreased the formation of 3 beta-diol whereas that of 3 alpha-diol increased. Treatment with F lead to an elevated formation of both diols. However, the 3 alpha/3 beta ratio did not change. As 3 beta-diol is considered to be an index of tubular function in the human testis it is concluded that CA has a direct inhibitory effect upon this testicular compartment whereas F has none.

The pharmacokinetics of flutamide and its major metabolites after a single oral dose and during chronic treatment.

Flutamide is a nonsteroidal antiandrogen used in the treatment of prostatic carcinoma. We have investigated the disposition of flutamide and its two major metabolites in ten urological in-patients without significant liver or renal disease. After oral administration flutamide is absorbed from the gastrointestinal tract with a tmax of about 2 h. Flutamide undergoes extensive first-pass metabolism, and its major metabolites are 2-hydroxyflutamide and the hydrolysis product 3-trifluoromethyl-4-nitroaniline. After the oral administration of a single dose of 250 mg or 500 mg maximum flutamide plasma concentrations of 0.02 and 0.1 micrograms.ml-1 respectively were observed. Maximum plasma concentrations of 2-hydroxyflutamide for the same flutamide doses were 1.3 and 2.4 micrograms.ml-1 (mean of n = 2 or n = 3). Steady-state concentrations of the biologically active metabolite 2-hydroxyflutamide (0.94 +/- 0.23 micrograms.ml-1, mean +/- SD, n = 5) were found at 2-4 days after the administration of 250 mg every 8 h. The area under the plasma concentration time curve for 2-hydroxyflutamide averaged 11.4 (10.6 and 12.1) and 24.3 (21.5-29.4, n = 3) micrograms.ml-1.h for 250 mg and 500 mg flutamide orally. 2-Hydroxyflutamide and 3-trifluoromethyl-4-nitroaniline were eliminated monoexponentially with half-times of 4.3-21.9 and 4.3-17.2 h (n = 5) respectively.

Monoclonal antibodies for characterization of the heterogeneity of normal and malignant transitional cells.

We present two monoclonal antibodies (mab), Mano 4/4 and 486 P3/12, directed against tumor-associated antigens of different subpopulations of transitional cell bladder carcinoma tumors. A detailed description is given concerning their reactivity patterns in an ELISA assay against different cell lines. Using immunohistochemical staining against normal bladder mucosa, mab Mano 4/4 reacts with distinct cells in the basal layer and mab 486 P 3/12 recognizes single cells in the superficial layer. Analysing different transitional-cell carcinomas, mab Mano 4/4 reacts with 17 of 20 and mab 486 P 3/12 with 17 of 19 bladder tumors. It is emphasized that both mabs may add new information in respect to a better characterization of the heterogeneity of bladder carcinoma. A biochemical characterization of the mabs and their corresponding antigens is given. Mab Mano 4/4 is directed against a 28 kD glycoprotein and mab 486 P 3/12 is reacting with a 200 kD glycoprotein belonging to the family of CEA-like proteins.

Comparative study of suprapubic sonography and computed tomography for staging of prostatic carcinoma.

In addition to the digital rectal examination, suprapubic transvesical ultrasonography and computed tomography were used to stage local tumor extension in 41 patients with histologically proven prostatic carcinoma. Although 22% of the cases revealed stage A/B disease on rectal examination, these numbers were 7% for ultrasonography and 37% for CT. For stage C/D disease the percentages were 73% for digital rectal staging, 81% for sonography, and 30% for CT. Compared with the digital examination, 22% versus 10% of the cases had to be upstaged by the results of ultrasound versus CT. A downstaging became necessary by sonography in 7% and by CT in 44%. The results are compared with the findings after prostatectomy, autopsy, and cystoscopy as well as with pathohistological data from the literature. From these results, suprapubic transvesical sonography is considered to be more reliable than CT for local tumor staging.

[Suprapubic prostatic sonography for staging prostatic carcinoma]. / Die suprapubische Prostatasonographie zum Staging von Prostatakarzinomen.

Sixty-one patients with histologically confirmed carcinomas of the prostate were examined by suprapubic, transvesical prostatic sonography in order to stage local tumor spread. The results were compared with rectal palpation. Compared with rectal palpation, sonography led to tumor up-staging in 16% and down-staging in 7%. The results showed good agreement with findings at prostatectomy, autopsy and cystoscopy. Additionally, the sonographic up-staging/down-staging figures were compared with "tumor staging after prostatectomy, as compared with clinical staging" quoted in the literature. It is concluded that suprapubic prostatic sonography is reliable supplementary method of examination for local tumor staging and that it improves the classification of local tumor extent.