Platinum(IV)-Estramustine Multiaction Prodrugs Are Effective Antiproliferative Agents against Prostate Cancer Cells.

Herein, we describe the synthesis, characterization, and biological properties of Pt(IV) derivatives of cisplatin with estramustine at the first axial position, which is known to disrupt the microtubule assembly and act as an androgen antagonist, and varying the second axial position using an innocent ligand (acetate or hydroxyl) to prepare dual-action and triple-action prodrugs with known inhibitors of histone deacetylase, cyclooxygenase, and pyruvate dehydrogenase kinase. We demonstrate superior antiproliferative activity at submicromolar concentrations of the prodrugs against a panel of cancer cell lines, particularly against prostate cancer cell lines. The results obtained in this study exemplify the complex mode of action of "multiaction" Pt(IV) prodrugs. Interestingly, changing the second axial ligand in the Pt-estramustine complex has a significant effect on the mode of action, suggesting that all three components of the Pt(IV) prodrugs (platinum moiety and axial ligands) contribute to the killing of cells and not just one dominant component.

Expanding the Arsenal of PtIV Anticancer Agents: Multi-action PtIV Anticancer Agents with Bioactive Ligands Possessing a Hydroxy Functional Group.

Most multi-action PtIV prodrugs have bioactive ligands containing carboxylates. This is probably due to the ease of carboxylating the OH axial ligands and because following reduction, the active drug is released. A major challenge is to expand the arsenal of bioactive ligands to include those without carboxylates. We describe a general approach for synthesis of PtIV prodrugs that release drugs with OH groups. We linked the OH groups of gemcitabine (Gem), paclitaxel (Tax), and estramustine (EM) to the PtIV derivative of cisplatin by a carbonate bridge. Following reduction, the axial ligands lost CO2 , rapidly generating the active drugs. In contrast, succinate-linked drugs did not readily release the free drugs. The carbonate-bridged ctc-[Pt(NH3 )2 (PhB)(Gem-Carb)Cl2 ] was significantly more cytotoxic than the succinate-bridged ctc-[Pt(NH3 )2 (PhB)(Gem-Suc)Cl2 ], and more potent and less toxic than gemcitabine, cisplatin, and co-administration of cisplatin and gemcitabine.

Dendrimer conjugated estramustine nanocrystalline 'Dendot': An effective inhibitor of DMBA-TPA induced papilloma formation in mouse.

Clinically approved anticancer drug estramustine mediates its function by impairing microtubule polymerization. However, the low aqueous solubility and high toxicity limit its anticancer activity via the oral route. Previously, efforts have been made to develop an enhanced water soluble form of estramustine as estramustine phosphate (EM) but acidic gastrointestinal pH breaks the phosphate derivative via oral administration. As an alternative approach, we have made an effort to enhance solubility and minimize toxicity in vivo by conjugating EM to a poly(amidoamine) (PAMAM) dendrimer, which generated the sustained release of dendrimer conjugate (DEM). To the best of our knowledge, for the first time, we report the direct proof of the nano-crystalline 'DenDot' of DEM on TEM image. The toxicity study showed that both EM and DEM were nontoxic up to 20mg/kg. A comparative anti-papilloma study was also performed with EM and dendrimer conjugates (DEM) using a two-stage mouse skin carcinogenesis model. We found that DEM was more effective in inhibiting skin tumor formation than EM. Histopathology and immunohistochemistry studies further indicated that DEM treatment increased cell apoptosis, and reduced epithelial hyperplasia, cell proliferation and inflammation in skin tissues of mice. In addition, the synthetic DEM conjugate inhibited skin tumor progression more effectively than EM.

Comparative study of microtubule inhibitors--estramustine and natural podophyllotoxin conjugated PAMAM dendrimer on glioma cell proliferation.

The synthetic estramustine (EM) and natural podophyllotoxin (PODO) anti-mitotic agents that inhibit tubulin polymerization are known anticancer agents. As low bioavailability limits their anticancer properties, we investigated whether conjugation with PAMAM dendrimer (D) could enhance the activity of D-EM and D-PODO by altering their release pattern. Release kinetics indicated synthesized conjugates to be stable against hydrolytic cleavage and showed sustained release characteristics. However, release of D-EM was slow compared to D-PODO conjugate. Antitumor effect of these conjugates on glioma cells revealed (i) increased cell death and cell cycle arrest (ii) decreased migration and (iii) increased tubulin depolymerization as compared to free drug. Importantly, the effects of natural PODO conjugate on glioma cell survival and migration is more pronounced than D-EM.

Enzyme-mediated precipitation of parent drugs from their phosphate prodrugs.

Many oral phosphate prodrugs have failed to improve the rate or extent of absorption compared to their insoluble parent drugs. Rapid parent drug generation via intestinal alkaline phosphatase can result in supersaturated solutions, leading to parent drug precipitation. The purpose was to (1) investigate whether parent drugs can precipitate from prodrug solutions in presence of alkaline phosphatase; (2) determine whether induction times are influenced by (a) dephosphorylation rate, (b) parent drug supersaturation level, and (c) parent drug solubility. Induction times were determined from increases in optical densities after enzyme addition to prodrug solutions of TAT-59, fosphenytoin and estramustine phosphate. Apparent supersaturation ratios (sigma) were calculated from parent drug solubility at intestinal pH. Precipitation could be generated for all three prodrugs. Induction times decreased with increased enzyme activity and supersaturation level and were within gastrointestinal residence times for TAT-59 concentration>/=21microM (sigma>/=210). Induction times for fosphenytoin were less than the GI residence time (199min) for concentrations of approximately 352 microM (sigma=4.0). At approximately 475 microM (sigma=5.3) the induction times were less than 90min. For estramustine-phosphate, no precipitation was observed within GI residence times. Enzyme-mediated precipitation will depend on apparent supersaturation ratios, parent drug dose, solubility and solubilization by the prodrug.

Estramustine resistance.

Estramustine (EM), a conjugate of nornitrogen mustard and estradiol, is a antimicrotubule drug currently in use for the treatment of advanced prostatic carcinoma. Experimental data are accumulating concerning the antitumor effect of EM in other malignancies, and clinical studies in other malignancies are ongoing. This review summarizes the information available to date concerning the effects of EM and the development of drug resistance. EM depolymerizes microtubules by binding to microtubule-associated proteins (MAPs) as well as tubulin. Because of the radiosensitizing effect of this drug there has been a recent increase in interest concerning estramustine and its clinical use. Recently, it was proposed that EM induces an apoptotic cell death in glioma cells in vitro and in a rat model. EM resistance is distinct from MDR phenotype; it has been used in combination with antimitotic agents which are part of the MDR phenotype. Observations made with estramustine-resistant cell lines show the acquisition of estramustine resistance is a function of multiple adaptation by changes at tubulin expression pattern, and is also associated with changes in tau expression and phosphorylation.

A new scalable purification procedure for prostatic secretory protein (PSP94) from human seminal plasma.

A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS-polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18-25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS-PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS-PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.

Molecular conformation of estramustine and two analogues.

The crystal and molecular structures of estramustine and two of its analogues have been determined by X-ray crystallographic techniques (a total of three different compounds). The compounds studied are estramustine [1,3,5(10)-estratriene-3,17 beta-diol-3-N,N-bis(2'- chloroethyl)carbamate] and its monohydrate, estromustine [17-oxo-1,3,5(10)-estratriene-3-yl-N,N-bis(2'-chloroethyl)carbamate], and 17-oxo-5-androsten-3 beta-yl-N,N-bis(2'-chloroethyl)carbamate. Three views of estramustine were obtained from the study of its two crystal forms. The main structural features found are as follows: (a) the geometries of the steroid moieties are closely similar to those of the parent steroids, (b) the bonds around the nitrogen atom of the nitrogen mustard grouping lie approximately in a plane in each structure, (c) the plane through the carbon atoms of the steroid A-ring lies approximately perpendicular to the plane through the carbamate atoms in each structure, (d) the carbonyl C-O of the carbamate points to the alpha side of the steroid moiety in each structure, and (e) one chlorine atom of the nitrogen mustard grouping makes a close contact [3.13 A], in each structure, to the nitrogen atom. Hydrogen bonding to the carbamate appears to occur from the alpha side of the steroid; there is no hydrogen bonding to the nitrogen atom of the carbamate group. These structural data provide some steric explanations for the resistance of the carbamate to enzymatic hydrolysis. The long in vivo half-life of the intact estramustine molecule is a result of this stability. This is responsible for the absence of alkylating ability and the propensity of the drug to bind microtubule-associated proteins and express an antimitotic mechanism of action.