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AAPS PharmSciTech Aug 2001The objective of this study was to develop and manufacture a stable parenteral formulation for Phase I clinical trials of VNP40101M...
The objective of this study was to develop and manufacture a stable parenteral formulation for Phase I clinical trials of VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2-methylamino)carbonyl] hydrazine), a novel antitumor agent. The solubility and stability of the drug was determined. Solubility studies suggested that VNP40101M exhibited poor aqueous solubility but showed appreciable solubility in nonaqueous solvents. The aqueous solubility of the drug could not be increased by adjusting the pH. At a pH above 7, base-catalyzed decomposition of VNP40101M occurred. The low octanol-water partition coefficient of 0.75 suggested poor solubility in lipophilic solvents. Based on these preformulation observations, a parenteral formulation containing 10 mg/mL of VNP40101M was prepared in a solvent system consisting of 30% ethyl alcohol and 70% polyethylene glycol-300 (PEG-300). To minimize base-catalyzed hydrolytic degradation, citric acid at 0.6% concentration was included to acidify the formulation. Rubber closures, filter membranes, and liquid transfer tubing were selected on the basis of compatibility studies and absence of loss of drug due to adsorption of these components. The formulation was subjected to accelerated stability studies and dilution studies with large volume parenteral (LVP) solutions, normal saline, and 5% dextrose injection (D5W). The results of the dilution study indicated that the formulation could be diluted in these solutions up to 2 mg/mL for 8 hours without drug precipitation and degradation. Accelerated stability studies suggested that the product should be kept at 2 degrees C to 8 degrees C for long-term storage. The developed formulation was successfully scaled up and manufactured for use in clinical trials.
Topics: Adsorption; Antineoplastic Agents; Dimethylpolysiloxanes; Drug Compounding; Drug Stability; Hydrazines; Hydrogen-Ion Concentration; Infusions, Parenteral; Injections; Rubber; Silicones; Solubility; Sterilization; Sulfonamides
PubMed: 14727873
DOI: 10.1208/pt020314 -
Biochemical Pharmacology Nov 2010O(6)-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O(6)-chloroethyl (Onrigin™ and carmustine) and...
O(6)-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O(6)-chloroethyl (Onrigin™ and carmustine) and O(6)-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O(6)-methylguanines/cell and ∼300 O(6)-chloroethylguanines/cell, respectively. AGT repaired O(6)-methylguanines until the AGT pool was exhausted, while its repair of O(6)-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O(6)-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O(6)-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.
Topics: Animals; Antineoplastic Agents; Carmustine; Cell Line, Tumor; DNA Damage; DNA Methylation; DNA Repair; Dacarbazine; Drug Resistance, Neoplasm; Guanine; Humans; Hydrazines; Mice; O(6)-Methylguanine-DNA Methyltransferase; Sulfonamides; Temozolomide
PubMed: 20654586
DOI: 10.1016/j.bcp.2010.07.022 -
Analytical Biochemistry Sep 2016Alkylating agents are a significant class of environmental carcinogens as well as commonly used anticancer therapeutics. Traditional alkylating activity assays have...
Alkylating agents are a significant class of environmental carcinogens as well as commonly used anticancer therapeutics. Traditional alkylating activity assays have utilized the colorimetric reagent 4-(4-nitrobenzyl)pyridine (4NBP). However, 4NBP based assays have a relatively low sensitivity towards harder, more oxophilic alkylating species and are not well suited for the identification of the trapped alkyl moiety due to adduct instability. Herein we describe a method using water as the trapping agent which permits the trapping of simple alkylating electrophiles with a comparatively wide range of softness/hardness and permits the identification of donated simple alkyl moieties.
Topics: Alcohols; Alkylating Agents; Carcinogens, Environmental; Chemistry Techniques, Analytical; Fresh Water
PubMed: 27188264
DOI: 10.1016/j.ab.2016.04.020 -
Blood Jan 2010
Topics: Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Double-Blind Method; Female; Humans; Hydrazines; Infusions, Intravenous; Leukemia, Myeloid, Acute; Male; Middle Aged; Recurrence; Remission Induction; Sulfonamides
PubMed: 20075171
DOI: 10.1182/blood-2009-09-244236 -
Biochemical and Biophysical Research... Jan 2009The antineoplastic prodrug Cloretazine exerts its cytotoxicity via a synergism between 2-chloroethylating and carbamoylating activities that are cogenerated upon...
The antineoplastic prodrug Cloretazine exerts its cytotoxicity via a synergism between 2-chloroethylating and carbamoylating activities that are cogenerated upon activation in situ. Cloretazine is reported here to inhibit the nucleotidyl-transferase activity of purified human DNA polymerase beta (Pol beta), a principal enzyme of DNA base excision repair (BER). The 2-chloroethylating activity of Cloretazine alkylates DNA at the O(6) position of guanine bases resulting in 2-chloroethoxyguanine monoadducts, which further react to form cytotoxic interstrand DNA crosslinks. Alkylated DNA is often repaired via BER in vivo. Inhibition of the polymerase activity of Pol beta may account for some of the synergism between Cloretazine's two reactive subspecies in cytotoxicity assays. This inhibition was only observed using agents with carbamoylating activity. Furthermore, while therapeutically relevant concentrations of Cloretazine inhibited the polymerase activity of Pol beta, the enzyme's lyase activity, which may also participate in BER, was not significantly inhibited.
Topics: Antineoplastic Agents; DNA Polymerase I; DNA Polymerase beta; DNA Repair; Humans; Hydrazines; Inhibitory Concentration 50; Prodrugs; Sulfonamides
PubMed: 19026985
DOI: 10.1016/j.bbrc.2008.11.042 -
Leukemia Research Oct 2008Cloretazine [1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute...
Cloretazine [1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia (AML) patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The numbers of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produces a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cross-Linking Reagents; Cytosine; DNA; Ethane; Guanine; HL-60 Cells; Humans; Hydrazines; Leukemia; Mice; Sulfonamides; U937 Cells
PubMed: 18479747
DOI: 10.1016/j.leukres.2008.03.005 -
Pediatric Blood & Cancer Sep 2008VNP40101M is a novel alkylating agent that yields two reactive compounds (a chloroethylating species and methylisocyanate) and has demonstrated activity against a wide...
VNP40101M is a novel alkylating agent that yields two reactive compounds (a chloroethylating species and methylisocyanate) and has demonstrated activity against a wide spectrum of tumor xenografts. VNP40101M was tested against an in vivo panel of five pediatric brain tumor xenografts at a dose of 18 mg/kg/day administered for 5 days. O-6-methylguanine-DNA methyltransferase (MGMT) levels of xenografts were assessed by Western blot analysis. Only one xenograft (GBM2), which lacked detectable MGMT expression, demonstrated an objective response to VNP40101M. VNP4010M antitumor activity was observed only in the absence of MGMT expression, with resistance to VNP4010M seen even with low MGMT expression.
Topics: Animals; Antineoplastic Agents, Alkylating; Brain Neoplasms; Drug Evaluation, Preclinical; Female; Glioblastoma; Glioma; Hydrazines; Mice; Mice, Inbred Strains; O(6)-Methylguanine-DNA Methyltransferase; Sulfonamides; Transplantation, Heterologous; Treatment Outcome
PubMed: 18493996
DOI: 10.1002/pbc.21620