Quantitative dose-response relations for the cytotoxic activity of chloroethylnitrosoureas in cell culture.

A dose-response relation for the cytotoxic activity of chloroethylnitrosourea cancer chemotherapeutic agents in cell culture has been developed. Data for the activity of 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea, and 1-(2-chloroethyl)-3-(piperidine-3,6-dion-3-yl)-1-nitrosourea against 9L rat brain tumor cells are presented. Cytotoxicity resulting from treatment schedules at different initial drug concentrations, exposure periods, and preincubation periods are correlated using a proposed dose function. Equations are presented that incorporate drug concentration, duration of exposure of cells, and the rate constant for conversion of the drug from an active intermediate into a single dose function. When compared in this way, all cell treatment schedules used were found to be equally effective in killing cells in culture. The cytotoxic activity of the four different chloroethyl-nitrosourea analogs were also found to be the same. These data demonstrate the application of quantitative dose-response relations to the activity of chloroethylnitrosoureas in cell culture and provide new insight into the mechanism of action and structure-activity relationships of these compounds.

Comparative pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea in rats and patients and extrapolation to clinical trials.

The plasma pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)1-nitrosourea (PCNU) were determined in ambulatory rats and in patients receiving PCNU chemotherapy in Phase 1 and II studies. After derivativization to the methyl carbamate, both rat and human PCNU plasma levels were measured by gas chromatography-mass spectrometry. Comparison of the tolerated dose levels and pharmacokinetics of PCNU to the values determined for 1,3-bis(2-chloroethyl)-1-nitrosourea in humans indicated that PCNU has a lower plasma drug area under the curve at equitoxic doses. We conclude that PCNU may show less clinical efficacy than 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of solid tumors.

An analysis of 1-(2-chloroethyl)-1-nitrosourea activity at the cellular level.

The effect of five different 1-(2-chloroethyl)-1-nitrosoureas on the growth of cultured P388 cells has been analyzed in terms of physical, chemical, and kinetic parameters that are related to the mechanism of action of this class of cancer chemotherapeutic agent. This study correlates structure with activity at the cellular level by using a dose function that is related to the amount of active species, the (2-chloroethyl)diazonium ion, that is formed during the period of exposure of cells to drug rather than to the initial drug dose. 1-(2-Chloroethyl)-1-nitrosourea analogues that rapidly enter the P388 cells are shown to have the same activity relative to the amount of active species formed. When analyzed in this way, activity is not influenced by the structure of the N-3 substituent, lipophilicity, or carbamoylating activity. The agents 1-(2-chloroethyl)-1-nitrosourea (CNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) all produce a 50% cell growth inhibition at 6 to 7 microM active species formed per cell volume. Chlorozotocin required a twofold higher effective dose to produce the same toxic effect. This decreased activity is attributed to the slow uptake of the water-soluble chlorozotocin into P388 and L1210 cells relative to the rate of chlorozotocin conversion to active species in medium. The yields to 2-chloroethanol from CNU, BCNU, and chlorozotocin were shown to be the same, indicating that these agents generate the same yield of alkylating intermediate at 37 degrees C and pH 7.4.

Reactions of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in aqueous solution.

Products formed from the reaction of two chloroethylnitrosoureas in neutral aqueous solution have been identified and quantified. Mixture components recovered after a 1-h incubation period accounted for 75--85% of the starting nitrosourea. Approximately 65--85% of the reaction products were formed by an initial cleavage of the nitrosourea to the proposed intermediates 2-chloroethyl azohydroxide and an isocyanate and by subsequent hydrolytic reactions. A minor pathway, 5--10% of products, involves denitrosation of the nitrosourea with oxazoline formation. Stable isotope labeling and mass spectrometry have been used to determine the reaction sequence and product origins. Reaction product identification has been made using high-performance LC isolation and comparison with synthetic material.

Metabolism of 1,3-bis(2-chloroethyl)-1-nitrosourea by rat hepatic microsomes.

The in vitro metabolism of the anticancer agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has been studied in male Fischer 344 rat liver microsomal preparations. The previously identified product. 1,3-bis(2-chloroethyl)urea (BCU), has been shown to be the major metabolite. Stable isotope labeling and mass spectral analysis of isolated metabolites indicate that BCU is formed exclusively from the metabolic denitrosation of BCNU. The rate of BCNU chemical decomposition in rat liver microsomal preparations deficient in NADPH and the metabolic disappearance rate in preparations containing added NADPH were measured and compared with the measured rate of metabolic formation of BCU under the same conditions. The rate of NADPH-dependent BCNU metabolism and BCU formation are equal within experimental error. BCNU was found to inhibit the rat liver 9000 g supernatant metabolism of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU).

Lack of prostaglandin F2 alpha metabolism by human ocular tissues.

The ability of human and rabbit ocular tissues to degrade prostaglandins (PGs) was compared by following the metabolic fate of PGF2 alpha. No metabolism was observed in vitro after 5.0 hr incubation with human cornea, iris/ciliary body, or sclera, as indicated by the absence of a decrease in [3H]-PGF2 alpha concentration or the appearance of [3H]-PGF2 alpha metabolites with time. Similarly, no metabolism of PGF2 alpha was observed in vitro after 5.0 hr incubation with these various rabbit ocular tissues or iris/ciliary body homogenate, or in vivo after topical application to rabbit eyes. The only detectable radioactive peak corresponded to PGF2 alpha. Therefore, it is concluded that both human and rabbit ocular tissues lack the enzymes that typically deactivate prostaglandins by 15-OH dehydrogenation, omega-oxidation, and beta-oxidation. In contrast to ocular metabolism, PGF2 alpha was metabolized in the presence of rabbit lung homogenate: [3H]-PGF2 alpha decreased with the simultaneous formation of a metabolite in a time-dependent manner. This metabolic transformation in lung homogenate was NADP+ dependent, and the radioactive metabolic peak had the same retention time as 13,14-dihydro-15-keto-PGF2 alpha.

The in vivo cytotoxic activity of procarbazine and procarbazine metabolites against L1210 ascites leukemia cells in CDF1 mice and the effects of pretreatment with procarbazine, phenobarbital, diphenylhydantoin, and methylprednisolone upon in vivo procarbazine activity.

An in vivo assay of the activity of procarbazine, N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride, and several metabolic intermediates against IP-implanted L1210 leukemia cells in CDF1 male mice is described. Treatment of tumor-bearing mice with procarbazine at doses of 300-500 mg/kg IP increased the mean lifespan of treated mice by 29%-32% relative to that of untreated animals. Procarbazine treatment with doses of 200-400 mg/kg/day given IP for 3 consecutive days increased mean lifespan by 39%-46%. The major circulating metabolite, azoprocarbazine (N-isopropyl-alpha-(2-methylazo)-p-toluamide), was as active as procarbazine when administered at equivalent doses for 3 consecutive days. A 2:1 mixture of azoxyprocarbazines (N-isopropyl-alpha-(2-methyl-ONN-azoxy)-: and N-isopropyl-alpha-(2-methyl-NNO-azoxy)-p-toluamide) was more active than procarbazine, increasing mean lifespan by 76% using the 3-consecutive-day dose schedule. The effects of pretreatment with procarbazine and drugs that are often co-administered with procarbazine, i.e., phenobarbital, diphenylhydantoin, and methylprednisolone, upon procarbazine anticancer activity against L1210 ascites leukemia cells was also determined. Pretreatment of CDF1 male mice with phenobarbital and diphenylhydantoin for 7 days was found to increase the antineoplastic activity of procarbazine by 13%-24%. Pretreatment with methylprednisolone did not significantly alter procarbazine activity. The effects of pretreatment with procarbazine, which is often administered daily for a period of 2-4 weeks, on procarbazine antineoplastic activity were varied. The results of these preliminary pretreatment studies combined with the finding that procarbazine metabolites have antitumor activity that is equal to or greater than that of the parent drug suggest that current clinical protocols that use procarbazine along with agents capable of altering procarbazine metabolism may involve drug interactions that alter the efficacy of procarbazine as an anticancer agent.

A method for measuring the lifetime of chemically and metabolically generated electrophilic species.

A method is presented for the characterization of the reaction order and rate constant for chemically and metabolically generated reactive electrophilic intermediates. The procedure employs flow kinetics and trapping of the electrophilic species. Reactive agents or metabolic intermediates are passed through a column containing a bound nucleophilic reagent. The electrophilic species reacts at a characteristic rate while moving through the column at a fixed pH, temperature and rate of flow. The alkylation products formed during this reaction are quantified in individual column segments which correspond to time intervals. This provides data for the calculation of the lifetime of the short-lived species. The method may be used for agents having half-lives of 1 s to 30 min. Metabolic intermediates need not be isolated. Data is presented for the reaction rates of 1-methyl-1-nitrosourea (MNU), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and iodomethane. A metabolic activation system is described for the conversion of acetoxy-methylmethylnitrosamine (DMN-OAc) to hydroxymethylmethylnitrosamine (DMN-OH) and measurement of the stability of that intermediate. Hydroxymethylmethylnitrosamine was found to have a half-life of 28s at pH 7.4, 37 degrees C.

Protein mediated chemical reactions of chloroethylnitrosoureas.

The rates of chemical degradation of chloroethylnitrosoureas in serum are significantly higher than in aqueous buffer at the same pH and temperature. This rate enhancement is shown to be produced by a non-specific protein mediated chemical reaction that involves the formation of a protein-chloroethylnitrosourea (CENU) complex. Purified human serum albumin catalyzed reactions have been studied and Vm- and Km-values obtained for 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU). The rate of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU) decomposition is not enhanced by serum proteins. Formation of a protein-BCNU complex can be inhibited by salicylic acid and dodecanoic acid, two compounds that have a high albumin binding affinity. Reaction product analysis indicates that the only BCNU reaction catalyzed by albumin is conversion to active 2-chloroethylazohydroxide and 2-chloroethylisocyanate intermediates. Formation of these reactive species at the protein surface leads to a high proportion of covalent bond formation to the protein. These results emphasize the complex and structure specific factors that may affect the biodistribution, antitumor activity and toxicity of members of the chloroethylnitrosourea class of cancer chemotherapeutic agents.

Quantitation of lipophilic chloroethylnitrosourea cancer chemotherapeutic agents.

A simple and rapid quantitative method for the derivatization and determination of lipophilic chloroethylnitrosoureas is described. This procedure involves the ether extraction of the chloroethylnitrosourea from plasma and conversion of the parent drug to an O-methylcarbamate by reaction in anhydrous methanol. The product O-methylcarbamate may be separated with gas chromatography (GC) and detected with nitrogen-specific GC detectors or with mass spectrometry using multiple-ion detection. The lower limit of detection for each method was approximately 100 ng/ml plasma.

Effects of four penetration enhancers on corneal permeability of drugs in vitro.

The usefulness of penetration enhancers in promoting drug permeation across the cornea was investigated for drugs varying from hydrophilic to lipophilic. Four purported penetration enhancers [Azone (laurocapram), hexamethylenelauramide, hexamethyleneoctanamide, and decylmethylsulfoxide] were employed. Corneal permeability coefficients of drugs that were either hydrophilic (acetazolamide, cimetidine, guanethidine, and sulfacetamide), moderately lipophilic (bunolol and prednisolone), or lipophilic (flurbiprofen and its amide analogue) were measured in the absence or in the presence of various Azone concentrations. The effects of penetration enhancers on the corneal penetration of cimetidine were also compared. The corneal penetration of hydrophilic compounds was enhanced by at least 20-fold at 0.1% Azone. For prednisolone and bunolol, the maximal enhancement was at 0.025-0.1% Azone and was marginal (two- to 5-fold), whereas Azone inhibited rather than enhanced the corneal penetration of the lipophilic flurbiprofen and its amide analogue. All four enhancers behaved similarly in enhancing corneal penetration of cimetidine and corneal hydration after incubation in vitro. Possible mechanisms of penetration enhancers on corneal drug penetration were discussed. Penetration enhancers may have clinical benefits in improving ocular drug delivery of hydrophilic compounds, however, their utility may depend on the toxicological profiles.

Competitive reactions of protonated polyamines in the gas phase.

The chemical ionization mass spectra of polyamines have been used to determine the importance of competing intramolecular reactions of protonated molecules in the gas phase. Multifunctional molecules react if possible in the order of intramolecular displacement-cyclization reactions, stabilization of the protonated molecular ion through collision stabilized intramolecular hydrogen bond formation and fragmentation of an isolated protonated function. Intramolecular nucleophilic displacement reactions closely parallel the relative rates of cyclization reactions in solution. These reactions may occur from protonated functions which are neither the initial nor the most stable sites of protonation.

Intracellular activation of cytotoxic agents: kinetic models for methylnitrosoureas and N-methyl-N'-nitro-N-nitrosoguanidine in cell culture.

The cytotoxic activity of N-methyl-N-nitrosourea (MNU), streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in cell culture by using a P388 cell growth rate inhibition assay. These agents appear to have very different activities when inhibition is related to the agent concentration in the culture medium: ED50(C0) = 40 microM for MNNG to 875 microM for streptozotocin. The mechanism of action of these three agents involves conversion to the active methanediazonium ion and subsequent methylation of cellular macromolecules. As a consequence, the rates of conversion of the parent agent to the methylating species in the medium and within the cell are important parameters that also need to be considered to reach a more detailed understanding of the mechanism of action. In order to do this, a kinetic model has been developed to calculate the concentration of drug that is converted to active methylating species within the cell during the assay incubation period. The use of cell culture kinetic models was extended from simple compounds activated through solvolytic reactions (nitrosoureas) to an agent that undergoes selective intracellular activation (MNNG). By use of measured values for initial drug concentration, incubation time, and cell volume, as well as extracellular and intracellular chemical activation rate constants, the intracellular concentration, [P4], which represents the cumulative intracellular reaction products formed during the incubation period, was calculated and related to cytotoxicity. All three agents showed an ED50[P4] between 140 and 180 microM, and for MNNG, this ED50 was independent of extracellular sulfhydryl concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A kinetic model of the cell culture cytotoxicity of metabolically activated agents: N-methyl-N-(acetoxymethyl)nitrosamine, methylnitrosourethane, and (methylazoxy)methanol acetate.

The activity of three metabolically activated methylating agents, N-methyl-N-(acetoxymethyl)nitrosamine (DMN-OAc), methylnitrosourethane (MNUT), and (methylazoxy)methanol acetate (MAM-Ac), were determined in cell culture by using a P388 cell growth rate inhibition assay. Experiments were conducted with normal P388 cells in Fischer's medium and under conditions in which the esterase-mediated activation was modified by pretreating cells with the irreversible esterase inhibitor paraoxon and by adding acetylcholinesterase to the medium. Inhibition of intracellular esterase had a much greater effect on activity than did addition of enzyme to medium. These experiments provided data that were used to assess the utility of kinetic models as a means to gain a more detailed understanding of the cytotoxicity process in cell culture. Growth rate inhibition was related to the amount of intracellular alkylation resulting from formation of metabolic intermediates and their subsequent chemical reaction to form methyldiazonium ion and methylation products by using kinetic rate laws and measured rate constants. The model is applicable to systems that form unstable metabolites that can, in part, partition between the cell volume and incubation medium. When growth rate inhibition effects were related to cumulative intracellular alkylation [P], the ED50 values were the same for all three agents and for three previously reported chemically activated methylating agents, N-methyl-N-nitrosourea, streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine, which are also thought to act through the methyldiazonium ion. This observation is consistent with a growth rate inhibition effect of the methyldiazonium ion that reflects the intrinsic activity of this species that is independent of the precursor molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of procarbazine, procarbazine metabolites and chemical degradation products with application to pharmacokinetic studies.

Quantitative analytical methods are described for the analysis of the anticancer drug procarbazine and eight known metabolites including those known to have cytotoxic activity. A direct sample insertion mass spectrometric assay for procarbazine and the urinary excretion product, N-isopropyl-terephthalamic acid, has been developed. This method employs stable isotope labeled variants in a procedure that minimizes analytical errors that may be encountered in the quantitation of the chemically unstable parent drug. a liquid chromatographic method is described for the analysis of seven known procarbazine metabolites. Use of these methods is demonstrated by the analysis of procarbazine metabolism during incubation in a 9000-g rat liver homogenate preparation. Procarbazine disappearance and metabolite appearance are also monitored in rat plasma following intraperitoneal administration of a 150 mg/kg bolus dose. Applications to patient pharmacokinetics is demonstrated using the liquid chromatographic assay to follow the appearance of active procarbazine metabolites on the first and fourteenth day of an oral 250 mg/kg/day course of therapy of a patient being treated for cancer.

The half-lives of alkylating intermediates from diethylnitrosamine and N-nitrosopyrrolidine: a method for the measurement of metabolically generated reactive species.

The half-times of the alpha-hydroxylated intermediates formed during metabolism of diethylnitrosamine and N-nitrosopyrrolidine have been determined. The method for determining half-times involved in vitro enzymatic conversion of the nitrosamine to a hydroxylated intermediate followed by trapping of the alkylating species generated from the chemical decomposition of the intermediate as it flowed through a column containing bound nucleophile. Half-times of less than 1 min at pH 7.4, 37 degrees C, were calculated from the distribution of alkylation on the column and the known flow rate of metabolic intermediates through the column. The half-times may be short enough to significantly limit organ distribution.

Determination of phenylbutazone, tolbutamide and metabolites in plasma and urine using chemical ionization mass spectrometry.

Quantitative analytical procedures for the analysis of phenylbutazone and tolbutamide levels in plasma have been developed which involve the addition of deuterium labeled internal standards to plasma followed by extraction and direct sample insertion into a mass spectrometer operating under chemical ionization conditions. Peak height ratios used to calculate plasma levels were determined by using either selected ion monitoring or repetitive scan data. The scan approach was used in a related procedure for the simultaneous determination of tolbutamide and two metabolites from urine. The accuracy, precision and sensitivity of the direct sample insertion approach to drug level measurement has been determined. Examples are given of data obtained in the course of pharmacokinetic studies in which this analytical approach appears to offer advantages in the analysis of multicomponent mixtures encountered in drug-drug interaction studies.

Pharmacokinetics of BCNU in man: a preliminary study of 20 patients.

Using the technique of direct sample insertion selected ion monitoring chemical ionization mass spectroscopy, BCNU levels were measured in vivo in patients and in vitro in serum, sera ultrafiltrates, and buffered Ringer's solution. The disappearance of BCNU in vitro was found to be first-order with a half-time of 11.6 minutes (+/- 0.5 SD) in volunteers and 15.6 minutes (+/- 2.3 SD) in patients. The disappearance from serum was catalyzed by a macromolecular component of the serum and was slowed by serum lipids. Analysis of the pharmacokinetics of BCNU in 20 patients using a two-compartment open model demonstrated a volume of distribution of 3.25 liters/kg (+/- 1.69 SD), a clearance of 56 ml/minute/kg (+/- 56 SD), and a transfer constant from the central compartment to the outside (K10) OF 0.0324 MINUTE-1 (+/- 41% SD), which was close to the decomposition rate observed for BCNU in serum in vitro. The pharmacokinetics of BCNU in patients may be affected by the percent of body fat and the lipid content of the serum.

Lipophilic drugs and lipoproteins: partitioning effects on chloroethylnitrosourea reaction rates in serum.

Chloroethylnitrosoureas are anticancer agents that undergo chemical reactions in vitro and in vivo to form active alkylating agents. The rate at which lipophilic chloroethylnitrosourease decompose in serum is faster than in aqueous solution and comparable to in vivo clearance rates. The increase in reaction rate is known to be caused by a reaction catalyzed by protein. Addition of lipoproteins to serum stabilizes chloroethylnitrosoureas to degradation. A model is presented that correlates serum decomposition rates to lipoprotein concentrations. This model involves partitioning of lipophilic chloroethylnitrosoureas into the hydrophobic core region of the lipoproteins where the chloroethylnitrosourea is chemically stable and is not free to undergo protein binding nor aqueous chemical degradation reactions. Normal interindividual variations of serum lipoprotein concentrations and hence partitioning may be a significant factor affecting the tissue distribution and pharmacokinetics of lipophilic drugs.