Relationship between glutathione levels and drug or radiation sensitivities in human gastric cancer cell lines in vitro

Summary Permanent cell lines and clones established from an untreated patient (AGS cells) with gastric carcinoma, and from a similar patient who had been treated with Adriamycin, 5FU and cytoxan (SII cells) were used in a study that compared their drug and radiation survival sensitivities to their glutathidine (GSH) values. The SII parental cell line was more resistant than the AGS cells in vitro to chlorambucil, ACT D, Adria, Bleo, and X-rays. This greater resistance was positively correlated with GSH values that were 1.77 times higher than in the AGS parental cell line. By contrast the SII parental cells were more sensitive than the AGS cells to MeCCNU and Melphalan. The drug and radiation sensitives expressed among the clones of the two cell lines were heterogeneous and did not correlate with their GSH values.Abbreviations GSH Glutathione - LD₉₉ Lethal Dose to 99% of the treated population - ACT D Actinomycin D - Mel Melphalan - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - Gal Galactitol - Bleo Bleomycin - Adria Adriamycin - 5FU 5-Fluorouracil - D₀ measure of the slope of the survival curve.     

Schedule dependent potentiation of antitumor drug effects by α-difluoromethylornithine in human gastric carcinoma cells in vitro

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with -difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.     

Heterogeneous survival responses of human gastric cancer clones to αDifluoromethylornithine in vitro

The effects of Difluoromethylornithine (DFMO)² on survival of human gastric cancer clones were studied in vitro. The responses were dose and time dependent. Treatments which lasted for less than 12 h were cytotoxic at only the highest doses used. The greatest effects on survival were observed only when cells were treated for 48 and 72 h. The effects on the clones produced by such prolonged treatment durations were heterogeneous, with survival values differing by as much as 460%. By contrast, the clonal survival responses to short DFMO treatments (12 h) were very uniform (dose differential of only 19%); however, this uniformity in response could be achieved only by using non-pharmacological doses of DFMO. The heterogeneity in survival responses in the clones might be slightly associated with their levels of intracellular spermidine. Clones with the smallest amounts of intracellular spermidine at the start of treatment were most sensitive to DFMO. However, this association may not hold up with further testing in other gastric cancer clones or when studied in other cancer lines in vitro.Abbreviations LD₉₀ dose lethal to 90% of the cell population - D₀ dose required to reduce survival by 63% on exponential portion of the survival curve - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - Bleo Bleomycin - 5FU 5 fluorouracil - DFMO Difluoromethylornithine - ODC ornithine decarboxylase     

In vitro cellular characteristics and survival responses of human astrocytoma clones to chloroethyl-nitrosoureas and dianhydrogalactitol

Three permanent clones were derived from a single astrocytoma cell line and were characterized for in vitro cell kinetics, chromosomal properties and for their responses to the anticancer drugs: 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU); 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU); and 1,2:5,6-dianhydrogalactitol (GAL); all of which have been shown to cross the blood brain barrier. The clones showed different population doubling times, saturation densities, plating efficiencies, chromosome counts, ploidy, cell cycle phase distributions and DNA indices. The only positive correlation among these parameters was between the population doubling times and the modal chromosome numbers; the lower the chromosome number, the shorter the doubling time. No correlation was observable between any of the cellular properties and responses to the four drugs. The clones showed a differential sensitivity to the nitrosoureas, seen maximally as a 600-fold difference in survival between two of the clones treated with the same dose of BCNU. In contrast, the clones exhibited almost identical and uniform sensitivities to galactitol, suggesting that this agent exerted its cytotoxic effects by similar mechanisms in each of the clones. By comparison BCNU (at the tested doses and duration of drug exposure used in this study) was found to be the most effective of the agents tested.     

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.     

Myeloid clonogenic assays for comparison of the in vitro toxicity of alkylating agents.

A battery of clonal assays for myeloid progenitor cells (HPP-CFC, CFU-gemm, CFU-gm, CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine, busulfan, melphalan, carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis, mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine, melphalan, carmustine and lomustine. Generally, there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays, the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents.     

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.     

Synthesis and in vitro evaluation of quaternary ammonium derivatives of chlorambucil and melphalan, anticancer drugs designed for the chemotherapy of chondrosarcoma

To enhance affinity for malignant cartilaginous tumors (chondrosarcomas), quaternary ammonium (QA) conjugates of chlorambucil and melphalan were prepared by linking the QA moiety to nitrogen mustards via an amide bond. They exhibited closely similar and sometimes more favorable values than their parent compounds. In the cell lines tested, the two QA conjugates displayed appreciable cytotoxicity, the QA conjugate of chlorambucil even showing an enhanced efficiency against chondrosarcoma compared with chlorambucil.     

Chemosensitivity of normal human trophoblasts evaluated by a newly developed ATP-based luminescence assay

Trophoblast injury may be one of the possible causes of fetal distress associated with chemotherapy administered during pregnancy. The purpose of this study was to investigate the ex vivo chemosensitivity of normal trophoblasts (NTB) against commonly used antineoplastic agents. Using the newly developed ex vivo ATP-based trophoblast assay (ATP-TBA), 31 NTB freshly sampled from human placentas (gestational week 7-42) were tested against dactinomycin (Act-D), 5-fluorouracil (5-FU), 4-OOH-cyclophosphamide (4-HC), vincristine (VCR) and methotrexate (MTX) alone or in combination with calcium folate (LV). All agents were studied at concentrations relevant to clinical dosages normally used for chemotherapy of solid neoplasms. Of 31 samples studied with the ATP-TBA, 20 (65%) were evaluable. VCR, Act-D and 4-HC were the most active drugs with 55, 45 and 45% of samples responding ex vivo. Antimetabolites were less active, producing ex vivo response rates of 25 (MTX) and 20% (5-FU), respectively. MTX activity was largely neutralized by adding LV. The chemosensitivity of NTB showed considerable inter-individual variations and did not decrease with increasing gestational age. We therefore conclude that NTB of any gestational age exhibit considerable ex vivo sensitivity against common anticancer agents which is comparable to that observed for various solid tumors. The ATP-TBA may be helpful in planning future trials with both single agents and drug combinations in order to standardize and optimize chemotherapy during pregnancy.     

In vitro activity of the novel cytotoxic agent CHS 828 in childhood acute leukemia

CHS 828, a pyridyl cyanoguanidine, is a new drug candidate now in phase I/II trials, that has shown promising anticancer activity in experimental tumor models and primary cultures of cancer cells from patients. In this study the fluorometric microculture cytotoxicity assay was used for evaluation of CHS 828 in primary cell cultures from children with acute leukemia. The activity of and interaction with the standard drugs, doxorubicin, melphalan, etoposide and cytosine arabinoside (Ara-C), were also assessed. Samples from 65 patients, 42 with acute lymphocytic leukemia (ALL) and 23 with acute myelocytic leukemia (AML) were tested with 72-h continuous drug exposure. There was 50% cell kill at very low CHS 828 concentrations; median IC50 was 0.01 microM in ALL and 0.03 in AML samples (NS) with large interindividual variability in both groups. ALL samples were significantly more sensitive than AML samples to melphalan, doxorubicin and etoposide, but not to Ara-C. In AML samples, combinations between CHS 828 and each of the four standard drugs resulted in significantly lower cell survival than either drug alone. This was also observed in ALL samples, except for Ara-C. Using the additive interaction model, CHS 828 showed a synergistic effect with melphalan in 67%, doxorubicin in 47%, etoposide in 38% and Ara-C in 14% of AML samples. In most ALL samples subadditive effects were found. Further exploration of CHS 828 in childhood leukemia is warranted, especially in AML.     

Histocultures of Patient Head and Neck Tumors for Pharmacodynamics Studies

This investigation was to establish a clinically relevant experimental model to evaluate the pharmacodynamics of drugs used for head and neck cancers. A total of 83 surgical samples of primary and lymph nodal metastatic tumors was obtained from 66 patients. Fragments of these tumors were cultured on a collagen gel matrix. The tumor cell labeling index (LI) was determined by [³H]thymidine incorporation and autoradiography. Seventeen tumors (20%) were contaminated. About 80% of the remaining 65 tumors were successfully cultured for at least 2 weeks. The cultured tumor fragments retained the morphology and architecture of the freshly removed specimens; both tumor and stromal cells were present. The tumor cell LI after 2–3 weeks in culture, determined from the most proliferative area of the tissue, averaged 77 ± 12% for primary tumors and 78 ± 12% for nodal metastases. The activity of three clinically active agents, 5-fluorouracil (FU), cisplatin (DDP), and mitomycin C (MMC), was evaluated in 47 tumors. All three drugs inhibited the tumor LI. The concentrations needed to produce a 50% inhibition of the tumor LI (IC₅₀) were within the clinically achievable concentration range. The intertumor variation in the IC₅₀ for FU (60-fold) was considerably greater than that for DDP and MMC (7- to 8-fold). The nodal metastatic tumors appeared to be less sensitive to FU than the primary tumors, while there were no apparent differences for DDP or MMC. Tumors from patients previously treated with chemotherapy and/or radiotherapy appeared less sensitive to FU and DDP than tumors from untreated patients, but the differences were not statistically significant. Interestingly, tumors from previously treated patients were significantly more sensitive to MMC than tumors from untreated patients. The maintenance of the morphology of the fresh tumor and the observed intertumor variability in IC₅₀ values indicate the preservation of intra- and intertumor heterogeneity in the histocultures. In summary the viability and high success rate of growth of human head and neck tumors in organ-like culture and the chemosensitivity of the cultured tumors to clinically active agents at clinically achievable concentrations support the use of this experimental model for pharmacodynamic evaluation.     

Correlations between the activities of 19 anti-tumor agents and the intracellular glutathione concentrations in a panel of 14 human cancer cell lines: comparisons with the National Cancer Institute data

The aim of this work was 2-fold: (i) to identify correlations between the activities of pairs of 19 anti-tumor agents in a mini-panel of 14 human cancer cell lines of diverse origins with the goal of validating the panel, and (ii) to look for correlations between the activities of 19 standard anti-tumor agents and the intracellular concentrations of glutathione (GSH). Validation with analogous data from the National Cancer Institute (NCI) Developmental Therapeutics Program was made. The cell growth inhibition potencies of the anti-tumor agents [cisplatin, carboplatin, oxaliplatin, DACH-Pt, melphalan, chlorambucil, thiotepa, busulfan, doxorubicin, etoposide, camptothecin, vinblastine, podophyllotoxin, colchicine, taxol, hydroxyurea, methotrexate, 5-azacytidine and 5-fluorouracil (5-FU)] were estimated in 14 cancer cell lines by their GI50 values. An enzymatic assay based on the method of Tietze was employed to measure intracellular total GSH concentrations. The Delta method was used to compare pairs of anti-tumor agents; similarities and differences in activity profiles (mean graphs) were evaluated by regression analysis. Most, but not all, of the correlations could be explained based on similarities in the mechanisms of action and many correlations/non-correlations were also observed in the NCI data. Some correlations were unexpected however, and not seen in the NCI data. For example, strong positive correlations (P < 0.01) were found between the GI50 values of melphalan/chlorambucil and the anti-mitotic agents. Similarly unexpected, a strong positive correlation was observed between methotrexate and cisplatin (P < 0.01). Interestingly, moderate to strong negative correlations (P < 0.01-0.05) were found between the GI50 values of 5-FU and the anti-mitotic agents/melphalan/chlorambucil. Significant positive correlations between intracellular GSH concentrations and GI50 values were found only for thiotepa (P < 0.05) and doxorubicin (P < 0.01). Data from a NCI panel of 34 cancer cell lines showed no correlations between GSH levels and the GI50 values of the same 19 compounds. In conclusion, a panel of 14 human cancer cell lines of diverse origin was used to identify similarities and differences in the activities of standard anti-tumor agents. The level of significance was stronger with the 34 cell lines of the NCI, however. Our results indicate that GSH intracellular concentrations correlate with resistance only with doxorubicin and thiotepa in these cell lines.     

Sensitivity of murine B- and T-lymphocytes to oxazaphosphorine and non-oxazaphosphorine nitrogen mustards

The relative sensitivities of murine B- and T-lymphocytes to the oxazaphosphorine nitrogen mustards, cyclophosphamide and ASTA Z 7557, and to the non-oxazaphosphorine nitrogen mustards, melphalan and chlorambucil, in vivo, were determined. B- and T-lymphocytes were defined by selective mitogen-induced proliferation. Lipopolysaccharide (LPS)-induced B-lymphocytes were approximately twice as sensitive to the cytotoxic effects of cyclophosphamide and ASTA Z 7557 as were phytohemagglutinin (PHA)- and concanavalin A (Con A)-induced T-lymphocytes. LPS-induced B-lymphocytes and PHA-induced T-lymphocytes were approximately equisensitive to the cytotoxic action of melphalan and chlorambucil, but the former were somewhat more sensitive to these agents than were Con A-induced T-lymphocytes. The relative sensitivities of murine B- and T-lymphocytes to ASTA Z 7557 and the non-oxazaphosphorine metabolite of cyclophosphamide, phosphoramide mustard, ex vivo, were also determined. LPS-induced B-lymphocytes were approximately twice as sensitive to the cytotoxic action of ASTA Z 7557 as were PHA- and Con A-induced T-lymphocytes. The three mitogen-induced lymphocyte populations were approximately equisensitive to the cytotoxic action of phosphoramide mustard. These observations suggest that the differential effect of cyclophosphamide on murine B- and T-lymphocytes is uniquely exhibited by oxazaphosphorine nitrogen mustards. Furthermore, the results suggest that 4-hydroxycyclophosphamide is the cyclophosphamide metabolite that mediates the differential immunotoxic effect of the parent compound.     

The effects of nitrogen mustards on the proliferative and Ig synthetic response of human peripheral blood lymphocytes

Maximal inhibition of pokeweed mitogen-stimulated Ig production and [3H]thymidine incorporation was shown to occur when unfractionated human peripheral blood mononuclear cells were cultured with concentrations of the nitrogen mustards melphalan, mechlorethamine or chlorambucil in the 20-100-microM range, whereas concentrations of microsome-activated cyclophosphamide (A-Cy) in the 2-mM range were required for equivalent inhibition. Around 400 microM A-Cy, IgM secretion was not inhibited, but secretion of IgA and IgG was. The [3H]thymidine incorporation of enriched populations of both large and small B and T cells all showed about 20-50-fold greater sensitivity to melphalan than to A-Cy, despite a difference of only 6-fold in alkylating activity between these drugs. Large (250 micron 3) B and T cells were only marginally more sensitive to melphalan and A-Cy than small (210 micron 3) T and B cells. Kinetic studies showed that IgG and IgA secreted by day 7 could be maximally inhibited by melphalan added as late as day 3, and IgM synthesis as late as day 2. In contrast, inhibition of Ig production by A-Cy steadily declined after the first day, especially IgM, which was no longer inhibitable by A-Cy on day 3. Inhibition of cumulative Ig production did not occur when A-Cy or melphalan was added on day 5 or later. Cell recombination experiments performed with drug pulsed and untreated monocytes plus B cells and irradiated T cells showed that inhibition of [3H]thymidine or Ig production was most striking when monocytes + B cells (rather than T cells) were exposed to melphalan in the first 16 h. When A-Cy was used in the first 16 h, inhibition of Ig production was partial and inconsistent, and inhibition of monocytes + B cell or T cell [3H]thymidine incorporation was not evident. We conclude that the nitrogen mustards melphalan and A-Cy can inhibit pokeweed mitogen-stimulated DNA synthesis by human T or B cells and Ig production in vitro, but that their mechanisms of action differ.     

Degradation and disposal of some antineoplastic drugs

Bulk quantities and pharmaceutical preparations of the antineoplastic drugs carmustine (BCNU), lomustine (CCNU), chlorozotocin, N-[2-chloroethyl]-N'-[2,6-dioxo-3-piperidinyl]-N-nitrosourea (PCNU), methyl CCNU, mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, uracil mustard, and spiromustine may be degraded using nickel-aluminum alloy in KOH solution. The drugs are completely destroyed and only nonmutagenic reaction mixtures are produced. Destruction of cyclophosphamide in tablets requires refluxing in HCl before the nickel-aluminum alloy reduction. Streptozotocin, chlorambucil, and mechlorethamine may be degraded using an excess of saturated sodium bicarbonate solution. The nitrosourea drugs BCNU, CCNU, chlorozotocin, PCNU, methyl CCNU, and streptozotocin were also degraded using hydrogen bromide in glacial acetic acid. The drugs were completely destroyed but some of the reaction mixtures were mutagenic and the products were found to be, in some instances, the corresponding mutagenic, denitrosated compounds.     

Changes in drug sensitivity of a human astrocytoma clone previously treated with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea in vitro

Summary We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3–4) to MeCCNU (10 g/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 g/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 g MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably.Abbreviations DAG Galactitol - Adria Adriamycin - PCNU 1-(2-Chloroethyl)-3-(2,6 dioxo-3-piperidyl-1-nitrosourea) - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea - FMF Flow microfluorometry Dedicated to the memory of Benjamin Drewinko, Physician, Scientist and friend.     

The effect of alkylating agents and other drugs on the accumulation of melphalan by murine L1210 leukaemia cells in vitro

The effect of cytotoxic and other drugs on the accumulation of melphalan by L1210 murine leukaemia cells was studied. We have confirmed that uptake is an active process competitively inhibited by l-leucine. In 36 experiments in amino acid-free medium the mean concentration of melphalan taken up was 225 pmoles/10⁶ cells. High pressure liquid Chromatographie analysis showed that the majority of the drug is present as free native melphalan. 1, 3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was the only drug that stimulated accumulation, but without significant effect on influx or efflux rates. Busulphan, chlorambucil, cyclophosphamide, interferon, methotrexate and prednisolone had no effect on accumulation after 30 min melphalan transport. Adriamycin, CCNU, methyl CCNU, mustine and vincristine all impaired melphalan accumulation as did the non-cytotoxic drugs aminophylline, chlorpromazine and ouabain. Adriamycin, aminophylline, chloropromazine, indomethacin and ouabain all reduced melphalan influx.     

Stability of allopurinol and of five antineoplastics in suspension

The stability of allopurinol, azathioprine, chlorambucil, melphalan, mercaptopurine, and thioguanine each in an extemporaneously prepared suspension was studied. Tablets of each drug were crushed, mixed with a suspending agent, and brought to a final volume of 10, 15, or 20 ml with a 2:1 mixture of simple syrup and wild cherry syrup. Suspensions were prepared in the following concentrations: allopurinol (20 mg/ml), azathioprine (50 mg/ml), chlorambucil (2 mg/ml), melphalan (2 mg/ml), mercaptopurine (50 mg/ml), and thioguanine (40 mg/ml). Using high-performance liquid chromatography or ultraviolet scans, duplicate assays were performed on each suspension periodically during storage for up to 84 days at ambient room temperature or 5 degrees C. The time required for the suspensions to drop below 90% of labeled strength was used as an indicator of drug stability. Allopurinol and azathioprine were stable for at least 56 days at room temperature and at 5 degrees C. Chlorambucil decomposed rapidly at room temperature but was stable for seven days when stored at 5 degrees C. Melphalan suspensions did not meet the stated criteria for stability even at the time of initial assay. Mercaptopurine and thioguanine were stable for 14 and 84 days, respectively, at room temperature; at 5 degrees C, assay values dropped below those obtained at room temperature. In the suspension formulation tested, allopurinol, azathioprine, mercaptopurine, and thioguanine are stable for at least 14 days at room temperature; chlorambucil suspensions should be refrigerated and discarded after seven days. Melphalan decomposes too rapidly to make this suspension formulation feasible for extemporaneous compounding.     

Concentration and time-dependent inter-relationships for cytotoxicities of nitrogen mustard drugs against lymphoblasts in-vitro

Peripheral lymphoblasts were exposed either to different initial concentrations of the alkylating agents (melphalan, chlorambucil or phenylacetic acid mustard) using a fixed incubation time or a constant [3H]methylthymidine incorporation into the trichloracetic acid-insoluble fraction of the cells. The concentration-time relationships were evaluated by calculating the amount of drug which had chemically reacted in the incubation system. Melphalan showed lower cytotoxicity at short exposure times and high drug concentrations, while chlorambucil exhibited higher cytotoxicity at longer exposure times. In the latter case the effect could be accounted for by the cytotoxic activity of monohydroxy chlorambucil which was formed in the incubation system.     

Relationships between resistance to cross-linking agents and glutathione metabolism, aldehyde dehydrogenase isozymes and adenovirus replication in human tumour cell lines

In a panel of 10 human tumour cell lines with no prior exposure to drugs in vitro, resistance to cisplatin correlated with resistance to the nitrogen mustard derivatives Asta Z-7557 (mafosfamide, an activated form of cyclophosphamide), melphalan and chlorambucil. Simultaneous treatment with ,R-sulfoximine did not enhance the toxicity of cisplatin or Asta Z-7557, and no correlation was found between drug resistance and cellular levels of metallothioneins (as judged by sensitivity to cadmium chloride), glutathione (GSH), GSH reductase, GSH transferase, or γ-glutamyltranspeptidase. The two cell lines most resistant to Asta Z-7557 expressed aldehyde dehydrogenase cytosolic isozyme 1, found also in normal ovary, but not isozyme 3. Treatment of resistant cells with cisplatin or Asta Z-7557 inhibited cellular DNA synthesis and replication of adenovirus 5 to a lesser extent than in sensitive cells. The virus could be directly inactivated by both drugs prior to infection, subsequent replication being inhibited to the same extent in sensitive and resistant cells. In contrast to Asta Z-7557 and other DNA damaging agents, cisplatin was much more toxic to adenovirus ( ₃₇ 0.022–0.048 μM) than to cells ( ₃₇ 0.25–2.5 μM). The adenovirus 5 mutant Ad5ts125 having a G → A substitution was even more sensitive to cisplatin ( ₃₇ 7–8 nM) than wild type virus and another mutant. Cisplatin was detoxified less by sonicated resistant cells than sensitive cells, as judged by inactivation of Ad5ts125 added to the reaction mixture. It can be inferred that (i) the major differences in cellular resistance to cisplatin and Asta Z-7557 in the present material did not involve enhanced DNA repair or protection by metallothioneins or GSH, but were associated with the ability to continue cellular and viral DNA synthesis during treatment, (ii) resistance was not associated with less template damage, and (iii) the adenovirus genome may be a suitable probe for predicting tumour resistance to cisplatin and for elucidating the DNA sequence dependence of cisplatin toxicity.