Phase I clinical and pharmacokinetic study of diethyldithiocarbamate as a chemoprotector from toxic effects of cisplatin.

Diethyldithiocarbamate (DDTC) has been shown to provide protection against most clinically significant toxic effects from cisplatin (DDP) without inhibiting tumor response in a variety of murine animal models. We conducted a phase I clinical and pharmacokinetic study of DDTC in combination with DDP to establish the types and severity of toxic effects and to determine whether protection of normal tissues and tumors occurs. Twenty-two courses of DDP plus DDTC were given to 10 patients. No nephrotoxic effects were seen at DDP doses of 50-120 mg/m2, and three patients had amelioration of nausea and vomiting. Objective antitumor responses were observed. Dose-limiting toxic effects from DDTC occurred at 150 mg/kg; these consisted of numbness in the infusion arm often accompanied by severe diaphoresis, chest discomfort, and agitation during DDTC infusion. These toxic effects resolved spontaneously, however, after termination of the infusion. The preliminary results suggest that plasma levels of DDTC that provide excellent protection in rodents were exceeded at the doses used in our clinical study without compromising antitumor response.

Quiescent LLC-PK1 cells as a model for cis-diamminedichloroplatinum(II) nephrotoxicity and modulation by thiol rescue agents.

Confluent LLC-PK1 cells express many characteristics of renal proximal tubule epithelia. We report here the use of this cell line to investigate the possible mechanisms of cis-diamminedichloroplatinum(II) (DDP)-induced nephrotoxicity and diethyldithiocarbamate (DDTC) amelioration. Cells were exposed to platinum-based drug for 1 h and then incubated in drug-free medium until assayed. There was a 10-h delay between DDP exposure and onset of toxicity which then continued to develop. Viability at 72 h was 97 +/- 2 (SD), 68 +/- 3, 33 +/- 3, and 10 +/- 2% of control after treatment with 100, 200, 300, and 400 microM DDP, respectively. trans-Diamminedichloroplatinum(II) was 5-fold less toxic than DDP and diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) was nontoxic at concentrations up to 2.0 mM. Incubation of cells with DDTC (3 mM) for 1 h immediately following DDP exposure resulted in chelation of 43% of total intracellular platinum by DDTC and significantly increased 72 h viability; 97 +/- 2, 88 +/- 3, and 42 +/- 2% of control for 200, 300, and 400 microM DDP, respectively. DDP treatment of L1210 cells yielded equivalent total intracellular platinum levels, but Pt(DDTC)2 concentrations were one-tenth those in LLC-PK1 cells after subsequent treatment with DDTC (3 mM). Immediate reduction of protein and RNA, but not DNA, synthesis by DDP was concentration dependent over the same range as viability. DDP-induced increases in unscheduled DNA synthesis also did not correlate with cytotoxicity. The inhibition of protein synthesis was unchanged by pretreatment with the RNA synthesis inhibitor actinomycin D.DDTC (3 mM) exposure produced an immediate and persistent DDP dose modification of 1.6 in protein but not RNA synthesis. CBDCA (0.5 to 1.0 mM) had no effect on protein, RNA, or DNA synthesis and only slight stimulatory effects on unscheduled DNA synthesis. DDTC alone (3-3000 microM) caused significant reduction in DNA synthesis and unscheduled DNA synthesis. Neither sodium thiosulfate nor 2-mercaptoethane-sulfonate had any effect on DDP-induced cytotoxicity or inhibition of protein, RNA, or DNA synthesis when incubated immediately after DDP, even though these drugs achieved intracellular concentrations at which DDTC was protective. These data indicate that quiescent LLC-PK1 cells are a good in vitro model for the study of DDP-induced nephrotoxicity and its modulation by thiol rescue agents and that DDP inhibition and DDTC rescue of posttranscriptional processes correlate best with viability in LLC-PK1 cells.

Diethyldithiocarbamate modulation of murine bone marrow toxicity induced by cis-diammine(cyclobutanedicarboxylato)platinum (II).

Diethyldithiocarbamate (DDTC) has been shown to ameliorate the myelosuppression induced by the platinum cancer chemotherapeutic drugs in mice. Optimal drug scheduling for DDTC and cis-diammine(cyclobutanedicarboxylato)platinum(II) (CBDCA) has been determined in C57BL/6 x DBA/2 F1 mice, using the pluripotent stem cell assay to assess hematological toxicity. DDTC, at doses of 0.3 to 300 mg/kg given 3 h after 60 mg/kg CBDCA, tripled the number of proliferating spleen colony-forming units compared to treatment with CBDCA alone. No significant difference in efficacy was noted among these doses. DDTC, at the lowest myeloprotective dose (0.3 mg/kg), was most active when administered from 1 to 3 h after CBDCA. The combination of DDTC with CBDCA in vivo did not alter the clonogenic survival of L1210 cells compared to CBDCA alone. CBDCA depressed both bone marrow and tumor cell DNA synthesis. DDTC given 3 h after CBDCA hastened the recovery of DNA synthesis only in marrow cells; the addition of DDTC to CBDCA did not alter DNA synthesis in tumor cells. DDTC alone did not significantly affect DNA synthesis in either normal or tumor cells. These results suggest that the mechanism of DDTC myeloprotection involves stimulation of bone marrow cell proliferation and that the selectivity of DDTC is based upon the absence of stimulation in tumor cells.

Myeloprotective effect of diethyldithiocarbamate treatment following 1,3-bis(2-chloroethyl)-1-nitrosourea, adriamycin, or mitomycin C in mice.

The effect of diethyldithiocarbamate (DDTC) on myelotoxicity induced by 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, or mitomycin C in C57BL/6J x DBA/2J mice is reported here. All drugs were administered i.v. Myelotoxicity was assessed, 24 h after administration of the myelotoxic drug, using bone marrow stem cell (spleen colony-forming unit) and granulocyte/macrophage progenitor cell (granulocyte/macrophage colony-forming unit in culture) clonogenic assays. Administration of DDTC alone had no effect on spleen colony-forming units or granulocyte/macrophage colony-forming units in culture. 1,3-Bis(2-chloroethyl)-1-nitrosourea showed a dose-dependent toxicity for both cell types, and subsequent treatment with DDTC (300 mg/kg i.v. 3 h after 1,3-bis(2-chloroethyl)-1-nitrosourea) ameliorated this toxicity. The same dosing regimen of DDTC ameliorated Adriamycin-induced toxicity to bone marrow stem cells at the two higher doses tested. However, the myelosuppressive effects of mitomycin C were not altered by DDTC administration (300 mg/kg i.v. 3 h after or 30 min before mitomycin C). These results demonstrate that DDTC ameliorates myelotoxicity induced by several, but not all, chemotherapeutic agents and suggest a broad role for DDTC in cancer chemotherapy.

Mechanism of diethyldithiocarbamate modulation of murine bone marrow toxicity.

Sodium diethyldithiocarbamate (DDTC) has been shown to modulate the myelosuppression that commonly occurs following treatment with anticancer drugs in mice. In order to investigate the mechanism of action of this myeloprotector, murine long-term bone marrow cultures were treated with DDTC alone or were preceded by the anticancer drug cis-diammine(cyclobutanedicarboxylato)platinum(II) (CBDCA), and the granulocyte/macrophage colony-stimulating activity of the supernatants was measured. The supernatants harvested from DDTC-treated cultures enhanced proliferation of granulocyte/macrophage progenitor cells almost 4-fold compared to cultures treated with no drug or with CBDCA alone. Pretreatment of cultures with CBDCA neither enhanced nor inhibited DDTC-induced colony-stimulating activity. Similar results were obtained by using marrow stromal cell cultures free of hematopoietic cells. Thus, DDTC may hasten bone marrow recovery by augmenting stromal cell production of a factor(s) with hematopoietic colony-stimulating activity.

Diethyldithiocarbamate inhibition of murine bone marrow toxicity caused by cis-diamminedichloroplatinum(II) or diammine-(1,1-cyclobutanedicarboxylato)platinum(II).

We report here the effects of diethyldithiocarbamate (DDTC) rescue on myelotoxicity caused by carboplatin (CBDCA) and cisplatin (DDP) in C57BL/6 x DBA/2 F1 mice. All drugs were administered by injection into the tail vein. Myelotoxicity was assessed by WBC, bone marrow cellularity, and assays for pluripotent bone marrow stem cells (spleen colony forming unit) and granulocyte/macrophage progenitor cells (granulocyte/macrophage colony forming unit in culture). The most significant protection occurred in stem cells, where a single dose of DDTC (300 mg/kg) produced a platinum-drug dose modification factor of 3.3; i.e., the addition of DDTC reduced stem cell toxicity to the level produced by approximately one-third the dose of platinum drug alone. On a molar basis, DDP was 2.4 times as toxic to stem cells as CBDCA. The response of the stem cells to CBDCA and DDP was linear both with and without rescue, and the dose modification factor remained constant for doses of CBDCA up to 120 mg/kg and doses of DDP up to 15 mg/kg. Moreover, stem cell rescue appeared to be independent of DDTC dose (100-750 mg/kg) and time of administration (1.5 h before to 5 h after platinum drug). DDTC protection was less impressive for more mature hematological cells (granulocyte/macrophage colony forming units in culture). In studies of bone marrow cellularity, addition of DDTC (300 mg/kg) to DDP treatment (10 mg/kg) produced a 50% increase in the granulocyte-predominant cell population but had no effect on the lymphocyte population. Peripheral WBC showed no significant difference between rescued and unrescued groups and did not reflect the toxicity observed directly in the bone marrow.

Further studies on the conversion of 4-hydroxyoxazaphosphorines to reactive mustards and acrolein in inorganic buffers.

The rates at which the 4-hydroxyoxazaphosphorines, 4-hydroxycyclophosphamide and 4-hydroxyifosfamide, are converted to reactive mustards and acrolein in phosphate and bicarbonate buffers were determined as a function of pH, ionic strength, temperature, and buffer concentration. Conversion was first-order with respect to both the 4-hydroxyoxazaphosphorine and phosphate or carbonate serving as a catalyst. The catalytic constant for dianionic phosphate-catalyzed conversion of 4-hydroxyifosfamide to isophosphoramide mustard and acrolein at 37 degrees was 0.189 min-1; a value of 0.194 min-1 M-1 was obtained when dianionic phosphate-catalyzed conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was examined. A catalytic constant of 3.09 min-1 M-1 was obtained for carbonate-catalyzed conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein. Hydroxyl ion and water also catalyzed the reaction; catalytic constants were 2710 and 0.000006 min-1 M-1, respectively. The rate at which the 4-hydroxyoxazaphosphorines were converted to reactive mustards and acrolein in phosphate buffer increased as the pH, ionic strength, and temperature increased. The energy of activation was about 20 kcal/mol. Dianionic phosphate, carbonate, hydroxyl ion, and water were apparently acting as general base catalysts of the rate-limiting step (probably the conversion of the intermediate aldehyde to the corresponding reactive mustard and acrolein) of the overall reaction, although specific base-general acid catalysis could not be ruled out. Bifunctional catalysis of the rate-limiting step did not appear to be significant and certainly was not obligatory as concluded previously by our laboratory. Assuming that the oncotoxic specificity of the oxazaphosphorines resides with the 4-hydroxyoxazaphosphorines and that their cytotoxic effect at therapeutic doses is largely mediated by the reactive mustards released within cells, these observations offer the possibility that intracellular general base catalytic activity serves as an important determinant with regard to the oncotoxic potential and specificity of the oxazaphosphorines. General base catalytic activity varies directly with pH, ionic strength, temperature, and the concentration of the base. The influence of some of these factors on the development of cyclophosphamide-induced bladder toxicity has already been demonstrated.

Conversion of 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein mediated by bifunctional catalysis.

The rates at which 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide are converted to phosphoramide mustard and acrolein were determined as a function of buffer composition, buffer concentration, and pH. Conversion of 4-hydroperoxycyclophosphamide to 4-hydroxycyclophosphamide in 0.5 M Tris buffer, pH 7.4, 37 degrees, was first-order (k = 0.016 min-1), but subsequent conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein under these conditions was negligible. Phosphoramide mustard and acrolein were readily generated from 4-hydroperoxycyclophosphamide or 4-hydroxycyclophosphamide when either of these agents was placed in phosphate buffer. Conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was first-order with respect to 4-hydroxycyclophosphamide (k = 0.126 min-1 in 0.5 M phosphate buffer, pH 8, 37 degrees) as well as first-order with respect to phosphate serving as a catalyst. The rate-determining step in the reaction was pH dependent only insofar as the hydrogen ion concentration governed the relative amounts of monobasic and dibasic phosphate present. Pseudo-first-order rate constants were 0.045 M-1 min-1 for monobasic phosphate and 0.256 M-1 min-1 for dibasic phosphate. The role of phosphate in this reaction was as that of a bifunctional catalyst. The reaction was not subject to specific or general, acid or base, catalysis. Other bifunctional catalysts such as glucose-6-phosphate and bicarbonate also catalyzed the reaction, albeit less efficiently. Aldophosphamide apparently exists only transiently; its presence could not be established by 31P nuclear magnetic resonance spectroscopy. We conclude that, in the reaction sequence 4-hydroxycyclophosphamide leads to aldophosphamide leads to phosphoramide mustard + acrolein, the conversion of 4-hydroxycyclophosphamide to aldophosphamide is rate limiting and is subject to bifunctional catalysis; this reaction can proceed efficiently only in the presence of a bifunctional catalyst. Assuming that the oncotoxic specificity of cyclophosphamide resides with 4-hydroxycyclophosphamide and that its cytotoxic effect at therapeutic doses is largely mediated by phosphoramide mustard released within cells, these observations offer the possibility that the intracellular concentration of bifunctional catalysts, whether in the form of inorganic phosphates, organic phosphates, enzymes, or other species, serve as important determinants with regard to the oncotoxic potential and specificity of cyclophosphamide. Similarly, the concentration of bifunctional catalysis in the urine as well as the pH of the urine may be important with regard to the potential of cyclophosphamide to induce, via acrolein, hemorrhagic cystitis.

Accelerated decomposition of 4-hydroxycyclophosphamide by human serum albumin.

Cyclophosphamide, a widely used anticancer agent, requires initial metabolic activation to 4-hydroxycyclophosphamide (4-OHCP) to elicit its activity. The rate of decomposition of cis-4-OHCP was much faster in plasma than in buffer at pH 7.4. This plasma activity was not affected by treatment with acid (pH 1.3) or heat (60 degrees C for 30 min). The activity was retained in the macromolecular fraction (greater than 10,000) but not in the filtrate. Serum albumin was identified as the catalyst for the elimination step that generates phosphoramide mustard from aldophosphamide; albumin had no effect on the rate of ring opening of cis-4-OHCP to aldophosphamide. This catalytic activity was dependent on serum albumin concentration and independent of pH over the range of 6.5 to 7.5, in contrast to the buffer-catalyzed reaction. The catalytic rate constants kcat (pH 7.4, 37 degrees C) for phosphate buffer, human serum albumin, and bovine serum albumin were 1.13, 285, and 83 M-1 min-1, respectively. Pretreatment of cis-4-OHCP with serum albumin resulted in a time-dependent decrease in cytotoxic activity against L1210 tumor cells in vitro. These data suggest that the albumin-catalyzed reaction of cis-4-OHCP in plasma represents an important pathway for the transformation of cyclophosphamide metabolites and further emphasize the importance of considering phosphoramide mustard generated extracellularly versus intracellularly and the respective contributions of extracellular and intracellular phosphoramide mustard to cyclophosphamide cytotoxicity in vivo.

Effect of diethyldithiocarbamate on cis-diamminedichloroplatinum(II)-induced cytotoxicity, DNA cross-linking, and gamma-glutamyl transpeptidase inhibition.

Diethyldithiocarbamate (DDTC) has been shown to protect against the toxicity of cis-diamminedichloroplatinum(II) (DDP) without inhibition of antitumor effect. We report here that DDTC is unreactive toward DDP complexes in which both chlorides have been replaced by guanine residues but removes platinum from a variety of other ligands, and that this difference in reactivity may provide the basis for the selective protection observed with DDTC. Platinum-DNA complexes were unreactive toward DDTC (10 mM, greater than 4 h) when the platinum:base ratio r less than 0.02. DDTC did not react with the 1:2 complex of DDP:guanosine but reacted rapidly with the 1:1 complex and with the 1:2 complexes of DDP:adenosine. Reaction of DDP with DDTC was second order with a rate constant k = 4.4 M-1 min-1 at 37 degrees C, corresponding to a t 1/2 = 150 min at [DDTC] = 1 mM. Treatment of L1210 cells with DDTC (0.5-1 mM) after exposure to DDP indicated that DDTC had no effect on cell kill if DDTC treatment was delayed for 1 h after DDP. The effect of DDTC on DDP-induced DNA interstrand cross-links was also examined in L1210 cells. Interstrand cross-links were decreased by approximately 50% when cells were treated with DDTC immediately after DDP; no change in DNA interstrand cross-links was observed when DDTC treatment occurred 3 h after DDP. A modified alkaline elution procedure was used to evaluate the effects of high concentrations of DDTC, thiourea, and cyanide on platinum:DNA cross-links from L1210 DNA. Exposure to DDTC (0.5 M, 4 h) did not alter interstrand cross-links, but both thiourea and cyanide caused extensive reversal of cross-links at concentrations as low as 10 and 1 mM, respectively. Both commercial and rat kidney brush border preparations of gamma-glutamyl transpeptidase were inhibited by exposure to 2 mM DDP; exposure of the inhibited enzyme to DDTC (1 or 10 mM) resulted in significant restoration of enzyme activity. These data indicate that DDTC has unique chemical specificity in its reactions with platinum complexes and that this specificity is ideal for application as a chemoprotective drug against cis-platinum toxicity.

Selective protection against cis-diamminedichloroplatinum(II)-induced toxicity in kidney, gut, and bone marrow by diethyldithiocarbamate.

Diethyldithiocarbamate (DDTC) has been shown to inhibit nephrotoxicity induced by cis-platinum (DDP) without inhibition of tumor response in the rat. We report here that DDTC at doses of 25-300 mg/kg inhibits DDP-induced nephrotoxicity and bone marrow toxicity in C57BL/6 X DBA/2F1 (hereafter called B6D2F1) mice, F344 rats, and beagle dogs and is also antiemetic in the dog. DDTC doses which afford excellent protection do not decrease median survival time following DDP treatment in L1210 and P388 leukemias, B16 melanoma, and Lewis lung and colon 26 carcinomas in B6D2F1 mice when DDTC is given 2 h after DDP. Preliminary experiments indicate that DDTC does not alter median survival time after treatment of P388 leukemia with the platinum analogues diammine(1,1-cyclobutanedicarboxylato)platinum(II) and cis-diisopropylamine-cis-dichloro-trans-dihydroxyplatinum(IV ). Maximum blood urea nitrogen levels after DDP treatment are reduced significantly by DDTC in all species; blood urea nitrogen elevation, total kidney platinum, weight loss, and leukopenia correlate with DDP-DDTC interval in the rat and indicate optimum protection at 2 h, the shortest interval examined. Bone marrow toxicity was assessed by peripheral white blood cell counts in all species and by marrow cellularity in the mouse. White blood cell nadirs were higher and bone marrow recovered more rapidly after DDTC compared with DDP given alone. DDP reduced marrow cellularity 50-60% in the mouse; administration of DDTC 2 h after DDP afforded no protection to the lymphocytes in the marrow but maintained the granulocyte + precursor population near control levels. DDTC plasma pharmacokinetic values have been determined after s.c., i.p., and i.v. administration in the mouse, rat, and dog. Peak plasma levels of 0.3-1.2 mM are observed after a 250-mg/kg dose, with a plasma half-life of 10-20 min. Our data indicate that DDTC may provide protection against most clinically significant toxicities arising from cis-platinum at doses which do not inhibit tumor response.

Tumor efficacy and bone marrow-sparing properties of TER286, a cytotoxin activated by glutathione S-transferase.

TER286 is a latent drug activated by human glutathione S-transferase (GST) isoforms P1-1 and A1-1 to produce a nitrogen mustard alkylating agent. M7609 human colon carcinoma, selected for resistance to doxorubicin, and MCF-7 human breast carcinoma, selected for resistance to cyclophosphamide, both showed increased sensitivity to TER286 over their parental lines in parallel with increased expression of GST P1-1. In primary human tumor clonogenic assays, the spectrum of cytotoxic activity observed for TER286 was both broad and unusual when compared to a variety of current drugs. In murine xenografts of M7609 engineered to have high, medium, or low GST P1-1, responses to TER286 were positively correlated with the level of P1-1. Cytotoxicity was also observed in several other cell culture and xenograft models. In xenografts of the MX-1 human breast carcinoma, tumor growth inhibition or regression was observed in nearly all of the animals treated with an aggressive regimen of five daily doses. This schedule resulted in a 24-h posttreatment decline in bone marrow progenitors to 60% of control and was no worse than for a single dose of TER286. These studies have motivated election of TER286 as a clinical candidate.

Modification of cisplatin toxicity with diethyldithiocarbamate.

Diethyldithiocarbamate (DDTC), a heavy metal-chelating agent, has been shown to decrease cisplatin (CP) toxicity in preclinical studies. This phase I dose-escalation study was undertaken to investigate DDTC as a chemoprotector in patients with advanced cancer. Thirty-five courses of CP in doses ranging from 120 to 160 mg/m2 were given intravenous (IV) bolus to 19 patients. DDTC at 4 g/m2 was infused over 1 hour, starting 45 minutes after CP. There was minimal nephrotoxicity with a mean creatine clearance of 99 mL/min +/- 4 pretreatment and 86 mL/min +/- 4 on day 21. Two courses were associated with a WBC count less than 2,000/mm3 and one course with a platelet count of 15,000/mm3. Two patients had grade 2 neurotoxicity. Hearing loss occurred in 11 patients: five greater than or equal to 20 dB, five greater than or equal to 40 dB, and one greater than or equal to 60 dB. All patients who received cranial irradiation had ototoxicity compared with 43% of those without radiation (P less than .05). All patients experienced toxicity during the DDTC infusion, including hypertension, flushing, diaphoresis, agitation, and local burning. We conclude that DDTC can protect against CP nephrotoxicity at doses up to 160 mg/m2. Ototoxicity became the dose-limiting factor.

Biochemical modulation of cisplatin toxicity.

The biochemical modulation of cisplatin toxicity has contributed substantially to the safe and effective administration of cisplatin in the clinic. In most cases, however, the demonstration of clinical efficacy has preceded the understanding of mechanisms by which these agents provide protection to normal tissues. A number of protocols are now available to ameliorate cisplatin-induced nephrotoxicity; these approaches have been so successful that renal toxicity is rarely of clinical significance today. However, the dramatic escalation in cisplatin dose engendered by the use of modulators has resulted in new dose-limiting toxicities involving the nervous system and the bone marrow. Although preliminary data suggests that certain modulators may reduce these toxicities, extensive clinical trials will be necessary to substantiate the beneficial effects in the clinic. Further clinical studies will also be necessary to determine the optimum scheduling for the newer agents and to provide convincing evidence of efficacy in man. Finally, modulator combinations (i.e. DDTC and hypertonic saline) have the potential to provide the broadest coverage of normal tissue protection and appear to merit more extensive clinical investigation. Cisplatin has demonstrated remarkable clinical efficacy at currently tolerated doses; further advances in the biochemical modulation of cisplatin toxicity should provide significant therapeutic gains for this important drug in the future.

Low-dose X-irradiation and 4-hydroperoxycyclophosphamide distinguishes three Lyt-1+ lymph node T-cell subtypes that respond to specific antigen in vitro.

We investigated whether preculture exposure to low-dose X-irradiation and culture treatment with 4-hydroperoxycyclophosphamide (4-HPCY), a synthetic derivative of cyclophosphamide (CY), might distinguish subtypes of thymic-dependent (T) lymphocytes that respond to specific antigen in vitro. Lymph node (LN) cells were obtained from mice pretreated with CY and immunized with aggregated (A) human IgG (HGG) in Freund's complete adjuvant (CFA), and proliferation was assessed by incorporation of tritiated thymidine. Primed LN cells were untreated or exposed to low-dose irradiation before being cultured in medium alone and in medium containing 4-HPCY. The results show that these agents (irradiation and 4-HPCY) distinguished, in a dose-dependent manner, subtypes of T-cells which contribute to the specific antigen-stimulated proliferative response in vitro. For LN T-cells and LN Lyt-1+ T-cells, 20-25 rads and 1.0 microM 4-HPCY inactivated non-overlapping cell subtypes that respectively accounted for 26% and 28% of the response to HGG. The remaining 46% of HGG-responding cells were not affected by either agent. Although similar cell subtypes were discerned in unseparated LN cells, it required use of higher agent-doses. Cell cycle analysis revealed that treatment with irradiation, 4-HPCY, and the combination (both agents) caused S-phase arrest of 29%, 30%, and 55% of HGG-responding cells, respectively. Thus, identification of these cell subtypes could not be attributed to agent-mediated inactivation of HGG-responding cells that might be in exclusively different phases of the cell cycle.

Evidence that X-irradiation and 4-hydroperoxycyclophosphamide affects different lymphocytes that respond to specific antigen in vitro.

The present study examined the effect of pulse treatment with the in vitro active synthetic derivative of cyclophosphamide (CY), 4-hydroperoxycyclophosphamide (4-HPCY), and exposure to X-irradiation on the in vitro Concanavalin A (ConA), lipopolysaccharide (LPS), and antigen-specific blastogenic responses of in vivo-primed lymph node cells. Primed lymph node cells from CY-pretreated, aggregated (A) human IgG-complete Freund's adjuvant (AHGG-CFA)-immunized mice were untreated, exposed to various doses of irradiation, or pulse treated with different concentrations of 4-HPCY before being cultured in medium alone or in medium containing HGG, ConA, or LPS. The results show that HGG-responding and LPS-responding cells exhibited similar dose-inactivation profiles following exposure to irradiation or pulse treatment with 4-HPCY. More than 75% of reactivity was eliminated by exposure to 100 rads or pulse treatment with 20 microM 4-HPCY. In contrast to preculture pulse treatment with 4-HPCY, however, when primed lymph node cells were cultured in medium containing 4-HPCY (culture treatment) LPS-responding cells were shown to be more sensitive to inactivation than HGG-responding cells. The data further show that the effect of low-dose irradiation and of culture treatment with 4-HPCY on the HGG-specific response of primed lymph node cells was additive, suggesting that these agents inactivate different cell subtypes that contribute to the HGG-specific response in vitro.

IL-1beta mediates diethyldithiocarbamate-induced granulocyte colony-stimulating factor production and hematopoiesis.

Diethyldithiocarbamate (DDTC) exhibits chemoprotective effects via reduced myelosuppression in mice treated with various chemotherapeutic agents. The mechanism of DDTC-mediated chemoprotection is believed to involve the induction and release of several cytokines, including interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte colony-stimulating factor (G-CSF). In the present study the roles of IL-1beta and TNF-alpha in DDTC-mediated G-CSF induction were examined using human long-term bone marrow cultures (hLTBMCs). Administration of IL-1 receptor antagonist (IL-1ra) to DDTC-treated hLTBMCs obviated the G-CSF induction profile and blocked the resultant colony proliferation, indicating that IL-1beta mediates DDTC-induced G-CSF release and hematopoiesis. IL-1beta mRNA levels were increased threefold over control following DDTC treatment of hLTBMCs, implying that DDTC induces IL-1beta at the level of transcription. Conversely, studies involving inhibition of DDTC-induced TNF-alpha synthesis, with the inhibitor MNX 160, had no effect on DDTC-induced G-CSF release or colony proliferation. These findings taken together strongly suggest that IL-1beta mediates the chemoprotective effects of DDTC.

31P NMR and chloride ion kinetics of alkylating monoester phosphoramidates.

31P NMR spectroscopy was used to study the solvolysis kinetics of a novel series of alkylating monoester phosphoramidates (4a-d) under model physiologic conditions. Halide ion kinetics were used to determine the rate of aziridinium ion formation. The solvolysis rates showed the expected dependence upon substitution at the reactive nitrogen; comparison of 4a with phosphoramide mustard (1a) indicated that replacement of the amino group by alkoxy decreased the solvolysis rate by approximately 10-fold. The rate of conversion of starting compound (4a-d) to solvolysis product was essentially equal to the rate of halide ion release, suggesting that the aziridinium ion is a short-lived intermediate. 1H NMR and 31P NMR kinetics experiments performed in the absence and presence of trapping agent (dimethyldithiocarbamate) confirmed that the aziridinium ion was too short-lived to be observed via NMR. These compounds were also tested for cytotoxicity against L1210 leukemia and B16 melanoma cells in vitro; the monoalkylators 4c and 4d showed no activity, 4a was weakly cytotoxic, and 4b was comparable in activity to phosphoramide mustard.

Synthesis and antitumor properties of activated cyclophosphamide analogues.

A series of 5- and 6-substituted cyclophosphamide analogues has been prepared, and their 31P NMR kinetics of phosphoramide mustard (PDA) release and in vitro and in vivo cytotoxicity have been evaluated. cis-4-Hydroxy-5-methoxycyclophosphamide equilibrated very slowly and to a minor extent with the ring-opened aldophosphamide analogues in aqueous buffer; release of PDA was observed to a minor extent and only at high (1 M) buffer concentrations. This analogue was essentially inactive in vitro against L1210 and P388 leukemia cells. 6-Phenylcyclophosphamide and its 4-hydroperoxy derivative were potent inhibitors of blood acetylcholinesterase and were lethal at therapeutic doses in mice. In contrast, 4-hydroperoxy-6-(4-pyridyl)cyclophosphamide did not inhibit acetylcholinesterase and showed significant antitumor activity in vitro and in vivo against both wild-type and cyclophosphamide-resistant L1210 leukemia. The 4-hydroperoxy-6-arylcyclophosphamides were generally active in vitro against both wild-type and cyclophosphamide-resistant L1210 and P388 cells, and several analogues showed significant activity in vivo. Surprisingly, there was no correlation between antitumor activity in vitro and the rate of PDA release in aqueous buffer. Several compounds that showed essentially no release of PDA in aqueous buffer over several hours were highly cytotoxic to leukemia cells following a 1-h exposure in vitro. These results show that activated cyclophosphamide analogues substituted at the 6-position are not cross-resistant in these leukemia cell lines, and that a specific intracellular activation mechanism may be catalyzing PDA release in these analogues.

Synthesis, activation, and cytotoxicity of aldophosphamide analogues.

A series of perhydrooxazine analogues of aldophosphamide has been prepared, and their 31P NMR kinetics and in vitro cytotoxicity have been evaluated. These compounds were developed on the basis of the idea that ring opening and tautomerization to an enamine intermediate might provide a mechanistic alternative to the beta-elimination reaction for release of phosphoramide mustard. The 4,4,6-trimethyltetrahydro-1,3-oxazine moiety was selected on the basis of its rapid rate of iminium ion generation and relatively slow rate of hydrolysis. These analogues underwent phosphorodiamidate release by three distinct mechanisms: hydrolysis to aldophosphamide and subsequent beta-elimination; cyclization to produce the 4-hydroxycyclophosphamides, which release phosphorodiamidate by ring opening and elimination; and tautomerization to the enamine with rapid expulsion of phosphorodiamidate. Kinetic studies demonstrated that hydrolysis to the aldehyde contributed minimally to the overall activation process and that the enamine pathway represented the major route of activation. For those analogues that could undergo cyclization, this pathway competed effectively with enamine release, and these analogues were essentially equivalent to their 4-hydroxycyclophosphamide counterparts in cytotoxicity. A series of tetra-N-substituted phosphorodiamidates that cannot undergo cyclization was prepared to explore the effects of cyclization on the cytotoxicity of these analogues. The tetrakis(chloroethyl)phosphorodiamidates were highly potent in vitro against both cyclophosphamide-sensitive and -resistant L1210 and P388 cell lines, and one of these analogues had significant antitumor activity against L1210 leukemia in vivo. These results demonstrate that the enamine mechanism provides a viable pathway for delivery of phosphorodiamidates and that this approach can be used to deliver phosphorodiamidates that are non-cross-resistant in cyclophosphamide-resistant cell lines.