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PubMed1979

The disposition of cyclophosphamide in a group of myeloma patients.

The disposition of cyclophosphamide and its alkylating metabolites was investigated in a group of myeloma patients with varying degrees of renal function impairment. No correlation between renal function and clearance of cyclophosphamide or its alkylating metabolites was found. No evidence of accumulation of cyclophosphamide or alkylating activity was found in four patients receiving radiolabelled cyclophosphamide. Renal function was found to be related to the reciprocal of the area under curve of alkylating activity, indicating that this area increased as renal function decreased. In view of the large nonrenal component of alkylating activity elimination and the large inter-subject variability, it is recommended that dose of cyclophosphamide is not altered in moderate impairment of renal function.

Alkylating Agents↗

PubMed1985

Prevention of cyclophosphamide-induced urotoxicity by reduced glutathione and its effect on acute toxicity and antitumor activity of the alkylating agent.

The effect of reduced glutathione on acute lethal toxicity and urotoxicity induced by cyclophosphamide was studied on both mice and rats. The results of this investigation indicate that reduced glutathione is an effective protective agent against bladder damage from treatment with the alkylating agent. The timing of glutathione administration (IV) with respect to cyclophosphamide treatment influenced the uroprotective efficacy of the thiol compound. A schedule-dependent protective effect of glutathione against acute lethal toxicity of the antitumor drug was also observed. This partial protection was accompanied by a reduction in body weight loss following cyclophosphamide treatment. The therapeutic activity of cyclophosphamide on two experimental tumor systems (L1210 and Gross leukemia) was not impaired by combined treatment with glutathione, even at a relatively high dose of glutathione compared with cyclophosphamide.

Animals↗

PubMed1988

The effect of fluosol-DA and oxygenation status on the activity of cyclophosphamide in vivo.

The addition of Fluosol-DA followed by carbogen breathing increased the antitumor effect of cyclophosphamide as measured by both tumor growth delay and tumor cell survival assays. Under air breathing condition, cyclophosphamide (100 mg/kg) administered i.p. five times on alternate days produced a tumor growth delay in the FSaIIC fibrosarcoma of 8.0 +/- 0.8 days. Adding Fluosol-DA (0.3 ml) to treatment with cyclophosphamide followed by carbogen breathing increased tumor growth delay to 11.4 +/- 3.6 days, which was not statistically significantly different from that obtained with the drug plus carbogen breathing without Fluosol-DA. As the dose of Fluosol-DA was increased and administered with drug treatment followed by carbogen breathing for 6 h, increasing tumor growth delays of 15.0 +/- 1.5 days, 18.1 +/- 1.7 days and 29.4 +/- 2.2 days were observed with 0.1 ml, 0.2 ml and 0.3 ml Fluosol-DA, respectively. When 0.1 ml Fluosol-DA was administered in combination with cyclophosphamide and immediately followed by 1 h of hyperbaric oxygen (3 atm), a tumor growth delay of 13.7 +/- 1.2 days was observed. With 0.2 ml Fluosol-DA under these conditions, the tumor growth delay increased to 23.2 +/- 1.6 days, and with 0.3 ml Fluosol-DA the tumor growth delay was 35.6 +/- 3.2 days. Single doses of cyclophosphamide with and without Fluosol-DA (0.3 ml) and various conditions of oxygenation were used in an FSaIIC fibrosarcoma tumor cell survival assay. The addition of Fluosol-DA to this single-dose protocol produced a five- to tenfold increase in tumor cell kill compared to air-breathing drug-treated animals. There was no significant difference in the toxic effect of any of the treatment conditions on bone marrow.

Animals↗

PubMed1975

[The effect of cyclophosphamide on tumors in the sensitivity-test (author's transl)].

The sensitivity of tumors to cyclophosphamide was tested in vitro. For this purpose, the urine of cyclophosphamide-treated rats (90 min., 500 mg/kg) was used. This method of activating cyclophosphamid proved more advantageous than other methods; all cytostatically effective metabolites can be used in the test and the production of metabolites can easily be standardized. Consequently results with good reproducibility can be obtained. In various long-term and short-term test models with animal transplantation-tumors in vitro, the effects of the cyclophosphamide metabolites depended on the dose used. This test results in the various test models are comparable. In the tissue-culture of tumors (long-term test) sensitivity was evaluated by using morphological criteria as well as by counting cell numbers. In the short-term test the sensitivity of tumors was found out by influencing the nucleic acid syntheses (3-H-thymidine-, 3-H-uridine-incorporation). As the tissue-culture meets with difficulties as a routine clinical examination, the use of the short-term test is recommended. Using as model the fast proliferating WALKER-256-carcinosarcoma and a slowly proliferating rat adenocarcinoma, the results of the sensitivity-test in vitro were compared with the effects of therapy in animal tests. Tumor remissions corresponding with the results reached in vitro, which depended on dosis, could also be achieved in both transplantation-tumors. Human tumors, tested in in vitro studies (short-term-test), show a different sensitivity to cyclophosphamide.

Adenocarcinoma↗

PubMed1994

Intravenous pulse cyclophosphamide--a new regime for steroid-resistant minimal change nephrotic syndrome.

The treatment of steroid-resistant minimal change nephrotic syndrome (MCNS) continues to pose a therapeutic challenge. We conducted a randomised prospective controlled trial to evaluate the efficacy of i.v. cyclophosphamide compared with oral cyclophosphamide in 13 children with biopsy-proven steroid-resistant MCNS. All 7 patients receiving i.v. cyclophosphamide achieved remission; this was sustained in 4 patients, while 3 relapsed. However, even these 3 patients subsequently became steroid sensitive. Of the 6 patients who received oral cyclophosphamide, 2 dropped out, 1 responded and 3 children continued to remain steroid resistant. The children who received IV cyclophosphamide had more sustained remissions, longer periods without proteinuria and fewer significant side effects; this was achieved at a lower cumulative dose.

Administration, Oral↗

PubMed1996

Phase I trial of intravenous carboplatin added to oral etoposide and oral cyclophosphamide for stage IV non-small cell lung cancer.

The combination of oral etoposide and oral cyclophosphamide is an active and easily administered outpatient regimen for non-small cell lung cancer with leukopenia as the most common severe toxicity. To maintain ease of outpatient administration and to take advantage of a differing dose-limiting toxicity, we attempted to add escalating doses of intravenous carboplatin to full-dose oral etoposide and oral cyclophosphamide for chemotherapy-naive patients with Stage IV non-small cell lung cancer. The first 4 patients received etoposide and cyclophosphamide (each at 50 mg PO BID Days 1-12 every 28 days) with intravenous carboplatin on Day 1 at a dose calculated by the Calvert formula to achieve AUC 4. With this regimen dose-limiting toxicity (2 patients with Grade 4 leukopenia/granulocytopenia) was noted. An additional 3 patients therefore received etoposide and cyclophosphamide at a 25% reduced dose (each at 50 mg PO BID Days 1-9 every 28 days) with intravenous carboplatin on Day 1 at a dose calculated to achieve AUC 4. Dose-limiting toxicity (2 patients with Grade 4 leukopenia/granulocytopenia) was again noted. One patient achieved a partial response maintained for 6 months. However potentiation of leukopenia/granulocytopenia by carboplatin prevents full-dose use of either cyclophosphamide and etoposide or of carboplatin in this regimen.

Administration, Oral↗

PubMed1989

Induction of cytotoxicity from fresh splenocytes after in vivo administration of cyclophosphamide. Importance of long-term culture with high-dose recombinant interleukin-2.

Cyclophosphamide, combined with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), is known to mediate regression of tumors, but the effects of cyclophosphamide on the subsequent generation of LAK cells are unclear. It was the aim of the experiments in this paper to determine whether fresh splenocytes cultured with rIL-2 would maintain or regain their cytotoxicity in vitro after being exposed to the cytotoxic agent cyclophosphamide in vivo. Functional monitoring of splenocytes after in vitro incubation with rIL-2 was performed at various times through chromium-release assays, thymidine assays and cell-cycle analysis. Chromium-release assays determined that the cytotoxicity of cultured splenocytes returned to normal after 12 days of in vitro culture with rIL-2. The thymidine assays indicated a normal rate of uptake of thymidine after 7 days in culture, while the cell cycle was still abnormal by day 12 of culture. The growth and expansion of rIL-2-activated splenocytes after different times of in vitro culture indicated a return to normal compared to control animals after 7 days of continuous in vitro exposure to rIL-2. It is concluded that murine splenocytes can demonstrate cytotoxicity after exposure to cyclophosphamide, through prolonged continuous in vitro culture with rIL-2. Since cyclophosphamide did not jeopardize the production of splenocyte cytotoxic effectors generated with rIL-2, it appears to be a strong contender for use in chemoimmunotherapy protocols.

Animals↗

PubMed1994

Ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression.

BACKGROUND: Lymphocytes obtained from tumor-draining lymph nodes (DLN) can have potent in vivo antitumor activity after in vitro activation with bryostatin 1 and ionomycin. However, the presence of visceral metastases in the donor can inhibit the effectiveness of such lymphocytes. In the present study, we tested the ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression in a murine model. METHODS: Mice were injected with MCA-105 sarcoma cells in the footpad alone or in the footpad and the tail vein to establish lung metastases. Cyclophosphamide was given i.p. 1 day before harvesting the draining popliteal lymph nodes. For all donor groups, DLN cells were activated with 5 nM bryostatin 1 and 1 microM ionomycin and cultured for 7 days in 20 U/ml IL-2. Activated DLN cells were then adoptively transferred to syngeneic mice with 3-day lung metastases. RESULTS: The adoptive transfer of DLN cells from mice with footpad tumors only significantly reduced the number of lung metastases compared to untreated mice. However, activated DLN cells obtained from mice with both footpad and lung tumors were significantly less effective. Treatment of similar donor mice with 10 mg/kg cyclophosphamide significantly improved the anti-tumor activity of adoptively transferred cells. This dose of cyclophosphamide did not reduce the number of cells obtained from each lymph node or the expansion of cell numbers in vitro. CONCLUSIONS: These results suggest that the administration of low-dose cyclophosphamide prior to harvesting DLN cells may improve the success of adoptive immunotherapy in cancer patients.

Animals↗

PubMed2004

Allogeneic dendritic cell vaccination against metastatic renal cell carcinoma with or without cyclophosphamide.

In this phase I/II study, we evaluated the feasibility, safety and efficacy of allogeneic dendritic cells (DCs) with or without cyclophosphamide in the treatment of patients with metastatic renal cell carcinoma (RCC). Immunomagnetic beads were used to isolate CD14(+) monocytes from healthy donor leukapheresis products, and CD83(+) antigen-pulsed monocyte-derived DCs (moDCs) loaded with tumor lysate and keyhole limpet hemocyanin (KLH) were generated. Twelve patients were treated with allogeneic moDCs alone, while ten patients also received cyclophosphamide on days 4 and 3 prior to vaccination. Of the 22 patients enrolled, 20 received full treatment consisting of at least three vaccinations at monthly intervals. Two mixed responses with substantial tumor regression were observed. In 3 patients, disease stabilization occurred, in 13 patients disease progressed and 4 patients were lost to follow-up. Overall, immune responses against KLH and tumor lysate were weak or absent; however, the strongest increases in antigen-independent and KLH-specific responses were observed in the 2 patients with mixed responses. In addition, 1 of them showed a substantial increase in oncofetal antigen (OFA)-specific IFN-gamma production. Importantly, the 2 mixed responders and 1 patient with stable disease belonged to the cyclophosphamide group. Median overall survival in the cyclophosphamide group was 23.2 and 20.3 months in the group that received allogeneic moDCs alone. Allogeneic immunotherapy with moDCs is feasible and well tolerated. However, the immunogenicity of allogeneic moDCs is clearly less pronounced than that of autologous moDC immunotherapy. Cyclophosphamide may have the capacity to augment DC-induced antitumor immunity.

Adult↗

PubMed2006

Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients.

CD4+CD25+ regulatory T cells are involved in the prevention of autoimmune diseases and in tumor-induced tolerance. We previously demonstrated in tumor-bearing rodents that one injection of cyclophosphamide could significantly decrease both numbers and suppressive functions of regulatory T cells, facilitating vaccine-induced tumor rejection. In humans, iterative low dosing of cyclophosphamide, referred to as "metronomic" therapy, has recently been used in patients with advanced chemotherapy resistant cancers with the aim of reducing tumor angiogenesis. Here we show that oral administration of metronomic cyclophosphamide in advanced cancer patients induces a profound and selective reduction of circulating regulatory T cells, associated with a suppression of their inhibitory functions on conventional T cells and NK cells leading to a restoration of peripheral T cell proliferation and innate killing activities. Therefore, metronomic regimen of cyclophosphamide does not only affect tumor angiogenesis but also strongly curtails immunosuppressive regulatory T cells, favoring a better control of tumor progression. Altogether these data support cyclophosphamide regimen as a valuable treatment for reducing tumor-induced immune tolerance before setting to work anticancer immunotherapy.

Adult↗

PubMed2005

Significant induction of cyclophosphamide and thiotepa metabolism by phenytoin.

PATIENT AND METHOD: A 42-year-old male patient with relapsing germ-cell cancer was enrolled in a salvage protocol that employed two 4-day courses of CTC high-dose chemotherapy with cyclophosphamide (1,500 mg m(-2) day(-1)), thiotepa (120 mg m(-2) day(-1)), and carboplatin, followed by peripheral blood progenitor cell support. From five days before the start of the second CTC course the patient received phenytoin for generalized epileptic seizures. Blood samples were collected on day 1 of both CTC courses and analyzed for cyclophosphamide and its activated metabolite 4-hydroxycyclophosphamide, and for thiotepa and its main active metabolite tepa. RESULTS: Exposure (expressed as area under the plasma concentration vs time curve) to 4-hydroxycyclophosphamide and tepa in the second CTC course was increased by 51% and 115%, respectively, compared with the first CTC course, whereas exposure to cyclophosphamide and thiotepa was significantly reduced (67% and 29%, respectively). Because high exposure to 4-hydroxycyclophosphamide and tepa correlates with increased toxicity, the treatment risk of this patient was significantly increased. Therefore doses were reduced on the third day of the second course. CONCLUSION: It was concluded that phenytoin significantly induces both cyclophosphamide and thiotepa metabolism, most probably by induction of the cytochrome p450 enzyme system. This potential clinical significant interaction should be taken into account when phenytoin is administered in combination with cyclophosphamide and thiotepa in clinical practice.

Adult↗

PubMed1998

Pharmacokinetic interaction between ondansetron and cyclophosphamide during high-dose chemotherapy for breast cancer.

PURPOSE: Both ondansetron and cyclophosphamide are thought to be metabolized by hepatic microsomal processes. The purpose of this study was to evaluate the potential pharmacokinetic interactions between ondansetron and high-dose alkylating agent chemotherapy. METHODS: A total of 54 breast cancer patients receiving high-dose cyclophosphamide, cisplatin and carmustine were treated prospectively in four sequential cohorts. Cohorts I and II received continuous infusions of both ondansetron and prochlorperazine, and cohorts III and IV received a continuous infusion of ondansetron alone at the same doses. All patients received lorazepam every 4 h. A group of 75 matched historical controls had received a continuous infusion of prochlorperazine with lorazepam. Pharmacokinetic monitoring of each drug used in the high-dose chemotherapy regimen was conducted. RESULTS: Median AUCs of cyclophosphamide in patients receiving ondansetron (73.6 mg/ml x min) were lower than those of the control patients (88.3 mg/ml x min, n = 75, P = 0.0004), but the median cisplatin AUC was approximately 10% higher and no difference in the disposition of carmustine was demonstrated. Patients treated with ondansetron displayed a higher frequency of headaches than the controls. The frequency of achieving complete emetic control was greater in the ondansetron + prochlorperazine groups compared to the ondansetron alone groups and was greater in both these groups than in the prochlorperazine alone group on the first day of therapy only. CONCLUSION: Ondansetron altered the systemic exposure to cyclophosphamide when these agents were administered concomitantly. Ondansetron did not substantially improve overall emetic control when used alone but may improve control in combination with prochlorperazine. Future randomized studies are needed to delineate the effect of ondansetron on the disposition of the active cyclophosphamide metabolites so that clinical implications can be addressed.

Antiemetics↗

PubMed2003

Intravenous cyclophosphamide in steroid-resistant nephrotic syndrome.

We prospectively examined the effect of treatment with intravenous cyclophosphamide in patients with steroid-resistant nephrotic syndrome. Twenty-four patients (minimal change disease in 11, focal segmental glomerulosclerosis in 9, and mesangioproliferative glomerulonephritis in 4), who did not show remission of proteinuria despite treatment with 8 weeks of oral prednisolone and six intravenous pulses of dexamethasone, were studied. Cyclophosphamide was administered intravenously, at a dose of 750 mg/m(2) once a month for 6 months; therapy with alternate-day prednisolone was continued. The mean (SD) age at treatment was 7.8 (4.0) years; 18 patients had initial resistance and 6 had late resistance. At the end of 6 months treatment, 7 (29.2%) patients each had complete remission (absent proteinuria, normal serum albumin) and partial remission (1-2+ proteinuria, normal serum albumin). Ten (41.6%) patients showed no response to therapy. The mean time to complete or partial remission, after initiation of treatment with cyclophosphamide, was 2.4+/-1.7 months and 2.7+/-1.8 months, respectively. Most responders (85.8% complete and 57.2% partial responders) achieved remission by the third dose of pulse cyclophosphamide. More patients with late resistance (50%) compared with initial resistance (22.2%) achieved complete remission. Partial remission was transient and lasted for a mean duration of 6.4+/-3.5 months. Serious infections were observed during therapy in 5 patients. On long-term follow-up, 5 (20.8%) patients were in remission, while nephrotic-range proteinuria or end-stage renal disease was seen in 17 (70.8%). Findings from the present study suggest that therapy with intravenous cyclophosphamide has limited efficacy in inducing sustained remission in patients with initial corticosteroid resistance. Sustained remission is likely to occur in a significant proportion of patients with late resistance and those with absence of significant tubulointerstitial changes on renal histology.

Adolescent↗

PubMed1997

Oral dolasetron mesilate (MDL 73,147EF) for the control of emesis during fractionated total-body irradiation and high-dose cyclophosphamide in patients undergoing allogeneic bone marrow transplantation.

The purpose of the present study was to evaluate the efficacy and safety of oral dolasetron mesilate in the prevention of nausea and vomiting that might otherwise be induced by total-body irradiation (TBI) and high-dose cyclophosphamide. In an open non-comparative study 20 patients who received TBI for 3 days and high-dose cyclophosphamide chemotherapy for 2 days as part of their preparation for bone marrow transplantation were given oral dolasetron mesilate at dosages ranging from 50 to 200 mg 1 h before each fraction of radiotherapy and cyclophosphamide administration. Initial rescue therapy consisted of intravenous dolasetron mesilate. If nausea and vomiting remained uncontrolled, standard antiemetics were to be used. Of the 20 patients, 13 had only two emetic episodes or fewer in the 3-day TBI period. On days 1 and 2 of cyclophosphamide administration, 11 and 6 patients had fewer than two emetic episodes. From day 1 to day 3, 15 patients experienced no nausea or only mild nausea, and on the days of chemotherapy 8 and 7 patients had mild nausea or none at all. Rescue with i.v. dolasetron mesilate was needed by 3 and 6 patients during the TBI and the chemotherapy periods respectively. In 2 patients additional antiemetics were used on days 2-3 and 4-5. Mild to moderate headache was reported in 6 patients. No unexpected abnormalities were observed in haematology, biochemistry or urinalysis, and vital signs were unaffected throughout the study period. The data suggest that oral dolasetron mesilate is effective and safe for the prevention of nausea and vomiting during TBI and cyclophosphamide chemotherapy prior to bone marrow transplantation. Future controlled studies should evaluate combination antiemetic therapy with dolasetron mesilate for this indication.

Administration, Oral↗

PubMed2006

Improvement of lung function in patients with systemic sclerosis after 6 months cyclophosphamide pulse therapy.

This study aims to analyze the effects of cyclophosphamide pulse therapy on parameters of lung function in patients with systemic sclerosis. Nineteen patients with systemic sclerosis (15 women and four men, aged 25-67 years, mean disease duration 5 years and 9 months) were included in this study. The main reason for the beginning of cyclophosphamide therapy was the decrease of transfer-factor (DLCO) or diffusing coefficient for carbon monoxide (DLCO/VA) under 70% of predictive value. Intravenous cyclophosphamide was administered monthly in a dose of 500 mg/m(2 )body surface. The efficacy was evaluated by comparison of forced vital capacity (FVC), DLCO, and DLCO/VA at the baseline and 1 month after the sixth pulse. Statistical analyses were performed using Student's T test and Wilcoxon's test. The difference between FVC at the baseline (86.6%) and at the end of the follow-up period (89.2%) was not statistically significant (t=-1.25, p>0.05). However, a significant increase of DLCO (from 61.2% to 70.5%, z=-2.04, p=0.04) and DLCO/VA (from 57.8% to 72.5%, z=-2.67, p=0.008) was observed. Minor side effects were noticed in some patients. Two patients had nausea after cyclophosphamide infusion, two patients had insignificant decrease of creatinine clearance, and two patients had temporary and mild leukopenia. In patients with systemic sclerosis and lung involvement, an improvement of lung-diffusing capacity was noticed 6 months after the beginning of cyclophosphamide pulse therapy, with only minor side effects.

Adult↗

PubMed2005

Histopathological changes in insulin, glucagon and somatostatin cells in the islets of NOD mice during cyclophosphamide-accelerated diabetes: a combined immunohistochemical and histochemical study.

The cyclophosphamide model of accelerated diabetes in the NOD mouse is a useful model of insulin-dependent diabetes mellitus (IDDM). Knowledge on the progressive destruction of beta cells and the fate of other islet endocrine cell-types in this model is sparse. We employed immunohistochemistry and histochemistry, to study temporal changes in islet cell populations, insulitis and glucose transporter-2 expression during cyclophosphamide administration. Cyclophosphamide was administered to day 95 female NOD mice and the pancreas studied at days 0 ( = day 95), 4, 7, 11 and 14 after treatment and in age-matched control mice. At day 0, a majority of the endocrine cells were insulin-positive. Glucagon and somatostatin cells were mostly in the islet periphery and also internally. In the cyclophosphamide group, insulitis was moderate at day 0, declined at day 4 but increased progressively from day 7. The extent of insulitis in treated mice which were diabetes-free at day 14 was comparable to age-matched control mice. From day 11, the marked increase in insulitis correlated with a reciprocal decline in the extent of insulin immunostained islet area. At day 14, the mean insulin area per islet was markedly less in diabetic mice than in age-matched non-diabetic treated and controls. At diabetes, some islets showed co-expression of glucagon and insulin. Our studies suggest that the mean number of glucagon or somatostatin cells per islet does not vary during the study. Glucose transporter-2 immunolabelling was restricted to beta cells but declined in those adjacent to immune cells. We conclude that in the cyclophosphamide model, there is specific and augmented destruction of beta cells immediately prior to diabetes onset. We speculate that the selective loss of glucose transporter-2 shown in this study suggests the existence of a deleterious gradient close to the immune cell and beta cell surface boundary.

Animals↗

PubMed1984

Deoxyribonucleic acid crosslinking by 4-hydroperoxycyclophosphamide in cyclophosphamide-sensitive and -resistant L1210 cells.

4-Hydroperoxycyclophosphamide, a synthetic, activated form of cyclophosphamide, has been used to study DNA crosslinking in L1210 cell lines sensitive and resistant to cyclophosphamide. The time course of crosslink appearance and the proportion of inter-strand to DNA-protein crosslinks support the belief that phosphoramide mustard is the ultimate alkylating agent derived from cyclophosphamide. Cell survival and DNA crosslinking studies with a cyclophosphamide-resistant L1210 cell line indicate that resistance is associated with a failure of 4-hydroperoxycyclophosphamide to produce DNA crosslinks. The ability to reverse this situation by exposure of resistant cells to disulfiram points to a role of aldehyde dehydrogenase in this mechanism of cyclophosphamide resistance.

Aldehyde Dehydrogenase↗

PubMed1988

N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine, the putative major DNA adduct of cyclophosphamide in vitro and in vivo in the rat.

The anti-cancer agent, cyclophosphamide, metabolises to the cytotoxic alkylating agent phosphoramide mustard, which can be dephosphoramidated to give nornitrogen mustard. A rat liver mitochondrial supernatant system was used to study the binding of [chloroethyl 3H]cyclophosphamide to DNA. The reacted DNA was acid-hydrolysed and one major adduct was identified using Sephadex G-10 chromatography, followed by HPLC, using reversed-phase or ion-exchange systems. Further studies, using [14C]guanine as reaction substrate for [chloroethyl 3H]cyclophosphamide, phosphoramide mustard or nornitrogen mustard, demonstrated the main adduct from each reaction had identical chromatographic properties in these systems. The radiolabelled ratio in the [3H]cyclophosphamide-[14C]guanine reaction demonstrated a monoadducted product. From this evidence and from 1H NMR data, the common adduct was putatively identified as a hydroxylated nornitrogen mustard adduct (N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine). In in vivo studies, rats were injected intraperitoneally with 2.775 MBq [3H]cyclophosphamide. Total organ [3H] content and DNA binding levels were ascertained. Maximal levels of [3H] binding to DNA were seen between 1-4 hr with the highest binding levels observed in the bladder. The in vivo adduct was shown, using various HPLC systems, to co-chromatograph with the in vitro adduct and thus the main in vivo adduct was putatively identified as N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine.

Alkylation↗