Doses Of CCNU Research Articles

VAMANA: A phase 2 study of low-dose bevacizumab plus CCNU in relapsed/recurrent glioblastoma.

LBA2064 Background: There are limited systemic therapy options for recurrent glioblastomas (rGBM). CCNU and/or Bevacizumab are often used to treat rGBM not amenable to local therapy. The addition of Bevacizumab (10 mg/kg) to CCNU failed to improve overall survival in the EORTC 26101 study. The question is whether the failure was due to the high dose of Bevacizumab used in the study. High doses of Bevacizumab may lead to excessive pruning & destruction of blood vessels, hampering the delivery of CCNU. In vitro & in vivo studies showed that a lower dose of Bevacizumab (1-1.5 mg/kg) has the potential to normalize tumor vasculature leading to improved drug delivery & outcomes. To test this hypothesis, we conducted this study to evaluate the efficacy of low-dose Bevacizumab in combination with CCNU in rGBM. Methods: This was a phase 2 open-label, single-arm trial that included adults with rGBM with an ECOG PS 0-2 and adequate organ & marrow function. The participants received CCNU 110 mg/m2 PO once a day, on day 1 of a 42-day cycle (max. 8 cycles) with Bevacizumab 1.5 mg/kg intravenously every 3 weeks (max.16 cycles). Appropriate anti-emetic prophylaxis was given. Treatment continued until disease progression, clinical deterioration, or development of intolerable side effects. Response assessment was done with MRI Brain +/- spine at 2 monthly intervals till disease progression. The modified RANO criteria were used for response assessment. Safety assessments were done on day 8 of cycle 1, & days 21 & 42 of each cycle. The primary end-point of this study was overall survival (OS) and secondary end-points were progression-free survival (PFS) & safety. A 6-month OS ≥ 40% was the signal to explore this regimen further and if the 6-month OS <20% it would be considered futile. Descriptive statistics were performed and the Kaplan-Meier method was used for time-to-event analysis. Results: Forty-six patients were enrolled in this study. The median age was 42 years (IQR 33-52.75) and 78.3% (36/46) were males. Most patients (76.1%, 35/46) had an ECOG PS of 1. The median follow-up duration was 15.27 months (95% CI 13.47-17.06). The median number of doses of Bevacizumab and CCNU were 4 and 2 respectively. Three (6.5%) patients completed all planned doses of Bevacizumab and CCNU. The objective response rate (ORR) was 15.2 % (7/46). The median OS was 6.133 months (95% CI 5.474-6.793). The 6-month OS was 57.1%. The median PFS was 3.267 months (95% CI 0.850-5.684). The most frequent grade 3 or higher toxicities seen were neutropenia (7/46, 15.2%), thrombocytopenia (5/46,10.8%), elevated ALT levels (3/46, 6.5%), anemia (2/46, 4.3%) and hyponatremia (2/46, 4.3%). Conclusions: This study demonstrates the efficacy of low-dose Bevacizumab in combination with CCNU in rGBM (the median OS exceeded the preplanned cut-off of 40%) with an acceptable toxicity profile and no new safety signals. This combination should be explored further in a phase III randomized study. Clinical trial information: CTRI/2020/07/026696 .

Risk factors associated with the onset of lomustine-induced neutropenia in tumour-bearing dogs.

Lomustine (1-[2-chloroethyl]3-cyclohexyl-1-nitrosurea, CCNU) is an oral alkylating agent in the nitrosourea subclass that can cause myelosuppression, with neutropenia being the main dose-limiting toxicity. The aim of this study was to define the frequency of neutropenic events and to identify predisposing risk factors in tumour-bearing dogs treated with CCNU. Dogs receiving CCNU for various malignancies were identified following a search of hospital databases. Variables analysed for correlation with neutropenia included signalment, body weight, tumour type, CCNU total dose, steroid use, protocol type, use of L-asparaginase, previous anthracycline administration and use of the drug as first-line or in the rescue setting. One-hundred and fifteen cases were included; median age was 7 years (range 1-14 years) and median body weight 27.6kg (range 3-74 kg). The median CCNU dose was 63.5mg/m2 (range 27.7-84.9mg/m2 ). Neutropenia occurred in 75 cases (65%) and was comprised of grade 1 (28%), 2 (16%), 3 (29.3%) and 4 (26.7%) events. Tumour type (histiocytic sarcoma [HS]), use of CCNU first line, dose >70 mg/m2 , absence of co-morbidities and previous anthracycline administration, were significantly associated with an increased risk of developing neutropenia, including high-grade events. There was a 1.7% reported mortality rate. When CCNU is used in dogs with HS, first-line, at a starting dosage >70 mg/m2 , in patients with no co-morbidities or with a history of previous anthracycline administration, there may be an increased risk of developing neutropenia. These data may help guide treatment decisions and minimize treatment delays or potentially life-threatening complications.

Outcome in dogs with curative-intent treatment of localized primary pulmonary histiocytic sarcoma.

Primary pulmonary histiocytic sarcoma (PHS) is a rare form of dendritic cell or macrophage neoplasia originating within the pulmonary parenchyma. There is limited literature describing prognosis in dogs with PHS receiving curative-intent treatment consisting of surgical excision and adjuvant chemotherapy. The primary objective of this study was to report outcomes in dogs with localized PHS treated with standardized local and systemic therapy. A secondary objective was to identify prognostic factors in this population. A multi-institutional retrospective study was performed and medical records including all surgical and histopathologic reports were retrospectively reviewed. For inclusion, dogs were required to have confirmed localized PHS and they must have undergone curative-intent surgery with resection of all gross primary tumour and enlarged tracheobronchial lymph nodes; additionally, they must have received curative-intent treatment with adjuvant single-agent CCNU chemotherapy. Twenty-seven dogs from six veterinary teaching hospitals and five private practices treated from 2008-2019 were included. The overall median survival time was 432 days. Higher CCNU dose was demonstrated to have a negative impact on survival on univariate, but not multivariable, analysis. Factors that were not found to be associated with survival on univariate analysis included body weight, breed, clinical signs at the time of diagnosis, hypoalbuminaemia, tumour size, lung lobe affected, lymph node metastasis, surgical margins and CCNU dose reductions. This study supports a favourable prognosis for dogs diagnosed with localized PHS treated with curative-intent surgery in addition to adjuvant CCNU chemotherapy and suggests that multimodal treatment may be advisable to attempt to prolong survival.

CN-18 * RELATIONSHIPS BETWEEN DOSE INTENSITY, TOXICITY, AND OUTCOME IN OLIGODENDROGLIAL TUMORS (OG) TREATED WITH PCV REGIMEN

BACKGROUND: In grade II and codeleted grade III gliomas, the procarbazine-CCNU-vincristine (PCV) combination increase survival when added to radiotherapy as first line treatment, despite the important toxicity of this treatment schedule. Our objective was to analyze the tolerance, feasibility and impact of dose intensity of the PCV regimen on outcome for patients with OG. METHODS: We retrospectively reviewed all grade III OG patients receiving PCV (CCNU:110mg/m2) who were referred to our two institutions. The total dose and dose adaptation, cycle delay, dose intensity, toxicity and premature discontinuation of CCNU were analyzed. Impact of these factors on patient outcome was evaluated. RESULTS: Between 2007 and 2011, 89 patients received PCV. Only 37% completed 6 cycles, whereas 13.4% prematurely discontinued PCV because of toxicity. Cycle delay and dose reduction were observed for 62% and 70% patients, respectively. Grade 3 and 4 toxicities were observed in 38% and 8% patients, respectively. Among patients who did not progress under PCV regimen, by multivariate analysis, premature discontinuation for toxicity was significantly correlated with poor PFS (p = 0.023, Hazard ratio (HR):2.354) and OS (p = 0.021, HR:5.093). By univariate analyses, absence of CCNU dose adaptation was correlated to poor PFS (p = 0.032). For OS, pejorative factors were high total CCNU dose (p = 0.029), absence of cycle delay (p = 0.009), absence of CCNU dose adaptation (p = 0.020) and grade 3/4 toxicities (p = 0.013). High CCNU dose-intensity tended to poorly impact PFS (p = 0.053) and OS (p = 0.112). By multivariate analysis, absence of CCNU dose adaptation remained significant for PFS (p = 0.001), while OS was negatively impacted by the absence of cycle delay (p = 0.049) and grade 3/4 toxicities (p = 0.045). CONCLUSION: Despite the efficacy of the PCV regimen, significant toxicity is associated with this schedule, which appears to impact its feasibility and efficacy. The optimal PCV schedule should be redefined taking into account this finding.

Low apparent risk of CCNU (lomustine)-associated clinical hepatotoxicity in cats.

The purpose of this study was to evaluate the prevalence of serum alanine transaminase (ALT) increases in cats treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU, lomustine). The medical records of 95 cats treated with CCNU were reviewed, 29 of which met study criteria (at least one treatment with CCNU as a single agent, and at least one pretreatment and one post-treatment complete biochemical profile). Cats that received concurrent prednisone or dexamethasone were included, but those that received concurrent hepatoprotective or hepatotoxic medications were excluded. Cats included in the study were diagnosed with hepatic carcinoma, mammary carcinoma, lymphoma, mast cell tumor, plasma cell tumor and gastrointestinal leiomyoma. CCNU was given as a single agent at 31-60 mg/m(2), once every 4-8 weeks. Serum alanine transaminase (ALT) activity was measured after at least one dose of CCNU. Four cats (13.7%) had increased ALT activity above the reference interval before starting treatment. Two additional cats (6.8%) developed increased ALT activity above the reference interval 1 month after treatment with CCNU. One cat developed clinical signs potentially associated with hepatotoxicity, without a concurrent increase in ALT, 3 weeks following the final dose of CCNU. No association between dosing frequency, cumulative dose, initial starting dose or concurrent medications, and increases in ALT were found. Clinically significant hepatic injury is seemingly uncommon in cats treated with CCNU.

Prophylactic intrathecal chemotherapy in primary CNS lymphoma

The role of prophylactic intrathecal chemotherapy in the treatment of primary central nervous system lymphoma remains controversial. We report a retrospective single center study of a cohort of 69 patients with primary central nervous system lymphoma who had been treated with a regimen that combined high intravenous doses of Methotrexate, CCNU, procarbazine and methylprednisolone. Before 2000, patients systematically received intrathecal prophylaxis including Methotrexate, cytarabine, and hydrocortisone delivered either by intraventricular or lumbar injection along with the systemic chemotherapy (group A, n = 39). After this date, the procedure was changed and intrathecal chemotherapy was withdrawn from the protocol (group B, n = 30). The median age and Karnofsky index were comparable in both groups. At the time of analysis, we found no significant difference between patients with and without intrathecal prophylaxis in terms of objective response rate, patterns of relapse, progression-free survival or overall survival. In our study, intrathecal prophylaxis withdrawal from a high dose intravenous Methotrexate-based chemotherapy regimen did not influence disease control and outcome of primary central nervous system lymphoma. Further studies prospectively investigating the role of intrathecal chemoprophylaxis are warranted for this disease.

Prospective Randomized Clinical Trial Assessing the Efficacy of Denamarin for Prevention of CCNU‐Induced Hepatopathy in Tumor‐Bearing Dogs

Increases in liver enzymes occur in up to 86% of dogs receiving CCNU and can result in treatment delay or early discontinuation of treatment. Denamarin contains S-adenosylmethionine and silybin, both of which have been investigated as treatments for various liver diseases. Dogs on CCNU receiving Denamarin have lower alanine aminotransferase (ALT) activity than dogs not receiving Denamarin. Dogs on Denamarin are less likely to require treatment delay because of hepatopathy and are more likely to complete their prescribed course of CCNU. Dogs with lymphoma, mast cell tumor, or histiocytic sarcoma that were prescribed CCNU with or without corticosteroids and with normal ALT activity were eligible for enrollment. Dogs were prospectively randomized to receive either concurrent Denamarin during CCNU chemotherapy or to receive CCNU alone. Liver-specific laboratory tests were run before each dose of CCNU. Increased liver enzyme activity occurred in 84% of dogs receiving CCNU alone and in 68% of dogs on concurrent Denamarin. Dogs receiving CCNU alone had significantly greater increases in ALT, aspartate aminotransferase, alkaline phosphatase, and bilirubin and a significantly greater decrease in serum cholesterol concentrations than dogs receiving concurrent Denamarin. Dogs receiving CCNU alone were significantly more likely to have treatment delayed or discontinued because of increased ALT activity. Increased liver enzyme activity occurs commonly in dogs receiving CCNU chemotherapy. These results support the use of concurrent Denamarin to minimize increased liver enzyme activity in dogs receiving CCNU chemotherapy. Denamarin treatment also increases the likelihood of dogs completing a prescribed CCNU course.

Prevalence of elevated alanine transaminase activity in dogs treated with CCNU (Lomustine)*

The purpose of this study was to evaluate prevalence of serum alanine transaminase (ALT) elevation in dogs receiving lomustine (CCNU) and to analyse the pattern of occurrence and potential risk factors. Serum ALT activity in 109 dogs during single-agent CCNU chemotherapy was retrospectively analysed. The median initial dose, dose-intensity and cumulative dose of CCNU were 64 mg m(-2), 21 mg m(-2) week(-1) and 171 mg m(-2), respectively. The overall prevalence of major ALT elevation [> 5-fold upper reference limit (URL)] was 29% (32/109) and developed most commonly after one to three doses of CCNU. These ALT elevations occurred without preceding mild ALT elevation in 53% (17/32) of the cases. Three dogs (2.8%) developed clinical hepatopathy. For severe ALT elevation (>10-fold URL), age < or =5-year-old was associated with higher risk. The findings of this study showed that elevation of ALT is common during CCNU chemotherapy in dogs and severe elevation can develop on a sudden onset.

Pulmonary fibrosis after high cumulative dose nitrosourea chemotherapy in a cat

Small to intermediate cell alimentary lymphoma was diagnosed in a cat after abdominal exploratory surgery with no prior history of pulmonary disease. Initial response to several chemotherapy regimens was poor, but a long-term remission was achieved with CCNU (lomustine) and corticosteroid therapy. After receiving a total cumulative CCNU dose of 552 mg m(-2) over 12 months, an acute episode of respiratory distress occurred and the cat died. Necropsy identified severe diffuse pulmonary fibrosis and no signs of lymphoma. This is the first report of pulmonary fibrosis following high cumulative dose nitrosourea chemotherapy in a cat.

Radiotherapy plus concurrent and adjuvant procarbazine, lomustine, and vincristine chemotherapy for patients with malignant glioma

We analyzed the clinical efficacy and toxicity of concurrent therapy as a first line modality for malignant glioma patients. From 1998 to 2004, 39 patients, 22 with glioblastoma (GM), nine with anaplastic astrocytoma (AA), 7 with anaplastic oligodendroglioma (AO) and 1 with anaplastic oligodendro-astrocytoma (AOA) were enrolled in this study. The median age was 46.2 years (range 8-67). Both external involved field radiotherapy and chemotherapy, composed of CCNU (75-110 mg/m(2)), procarbazine (60 mg/m(2)) and vincristine (1.4 mg/m(2)), were started simultaneously two weeks after surgery. The median progression-free survival time for the GM, AA, and AO patients was 6, 26, and 31 months, respectively. The median survival of the patients with GM and AA was 27 and 41 months. The two-year survival rate of the GM and AA patients was 50.4 and 66.7%, respectively. Grade III/IV hematological toxicity was reduced from 25.6 to 13% after reduction of the dose of CCNU (75 mg/m(2)). Radiation necrosis was confirmed by pathologic examination in four patients (10.3%). The median interval from the completion of radiotherapy to the diagnosis of necrosis was 19 weeks. Modified concurrent chemoradiotherapy may be a feasible option for treating malignant glioma with acceptable toxicity.

Temozolomide (TMZ) and lomustine (CCNU) in high grade glioma (HGG) patients: Phase I study

1577 Background: TMZ has activity in HHG and same new combinations are being studied to evaluate an increase of this activity. The combination of TMZ and nitrosureas were evaluated preclinically. These experimental results indicated synergistic killing when TMZ is combined with nitrosureas in resistant human glioma cell lines. Based on these finding, as primary endpoint, this phase I study was performed to determine the maximum tolerated dose (MTD), safety and feasibility of new treatment schedule of temozolomide when administered with a fixed dose of CCNU, and secondary, was evaluated the preliminary data of efficacy. Methods: Recurrent HHG patients (10 with glioblastoma multiforme and two with anaplastic astrocytoma) were treated with oral TMZ 75 mg/mq for 21 or 28 consecutive days or TMZ 100 mg/mq for 28 consecutive days together with orally CCNU 100 mg/mq on day 1 every 6 weeks. This schedule was approved by ethics committe and a informed consent was obtained for all subjects. Results: Eleven patients received a total of 35 courses of TMZ/CCNU. The main toxicities of the regimen consisted of vomiting, costipation, neutropenia and thrombocytopenia. The myelotoxicity was dose-limiting in two of six new patients treated with TMZ 100 mg/mq x 28 days plus CCNU 100 mg/mq d1 dose level. None dose-limiting toxicity was evaluated in 15 courses in the patients at the lower dose level. Preliminary data about toxicity are ongoing. For ongoing phase II clinical trial we recommended safe dose is TMZ 75 mg/mq for 28 consecutive days and CCNU 100 mg/mq d 1 every 6 ws. Conclusions: The schedule didn't reduce the TMZ dose-intensity and the addition of CCNU didn't substantially modify the tolerability of TMZ respect to standard single-agent TMZ. The preliminary results about TTP disease seem to be very promising. No significant financial relationships to disclose.

Temozolomide (TMZ) and lomustine (CCNU) in high grade glioma (HGG) patients: Phase I study

1577 Background: TMZ has activity in HHG and same new combinations are being studied to evaluate an increase of this activity. The combination of TMZ and nitrosureas were evaluated preclinically. These experimental results indicated synergistic killing when TMZ is combined with nitrosureas in resistant human glioma cell lines. Based on these finding, as primary endpoint, this phase I study was performed to determine the maximum tolerated dose (MTD), safety and feasibility of new treatment schedule of temozolomide when administered with a fixed dose of CCNU, and secondary, was evaluated the preliminary data of efficacy. Methods: Recurrent HHG patients (10 with glioblastoma multiforme and two with anaplastic astrocytoma) were treated with oral TMZ 75 mg/mq for 21 or 28 consecutive days or TMZ 100 mg/mq for 28 consecutive days together with orally CCNU 100 mg/mq on day 1 every 6 weeks. This schedule was approved by ethics committe and a informed consent was obtained for all subjects. Results: Eleven patients received a total of 35 courses of TMZ/CCNU. The main toxicities of the regimen consisted of vomiting, costipation, neutropenia and thrombocytopenia. The myelotoxicity was dose-limiting in two of six new patients treated with TMZ 100 mg/mq x 28 days plus CCNU 100 mg/mq d1 dose level. None dose-limiting toxicity was evaluated in 15 courses in the patients at the lower dose level. Preliminary data about toxicity are ongoing. For ongoing phase II clinical trial we recommended safe dose is TMZ 75 mg/mq for 28 consecutive days and CCNU 100 mg/mq d 1 every 6 ws. Conclusions: The schedule didn't reduce the TMZ dose-intensity and the addition of CCNU didn't substantially modify the tolerability of TMZ respect to standard single-agent TMZ. The preliminary results about TTP disease seem to be very promising. No significant financial relationships to disclose.

Hepatotoxicity Associated with CCNU (Lomustine) Chemotherapy in Dogs

One hundred seventy‐nine tumor‐bearing dogs were treated with 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea (CCNU) between 1995 and 2001. CCNU was given as a single dose of 50–110 mg/m2body surface area PO. Treatment interval varied, but the minimal interval between CCNU doses was 3 weeks. After treatment, 11 dogs (6.1%) developed hepatic toxicity. The median number of CCNU doses and the median total cumulative CCNU dose were significantly higher in dogs that developed hepatic toxicity (4 doses; 350 mg/m2) than in dogs without hepatic damage (3 doses; 230 mg/m2). Median duration to detection of hepatic toxicity from the last dose of CCNU was 11 weeks (range 2–49 weeks). Common biochemical abnormalities were abnormally high serum liver enzyme activities and hypoalbuminemia. Six dogs with CCNU‐associated hepatic toxicity had ascites, and 3 dogs had concurrent pleural effusion. Serum concentrations of bile acids were abnormally high in 4 of 5 dogs tested. Percutaneous ultrasound‐guided liver biopsies were performed in 10 dogs, and findings were nonspecific and chronic in nature. Seven dogs were euthanized because of progressive liver failure, and their median survival from diagnosis of liver disease was 9 weeks. Three dogs died of other causes and 1 dog of unknown cause. Although clinical signs resolved in 3 dogs, biochemical abnormalities and histopathologic lesions persisted 4 to 38 months from the time of diagnosis of liver disease. Our findings suggest that CCNU can cause delayed, cumulative dose‐related, chronic hepatotoxicity that is irreversible and can be fatal.

Hepatotoxicity associated with CCNU (lomustine) chemotherapy in dogs.

One hundred seventy-nine tumor-bearing dogs were treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) between 1995 and 2001. CCNU was given as a single dose of 50-110 mg/m2 body surface area PO. Treatment interval varied, but the minimal interval between CCNU doses was 3 weeks. After treatment, 11 dogs (6.1%) developed hepatic toxicity. The median number of CCNU doses and the median total cumulative CCNU dose were significantly higher in dogs that developed hepatic toxicity (4 doses; 350 mg/m2) than in dogs without hepatic damage (3 doses; 230 mg/m2). Median duration to detection of hepatic toxicity from the last dose of CCNU was 11 weeks (range 2-49 weeks). Common biochemical abnormalities were abnormally high serum liver enzyme activities and hypoalbuminemia. Six dogs with CCNU-associated hepatic toxicity had ascites, and 3 dogs had concurrent pleural effusion. Serum concentrations of bile acids were abnormally high in 4 of 5 dogs tested. Percutaneous ultrasound-guided liver biopsies were performed in 10 dogs, and findings were nonspecific and chronic in nature. Seven dogs were euthanized because of progressive liver failure, and their median survival from diagnosis of liver disease was 9 weeks. Three dogs died of other causes and 1 dog of unknown cause. Although clinical signs resolved in 3 dogs, biochemical abnormalities and histopathologic lesions persisted 4 to 38 months from the time of diagnosis of liver disease. Our findings suggest that CCNU can cause delayed, cumulative dose-related, chronic hepatotoxicity that is irreversible and can be fatal.

Induction of specific-locus and dominant lethal mutations in male mice by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) induced dominant lethal and specific-locus mutations in male mice. For both compounds the germ cell stage sensitive to the induction of dominant lethal mutations was dose dependent. A dose of 5 mg BCNU per kg b.wt. induced dominant lethal mutations primarily in spermatocytes, whereas higher doses of BCNU induced dominant lethals in spermatids and spermatocytes. Following doses of 5 and 10 mg CCNU per kg b.wt. dominant lethals were induced in spermatids and spermatocytes similar to the results for higher doses of BCNU. Higher dose exposure to BCNU and CCNU was associated with dominant lethals expressed as pre-implantation loss (reduction in total number of implants). In addition, higher doses of CCNU showed a cytotoxic effect in differentiating spermatogonia. Both compounds induced specific-locus mutations in post-spermatogonial germ cell stages of mice. However, CCNU increased also the specific-locus mutation frequency in spermatogonia in two out of three experiments. We conclude in analogy with criteria developed by IARC, that BCNU and CCNU are potential human mutagens.

Pharmacokinetics of high-dose oral CCNU in bone marrow transplant patients.

CCNU (lomustine) and other nitrosourea compounds are rapidly metabolized to alkylating moieties, which are active against various malignancies. In humans, CCNU undergoes biotransformation to the geometric isomers of 4'-hydroxyCCNU. The pharmacokinetics of trans-and cis-4'-hydroxyCCNU were determined in five patients with Hodgkin's or non-Hodgkin's lymphoma receiving sequential doses of CCNU (15 mg/kg), etoposide (60 mg/kg) and cyclophosphamide (100 mg/kg) prior to autologous bone marrow transplantation. Plasma concentrations of the isomeric forms of the metabolites were determined by high-performance liquid chromatography. Data were analysed using noncompartmental pharmacokinetic methods. Formation of the trans-isomer predominated over that of the cis-isomer, with an average exposure ratio of 1.4 (range 1.0-2.1). Peak plasma concentrations occurred between 1 and 4.1 h postdosing and averaged 1.56 mg/l for the trans-isomer and 1.10 mg/l for cis isomer. Peak plasma concentrations and systemic exposures varied approximately six- and ninefold, respectively, reflecting significant interindividual variability in alkylating activity after high doses of CCNU. Plasma half-lives of the metabolites were 3.1 h (range 1.1-4.5 h) for the trans-isomer and 3.5 h (range 1.3-6.4 h) for the cis- isomer, and varied linearly with increasing patient body weight. There was no significant difference in plasma half-lives after high-dose CCNU administration observed in this study and those reported previously after the administration of substantially lower doses of CCNU. Despite linearity in the pharmacokinetics of the isomeric forms of 4'-hydroxyCCNU at high doses, large interindividual variability in exposure to the CCNU metabolites was observed. Potential saturation of metabolic pathways to the isomers at these doses may manifest as toxic symptoms since alkylating moieties formed directly from the parent, CCNU, may be associated with greater toxicity than those formed from the isomeric forms of the 4'-hydroxylated metabolite.

Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in the Escherichia coli lacl gene of O6‐alkylguanine‐DNA alkyltransferase‐proficient and ‐deficient strains

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the lacl gene of Escherichia coli were recovered from bacteria proficient (Ogt+ Ada+) and deficient (Ogt- Ada-) in O6-alkylguanine-DNA alkyltransferase activity. A CCNU dose of 1 mM was selected for DNA sequence analysis. A total of 245 induced mutations were characterized. The mutations were almost exclusively (95%) GC-->AT transitions, indicating that CCNU-induced mutations arose in bacteria primarily from misreplication of O6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents. The distribution of CCNU-induced GC-->AT mutations was significantly altered by the presence of DNA alkyltransferase activity (P = 0.01). In the Ogt+ Ada+ mutational spectrum, guanines flanked on both sides by A:T base-pairs were on average 2.8 times more likely to mutate than those flanked by G:C base-pairs on at least one side. This bias disappeared in the Ogt- Ada- genetic background, thereby providing evidence that O6-chloroethylated guanines adjacent to G:C base-pairs are better targets for bacterial alkyltransferase than those not adjacent to G:C base-pairs. We recently reported a similar bias for ethyl methanesulfonate, strengthening the idea that CCNU is acting as a simple ethylating compound. In summary, this paper presents for the first time evidence that DNA repair by O6-alkylguanine-DNA alkyltransferases plays a major role in removing lesions responsible for GC-->AT transitions induced by CCNU, influencing their ultimate distribution with respect to sequence context.

Chemosensitization of CCNU in KHT murine tumor cells in vivo and in vitro by the agent RB 6145 and its isomer PD 144872

The cytotoxicity and chemosensitizing potential of the nitrohetercyclic agent RB 6145 and its R enantiomer PD 144872 were determined in rodent tumor cells grown in tissue culture or as solid tumors. Using a clonogenic cell survival assay the degree of selective cytotoxicity of these bioreductive drugs was first determined in KHT/iv cells. Cells treated under hypoxic conditions were observed to be ~50–80-fold more susceptible to the action of RB 6145 or PD 144872 than were cells exposed under air. To assess the in vitro chemosensitizing potential of RB 6145 and PD 144872, doses of these agents which led to survival values between 0.5 and 1.0 under hypoxic conditions were administered, and were then combined concomitantly with variable doses of the nitrosourea CCNU. Exposure to the nitrosourea was for 1 h. The results showed that inclusion of either sensitizer enhanced the cell killing of the chemotherapeutic agent 2.4-2.6-fold. Subsequent experiments evaluated the therapeutic benefit of combining these bioreductive agents with CCNU in KHT sarcoma-bearing C3H/HeJ mice. When given at times ranging from 90 min before to 60 min after CCNU exposure, these bioreductive drugs increased the tumoricidal activity of the chemotherapeutic agent. Complete dose response curves combining RB 6145 and PD 144872 and a range of CCNU doses also were evaluated. The sensitizers (0.75 mmol/kg) were administered 30 min prior to the chemotherapeutic agent and survival of clonogenic tumor cells 22–24 h after treatment was used to assay tumor response. Normal tissue toxicity under similar treatment conditions was assessed using a bone marrow stem cell (CFU-GM) assay. When combined with CCNU, the bioreductive agents increased tumor cell kill by a factor of ~1.7. Bone marrow toxicity was not, however, enhanced by the addition of either agent to the CCNU treatment. These data suggest that further consideration should be given to the clinical application of these bioreductive agents.

In vivo therapeutic potential of combination thiol depletion and alkylating chemotherapy

The effect of administering the thiol modulating agent buthionine sulfoximine (BSO) in conjunction with alkylating chemotherapy was investigated in vivo in the mouse KHT sarcomas and bone marrow stem cells. Tumour response to treatment was assessed by an in vivo to in vitro excision assay and bone marrow survival was determined in vitro by CFU-GM. Glutathione (GSH) depletion and recovery kinetics were determined at various times after treatment using high performance liquid chromatography (HPLC) techniques. Following a single 2.5 mmol kg-1 dose of BSO, tumour GSH reached a nadir of approximately 40% of control 12-16 h after treatment. Bone marrow GSH was depleted to approximately 45% of control 4-8 h after treatment but recovered to normal by 16 h. When a range of doses of CCNU, mitomycin C, cyclophosphamide or melphalan (MEL) were given 16 h after mice were exposed to a 2.5 mmol kg-1 dose of BSO, only the antitumour efficacy of MEL was effectively enhanced (by a factor of approximately 1.4). This BSO-MEL combination appeared to be selective for the tumour as the bone marrow toxicity was not increased beyond that seen for MEL alone. Since increasing the administered dose of BSO neither increased the extent of thiol depletion in the tumour nor enhanced the antitumour efficacy of MEL, three other protocols for delivering the thiol depletor were explored. BSO was given either as multiple 2.5 mmol kg-1 doses administered at 6 or 16 h intervals or continuously at a concentration of 30 mM supplied in the animals' drinking water. Both multi-dose BSO pretreatments were found to increase both the antitumour efficacy and normal tissue toxicity of MEL such that no advantage compared to the single dose combination was achieved. In contrast, maintaining the thiol depletor in the drinking water led to an approximately 1.7-fold increase in the antitumour efficacy of MEL without any corresponding increase in bone marrow stem cell toxicity. For the various pretreatment strategies it was possible, in all cases, to account for the presence or absence of a net therapeutic benefit on the basis of the tumour and bone marrow GSH depletion and recovery kinetics.

Potentiation of alkylating chemotherapy by dual function nitrofurans in multi-cell spheroids and solid tumors

The cytotoxicity and chemosensitizing potential of four dual function nitrofurans was determined in human HT-29 multi-cell spheroids and rodent KHT sarcoma solid tumors. Spheroids were treated with a range of doses of the bioreductive drugs for a period of up to 48 h and the extent of cell kill was assessed at various times after treatment. Cytotoxicity was determined using a clonogenic cell-survival assay. The results demonstrated that two of the nitrofurans were even more toxic to spheroid cells than was the potent bioreductive nitroimidazole aziridine RSU 1069. The dose of the nitrofuran which, after a 24-h exposure, led to a survival value between 0.5 and 1.0, then was chosen for subsequent studies aimed at assessing the ability of these agents to potentiate the efficacy of the nitrosourea CCNU. Exposure to this chemotherapeutic agent was for a period of 1 h. The results indicated that all four dual function nitrofurans enhanced the cell killing of the conventional chemotherapeutic agent by factors ranging from 1.1 to 1.7. Subsequent studies evaluated the therapeutic benefit of combining these bioreductive agents and CCNU in KHT sarcoma-bearing C3H/HeJ mice. The nitrofurans were administered i.p. 0.5 h prior to the chemotherapy and tumor response was assessed by measuring the survival of clonogenic KHT cells 22–24 h after treatment. Normal tissue toxicity was determined using a bone marrow stem cell (CFU-GM) assay. Combining these bioreductive agents with CCNU increased the tumor cell kill by factors of 1.2 to 1.7. Bone marrow toxicity increased with CCNU dose, but the addition of the nitrofurans failed to enhance this endpoint of normal tissue toxicity.