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Purpose The purpose of this investigation was to synthesize a series of carbonate and carbamate derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma non-toxic metabolite of penclomedine (PEN) seen in patients. DM-PEN has been observed to be an active antitumor agent in mouse human xenograft tumor models and non-neurotoxic in a rat model, however, activity in intracranially implanted human glioma xenograft models have not been reported. The major goal was to identify derivatives that are active in brain tumors. Methods Derivatives were prepared from DM-PEN and evaluated in vivo against human U251 glioblastoma, D54 glioblastoma and MX-1 breast tumor xenografts and mammary tumor 16/C that were implanted in the mammary fat pad or intracranially (IC). Results Carbonate and carbamate derivatives were found to be superior to DM-PEN against IC growing human glioblastoma xenografts. Conclusion The activity of the carbonates and carbamates against human tumor xenografts in vivo suggests consideration of these two series of derivatives of DM-PEN for clinical development.

Isophosphoramide mustard (IPM) is known to have substantial anti-cancer activities in various animal models. Liquid chromatography–electrospray mass spectrometry (LC–ES–MS) and LC–ES–MS/MS methodologies have been developed and applied to the analysis of synthesized preparations of IPM. Our studies reveal that the principal impurity in IPM is N -(2-chloroethyl)- N ′-ethylphosphorodiamidic acid (MC-IPM) formed by dehydrochlorination of IPM with subsequent hydrogenation during synthesis. This impurity is present at levels in the range of 2–5% depending upon synthesis conditions. In addition, a second IPM derivative has been characterized by LC–ES–MS/MS and has been shown to be the product of a reaction of IPM with the dilute perchloric acid mobile phase used for liquid chromatography separations. The LC–ES–MS/MS method has been successfully employed to detect IPM spiked into a blood plasma sample. This work establishes that LC–ES–MS/MS is a viable tool for the detailed characterization of IPM and related products.

Purpose: The purpose of this investigation was to compare the antitumor activities of a series of acyl derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) observed to be an active antitumor agent in vivo and non-neurotoxic in a rat model with that of DM-PEN. Methods: Acyl derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted subcutaneously (s.c.) or intracerebrally (i.c.). Several derivatives were also evaluated against other human tumor xenografts and murine P388 leukemia cell lines. Results: Several of the acyl derivatives were found to be superior to DM-PEN against MX-1, human ZR-75-1 breast tumor, human U251 CNS tumor and the P388 leukemia parent cell line and lines resistant to cyclophosphamide and carmustine. 4-Demethyl-4-methoxyacetylpenclomedine showed inferior activity to current clinical brain tumor drugs against a glioma cell line, superior activity to temozolomide and procarbazine against the derived mismatch repair-deficient cell line, and superior activity to cyclophosphamide and carmustine but inferior activity to temozolomide against two ependymoma cell lines, all of which were implanted s.c. Conclusion: Proposed mechanisms of activation and action of DM-PEN and the acyl derivatives support the potential clinical superiority of the acyl derivatives.

Purpose: The purpose of this investigation was to determine the base sequence specificity of isophosphoramide mustard (IPM), the alkylating metabolite of ifosfamide, by crosslinking of designed DNA oligomers in comparison with the clinical alkylating agents mechlorethamine (ME) (nitrogen mustard) and phosphoramide mustard (PM), the alkylating metabolite of cyclophosphamide. Methods: IPM, as well as PM and ME were each reacted with three dodecameric duplexes, which were designed to detect interstrand crosslinking between guanines in 5′-GC-3′ (I), 5′-GNC-3′ (II) or 5′-GNNC-3′ (III) sequences (N=A or T). Results: All three agents preferentially react with 5′-GNC-3′ target sequences. The 5′-GNNC-3′ target sequence is less reactive by a factor of approximately 2.5- to 10-fold, while 5′-GC-3′ is of even lower reactivity. Conclusion: These results indicate that all three agents show approximately equal preference for reaction with a 5′-GNC-3′ target sequence in spite of the fact that IPM yields a 7-atom crosslink, while the other two agents yield 5-atom crosslinks.

Purpose: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced␣repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, but partial sensitivity is restored after elevated levels of O 6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O 6-benzylguanine (O 6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O 6-BG. Methods: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O 6-BG. Results: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O 6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. Conclusions: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC50 values in the range of 1−10 μg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4,6-tetra-O-acetyl-1-α,β-d-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.

We previously reported that cimetidine but not ranitidine significantly enhances cyclophosphamide-induced bone marrow toxic effects and the appearance of cyclophosphamide alkylating species in a murine leukemia mouse model, and we advised caution in the use of cimetidine with microsomally metabolized anticancer drugs. Both drugs have been used for the treatment of gastric complications of chemotherapy. Using a randomized, double-blind, crossover study design, we have now evaluated the potential interaction of ranitidine with cyclophosphamide in seven cancer patients, who received two courses of cyclophosphamide, one with ranitidine and one with placebo. Four patients received ranitidine in the first course, and three received placebo. Ranitidine or placebo was started 3 days before a single dose of cyclophosphamide and given for 17 consecutive days. Ranitidine or placebo was given orally (300 mg/d), and cyclophosphamide (600 mg/m2) was given intravenously with [3H]cyclophosphamide (1000 muCi). Cyclophosphamide treatment was repeated at 4 weeks plus or minus 4 days. Blood samples were collected at intervals from 5 minutes to 24 hours after cyclophosphamide treatment and analyzed by thin-layer chromatography and radioassay for the drug and its metabolites. On days 0, 7, 14, and 21 after cyclophosphamide administration, complete blood cell counts, white blood cell differential counts, platelet counts, and SMA-17 were determined. The differences in mean nadir white blood cell counts, granulocyte counts, hemoglobin levels, and hematocrit values during ranitidine versus placebo treatment were not statistically significant. In a statistical but not a clinical sense, mean nadir platelet counts were significantly lower with ranitidine. There was a statistically significant increase in area under the curve for drug concentration in plasma x time (AUC) with ranitidine as well as a statistically significant decrease in the total-body clearance rate of the cyclophosphamide molecule. However, the effect on AUC for the major oncolytic metabolites 4-hydroxycyclophosphamide and phosphoramide mustard was not statistically significant. The lack of toxicologic or metabolic interaction between ranitidine and cyclophosphamide suggests that ranitidine can be used safely with cyclophosphamide.

The cyclophosphamide metabolite, acrolein, was reacted with 2'-deoxyguanosine-3'-monophosphate, and two adducts were detected by high performance liquid chromatography and 32P-postlabeling assay. These adducts were resistant to dephosphorylation by nuclease P1 and could be isolated and detected from calf thymus DNA that had been reacted in vitro with acrolein. A combination of HPLC purification and enzymatic digestion of normal nucleotides by nuclease P1 allowed for the detection of these adducts in hepatic DNA from mice treated with cyclophosphamide. The level of the two adducts in the hepatic DNA, as determined by 32P-postlabeling, was one adduct per 2.7-4.1 x 10(7) normal nucleotides.

Purpose: Ifosfamide is becoming an important clinical anticancer drug. Meaningful pharmacology studies require quantification of its activated and active metabolites, 4-hydroxyifosfamide (HOIfos) and isophosphoramide mustard (IPM), respectively. Methods: Current methodology for quantifying the unstable HOIfos in biological fluids consists of trapping acrolein as it is produced during the decomposition of this metabolite. However, unlike cyclophosphamide, ifosfamide is extensively metabolized to two dechloroethylated metabolites, which are susceptible to 4-hydroxylation and similarly are capable of yielding acrolein upon decomposition. Because the current method has the potential to yield higher than actual values for HOIfos, it was compared with an HOIfos-specific method that traps the first stage degradation product of HOIfos, aldoifosfamide, as its semicarbazone, and depends on the use of radiolabeled drug for quantification. Six experiments in mice were conducted with blood collection 15 or 30 min after drug treatment followed by determination of HOIfos in plasma by the two methods. Results: Comparison of plasma levels of HOIfos determined by the two methods indicated only minor differences between the two. Conclusion: These results suggest the possibility that the nonspecific method may be acceptable as a first estimation of levels of this metabolite in biological fluids until the development of a specific method that does not require radiolabeled drug, such as high-performance liquid chromatography or gas chromatography/mass spectrometry, has been developed.

Purpose: The purpose of this investigation was to synthesize a series of thiolo-, thiono- and dithiocarbonate and thiocarbamate derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) in patients observed subsequently to be an active antitumor agent and non-neurotoxic in a rat model, in order to compare their antitumor activity with that of DM-PEN. Methods: Derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted in the mammary fat pad, several of which were also evaluated against human brain tumor xenografts. Results: Thiolocarbonate and thiocarbamate derivatives were found to be superior to DM-PEN against MX-1 tumor and modestly active against glioblastoma. Conclusion: The activity of the thiolocarbonates and thiocarbamates against human tumor xenografts in vivo suggests consideration of these two series of derivatives of DM-PEN for clinical development.

Purpose. Penclomedine (PEN), a multichlorinated α-picoline derivative which is metabolized to highly reactive alkylating species, was selected for clinical development due to its prominent activity against a wide range of human tumor xenografts when administered either parentally or orally. Its principal dose-limiting toxicity in preclinical and clinical studies has been neurocerebellar toxicity, which has been related to the magnitude of peak plasma PEN concentrations, but not to plasma concentrations of its putative principal alkylating metabolite, 4,o-demethylpenclomedine (DMPEN). These observation, as well as PEN's toxicologic, pharmacologic, and tissue distribution profiles, have suggested that the parent compound is primarily responsible for cerebellar toxicity. The studies described in this report were undertaken to characterize the neuropathology of PEN neurotoxicity, with a long-term goal of developing strategies to maximize its therapeutic index. Design. Male Sprague–Dawley rats were treated with therapeutically relevant doses of PEN, orally and intraperitoneally (i.p.), on various administration schedules, and DMPEN administered i.p. The animals were monitored for neurotoxicity, and brain sections were examined for neuropathology, particularly Purkinje cell loss and neuronal injury. Brain sections were stained using standard histochemical techniques and immunostained with OX-42 to detect microglial cells that are activated following neuronal damage, and calbindin D28K, a calcium-binding protein expressed by cerebellar Purkinje cells. Results. Dose-related neurocerebellar toxicity associated with parasagittal bands of Purkinje cell degeneration and microglial activation in the cerebellar vermis were evident in rats treated with PEN 100–400 mg/kg i.p. as a single dose. Neuronal injury was not observed in other regions of the brain. Furthermore, neither clinical nor histopathological evidence of cerebellar toxicity was apparent in rats treated with similar total doses of PEN administered i.p. on a daily×5-day dosing schedule. Similar histological findings, in an identical neuroanatomical distribution, were observed in rats treated with PEN orally; however, the magnitude of the neuronal toxicity was much less than in animals treated with equivalent doses of PEN administered i.p. Although acute lethality occurred in some rats treated with equimolar doses of DMPEN as a single i.p. treatment, surviving animals exhibited neither signs nor histopathological evidence of neurocerebellar toxicity. Conclusions. PEN produces selective dose- and schedule-dependent Purkinje cell degeneration in the cerebellar vermis of rats, whereas therapeutically relevant doses of PEN administered orally are better tolerated and produce less neurocerebellar toxicity. In addition, roughly equivalent, albeit intolerable, doses of the major active metabolite DMPEN, which was lethal to some animals, produced neither clinical manifestations of neurocerebellar toxicity nor Purkinje cell loss. These results support a rationale for investigating whether PEN administered orally, which may undergo significant first-pass metabolism to DMPEN and other less toxic intermediates, or treatment with DMPEN, itself, may result in less neurocerebellar toxicity and superior therapeutic indices than PEN administered parenterally.

Isophosphoramide mustard (IPM) is the cytotoxic alkylating metabolite of Ifosfamide (IFOS). IPM is being readied for a phase I clinical trial. In the present preclinical study, IPM was evaluated for usage in multidose intravenous (IV) infusion protocols.Mice and dogs received IV IPM daily for 3 days. Single-day dosing-oral and IV-to mice, rats, and monkeys is also reviewed for comparison. Complete toxicology studies were completed in the mice and dogs. For mice, dogs and monkeys, IV pharmacokinetic studies were conducted and compared.For mice, the LD(10) for the 3-day IV schedule for IPM was calculated to be 119 mg/kg (with 95% confidence limits of 87-134 mg/kg) (combined sexes), and for adult male dogs the maximum tolerated dose (MTD) was 5 mg/kg. Pharmacokinetic studies in mice, dogs and monkeys were compared and projected to human dosing. For dogs that received 10 mg/kg of IPM, T(1/2beta) was 0.99 h, and clearance was constant (1.01 l/h/kg). IPM was detected from 0 h to 1.5 h after the 5 mg/kg dose and from 0 h to 2 h after the 10 mg/kg dose; none was detected after 2 h. The IV MTD in dogs was 5 mg/kg per day for 3 days. Renal tubular necrosis and bone marrow failure were the causes of death. Transient liver, renal and bone marrow toxicity and gastrointestinal dysfunction were seen at low doses (5 mg/kg) in dogs. In mice (receiving 100 mg/kg IV) plasma concentrations disappeared in less than 1 h (T(1/2alpha) 2 min), with a clearance of 8.44 l/h/kg. For monkeys, the mean T(1/2) was 4.2 h. Median clearance was 1.65 l/h/kg and no IPM was detected 4 h after dosing. No potential IPM metabolites could be detected in any of the studies. In vitro, plasma protein bound 90% of IPM within 5 min of incubation.Predictions for human pharmacokinetic parameters and dosing are made from allometric analysis using the above three species. Data predicted an acceptable starting dose of 30 mg/m(2) with a clearance of 39.5 l/h, and a T(1/2) of 1 h 45 min for a 70-kg patient.

The main objective of this investigation was to identify the reactive pharmacophore in penclomedine (PEN, 3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl) pyridine) for in vivo antitumor activity and also to discover related ring structures and sulfur analogues that might exhibit superior antitumor activity in vivo. Several new analogues of PEN and related structural variants have been synthesized and evaluated in vivo against MX-1 human breast tumor xenograft implanted subcutaneously (sc), although none of them demonstrated significant activity.

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC(50) values in the range of 1-10 microg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4, 6-tetra-O-acetyl-1-alpha,beta-D-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.

The relative mutagenicity, nature of the mutations and the sequence specificity of mutations induced by the bifunctional alkylating agent, phosphoramide mustard (PM) and a monofunctional derivative, dechloroethyl phosphoramide mustard (dePM), were analyzed by the Ames test and by an in vitro shuttle vector mutagenesis assay. Both PM and dePM increased the mutation frequency above background in either assay. However, on an equimolar basis, dePM was less mutagenic than PM. In the in vitro shuttle vector mutagenesis assay, sequencing demonstrated that about 40% of the mutant plasmids contained more than one mutation in the supF tRNA gene segment of the plasmid. About 70% of the mutations observed in dePM-treated plasmids were single base substitutions with A:T and G:C base pairs being mutated at equivalent rates. In contrast, only about 50% of the mutations observed in PM-treated plasmids were single base substitutions, 80% of which involved G:C base pairs. Single base deletions and insertions were found in approximately equal proportions with both compounds; however, these lesions were in greater abundance in PM-treated plasmids. Putative hot-spots for mutation in the supF tRNA gene included base pairs at positions 102 and 110 for PM and positions 170 and 171 for dePM.

A series of halogen analogs of phosphoramide mustard, isophosphoramide mustard, and triphosphoramide mustard, the cytotoxic metabolites of the antitumor drugs cyclophosphamide, ifosfamide, and trofosfamide, respectively, was evaluated in vitro against human tumor cell lines and in vivo against experimental tumors to investigate the effect of replacement of chlorine with bromine or fluorine on the antitumor activity of the parent phosphoramide mustards. In the experimental tumors L1210 leukemia, B16 melanoma, mammary adenocarcinoma 16/C, and ovarian sarcoma M5076, the antitumor activity of the analogs was observed to be generally comparable with that of the parent mustards when chlorine was replaced by bromine but uniformly lower when chlorine was replaced by fluorine. Furthermore, the monobromo analog of isophosphoramide mustard displayed equal or somewhat greater activity in comparison with cyclophosphamide when evaluated against subcutaneously implanted L1210 leukemia with intraperitoneal drug treatment and against mammary adenocarcinoma 16/C.

Introduction: 4-Demethyl-4-cholesteryloxycarbonyl-penclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate, which is in Phase I clinical trials in patients with advanced cancer - IND 68,876. DM-CHOC-PEN is active vs. intracranially (IC) implanted human xenograft models - U251 and D54 glioblastoma and MX-1 breast cancer and recently found to be active vs. B-16 melanoma - 142% ILS. DM-CHOC-PEN's MOA is via alkylation of DNA @ N7 - guanine, as well as converting melanoma cells into a melanotic Go phase with cell death. Not all cells were converted into Go phase and senescence, thus the interest in designing a binary drug approach for DM-CHOC-PEN, as an improvement in treatment for melanoma. A number of agents are cytotoxic vs. B-16 cells in vitro and in vivo; however, 4-hydroperoxyifosfamide (HOOI), which is converted to isophosphoramide mustard (IPM) in vivo, demonstrated the best %ILS. The latter drug has been chosen as a companion with DM-CHOC-PEN in the present binary drug study vs. B-16 mouse melanoma. Methods: B-16 melanoma cells were cultured using RPMI media with 10% FBS and pen/strep @ 37° C in a CO2 incubator. Drugs were added to the cells in a growth phase and removed after 8–12 h. Adult female C57BL mice in groups of 5–6 mice were implanted subcutaneously (SC) with B-16 mouse melanoma (106 cells) and when SC nodules were palpable the mice were dosed IP daily (200 mg/kg) for 5-days with DM-CHOC-PEN followed by HOOI administered IP @ varying doses and daily/weekly schedules and monitored daily until death. Mice with SC B-16 melanoma were dosed with single agents - DM-CHOC-PEN, HOOI, cis-platinum and temozolamide, which were used as controls. Tumor tissue was extracted with dichloromethane, and assayed per HPLC and NMR. Results: In vitro, DM-CHOC-PEN and HOOI, as single agents, had IC50 of 0.5 and 0.8 μg/mL vs. B-16 melanoma cells, resp. Mice bearing SC B-16 melanoma treated with DM-CHOC-PEN (200 mg/kg/d × 5d, IP) alone vs. saline controls demonstrated %ILS of 142%; thus supporting the in vitro observations. In vivo in the B-16 melanoma model, cis-platinum as a single agent had a %ILS = 0%, but for HOOI it was 85%. In 2-drug studies, DM-CHOC-PEN plus cis-platinum together (in theoretical therapeutic ranges) were too toxic in combination, however, the %ILS for DM-CHOC-PEN plus HOOI was 173%. Tumor tissue was removed within 2-days of treating mice with DM-CHOC-PEN, extracted and revealed 75 μg/g tumor tissue of DM-PEN, a metabolite. Discussion: HOOI is a S-phase alkylating agent that is an appropriate 2nd agent to kill cells escaping from the DM-CHOC-PEN - induced Go phase induction. To date, the best treatment regimen was DM-CHOC-PEN (200 mg/kg/d × 5d) followed by HOOI (90 mg/d × 3d) - %ILS = 173. The finding that melanoma tissue extracts resulted in μg of DM-PEN (the metabolite) is in agreement with the pharmacokinetic findings observed for DM-CHOC-PEN in rats and humans, which will be reviewed. The binary drug combination will be reviewed with the FDA. Supported in part by: NCI SBIR grants - R43/44CA85021. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B219.

HOOI is a hydroperoxy-derivative of ifosfamide (IFOS) and a pro-drug of isophosphoramide mustard (IPM) [the active metabolite of IFOS] – a bi-functional alkylator that cross-links with G/C DNA base sequences resulting in irreparable inter-strand DNA cross-linking and cell death. HOOI has been an unstable laboratory curiosity for years; however, HOOI.L-lysine is a stable salt complex that has allowed the development of HOOI for clinical trials. HOOI spontaneously releases acrolein and chloroacetaldehyde in situ in cancer cells not extracellular in the general circulation (as does IFOS) and has not been associated with cystitis, renal tubular necrosis and/or CNS toxicity. HOOI has been screened in 20+ human xenograft tumor and murine tumor models and has significantly improved %ILS in intracranially implanted human xenografts – MX-1, U251; ZR-75-1 and in P399/CPA leukemia vs. IFOS and IPM. The U251 data (%ILS – 84+) is impressive considering – BCNU produced a 72% ILS. The ZR-75-1 breast cancer had a %ILS – 83. Bone marrow failure was the DLT in mice – LD10 100 mg/kg (for M/F). Dogs (M/F) were treated with 10, 15, 20 and 30 mg/kg. For both sexes, the LD10 was calculated to be 17.2, while the LD50 was calculated to be 17.3 mg/kg. PK values in dogs revealed the following profile for groups dosed with 30 mg/kg: AUC0- t = 1.53 (mg h/L), T1/2α = 0.93 (h), T1/2β = 6.1 (h) & CL = 19.5 (L/ h) [a two compartment model]. The AUC was linear for the 10 and 30 mg/kg doses. HOOI did not generate any detectable plasma chloroacetaldehyde vs. IFOS (2.12 µg/mL from 400 mg/kg) and 25% of the acrolein generated by the MTD of CPA & IFOS. No convulsions, neuropathies or renal dysfunction were observed in either specie. Unlike IFOS/IPM, HOOI is lipophilic, activated intracellular (with < extracellular acrolein and no chloroacetaldehyde released); no IFOS/IPM-associated CNS or GU toxicity was noted. HOOI may increase the safety and efficacy margins of this class of alkylators in advanced CPA- and IFOS-resistant cancers and broaden the target-range (CNS-gliomas). The L-lysine salt stability supports developing HOOI for clinical trials. Supported by grant R44 CA094566 from the NCI/SBIR program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3222. doi:10.1158/1538-7445.AM2011-3222

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.

Evidence at the molecular level is presented in support of alkylation of O6-guanine moieties of DNA as the mechanism of cytotoxicity of Clomesone to HT-29 cells and consists in the isolation and identification of a product resulting from alkylation of calf thymus DNA with Clomesone, followed by depurination to yield 7-(2-hydroxyethyl)guanine, whose formation is reasonably explained by O6-guanine chloroethylation followed by intramolecular alkylation at N7 of guanine and subsequent hydrolysis to the hydroxyethylguanine.

Six female rabbits were given 20 mg/kg cyclophosphamide (containing 100 microCi [3H-chloroethyl]-cyclophosphamide) alone or 1 h following 100 mg/kg cimetidine. Serial plasma and urine specimens were collected and levels of cyclophosphamide and its metabolites (4-hydroxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard, and carboxyphosphamide) were measured. 4-Ketocyclophosphamide was the major metabolite present in rabbit plasma and urine, with lesser amounts of 4-hydroxycyclophosphamide, carboxyphosphamide, and phosphoramide mustard also being identified. Cimetidine pretreatment resulted in prolongation of cyclophosphamide's half-life from 24.3 +/- 7.3 to 33.5 +/- 9.5 min (mean +/- SD; P = 0.036) but did not significantly alter the AUC0-8 h for the latter drug. Cimetidine pretreatment resulted in a significantly greater AUC0-8 h for 4-hydroxycyclophosphamide (189.4 +/- 77 vs 364.6 +/- 126.7 mumol min/l-1; P = 0.016) as compared with control values. A higher AUC0-8 h value for phosphoramide mustard (53.7 +/- 69.2 vs 95.7 +/- 34.7 mumol min/l-1) was also observed after cimetidine dosing but the difference was not significant (P = 0.21). Kinetics of 4-ketocyclophosphamide and carboxyphosphamide were not significantly affected by cimetidine treatment. Cimetidine was added to hepatic microsomes isolated from phenobarbital-treated rabbits; it did not inhibit cyclophosphamide's metabolism in vitro, suggesting that its in vivo effect may be mediated through mechanisms other than cytochrome P-450 inhibition. Cimetidine pretreatment increases exposure to cyclophosphamide and its major activated metabolite, 4-hydroxycyclophosphamide. Potentiation rather than inhibition of cyclophosphamide's pharmacodynamic effect is to be predicted when cimetidine is given concomitantly with the former. Alterations in hepatic blood flow or mechanisms other than microsomal inhibition by cimetidine may explain this potentiation.

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. In vitro CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.

The antitumor 2-halo(chloro, bromo, and fluoro)-ethyl(methylsulfonyl) methanesulfonates, ethyl(methylsulfonyl) methanesulfonate, and chlorozotocin, a 2-chloroethylnitrosourea, were evaluated for their potential to induce SCEs in L1210 cells. The results indicate that all the compounds induced approximately 2-fold or greater increases in SCEs in a dose-related manner. 2-Chloroethyl(methylsulfonyl) methanesulfonate, a DNA-interacting agent and a drug selected for clinical trials, exhibited the highest SCE increase in these cells.

The potential immunosuppressant activity of the following compounds derived from cyclophosphamide (CPA) was evaluated: 8 known metabolites, oxidation products and hydrolytic products of CPA containing the mustard moiety; the mixture of alkylating products formed from CPA by chemical and biological oxidation in vitro; acrolein. Mechlorethamine (HN2) and mannomustine were included in this survey as reference compounds.

Some DDT analogs are estrogenic, particularly o,p'-DDT, which comprises approximately 15-20% of the commercial DDT mixture. Whether this compound or its metabolites are active has not been established. In fact, the data obtained thus far are more confusing than enlightening. For example, CCl4 pretreatment of immature female rats has been reported to inhibit or enhance estrogenic activity of o,p'-DDT, and SKF-525A pretreatment has been reported to enhance or not alter the estrogenic effect. Although o,p'-DDT inhibits binding of estradiol to the estrogen receptor from rat or human at low levels (approximately 1-10 micrometer) in vitro, higher levels are required to inhibit nuclear binding of [3H] estradiol in incubated whole uteri. Futhermore, o,p'-DDT appears to be neither estrogenic nor antiestrogenic in an in vitro estrogen assay. Methoxychlor appears to be "activated" by metabolism, and it is probable that phenolic metabolites are responsible for its estrogenic activity. Since chlorinated hydrocarbons often enhance the metabolism of steroids and may reduce circulating levels of steroids, interactions of the exogenous hormonally active agents with steroid receptors may be self-potentiating in vivo.

Because certain (2-chloroethyl)triazenes and (2-haloethyl)nitrosoureas have high antineoplastic activity, 2-chloroethyl and 2-fluoroethyl sulfonates were prepared to try to develop additional types of 2-haloethylating agents. In this initial study, it was demonstrated that antineoplastic activity much superior to that of the prototype, 2-chloroethyl methanesulfonate, could be found among 2-chloroethyl sulfonates. Among a variety of 2-chloroethyl alkane- and arenesulfonates, several substituted methanesulfonates displayed significant activity against P388 leukemia in mice; the chloromethanesulfonate showed high activity (T/C = 218%). None of the arenesulfonates were active in this test.

Analogous extracts of the flowers and buds of cotton, Gossypium hirsutum L., were compared as feeding stimulants for the boll weevil, Anthonomus grandis Boheman. The flower extracts and their fractions were examined by paper, column, thin-layer, and gel-permeation chromatography, and active fractions were obtained. A methanol-extractable active substance was concentrated by precipitation with lead acetate, and another highly active fraction was recovered by re-extraction with methanol. Examination of the acidic, ether-extractable components in methanol extracts of flowers and buds indicated that these fractions from buds were more stimulating than similar extracts of flowers.

The nonpolar feeding stimulant complex for Anthonomus Grandis Boheman found in the buds of cotton, Gossypium hirsutum L. (Deltapine Smooth Leaf), was fractionated and concentrated. Active fractions were extracted from buds and freeze-dehydrated bud powder with petroleum ether, chloroform, acetone, and chloroform-methanol. These fractions possessed widely different properties from those of the polar fractions studied previously. Silicic acid column chromatography produced a series of related compounds having the properties of the polar lipids. Thin-layer chromatography and countercurrent distribution studies produced compounds that were probably pheophytins. When pheophytins a and b were isolated from the bud, they gave high feeding activity after significant purification. Pheophytin a from spinach also gave modest activity, but cotton pheophytin a was inactive after hydrolysis and reesterification with phytol. These and previous studies of the polar feeding-stimulant complex suggest that a multicomponent system is responsible for optimum feeding activity of the boll weevil.

Techniques were used to concentrate and fractionate the polar feeding stimulant for the boll weevil, Anthonomus grandis Boheman, that is found in the buds of cotton, Gossypiun hirsutum L., Delta Pine Smooth Leaf. An active fraction could be extracted from freeze-dehydrated bud powder with water after successive extractions with several less polar solvents and, consequently, after removal of hydrocarbons, carotenoids, terpenoids, and flavonoids. The major active polar principle extractable with water was not absorbed on a weak cation exchange resin and was precipitated with lead ions. Studies by gel permeation suggested a moderate molecular weight

The 1 H and 13 C NMR spectra of 2-aminoperimidine hydrobromide, an example of a little-studied N -heterocyclic ring system, have been determined in dimethyl sulfoxide solution. In this solvent, contrary to some previous reports in the literature, both spectra show that the base is protonated on the ring nitrogen, rather than on the amino group. Chemical shifts and some coupling constants are reported.

Cyclophosphamide (CPA), the most commonly used alkylating agent in the treatment of a wide variety of hematologic and solid tumors, requires oxidation by hepatic microsomal enzymes to its active alkylating species. A number of alternative methods exist to simulate the in vitro cytotoxicity of CPA against animal and human tumors, including the co-incubation of CPA with the S-9 fraction of rat liver homogenates (S-9) and the use of either 4-hydroperoxy CPA (a stabilized form of a major blood-borne metabolite of CPA), phosphoramide mustard (PM, considered to be the ultimate intracellular alkylating metabolite of CPA), or ASTA Z 7557 [4-(2-sulfonatoethylthio)-CPA, a new oxazaphosphorine compound which after dissolution undergoes rapid spontaneous hydrolysis in vitro with liberation of 4-hydroxy-CPA]. Using a human tumor clonogenic assay (HTCA) we have quantitated the median molar inhibitory dose 50 (ID50) concentrations of S-9 activated-CPA, 4-hydroperoxy-CPA, PM, and ASTA Z 7557 against 107 previously untreated tumors, as well as determining the in vitro biological stability of the former three CPA metabolite preparations. 4-Hydroperoxy-CPA proved the most consistently cytotoxic (median molar ID50=5.77#x00D7;10−5M) compound, followed by ASTA Z 7557, S-9 activated-CPA and PM in that order. Of additional interest S-9 activated CPA and PM proved relatively unstable biologically when frozen at -120°C, whereas 4-hydroperoxy-CPA lost none of its cytotoxicity over a 36 day period during freezing. On the basis of these data 4-hydroperoxy-CPA appears the compound of choice for use in vitro to evaluate the activity that CPA is likely to express clinically against solid tumors. Since 4-hydroperoxy-CPA is not available for clinical use, ASTA Z 7557, which was slightly less cytotoxic to ovarian cancers and a wide variety of other tumors in the HTCA, appears an attractive agent to develop further clinically, especially for regional chemotherapy (e.g., intraperitoneal and intra-arterial treatment) of solid tumors.

Iminocyclophosphamide (4) has been identified by 1H NMR as a product from base treatment of 4-alkylthio-substituted cyclophosphamide derivatives, viz., cis-4-(propylthio)cyclophosphamide (cis-7). A maximum concentration of approximately 12% of total product was observed by treating cis-7 with ethyl propiolate and NaH or deuteriated dimsyl anion in anhydrous Me2SO-d6. Treatment of cis-7 with base alone established a rapid cis-/trans-7 equilibrium via the imine intermediate 4. Base-catalyzed expulsion of 1-propanethiol (8) from cis-7 and thiol trapping afforded formation of 4, which subsequently underwent elimination to the relatively more stable conjugated (vinylimino)-phosphamide (9). Iminocyclophosphamide (4) was also identified by fast atom bombardment mass spectrometry as a product generated upon analysis of cyclophosphamide derivatives substituted in the 4-position of the oxazaphosphorine ring with various leaving groups.

1. Studies on the metabolism of 14C-Michler's ketone (4,4'-bis-(dimethylamino)[carbonyl- 14C]benzophenone) in rats have revealed that this carcinogen is subject to demethylation, ring-hydroxylation and N-acetylation after adjacent methyl groups have been removed. 2 As identified by mass spectral analysis, microsomal metabolites are the mono-, di-, tri- and tetra-demethylated derivatives. 3. The major metabolites appearing in the bile are the di- and tri-demethylated derivatives and the N-acetylated tetra-demethylated compound; minor metabolites, tentatively identified, are the ring-hydroxylated derivatives of di- and tri-demethylated Michler's ketone and of N-acetylated tri- and tetra-demethylated Michler's ketone. 4. The major urinary metabolite is tentatively identified as a ring-hydroxylated derivative of N-acetylated, di-demethylated Michler's ketone; a minor urinary metabolite lacks the hydroxyl group. 5. Injection of 14C-Michler's ketone into rats resulted in the irreversible binding of radioactivity to liver proteins. 6. When the rats were pretreated with phenobarbital, this binding was increased and extended to proteins of some other tissues and to DNA and RNA of liver and DNA of kidney.

Analysis of blood from a dog given a 400 mg/m2 dose of 9-β- d -arabinofuranosyl-2-fluoroadenine (2-F-araA) led to the identification of parent drug and a major metabolite, 9-β- d -arabinofuranosyl-2-fluorohypoxanthine. 2-Fluoroadenine, a toxic derivative of 2-F-araA, was not detected in blood within the limits of detection, suggesting that parent drug was absorbed and distributed without systemic exposure to this toxic derivative. Parent drug, 2-fluoroadenine, and 9-β- d -arabinofuranosyl-2-fluorohypoxanthine were identified in urine of dog, monkey, and mouse.

We investigated the mechanism of cyclophosphamide (CTX)-induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on rat granulosa cells in vitro. Cells were obtained from PMSG-primed immature rats and incubated with PCTX at concentrations of 1, 10, 100, and 500 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. Cell viability before and after seven hours incubation was determined. Progesterone and prostaglandin E accumulation were measured by radioimmunoassay. Granulosa cell viability was significantly decreased at PCTX concentrations of 10 micrograms/mL or higher in a dose-related manner. PCTX at concentrations of 100 micrograms/mL and 500 micrograms/mL significantly decreased basal and LH-induced progesterone and prostaglandin E accumulation. The above findings demonstrate that cyclophosphamide metabolites decrease granulosa cell survival and function in vitro. These direct effects suggest a possible mechanism for CTX-induced premature ovarian failure.

Spirohydantoin mustard (SHM), a central nervous system directed nitrogen mustard with anticancer activity, was metabolized in the presence of mouse liver postmitochondrial supernatant (9000g fraction) to a nonpolar alkylating metabolite. The metabolite was isolated by thin-layer chromatography of chloroform or ethyl acetate extracts of incubation mixtures, and its structure was established by mass spectral analysis, synthesis, and cochromatography. The metabolite, spirohydantoin aziridine, was mutagenic for Salmonella typhimurium TA1535 in the Ames assay but inactive as an antitumor agent against P388 leukemia in vivo.

Thin-layer chromatography and mass spectrometry were used to isolate and identify cyclophosphamide metabolites present in blood of mice. Blood was removed 5, 15, 30 and 45 minutes after intraperitoneal administration and extracted with chloroform followed by methanol. Thin-layer chromatography of the two extracts and the residual solid with or without prior methylation, collection of resulting alkylating components, determination of radioactivity and mass spectral analysis, served to identify cyclophosphamide, 4-ketocyclophosphamide, alcophosphamide, N-dechloroethylcyclophosphamide, carboxyphosphamide, phosphoramide mustard and nor-HN2. The absence of detectable levels of 4-hydroxycyclophosphamide or aldophosphamide in the blood of cyclophosphamide-treated mice suggests that cyclophosphamide is converted rapidly in the liver to carboxyphosphamide, 4-ketocyclophosphamide, phosphoramide mustard and nor-HN2. Direct administration of synthetic 4-hydroxycyclophosphamide to mice and extraction of blood with chloroform demonstrated the recovery of this metabolite in vivo. Analysis of extracts of blood from mice treated with phosphoramide mustard indicated the presence of nor-HN2, 3-(2-chloroethyl)-1,3-oxazolidin-2-one and unchanged drug. Consideration of blood levels, cytotoxicity and alkylating activity of metabolites identified in, or inferred from, this study, implicates phosphoramide mustard as a leading candidate for the biologically active form of cyclophosphamide.

Publisher Summary Comparison of structure–activity relationships of nitrosoureas active against L1210 leukemia with their toxicities indicated that the neutral agents with octanol/water partition coefficients of -1.5 to -2.5 might have better therapeutic indices than those currently in use. Clinical demonstration that cross-resistance between mustard-type alkylating agents does not exist in some tumors was found when cyclophosphamide-resistant, advanced breast cancer responded to the treatment with peptichemio. Direct chemical evidence that DNA cross-linking is a product of alkylation with busulfan is presented int his chapter. Methotrexate polyglutamates, which are readily formed in human tumor cells and bind to dihydrofolate reductase, may be selectively retained in cells and may be important determinants of duration of action and cytotoxicity of methotrexate in human solid tumors. A study of the interaction between adriamycin and phospholipids indicate similar association constants for such complexes and for DNA complexes; the data suggest competitive behavior between a membrane site and target DNA and consequently, lipid components of cell membranes may be an important determinant in the behavior of this agents. Some amino acid and dipeptide derivatives of daunorubicin were more active than the parent against subcutaneously-inoculated LL. Chemical reduction to semiquinones and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal activation of adriamycin and daunorubicin resulted in covalent binding to DNA. A mitomycin C-dextran conjugate demonstrated increased antitumor activity in comparison with the free drug and cellular uptake of methotrexate-poly (L-lysine) conjugates far exceeded the uptake of free drug in drug sensitive and resistant lines.

Summary The reaction of 2′-deoxyguanosine 3′-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2′-deoxyguanosine 3′-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32 P from[γ 32 P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32 P-postlabeling assay.