Thiolo-, thiono- and dithiocarbonate and thiocarbamate derivatives of demethylpenclomedine as novel anticancer agents

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. This investigation was supported by USPHS Grant CA34200 from the National Cancer Institute, Bethesda, MD.     

Preclinical antitumor activity of an alpha-picoline derivative, penclomedine (NSC 338720), on human and murine tumors

Penclomedine, a synthetic alpha-picoline derivative, was identified as a potential antitumor agent in the P388 leukemia prescreen of the National Cancer Institute. Upon further evaluation in the National Cancer Institute in vivo tumor panel, the compound demonstrated good activity against two breast tumors. A single i.p. dose or five daily doses caused partial regressions of advanced-stage s.c. implanted mouse CD8F1 mammary adenocarcinomas. Also, penclomedine administered i.p. on Days 1,5, and 9 caused regression of the human MX-1 mammary carcinoma implanted under the renal capsule of athymic mice. In contrast, penclomedine demonstrated only marginal to moderate activity against the i.p. implanted L1210 leukemia and M5076 sarcoma and was inactive in three additional non-breast tumor models (i.p. B16 melanoma, i.v. Lewis lung carcinoma, and s.c. colon adenocarcinoma 38). Penclomedine administered p.o. and i.p. was equally effective against the subrenal capsule MX-1. Doses given p.o. every fourth day caused complete regression of 39 of 40 advanced-stage s.c. implanted MX-1 tumors but were much less effective against human H82 small cell lung carcinomas (13 of 80 complete regressions). Penclomedine p.o. also inhibited growth of the human MCF-7 and mouse 16/C breast adenocarcinomas. Further studies to support the development of penclomedine to clinical trial are in progress.     

Urinary metabolites of N-nitrosodibutylamine in the rat: identification of N-acetyl-S-butyl-L-cysteine derivatives

N-Acetyl-S-(butyl, 3-oxobutyl and 3-hydroxybutyl)-L-cysteines have been isolated and identified (as their methyl esters) from the urine of rats given N-nitrosodibutylamine (NDBA), N-nitrodibutylamine (NTDBA) and their corresponding alpha-acetoxy derivatives, N-nitroso-N-butyl(1-acetoxybutyl)amine and N-nitro-N-butyl(1-acetoxybutyl)amine, respectively. Greater amounts of these L-cysteine derivatives were detected in urine after administration of NDBA than of NTDBA. This suggests that the markedly different biological activities of NDBA and NTDBA might be due, in part, to a difference in their alkylating abilities in vivo.     

Preclinical antitumor activity of penclomedine in mice: cross-resistance, schedule dependence, and oral activity against tumor xenografts in brain

Penclomedine is 3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine (NSC 338720), an alpha-picoline derivative with p.o. antitumor activity in preclinical leukemia and solid tumor models. Described here are an in vivo cross-resistance profile of penclomedine, treatment schedule dependence studies, and studies exploring the effects of p.o. drug on human tumors xenografted into mouse brain. The latter studies exploited the apparent facile distribution of penclomedine to the central nervous system. Tumor models used included murine leukemia lines selected in vivo for acquired resistance to various antitumor drugs and the human mammary and lung tumor xenografts MX-1 and H82, respectively. The therapeutic effects of p.o. penclomedine against s.c. MX-1 and H82 xenografts were shown to be independent of treatment schedule. Therapeutic activity was comparable when p.o. and parenteral treatments were compared. Lines of P388 leukemia resistant to melphalan, cyclophosphamide, and carmustine were cross-resistant to penclomedine in vivo. Leukemia lines resistant to antimetabolites, DNA binders/intercalators, and vincristine were not cross-resistant to penclomedine. Intracerebrally implanted MX-1 xenografts retained their sensitivity to p.o. penclomedine, and therapeutic activity was at least comparable to that of carmustine, a drug known for its ability to cross the blood-brain barrier. These results demonstrate attributes of penclomedine that are relatively uncommon among currently available antitumor drugs and that are of interest for the anticipated clinical development of this drug.     

The mutagenicity of dibenz[a,j]acridine, some metabolites and other derivatives in bacteria and mammalian cells

Dibenz[a,j]acridine (DBAJAC) was studied because of its close structural relationship with a number of important carcinogenic polycyclic and azaaromatic hydrocarbons. It was of particular relevance to examine the mutagenicity of known or proposed 'bay-region' metabolites, which may be proximate or ultimate carcinogenic derivatives of DBAJAC. Trans-1,2-, 3,4- and 5,6-dihydrodiols, the 4- and 6-phenols, the 5,6-oxide and N-oxide derivatives, and anti- and syn-3,4-diol 1,2-epoxides of DBAJAC were examined for their mutagenicity in Salmonella typhimurium TA98 and TA100 and in V79 Chinese hamster lung cells. Of all the compounds studied which require metabolic activation, the 3,4-dihydrodiol was the most active in both TA100 and in V79 cells. The activity of the 3,4-dihydrodiol enantiomers was also tested in strain TA100 where no difference was observed from that of the racemic mixture. In V79 cells only the 3R,4R-dihydrodiol was active, the activity being about three times that of the racemic material. Salmonella strains TA98 and TA100 also differed in their sensitivity towards DBAJAC dihydrodiols, the 1,2-isomer being of greatest activity in TA98. The most mutagenic compounds in both mammalian and bacterial cells were the 'bay-region' diol epoxides of DBAJAC which did not require metabolic activation by S9 mix. The anti-DBAJAC 3,4-diol 1,2-epoxide was more mutagenic than the syn form in V79, TA98 and TA100 cells. Overall these results suggest that the in vivo biological activity of DBAJAC metabolites is likely to reflect previous findings with other similar polycyclic aromatic hydrocarbons.     

Successful non-neurotoxic therapy (without radiation) of a multifocal primary brain lymphoma with a methotrexate, vincristine, and BCNU protocol (DEMOB)

A 3.5-year-old boy with a multifocal primary lymphoma of the brain was treated successfully without neurotoxicity with a treatment regimen that did not include radiation. The protocol of Dexacort (dexamethasone), methotrexate, Oncovin (vincristine), and BCNU (carmustine) (DEMOB), which was developed with the use of MTX pharmacokinetic studies, was given over 7.5 months, and resulted in tumor disappearance on computerized tomography scans and marked improvement in clinical status. The patient remains in good health 3 years after diagnosis (March 1985).     

Murine pharmacokinetics and metabolism of penclomedine [3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine, NSC 338720]

Penclomedine, a highly substituted pyridine derivative, has been selected by the National Cancer Institute for evaluation as a potential anticancer agent based on antitumor activity observed in murine tumor models following i.v., p.o., and i.p. administration. We have developed a reverse-phase high performance liquid chromatography assay for PEN, and subsequently investigated murine pharmacokinetics and metabolism. Following rapid i.v. injection of PEN (300 mg/m2) to mice, plasma elimination was best described by a 2-compartment open model with an elimination phase half-life, total body clearance, and steady-state distribution volume of 69 min, 114 ml/min/m2, and 4800 ml/m2, respectively. While PEN displayed good p.o. absorption, bioavailability of PEN after p.o. administration was approximately 2% of that observed following i.v. administration. Metabolism contributed substantially to drug clearance, and total metabolites were slowly eliminated from plasma. After i.v. and p.o. administration of radiolabeled PEN, less than 0.2% of the parent drug was excreted in the 48-h urine, and 25-30% of the total radioactivity was recovered in urine. NADPH-dependent oxidative and reductive metabolism was observed when penclomedine was incubated with mouse microsomal preparations. Microsomal reductive metabolism of PEN led to formation of a metabolite tentatively identified as a molecule formed by dimerization of the radical species produced by cleavage of chlorine from the trichloromethyl moiety of penclomedine.     

Ketonitrosamines as metabolites of methyl-n-amylnitrosamine (MNAN) and its hydroxy derivatives in the rat

In a previous study of the metabolism of methyl-n-amylnitrosamine(MNAN) in the rat, 2- to 5-hydroxy-MNAN (HOMNAN) were provisionallyidentified as metabolites and the identity of 4-HO-MNAN wasconfirmed by mass spectrometry. We now describe syntheses andmass and other spectra for 2- to 5-oxo-MNAN. Two previouslyunidentified MNAN metabolites were shown to be 3- and 4oxo-MNAN.In addition to 4-HO-MNAN, we confirmed 3-HO-, 4oxo- and (lesscertainly) 2-HO-MNAN as urinary MNAN metabolites by GLC-MS ofHPLC fractions. Analysis with and without ß-glucuronidasetreatment showed that the urinary HO-MNANs occurred as theirß-glucuronides. MNAN (25 mg/kg injected i.p.) hada blood half-life of 21 min in adult male rats. The blood alsocontained 4-HO- and 4oxo-MNAN, which showed maximum levels thatwere 13 and 26% respectively of that for MNAN, and were clearedmore slowly than MNAN. On incubation for 3 h with MNAN, ratesophagus produced 3- and 4-oxo-MNAN in yields that were 5%of those for the corresponding HO-MNANs. For MNAN metabolism,the 4-oxo-/4-HO-MNAN ratio of metabolites was 5% for adult ratliver and was 22% for adult hamster liver and 9-day-old ratliver. On incubation with 4-HO-MNAN for 3 h, oxidation to 4oxo-MNANwas 16–25% for adult hamster or 9-dayold rat liver slicesand for adult hamster liver homogenate. Homogenate activitywas concentrated in the microsomal fraction, for which NAD wasa more effective co-factor than NADP. A bacterial alcohol dehydrogenaseoxidized 4-HO- to 4-oxo-MNAN in 38% yield/3 h. None of thesepreparations oxidized 2-HO- to 2-oxo-MNAN. It was concludedthat 3- and 4-oxo-MNAN were metabolites of MNAN, apparently(for 4-oxo-MNAN) via HO-MNAN oxidation by a microsomal NAD-dependentenzyme, that 4-HO- and 4-oxo-MNAN formation was a major routeof MNAN metabolism, and that 4-oxo-MNAN might play a role inMNAN carcinogenesis.     

Dose-limiting neurotoxicity in a phase I study of penclomedine (NSC 388720, CRC 88-04), a synthetic alpha-picoline derivative, administered intravenously.

3,5-Dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine (penclomedine, NSC 338720, CRC 88-04) is an alpha-picoline derivative with anti-tumour activity in preclinical models. Penclomedine administration by 1-h intravenous infusion on 5 consecutive days was repeated 3 weekly in the absence of dose-limiting toxicity (DLT) or disease progression. Five dose levels were investigated (22.5-340 mg m(-2) day[-1]). Eight men and eight women were entered, median age 59 years (range 39-73 years), with good performance status (ECOG 0/1) in 11 patients. A total of 13 out of 16 patients had received previous chemotherapy. Common toxicity criteria grade (CTCg) II vomiting was recorded at all dose levels. Neurotoxicity (cerebellar ataxia and dizziness) was the DLT, CTCg III toxicity occurring in three out of three patients treated at 340 mg m(-2) day(-1). CTCg III dizziness was noted in one out of three patients at 250 mg m(-2) day(-1). Neurotoxicity developed during the 1-h infusion and persisted for a variable period (maximum 5 h) after infusion. Prophylactic antiemetic drugs appeared to reduce associated vomiting but did not prevent ataxia. No antiproliferative toxicities were noted and no anti-tumour responses were documented. Penclomedine pharmacokinetic studies confirmed preclinical evidence of extensive apparent distribution (93 l m[-2]) and rapid clearance (41 l h[-1] m[-2]). Purkinje cell loss has been identified in preclinical models after intraperitoneal administration (O''Reilly et al, 1996a) and further clinical development of penclomedine will focus on oral administration.     

Properties of N-methyl-N-nitrosourea-resistant, Mex- derivatives of an SV40-immortalized human fibroblast cell line

We have selected two N-methyl-N-nitrosourea (MNU)-resistant derivatives of the SV40-transformed, alkyltransferase-deficient (Mex-) human fibroblast cell line MRC5V1. Both derivatives remain Mex-. They are cross-resistant to methylmethanesulphonate (MMS) and 6-thioguanine (6TG) but not 2,6-diaminopurine. They show increased sensitivity to the bifunctional chloroethylating agent mitozolomide (MTZ). We have transfected MRC5V1 and one of our MNU-resistant lines with the bacterial O6-methylguanine (O6-MeG)-DNA methyltransferase (ada) gene. Transfectants of MRC5V1 are significantly more resistant to MNU but exhibit only a small increase in resistance to MMS and MTZ. Transfectants of the MNU-resistant derivative exhibit only a small additional increase in resistance to MNU, no further increase in resistance to MMS and a large increase in resistance to MTZ. The pattern of resistance to cytotoxic agents of these transfectants suggests that a second mechanism of resistance to MNU, independent of alkyltransferase expression, is operating in our resistant lines. This mechanism apparently enables the cells to tolerate O6-MeG and 6TG, but not chloroethyl adducts in their DNA.     

Toxicity, pharmacokinetics, and in vitro hemodialysis clearance of ifosfamide and metabolites in an anephric pediatric patient with Wilms' tumor

Purpose: We evaluated the in vitro hemodialysis ratio and subsequent toxicity and pharmacokinetics of ifosfamide in an anephric patient with Wilms' tumor. Methods: An in vitro model was used to determine the extraction ratio of ifosfamide by dialysis. The toxicity and plasma concentrations of ifosfamide, chloroacetaldehyde, and 4-hydroxyifosfamide were then determined over 24?h after a single 1.6?g/m² dose of ifosfamide. Plasma concentrations were also measured before and after ten dialysis sessions during four courses of ifosfamide therapy. Results: The in vitro hemodialysis model showed that ifosfamide was cleared with an extraction ratio of 86.7?±?0.5% and remained constant even at low concentrations of drug. The mean decrease in vivo following hemodialysis for ifosfamide, chloroacetaldehyde, and 4-hydroxyifosfamide were 86.9%, 77.2%, and 36.2%, respectively. The pharmacokinetic parameters for ifosfamide using model-independent methods were calculated: Vd?=?0.23 l/kg, t_½?=?4.8?h, and Cl_T?=?3.30 l/h per?m². Ifosfamide-associated neurotoxicity was noted within hours of drug administration and improved rapidly following hemodialysis. Conclusions: The results of our study suggest that the pharmacokinetics of parent ifosfamide may not be substantially altered in patients with renal failure. Hemodialysis was shown to remove ifosfamide, chloroacetaldehyde, and 4-hydroxyifosfamide from the blood stream. Hemodialysis was also shown to reverse ifosfamide-related neurotoxicity.     

Phase I clinical and pharmacokinetic study of oral penclomedine (NSC 338720) in adults with advanced solid malignancy.

Penclomedine is a synthetic alpha-picoline derivative that has shown antitumor activity both in preclinical development and in Phase I work using an i.v. preparation. The main toxicities seen in those studies were dose dependent and mainly neurocerebellar, with hematological toxicity being far less severe. This Phase I trial of p.o. penclomedine was conducted to potentially alter the toxicity profile and to avoid the neurological side effects seen with i.v. penclomedine. Eligibility criteria included microscopic confirmation of a solid malignancy or lymphoma with a lack of effective anticancer therapy. Twenty patients were enrolled. The median age was 60.5 years, and the median performance status was one. All but one patient had received prior systemic therapy. The starting dose of penclomedine was 200 mg/m(2) p.o. for 5 days, and was escalated according to a traditional Fibonacci sequence until the maximum tolerated dose (MTD) was observed. No treatment-related deaths were observed during the study. The MTD was determined to be 800 mg/m(2) p.o. for 5 days. Dose-limiting toxicities included mainly neurocerebellar symptoms such as ataxia and dysmetria, but neurocortical symptoms, such as confusion, were seen as well. Myelosuppression was less common and resulted in the discontinuation of therapy in only two patients. Pharmacokinetics show that the observed MTD is consistent with the i.v. preparations, and that the bioavailability of p.o. penclomedine is 49 +/- 18%. This regimen can be considered for additional studies in patients with intracranial neoplasms, because good central nervous system penetration is evident. Further development of penclomedine metabolites, such as 4-O-demethylpenclomedine, should be considered to minimize dose-limiting neurotoxicity.     

A search for new metabolites of N,N',N'-triethylenethiophosphoramide.

An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N'-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N'-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.     

Chemical form of selenium, critical metabolites, and cancer prevention.

Methylated selenides are prominent metabolites at the dietary levels used for obtaining anticarcinogenic effects with selenium. The present study reports the chemopreventive activities of 2 novel selenium compounds, Se-methylselenocysteine and dimethyl selenoxide, in the rat dimethylbenz(a)anthracene-induced mammary tumor model. Other treatment groups were supplemented with either selenite or selenocystine for comparative purposes. Each selenium compound was tested at different levels and was given to the animal starting 1 week before dimethylbenz(a)anthracene administration and continued until sacrifice. Results of the carcinogenesis experiments showed that the relative efficacy with the four compounds was Se-methylselenocysteine greater than selenite greater than selenocystine greater than dimethyl selenoxide. In correlating the chemical form and metabolism of these selenium compounds with their anticarcinogenic activity, it is concluded that: (a) selenium compounds that are able to generate a steady stream of methylated metabolites, particularly the monomethylated species, are likely to have good chemopreventive potential; (b) anticarcinogenic activity is lower for selenoamino acids, such as selenocysteine following conversion from selenocystine, which have an escape mechanism via random, nonstoichiometric incorporation into proteins; and (c) forms of selenium, as exemplified by dimethyl selenoxide, which are metabolized rapidly and quantitatively to dimethyl selenide and trimethylselenonium and excreted, are likely to be poor choices. We also undertook a separate bioavailability study using Se-methylselenocysteine, dimethyl selenoxide, and trimethylselenonium as the starting compounds for delivering selenium with one, two, or three methyl groups, and measured the ability of these compounds to restore glutathione peroxidase activity in selenium-depleted animals. All three compounds were able to fully replete this enzyme, although with a wide range of efficiency (Se-methylselenocysteine greater than dimethyl selenoxide greater than trimethylselenonium), suggesting that complete demethylation to inorganic selenium is a normal process of selenium metabolism. However, the degree to which this occurs under chemoprevention conditions would argue against the involvement of selenoproteins in the anticarcinogenic action of these selenium compounds.     

DNA adducts in rat lung, liver and peripheral blood lymphocytes produced by i.p. administration of benzo[a]pyrene metabolites and derivatives

DNA adducts produced in vivo in rat lung, liver and peripheral blood lymphocytes following the i.p. administration of several synthetic benzo[a]pyrene (B[a]P) metabolites and ring-substituted derivatives have been analyzed by the nuclease P1 version of the 32P-postlabeling assay. These include 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11- and 12-hydroxy-B[a]P, (+/-)-B[a]P-trans-4,5-dihydrodiol, (+/-)-B[a]P-trans-7,8-dihydrodiol, (+/-)-B[a]P-trans-9,10-dihydrodiol and B[a]P-7,8-dione. Among the monohydroxy derivatives, only 2-, 9- and 12-hydroxy-B[a]P produced detectable adducts. The only disubstituted derivative studied that produced adducts was the trans-7,8-dihydrodiol. The resulting DNA adducts were compared to those produced in each tissue by administration of B[a]P. 9-Hydroxy-B[a]P and B[a]P-trans-7,8-dihydrodiol each lead to the formation of major B[a]P adducts seen in lung and liver respectively. None of the adducts derived from either 2-hydroxy-B[a]P or 12-hydroxy-B[a]P were observed following administration of B[a]P alone.     

Correlation of nucleic acid binding by metabolites of trans-4-aminostilbene derivatives with tissue specific acute toxicity and carcinogenicity in rats

The reaction with macromolecules was determined in various tissues of female Wistar rats 24 h following a single oral administration of [3H]-trans-4-dimethylamino- and [3H]-trans-4-acetylaminostilbene. Total binding to proteins was 4-9 times greater than to nucleic acids in most tissues. Binding to RNA and DNA was very similar and greatest in liver, about 1/4 in kidney and 1/8 - 1/10 in lung, glandular stomach (target tissue for acute toxicity), bladder, mammary glands and Zymbal glands (target tissue for carcinogenicity). The target tissues, therefore, appear not to be notably exposed to reactive metabolites. The pattern of adducts was analyzed by Sephadex LH20 chromatography of RNA and DNA hydrolysates from liver, glandular stomach, lung and kidney. It was found to be very similar qualitatively. DNA-bound metabolites were very persistent in the non-target tissues liver and kidney, t 1/2 being 34 and 60 days, respectively. Some of the major adducts in liver were not eliminated at all within 4 weeks. It thus appears that aminostilbene derivatives represent examples of strong and genotoxic carcinogens for which the extent and persistence of primary DNA lesions are not correlated with the biological effect.