The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.     

Autoradiographic distribution of [14C]-labelled pimonidazole in rhabdomyosarcoma-bearing rats and pigmented mice

Summary The hypoxic cell radiosensitizer [2-¹⁴C] pimonidazole (2-nitro--(piperidinomethyl)-l-imidazole ethanol) was injected i.p. into pigmented mice and rats bearing transplanted rhabdomyosarcoma. The injected dose level was 200 mg/kg, and the delivered activity was 96 Ci/kg. Whole-body autoradiography was carried out on all animals. We noted an extensive whole-body distribution of radioactivity. At short intervals, the autoradiograms were characterized by an accumulation of radioactivity in the metabolic and exretory organs (liver, kidney, urinary tract, and intestinal content) as well as in lymphomyeloid tissues (thyroid gland, suprarenal gland, and hypophysis) and salivary glands. In pigmented mice, the uveal and biliary tracts were the highest labelled. The liver and particularly the renal medulla were identified as sites of retention of radioactivity. In the tumor the radioactivity was detected only in peripheral regions, with higher uptake in viable zones than in necrotic islets.Abbreviations used MISO misonidazole - [¹⁴C] ou [¹⁴C] Ro, [¹⁴C] Ro 03-8799 plus Ro 03-8799-derived [¹⁴C]-labelled metabolites     

Autoradiography of [14C]N-nitrosodiethanolamine in Sprague-Dawley rats

Whole-body autoradiography in Sprague-Dawley rats injected i.v. with [14C]N-nitrosodiethanolamine ([14C]NDELA) showed a localization of tissue-bound radioactivity in the liver and the nasal olfactory mucosa. Microautoradiography of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands (Bowman's glands) in the lamina propria mucosae. Experiments in vitro showed a capacity of the liver and the nasal mucosa to form 14CO2 from the [14C]NDELA. Most of the injected [14C]NDELA was recovered in the urine in a non-metabolized form. A small proportion of the dose was exhaled as 14CO2. The target tissues for the NDELA carcinogenesis in Sprague-Dawley rats are the liver and the nasal mucosa. Our results indicate that a bioactivation of the NDELA will take place in the nasal mucosa, as well as in the liver.     

Disposition and metabolism of [14-14C] 4-demethoxydaunorubicin HCl (idarubicin) and [14-14C]daunorubicin HCl in the rat

Summary The disposition of [14-¹⁴C]4-demethoxydaunorubicin HCl ([14-¹⁴C]idarubicin HCl, [¹⁴C]IDR) and of [14-¹⁴C]daunorubicin HCl ([¹⁴C]DNR) was studied in male Sprague Dawley rats. [¹⁴C]IDR was administered either IV at 0.25 mg/kg body weight or PO at 1 mg/kg body weight, whereas [¹⁴C]DNR was dosed IV at 1 mg/kg body weight. The main elimination route for both compounds was the bile, fecal excretion representing 0.75–0.8 times the total dose at 72 h. Radioactivity due to [¹⁴C]IDR-derived species is released by the tissues at a slower rate than activity derived from [¹⁴C]DNR. After IV treatment comparable plasma levels are obtained, but tissue radioactivity is markedly lower with [¹⁴C]IDR, in keeping with the lower dosage. The ratio of plasma to tissue radioactivity is even higher in animals treated PO with [¹⁴C]IDR, because of the more extensive metabolism after this route of administration. The 13-dihydro derivatives of both [¹⁴C]IDR and [¹⁴C]DNR are the main metabolites in tissues, but in the case of the former, products of phase II reactions become more important at later times in liver and kidney and in excreta.     

Autoradiographic distribution of 14C-labeled 3H-imidazo[4,5-f]quinoline-2-amines in mice

The highly mutagenic heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), are formed during heating of protein-rich foods. In order to gain information about the distribution and fate of IQ and MeIQ in vivo, a whole-body autoradiographic study of i.v.-injected 14C-labeled IQ and MeIQ has been performed in male NMRI, pregnant NMRI, and female C3H mice. IQ and MeIQ showed similar distribution patterns. At short survival times, the autoradiograms were characterized by an accumulation of radioactivity in metabolic and excretory organs (liver, kidney, bile, urine, gastric and intestinal contents, salivary glands, nasal mucosa, and Harder's gland), as well as in lymphomyeloid tissues (bone marrow, thymus, spleen and lymph nodes) and in endocrine and reproductive tissues (adrenal medulla, pancreatic islets, thyroid, hypophysis, testis, epididymis, seminal vesicles, ampulla, and prostate). The liver and kidney cortex were identified as sites of retention of nonextractable radioactivity. IQ and MeIQ showed a strong affinity for melanin. IQ and MeIQ passed the placenta, but no radioactivity was retained in fetal tissues. The results pinpoint the liver as a site of IQ- and MeIQ-mediated toxicity. Future studies of IQ and MeIQ may be guided by and clarify the role of other tissue localizations in the toxicity of IQ and MeIQ.     

Synthesis and excretion profile of 1,4- [14C]phenylenebis(methylene)selenocyanate in the rat

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits chemically induced tumors in several laboratory animal models. To understand its mode of action, we synthesized p-[14C]XSC, examined its excretion pattern in female CD rats and also the nature of its metabolites. p- [14C]XSC was synthesized from alpha,alpha-dibromo-p-[ring-14C]xylene in 80% yield. The excretion profile of p-[14C]XSC (15.8 mg/kg body wt, 200 microCi/rat, oral administration, in 1 ml corn oil) in vivo was monitored by measuring radioactivity and selenium content. On the basis of radioactivity, approximately 20% of the dose was excreted in the urine and 68% in the feces over 3 days. The cumulative percentages of the dose excreted over 7 days were 24% in urine and 75% in feces, similar to excretion rates of selenium. According to selenium measurement, <1% of the dose was detected in exhaled air; radioactivity was not detected. Only 15% of the dose was extractable from the feces with EtOAc and was identified as tetraselenocyclophane (TSC). Most of the radioactivity remained tightly bound to the feces. Approximately 10% of this bound material converted to TSC on reduction with NaBH4. Organic soluble metabolites in urine did not exceed 2% of the dose; sulfate (9 % of urinary metabolites) and glucuronic acid (19.5% of urinary metabolites) conjugates were observed but their structural identification is still underway. Co-chromatography with a synthetic standard led to the detection of terephthalic acid (1,4- benzenedicarboxylic acid) as a minor metabolite. The major urinary conjugates contained selenium. Despite the low levels of selenium in the exhaled air, the reductive metabolism of p-XSC to H2Se cannot be ruled out. Identification of TSC in vivo indicates that a selenol may be a key intermediate responsible for the chemopreventive action of p- XSC.      

Binding of [14C]azaserine to DNA and protein in the rat and hamster

The binding of [¹⁴C]azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various times after treatment with [¹⁴C]azaserine. The specific activity of ¹⁴C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.     

Characterization of nonexchangeable radioactivity in L1210 cells incubated with [14C]thiotepa: labeling of phosphatidylethanolamine

N,N',N'-Triethylenethiophosphoramide ([14C]thiotepa) accumulation by L1210 cells is a biphasic process. A very rapid initial phase is followed by a much slower second phase that reflects accumulation of radioactivity in a form that is not lost or exchanged when cells are resuspended and incubated in drug-free medium for up to 8 h. In this study we attempted to characterize this nonexchangeable radioactivity. Nuclei (10(7)) isolated from L1210 cells and incubated with [14C]thiotepa did not accumulate 14C during incubations of up to 5 h. Similarly, nuclei isolated from 10(7) L1210 cells that had been shown to accumulate nonexchangeable 14C after incubation with [14C]thiotepa did not show an increase in nuclear-associated 14C. Eighty to 85% of nonexchangeable 14C in L1210 cells incubated with [14C]thiotepa was soluble in ethanol or chloroform:methanol (2:1, v/v), and although most of this cell-associated nonexchangeable 14C was precipitated by trichloroacetic acid, subsequent treatment of that precipitate with methanol solubilized most of the 14C so that only 15 to 20% remained with the final precipitate. When chloroform:methanol-soluble nonexchangeable 14C was analyzed with thin-layer chromatography systems suitable for thiotepa or simple lipids, all radioactivity remained at the origin. In contrast, when analyzed with one- and two-dimensional thin-layer chromatographic systems suitable for complex lipids, all chloroform:methanol-soluble radioactivity was associated with a single lipid spot. This lipid cochromatographed with phosphatidylethanolamine, reacted with ninhydrin but not with 4-(p-nitrobenzyl)pyridine or the Dragendorff choline reagent, and was digested by phospholipases C and D, all of which lead to its identification as phosphatidylethanolamine. This extensive labeling of phosphatidylethanolamine in L1210 cells incubated with [14C]thiotepa can be explained by liberation of [14C]aziridine from [14C]thiotepa, hydrolysis of the [14C]aziridine to [14C]ethanolamine, and incorporation of that radiolabeled material into phosphatidylethanolamine via the normal cellular synthetic pathways for that lipid. This information implies that thiotepa serves, at least in part, as a prodrug for aziridine and has implications as to the mechanism of thiotepa-induced cytotoxicity in that aziridine is a monofunctional alkylating agent incapable of producing interstrand, intrastrand, or protein-DNA cross-links.     

The comparative disposition of [14C]-fotemustine in non-tumourous and tumourous mice

Summary The distribution and excretion of radioactivity from [¹⁴C]-fotemustine was examined in mice with melanomas at different stages of development to determine whether the disease state substantially alters the disposition of the drug and its metabolites. Normal BDF₁ mice and mice that had been subcutaneously grafted with B16 melanoma either 1, 3, 7, 14 or 21 days previously were used. The animals were killed at either 5 min, or at 3, 24 or 96 h after receiving an intravenous dose of [¹⁴C]-fotemustine (20 mg/kg) and were examined either by whole-body autoradiography or by liquid scintillation counting of excreta and tissues of interest. The majority of the [¹⁴C]-fotemustine dose was excreted in the urine, with similar amounts being measured in both non-tumourous animals (61.6%±13.1%) and tumourous mice grafted 14 days previously (67.2%±5.7%). Small amounts of radioactivity, again similar in both non-tumourous and tumourous mice, were recovered in the faeces (5.4%±5.6% and 3.6%±1.8%, respectively) and as carbon dioxide (7%±3.5% and 6.4%±1%, respectively), with minimal amounts being expired as chloroethanol (<1%). When mice were examined 5 min after dosing, there was extensive tissue distribution accounting for 75%±10% of the dose. The highest concentrations determined by both whole-body autoradiography and liquid scintillation counting were measured in the excretory organs, with 33 and 28 gEq/g being found in the liver and kidney, respectively. High levels were also seen in the lung and plasma (19.8 and 19.5 gEq/g, respectively). Analysis of variance indicated that groups of tissues, such as the excretory organs, blood and plasma or the pigmented tissues, showed distinct but inconsistent patterns. Only tumours at 14 and 21 days of development were suitable for examination, and these showed levels of 12.1 gEq/g; however, the tumourto-plasma ratio increased from between approx. 0.5 and 0.6 at 5 min to approx. 2 at 96 h after dosing, suggesting retention within the melanoma, whereas the ratio for the femur remained at approx. 1. Whole-body autoradiography showed that the distribution in the tumour was not uniform, but rather was concentrated in the peripheral area (presumably viable cells) as opposed to the central necrotic region. Thus, the high and sustained concentration of radioactivity found in the active cells of the melanoma may provide an explanation for the high efficacy of the drug.     

Radioactive species in rat urines and tissues after [14C] AD 32 administration

Summary [14-¹⁴C]N-Trifluoroacetyldoxorubicin-14-valerate ([¹⁴C]AD 32) was synthesized and administered IV to male rats at 9.09 mg/kg. Urinary radioactivity excreted in the 0–24 h interval was only 2.3% of the dose, N-trifluoroacetyldoxorubicin (AD 41), 13-dihydro-N-trifluoroacetyldoxorubicin (AD 92), and doxorubicin being the major urinary metabolites identified. Doxorubicin and AD 32 were the main radioactive species extracted from tissues at 24 h after treatment. The amount of doxorubicin present in the analysed tissue samples is in agreement with the relatively low toxicity of AD 32 compared with doxorubicin.     

Covalent binding of [2-14C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) to mouse DNA in vivo

Female BALB/c mice were administered intragastrically with equimolaramounts of either [2-¹⁴C]2-amino-3,8-dimethyl-[4,5-f]quinoxaline(MeIQx) or 2-acetylamino[9-¹⁴C]fluorene (2AAF). DNA was isolatedfrom tissues of mice killed either 6 or 24 h after administration.Analysis of liver DNA nucleotide digests by HPLC analysis revealedthat all of the radioactivity was attributable to adduct formation.The specific activities of DNA samples were converted to covalentbinding indices (CBI, µmol adduct per mol DNA nucleotides/mmolchemical applied per kg animal body weight). CBI values of 25and 9 were determined for 2AAF and MeIQx in the livers of micekilled 6 h after dosing. The values were in general agreementwith the moderate carcinogenic potency of these compounds. Thespecific activities of DNA preparations obtained from the kidneys,spleens, stomachs, small intestines and large intestines ofmice treated with MeIQx and killed 6 h after dosing were 5-to 35-times less than those obtained with the liver. DNA isolatedfrom the lungs (a target organ for MeIQx tumorigenicity) ofMeIQx-treated mice was not radiolabelled at the limit of detection(CBI <0.3). With the exception of the gastrointestinal tract,the specific activities of DNA samples isolated from mice killed6 h after administration were higher than those from mice killedafter 24 h.     

Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP- dG, PhIP-DNA and DiMeIQx-DNA from the rat

The heterocyclic aromatic amines, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are formed during frying of meat. PhIP and 4,8-DiMeIQx have, after metabolic activation, been shown to form adducts with DNA at the C8 of guanine both in vitro and in vivo. In order to investigate possible urinary biomarkers for estimation of the genotoxic dose of PhIP and 4,8-DiMeIQx, [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx- DNA were injected i.p. to rats and the excretion of radioactivity in urine and faeces were measured. For all three [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA 15-20% of the dose were excreted in the urine and 80-85% of the dose were excreted in the faeces. Urinary excretion showed maximum to 24 h (90%) with a rapid decline, 10% to 48 h and 0% to 72 h. Faecal excretion also showed maximum to 24 h (60%) with a slower decline, 30% to 48 h and 10% to 72 h. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-dG, showed that approximately 90% of the radioactivity co-eluted with PhIP- dG, indicating that PhIP-dG is excreted unmetabolized. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-DNA, showed that approximately 85% of the radioactivity co- eluted with PhIP-dG, indicating that PhIP-DNA adducts is mainly excreted as nucleoside adducts. Approximately 5% of the radioactivity excreted in the urine co-eluted with PhIP-G, indicating loss of deoxyribose. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [14C]4,8-DiMeIQx-DNA, showed that approximately 90% of the radioactivity co-eluted with 4,8-DiMeIQx-dG, indicating that 4,8-DiMeIQx-DNA adducts is mainly excreted as nucleoside adducts. Man is able to eliminate compounds of a higher mol. wt in the urine than the rat, the percentage of PhIP-dG and 4,8-DiMeIQx eliminated in the urine of man would therefore be expected to be higher than in the rat. Measurement of urinary nucleoside adducts of PhIP and 4,8-DiMeIQx could therefore provide a basis for the development of a biomonitoring strategy for the genotoxic dose of these food derived HAA.      

Metabolism and urinary excretion of mutagenic metabolites of benzo[a]pyrene in C57 and DBA mice strains

Groups of C57Bl/6 (B6) mice (responsive) and DBA/2 (D2) (non-responsive)mice received an i.p. dose of benzo[a]pyrene (BP) (100 mg/kgbody weight) with or without pretreatment with or without pretreatmentwith ß-naphthoflavone (BNF), an inducer of monooxygenases.The amount of mutagenic BP metabolites in the urine was determinedby the Salmonella/microsome assay (strain TA100) and excretionof BP metabolites was quantifled by h.p.l.c. After inducer treatment,the responsive mice excreted more mutagens and total oxidizedBP metabolites in the urine than did the non-responsive strain.The ratio of BP diols:total oxidized metabolites was 0.42 inB6 mice and 0.72 in D2 mice and was not affected by inducertreatment. After a dose of [¹⁴C]BP, radioactivity was measuredin the faeces for up to 7 days. The half-life of [¹⁴C]BP inthe non-responsive D2 mice was about twice that in the responsiveB6 mice. Our results agree with a previously proposed modelof the risk factors for toxic effects and/or the developmentof tumours after exposure to polycyclic aromatic hydrocarbonsin responsive and nonresponsive mouse strains.     

Fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats

Fischer 344 rats were given a single dose of 0.60 mg/animal of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by gavage; and radioactivity contained in feces, urine, blood, serum proteins, hemoglobin, and tissues was determined at 12, 24, 48 and 96 h after dosing. One major and four minor radioactivity-containing fractions were found in the urine and one major and two minor radioactivity-containing fractions were found in the feces. The feces was the major route of excretion, representing 78% of dose during the first 24 h, and unchanged PhIP in the feces accounted for 51% of the dose. Unmetabolized PhIP was also shown to be the major radioactive fraction in bile and feces from animals given a single dose by i.p. injection. Blood contained a small fraction of the dose and the major, persistently-bound form of PhIP in the blood was to hemoglobin. At 12 h after administration of the dose the colon and cecum contained the highest concentration of radioactivity, while at later times the kidney and liver showed the highest concentration. Of the tissue-contained radioactivity 80-90% was ethanol insoluble at time points later than 24 h, suggesting that it was covalently bound to macromolecules.     

Assessment of the absorption, metabolism and excretion of [14C]pasireotide in healthy volunteers using accelerator mass spectrometry

Purpose

Pasireotide (SOM230) is a multireceptor-targeted somatostatin analog designed to have a broader somatostatin receptor binding profile than other currently available somatostatin analogs. The purpose of this study was to evaluate the absorption, metabolism and excretion of pasireotide in healthy male subjects (N = 4) following a single, subcutaneous (sc), 600 μg dose of [¹⁴C]pasireotide.

Methods

Blood, plasma, urine and feces were collected for 240 h post-dose and analyzed for total ¹⁴C and metabolite profile by accelerator mass spectrometry (AMS) or high-performance liquid chromatography–AMS. Parent drug levels were analyzed by radioimmunoassay.

Results

[¹⁴C]pasireotide was rapidly absorbed, with a mean peak plasma ¹⁴C concentration of 16.6 ± 5.28 ngEq/mL at 0.5 h in plasma. The parent drug to total ¹⁴C AUC_0–24h ratio was 1.08, indicating that little metabolite was present in plasma up to 24 h post-dose. In pooled plasma samples (0–12 h), only unchanged [¹⁴C]pasireotide was detected. Unchanged [¹⁴C]pasireotide accounted for approximately 84 % of total excretion (feces and urine). Approximately 56 % of the administered radioactive dose was recovered within 240 h, eliminated primarily in feces (48.3 ± 8.16 %) and minimally in urine (7.63 ± 2.03 %). No serious adverse events were reported.

Conclusions

A single dose of [¹⁴C]pasireotide 600 μg sc administered to healthy male subjects was rapidly absorbed and excreted in its unchanged form primarily via the hepatic route.     

Distribution of radioactivity and anthracycline-fluorescence in tissues of mice one hour after [14C]-labeled AD 32 administration

Summary Levels of radioactivity and total anthracycline fluorescence in tissues of A/JAX mice were compared 1 h after IV administration of unlabeled or [¹⁴C]-labeled AD 32 (50 mg/kg). Highest levels of both fluorescence and radioactivity were found in the small intestine (including contents) and liver, a result consistent with the known hepatobiliary excretion of AD 32 and metabolites. Significant accumulations of radioactivity and fluorescence were found in kidney, spleen, large intestine (including contents), lung, and heart. Lesser levels were found in muscle and fat. Little radioactivity and fluorescence were found in brain. Liquid chromatographic analysis of extracts of small intestine and liver homogenates showed N-trifluoroacetyladriamycin (AD 41) as the major fluorescent species, and also revealed N-trifluoroacetyladriamycinol (AD 92) and occasional low levels of AD 32. In addition, there was a major peak of nonfluorescent radioactive material and two fluorescent nonradioactive signals (unknowns 1 and 2), indicative of cleavage of the radiolabel from the chromophore.The abbreviations used are AD 32 N-trifluoroacetyladriamycin-14-valerate - AD 41 N-trifluoroacetyladriamycin - AD 92 N-trifluoroacetyladriamycinol - ADR adriamycin - AD 48 adriamycin-14-valerate - AD 60 13-dihydro-N-trifluoroacetyladriamycin-14-valerate - AMNOL adriamycinol - HPLC high-performance liquid chromatography - TLC thin layer chromatography - SDS sodium dodecylsulfate     

Role of N-methylolpentamethylmelamine in the metabolic activation of hexamethylmelamine

Hexamethylmelamine (HMM) is metabolized by rat hepatic microsomal preparations to reactive species which covalently bind to microsomal protein and to calf thymus DNA added to microsomal incubation mixtures. Covalent binding to macromolecules is dependent on the presence of molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate and is catalyzed by cytochrome P-450 monooxygenases. Reduced nicotinamide adenine dinucleotide-dependent covalent binding of [methyl-14C]HMM to microsomal protein is greater than that of [ring-14C]HMM. Reduced nicotinamide adenine dinucleotide phosphate-dependent covalent binding of [ring-14C]HMM and [methyl-14C]HMM to calf thymus DNA added to microsomal incubation mixtures are approximately equal. The [ring-14C]-labeled carbinolamine intermediate in HMM demethylation, N-methylolpentamethylmelamine, covalently binds to microsomal protein and, to a much greater extent, to calf thymus DNA.     

A phase I, open-label, mass balance study of [14C] dacomitinib (PF-00299804) in healthy male volunteers

Purpose

This study aimed to characterize the primary routes of elimination of the pan-HER tyrosine kinase inhibitor, dacomitinib (PF-00299804), to evaluate the pharmacokinetics of total radioactivity and of dacomitinib and to identify the metabolites of dacomitinib in plasma, urine, and feces in the healthy volunteers.

Methods

Six male healthy volunteers (mean age 31.5 years) received a single 45-mg oral dose containing ~100 μCi [¹⁴C] dacomitinib. Whole blood, urine, and fecal samples were collected throughout the study and analyzed for total radioactivity by liquid scintillation counting. Safety evaluations included vital signs, 12-lead ECGs, safety laboratory tests, and monitoring of adverse events.

Results

78.8 % of the radiolabeled material was excreted in feces, and 3.2 % was recovered in urine. Peak concentrations of dacomitinib in plasma occurred 12 h (median) after oral dosing. Mean terminal plasma half-life was 55 and 182 h for dacomitinib and total plasma radioactivity, respectively. Geometric mean C _max was approximately 2-fold higher, and total exposure (AUC_inf) was almost 6-fold higher for total radioactivity than for dacomitinib in plasma. O-desmethyl dacomitinib (PF-05199265) was the major circulating metabolite. T _max of this metabolite occurred 6 h after oral dosing with dacomitinib. Plasma exposure for the metabolite was one-third that of the parent compound. There were no serious/severe adverse events or deaths during the study. Dacomitinib was well tolerated.

Conclusions

In humans, [¹⁴C] dacomitinib underwent oxidative and conjugative metabolism. Most of the administered dose was eliminated via the fecal route, and the major circulating metabolite was PF-05199265.     

Measurement of 7-methylguanine as an estimate of the amount of dimethylnitrosamine formed following administration of aminopyrine and nitrite to rats

We have demonstrated that there is a dose-related increase in the excretion of 7-[methyl-14C]methylguanine ( [14C]m7Gua) following p.o. administration of di[methyl-14C]methylnitrosamine to rats. Urine was collected for 24 hr after di[methyl-14C]methylnitrosamine administration, and the purines were precipitated from an aliquot of the urine with silver nitrate. Purines were released from the precipitate with HCl, and [14C]m7Gua was quantified by chromatography on an Aminex A-6 column. The excretion of [14C]m7Gua increased linearly with the dose of dimethylnitrosamine. This relationship was used to estimate the amount of di[methyl-14C]methylnitrosamine formed in the reaction of [14C]aminopyrine with sodium nitrite in rats gavaged with these compounds. The dose of dimethylnitrosamine was also estimated from the amount of alkylation of liver DNA in the same animals. These estimates usually differed by less than a factor of 2. [14C]aminopyrine and sodium nitrite were administered. The possibility of using this assay to obtain data on nitrosation in humans is discussed.     

Absorption, metabolism and excretion of [14C]pomalidomide in humans following oral administration

Purpose

To investigate the pharmacokinetics and disposition of [¹⁴C]pomalidomide following a single oral dose to healthy male subjects.

Methods

Eight subjects were administered a single 2 mg oral suspension of [¹⁴C]pomalidomide. Blood (plasma), urine and feces were collected. Mass balance of radioactivity and the pharmacokinetics of radioactivity, pomalidomide and metabolites were determined. Metabolite profiling and characterization was performed. The enzymes involved in pomalidomide metabolism and the potential pharmacological activity of metabolites were evaluated in vitro.

Results

Mean recovery was 88 %, with 73 and 15 % of the radioactive dose excreted in urine and feces, respectively, indicating good oral absorption. Mean C _max, AUC_0?∞ and t _max values for pomalidomide in plasma were 13 ng/mL, 189 ng*h/mL and 3.0 h. Radioactivity and pomalidomide were rapidly cleared from circulation, with terminal half-lives of 8.9 and 11.2 h. Pomalidomide accounted for 70 % of the circulating radioactivity, and no circulating metabolite was present at >10 % of parent compound. Pomalidomide was extensively metabolized prior to excretion, with excreted metabolites being similar to those observed in circulation. Clearance pathways included cytochrome P450-mediated hydroxylation with subsequent glucuronidation (43 % of the dose), glutarimide ring hydrolysis (25 %) and excretion of unchanged drug (10 %). 5-Hydroxy pomalidomide, the notable oxidative metabolite, was formed primarily via CYP1A2 and CYP3A4. The hydroxy metabolites and hydrolysis products were at least 26-fold less pharmacologically active than pomalidomide in vitro.

Conclusions

Following oral administration, pomalidomide was well absorbed, with parent compound being the predominant circulating component. Pomalidomide was extensively metabolized prior to excretion, and metabolites were eliminated primarily in urine.