Safety and Evidence of Off-Label Use of Approved Drugs at the National Cancer Center Hospital in Japan.

PURPOSE: In Japan, for pharmaceutical products to be covered by public medical insurance, their efficacy and safety must first be confirmed in clinical trials. To our knowledge, this study is the first investigation into the off-label use of pharmaceutical products at a high-volume cancer treatment center in Japan. The objective of this study is to explore the framework necessary for future pharmaceutical development and regulatory approval in the field of oncology by surveying the frequency of and indications for off-label use of pharmaceutical products at the National Cancer Center Hospital in Tokyo, Japan. MATERIALS AND METHODS: The pharmaceutical products used off-label in daily practice from 2003 to 2015 at the National Cancer Center Hospital were retrospectively examined based on applications that had been submitted to an internal review committee requesting off-label use. RESULTS: A total of 1,390 applications were submitted during the study period. The most frequently used supporting documents were the results of phase II trials, followed by case series and phase III trials. The cancer most frequently treated with off-label drugs was sarcoma (15.1%), followed by urologic cancer (9.2%) and GI cancer (7.6%). CONCLUSION: As reported in previous studies, pharmaceutical products were generally used off-label for the treatment of rare cancers, for which large-scale clinical trials are difficult to conduct. Continued discussion of the types of frameworks that are needed to guide pharmaceutical development is necessary.

Simple and sensitive HPLC method for determination of amrubicin and amrubicinol in human plasma: application to a clinical pharmacokinetic study.

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for determination of amrubicin and its metabolite amrubicinol in human plasma. After protein precipitation with methanol without evaporation procedure, large volume samples were injected and separated by two monolithic columns with a guard column. The mobile phase consisted of tetrahydrofuran-dioxane-water (containing 2.3 mM acetic acid and 4 mM sodium 1-octanesulfonate; 2:6:15, v/v/v). Wavelengths of fluorescence detection were set at 480 nm for excitation and 550 nm for detection. Under these conditions, linearity was confirmed in the 2.5-5000 ng/mL concentration range of both compounds. The intra- and inter-day precision and intra- and inter-day accuracy for both compounds were less than 10%. The method was successfully applied to a clinical pharmacokinetic study of amrubicin and amrubicinol in cancer patients.

Isolation and identification of a novel aromatic amine mutagen produced by the Maillard reaction.

To clarify the formation of mutagens in the Maillard reaction of glucose and amino acids, 20 amino acids were separately incubated with glucose in the presence or absence of hydroxyl radicals produced by the Fenton reaction. After 1 week at 37 degrees C and pH 7.4, the reaction mixtures of glucose and tryptophan with and without the Fenton reagent showed mutagenicity toward Salmonella typhimurium YG1024 in the presence of a mammalian metabolic system (S9 mix). To identify mutagens in the reaction mixture, blue rayon-adsorbed material from a mixture of glucose, tryptophan, and the Fenton reagent was separated by column chromatography using various solid and mobile phases, and one mutagen, which accounted for 18% of the total mutagenicity of the reaction mixture, was isolated. The chemical structure of the mutagen was determined to be 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ) on the basis of ESI mass, high-resolution APCI mass, (1)H NMR, (13)C NMR, and IR spectral analyses and chemical synthesis of the mutagen. The novel aromatic amine showed high mutagenicity toward S. typhimurium TA98 and YG1024 with S9 mix, inducing 857 revertants of TA98 and 6007 revertants of YG1024/microg, respectively. The mutagenicity of ABAQ was comparable to that of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, which is a mutagenic and carcinogenic hetrocyclic amine in cooked meat and fish formed through the Maillard reaction at high temperature.

Carcinogenic risk of heterocyclic amines in combination - assessment with a liver initiation model.

Carcinogenic potential of heterocyclic amines (HCAs) was investigated using an in vivo 5-week initiation assay with quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver. Numbers of GST-P positive foci were significantly increased with individual administration of six different HCAs, indicating utility of the assay. It was therefore applied to investigate risk with multiple HCAs in combination. Unexpectedly, concomitant treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) did not result in any additive carcinogenicity. In the rats taking MeIQx prior to PhIP the value was almost equal to the sum total of individual data, indicating additive initiation activities. In contrast, simultaneous or prior administration of PhIP rather exerted inhibitory effects on the carcinogenic potential of MeIQx. Moreover, microarray and quantitative RT-PCR assessment revealed that PhIP induced cytochrome P450 1A1, responsible for both activation and detoxification of HCAs, more strongly than MeIQx. It is noteworthy that complex exposure to multiple HCAs is not necessarily associated with increased risk of carcinogenesis because they are simultaneously and continuously ingested under normal circumstances.

Detection of aminophenylnorharman, a possible endogenous mutagenic and carcinogenic compound, in human urine samples.

Mutagenic/carcinogenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is formed from norharman and aniline in the presence of cytochrome P450 3A4/1A2. Because both precursors are widely distributed in the environment, human exposure is unavoidable. To clarify APNH formation in the human body, amounts of the compound in 24-h human urine collected from smokers and nonsmokers, eating a normal diet, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry. In addition, norharman and aniline were also analyzed by high-performance liquid chromatography and gas chromatography, respectively. APNH could be detected in all urine samples at levels 49 to 449 pg for smokers and 21 to 594 pg for nonsmokers per 24-h urine, respectively. The amounts of norharman and aniline were 46 to 185 ng and 0.70 to 8.10 microg for smokers and 52 to 447 ng and 0.49 to 5.72 microg for nonsmokers, respectively, per 24-h urine (none of the levels differing significantly between smokers and nonsmokers). To exclude exogenous exposure to norharman and aniline, we analyzed the levels of APNH, norharman, and aniline in urine samples collected from inpatients receiving parenteral alimentation. Similar to the healthy volunteers, all urine samples contained 12 to 338 pg of APNH, 6 to 75 ng of norharman, and 0.33 to 1.86 microg of aniline per 24-h urine. These results suggest that APNH should be considered as a novel endogenous mutagen/carcinogen; thus, it is very important to determine the biological significance of this carcinogen for human cancer development.

Structures and biological properties of DNA adducts derived from N-nitroso bile acid conjugates.

A kind of N-nitrosobile acid conjugate, N-nitrosotaurocholic acid (NO-TCA), was incubated with calf thymus DNA, and formation of an adduct was detected by the 32P-postlabeling method under nuclease P1 conditions. To examine the nucleotides containing the adduct from NO-TCA, each of 2'-deoxyribonucleotide 3'-monophosphates (3'-dAp, 3'-dGp, 3'-dCp, or 3'-Tp) was incubated with NO-TCA. The same adduct spot was detected in the reaction of NO-TCA with 3'-dCp. The structure of this adduct was determined to be 3-ethanesulfonic acid-dC by several spectrometry techniques. Moreover, bulky adducts containing bile acid moiety were also produced from the reaction of NO-TCA with 3'-dCp and 3'-dAp. From comparison with spectral data for authentic compounds, these adducts were concluded to be N4-cholyl-dC and N6-cholyl-dA. N4-Cholyl-dC and N6-cholyl-dA were also detected in calf thymus DNA treated with NO-TCA. In addition, 3-ethanesulfonic acid-dC and N4-deoxycholyl-dC were found to be produced from N-nitrosotaurodeoxycholic acid (NO-TDCA) with dC. NO-TCA and NO-TDCA induced mutations in Salmonella typhimurium TA100 but not in TA98. Mutational spectrum analysis revealed that NO-TCA induced G to A transitions predominantly. When NO-TCA (250 mg/kg) was singly administered to male Wistar rats by gavage, both ethanesulfonic acid-dC and N4-cholyl-dC could be detected in the glandular stomach and colon. The levels of ethanesulfonic acid-dC were 0.22-0.29 per 10(6) nucleotides, but values for N4-cholyl-dC were about 500-fold lower. These observations suggest that N-nitroso bile acid conjugates, NO-TCA and NO-TDCA, may induce G to A base substitutions in genes via DNA adduct formation, producing ethanesulfonic acid- and/or (deoxy)cholic acid-DNA and, therefore, may be related to human carcinogenesis as endogenous mutagens.

Identification of cytochrome P-450s involved in the formation of APNH from norharman with aniline.

Mutagenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), formed from norharman and aniline in the presence of S9 mix, is thought to be accountable for the co-mutagenic action of norharman. Our previous studies suggest that cytochrome P-450s (CYPs) are involved in the generation of APNH. In order to identify the responsible CYP species in the present study, norharman (8 mg) and aniline (4 mg) were incubated with individual recombinant human CYPs (2 nmol) at 37 degrees C for 20 min. Formation of APNH was observed with CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2D6, CYP2E1 and CYP3A4, but not with CYP2A6, CYP2C9 and CYP2C19. The amounts of APNH from norharman and aniline were 33 ng for CYP1A1, 15 ng for CYP3A4, 7 ng for CYP2D6, 6 ng for CYP1A2 and 5 ng for CYP2B6. APNH formation in the presence of CYP1B1 and CYP2E1 was very low at around one fiftieth of that with CYP3A4. When CYP selective chemical inhibitors, such as furafylline for CYP1A2 and ketoconazole for CYP3A4, were added to the reaction mixture of norharman, aniline and human microsomes, formation of APNH was decreased to 14 and 16% of the control level, respectively. Moreover, human lung microsomes also showed the activity of APNH formation from norharman and aniline, albeit at only one hundredth of that with liver microsomes. In general, content in human liver microsomes is rather high for CYP3A4 and CYP1A2 but relatively low for CYP2D6 and CYP2B6, at about 30, 10, 1.5% and less than 1% of the total CYP, respectively. Although CYP1A1 showed the highest APNH formation activity, its expression in human liver is reported to be below the level of detection. Based on these observations, it is suggested that the practical major contributors to the formation of APNH from norharman and aniline are CYP3A4 and CYP1A2, the responsible reactions mainly occurring in the liver.

Analysis of HPRT and supF mutations caused by pierisin-1, a guanine specific ADP-ribosylating toxin derived from the cabbage butterfly.

Pierisin-1, an ADP-ribosylating toxin derived from the cabbage butterfly, Pieris rapae, induces apoptosis in various mammalian cell lines. We recently reported that the target for ADP ribosylation by pierisin-1 is the 2'-deoxyguanosine residue in DNA. To examine whether pierisin-1 would induce mutations in mammalian cell genes, we conducted a mutational analysis for the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in pierisin-1-treated Chinese hamster lung (CHL) cells. N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine was detected by the (32)P-postlabeling method in CHL cells after treatment with pierisin-1 at doses of 2-32 ng/mL; adduct levels were 1.1-12.0 per 10(6) nucleotides. Pierisin-1 induced mutations in the HPRT gene dose-dependently, and the frequency was 38 times higher than the control, at a dose of 32 ng/mL. To confirm that mono(ADP-ribosyl)ated dG itself leads to mutations, the pierisin-1-treated DNA of plasmid pMY189 bearing the supF gene was used for mutational analysis. The mutation frequency of the supF gene treated with 2-8 micro g/mL of pierisin-1 was 17-40-fold the control value. Mutation spectrum analysis showed that single base substitutions dominated in both HPRT and supF genes. Among these, transversions were predominant, and more than 70% of the base substitutions occurred at G:C base pairs in both genes. The most frequent mutations were G:C to C:G, followed by G:C to T:A in HPRT gene, whereas G:C to T:A transversions dominated in the supF gene. Our results indicate that pierisin-1 produced N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine and this guanine-adduct could lead to mutations in the HPRT and supF genes. These findings could provide very useful information for understanding the biological significance of pierisin-1.

Structure of DNA adduct formed with aminophenylnorharman, being responsible for the comutagenic action of norharman with aniline.

A mutagenic heterocyclic amine (HCA), 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), is produced in the presence of S9 mix by the reaction of norharman and aniline, both of which are nonmutagenic and abundantly present in our environment. It has been previously reported that APNH-DNA adducts were detected in DNA of Salmonella typhimurium strain incubated with APNH and S9 mix. In the present study, we examined the structures of APNH-DNA adducts using the (32)P-postlabeling method and various spectrometry techniques. When the reaction mixture of N-acetoxy-APNH and 2'-deoxyguanosine 3'-monophosphate (3'-dGp) was analyzed, three adduct spots (two major and one minor) were observed by (32)P-postlabeling under modified-standard conditions. No adduct formation was observed for reaction mixtures of N-acetoxy-APNH with 3'-dAp, 3'-dTp, or 3'-dCp. The two major adduct spots (spots 1 and 2) detected by TLC were extracted and subjected to HPLC along with the standards 3',5'-pdGp-C8-APNH and 5'-pdG-C8-APNH, which were independently chemically synthesized. On the basis of the results of co-chromatography, spots 1 and 2 were identified to be 5'-monophosphate and 3',5'-diphosphate forms of dG-C8-APNH. When the extract of spot 2 (3',5'-pdGp-C8-APNH) was further digested with nuclease P1 and phosphodiesterase I, a spot corresponding to spot 1 (5'-pdG-C8-APNH) was newly observed on TLC. From these observations, both of the two major spots were concluded to be dG-C8-APNH. A similar DNA adduct pattern to that apparent in vitro was observed in various organs of F344 rats fed 40 ppm of APNH for 4 weeks. The levels of APNH-DNA adducts were highest in the liver and colon, with RAL values of 1.31 +/- 0.26 and 1.32 +/- 0.11 adducts/10(7)nucleotides, respectively. Thus, APNH was demonstrated to form DNA adducts primarily at the C-8 position of guanine residues in vitro and in vivo, like other mutagenic and carcinogenic HCAs.