ABSTRACT
The antitumor effect of IHL-305, a novel pegylated liposome containing irinotecan, was investigated in human xenograft models. After subcutaneous transplantation of several human cancer cell lines (colorectal, non-small cell lung, small cell lung, prostate, ovarian and gastric cancer cells) to nude mice, IHL-305 or CPT-11 was administered intravenously 3 times at 4-day intervals. In all xenograft models tested, IHL-305 showed superior antitumor activity to that of CPT11 and a comparable tumor-growth-inhibitory effect at one-eighth or less of the dose of CPT-11, even against HT-29 colorectal and NCI-H460 non-small cell lung cancer cell lines, which show intrinsic resistance to CPT-11. A single injection or 2 injections of IHL-305 on several dosing schedules also resulted in a significant antitumor effect compared to that of vehicle control in a dose-dependent manner and showed comparable antitumor activity at about one-fifth the dose of the maximum tolerated dose of CPT-11. The analysis of the concentrations of irinotecan and SN-38, an active metabolite of CPT-11, in plasma and tumors revealed that irinotecan was maintained at high concentrations, and the prolonged presence of SN-38 in plasma and tumors in IHL-305 treated mice compared with CPT-11-treated mice. Therefore, the stronger tumor inhibitory effect of IHL-305, as compared to CPT-11, was associated with the difference in the concentration of irinotecan in plasma or tumors after each agent was administered and with the maintainance of a higher concentration of SN-38. These results indicate that IHL-305 demonstrated superior antitumor activity against a wide range of tumors at lower doses than CPT-11.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Camptothecin/analogs & derivatives , Neoplasms, Experimental/drug therapy , Animals , Camptothecin/administration & dosage , Camptothecin/pharmacokinetics , Cell Line, Tumor , Humans , Irinotecan , Liposomes , Male , Mice , Mice, Nude , Polyethylene Glycols , Xenograft Model Antitumor AssaysABSTRACT
We previously reported that phenanthroindolizidine alkaloid 3 and its derivatives had markedly potent in vitro cytotoxicity. However, they had low in vivo antitumor activities and high in vivo toxicities, which was a serious problem. To address this problem, new phenanthroindolizidine derivatives were synthesized and their antitumor activities and toxicities were evaluated. This study describes the relationship between the chemical structures, antitumor activities, and toxicities of these phenanthroindolizidine derivatives. Based on its properties, compound 8 was found to be the most suitable potential antitumor agent.
Subject(s)
Alkaloids/chemical synthesis , Alkaloids/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Drug Design , Indolizines/chemical synthesis , Indolizines/pharmacology , Phenanthrolines/chemical synthesis , Phenanthrolines/pharmacology , Alkaloids/chemistry , Alkaloids/toxicity , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Line, Tumor , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HT29 Cells , Humans , Hydroxides/chemistry , Hydroxides/metabolism , Indolizines/chemistry , Indolizines/toxicity , Male , Mice , Molecular Structure , Phenanthrolines/chemistry , Phenanthrolines/toxicity , Stereoisomerism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
In order to develop a method for estimating the success/failure rates of reproductive processes, especially those of ovulation and neonate nurturing, in the Japanese black bear (Ursus thibetanus japonicus), we examined offspring status, corpora lutea (CLs), placental scars (PSs) and corpora albicantia (CAs) in 159 females (0-23 years old) killed as nuisances on Honshu Island of Japan during 2001-2009. PSs were found to remain in the uterus at least until November of the year of parturition. CA detectability began to decline after September of the year of parturition. Monthly and age-specific proportions of CL-present females revealed that the post-mating season starts in August, and that the age of first ovulation is 4 years. These results indicate that the success rate of ovulation (SRO: the probability that solitary/non-lactating mature females actually succeed in ovulation) can be estimated by calculating the proportion of CL-present females among > or = 4-year-old females without PSs captured from August to November; the early litter loss rate (ELLR: the probability that parenting females lose all of their cubs [0-year-old offspring] before mating season) can be estimated by calculating the proportion of CL-present females among those with PSs and CAs captured in August or later. The estimated values of SRO and ELLR were 0.93 (62/67) and 0.27 (6/22), respectively.
Subject(s)
Corpus Luteum/physiology , Ovulation Detection/methods , Placenta/physiology , Ursidae/physiology , Animals , Female , Japan , Ovulation Detection/veterinary , Pregnancy , ReproductionABSTRACT
The asymmetric total synthesis of the strongly cytotoxic phenanthroindolizidine alkaloid 3 was achieved. Using the same route, various derivatives were also synthesized. Cytotoxicity of those synthetic compounds was evaluated and compounds 19, 23, and 27 demonstrated potent cytotoxicities similar to that of 3. The in vivo antitumor efficacy of selected compounds was also evaluated and 23 demonstrated moderate antitumor efficacy.
Subject(s)
Alkaloids/chemistry , Antineoplastic Agents/chemical synthesis , Indolizidines/chemical synthesis , Indolizines/chemistry , Phenanthrolines/chemical synthesis , Alkaloids/therapeutic use , Alkaloids/toxicity , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/toxicity , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Indolizidines/therapeutic use , Indolizidines/toxicity , Neoplasms/drug therapy , Phenanthrolines/chemistry , Phenanthrolines/therapeutic use , Phenanthrolines/toxicity , Structure-Activity RelationshipABSTRACT
BACKGROUND: Gefitinib competes with topoisomerase I inhibitors at drug efflux pumps in vitro. We evaluated the effects of oral gefitinib on pharmacokinetic parameters of orally coadministered irinotecan. METHODS: We measured the serum pharmacokinetic parameters and biliary excretion of irinotecan, SN-38 and its glucuronide after irinotecan (50 or 100 mg/kg) was orally administered with or without gefitinib 100 mg/kg to rats. We measured the concentrations of irinotecan and SN-38 in the small intestine, liver, lungs and kidneys in each rat. RESULTS: The plasma area under the curve (0-24 h) of irinotecan and SN-38 was increased significantly, while the apparent elimination constant of irinotecan was decreased significantly. Gefitinib significantly increased the biliary cumulative amounts of irinotecan, but not of SN-38, and also influenced the enterohepatic circulation of irinotecan, but not of SN-38. CONCLUSIONS: Gefitinib increased the serum concentrations of irinotecan and SN-38 following oral administration of irinotecan without increasing the biliary excretion of SN-38 in vivo.
Subject(s)
Biliary Tract , Camptothecin/analogs & derivatives , Quinazolines/pharmacology , Administration, Oral , Animals , Biliary Tract/metabolism , Camptothecin/administration & dosage , Camptothecin/metabolism , Gefitinib , Irinotecan , Male , Rats , Rats, WistarABSTRACT
Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.
Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Polymorphism, Single Nucleotide/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/classification , Adenosine Triphosphatases/metabolism , Camptothecin/chemistry , Camptothecin/pharmacology , Cell Line , Chromosomes, Human/genetics , DNA, Complementary/genetics , Drug Resistance , Gene Expression/drug effects , Genome, Human/genetics , Glutamine/genetics , Glutamine/metabolism , Humans , In Situ Hybridization, Fluorescence , Molecular Structure , Mutation/genetics , Neoplasm Proteins/classification , Valine/genetics , Valine/metabolismABSTRACT
It is unknown whether the bioavailability of isoflavones is affected by the concomitant ingestion of glucosides or aglycones. This study was designed to investigate the effects of soymilk-based beverages containing different types of isoflavones on their absorption, excretion, and metabolism. Twelve healthy volunteers consumed 3 kinds of soymilk: untreated soymilk, beta-glucosidase-treated soymilk, and fermented soymilk. Blood samples were collected after 0, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h. Urine samples were collected from 0 to 48 h. Concentrations of isoflavones and daidzein metabolites in serum and urine were measured by liquid chromatography-mass spectrometry. After the ingestion of soymilk, the total concentration of isoflavones in serum rose slowly and reached a maximum of 0.94 +/- 0.39 micromol/L at 6.0 +/- 1.2 h. However, beta-glucosidase-treated soymilk and fermented soymilk increased the serum isoflavone concentration significantly more quickly with maximum concentrations at 1.0 h of 1.75 +/- 0.33 micromol/L and 2.05 +/- 0.32 micromol/L, respectively. The urinary excretion of isoflavones after ingesting of these aglycone-enriched preparations was significantly greater than after consumption of untreated soymilk up to 8 h after injection, but not thereafter. The total and individual concentrations of isoflavones in serum and urine did not differ when subjects consumed the 2 aglycone-enriched soymilks. However, in equol producers (n = 5), the ingestion of ESM tended to increase urinary excretion of equol compared with the consumption of FSM (P = 0.08). These results demonstrated that the isoflavone aglycones of soymilk were absorbed faster and in greater amounts than their glucosides in healthy adults and that the metabolism of isoflavones might be affected by the type of soymilk consumed.
Subject(s)
Isoflavones/pharmacokinetics , Soy Milk/administration & dosage , Soy Milk/chemistry , Adult , Biological Availability , Chromatography, High Pressure Liquid , Equol , Female , Fermentation , Food Handling/methods , Genistein/blood , Genistein/urine , Humans , Isoflavones/blood , Isoflavones/urine , Kinetics , Male , Mass Spectrometry , Middle Aged , beta-Glucosidase/administration & dosageABSTRACT
Acquired and intrinsic drug resistance in cancer is the major obstacle to long-term, sustained patient response to chemotherapy. Irinotecan (CPT-11) is a widely-used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, we have synthesized and characterized a total of fourteen new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition activity. In this context, we have synthesized new CPT analogues with different substitutions at positions 10 or 11 of the A ring. All of the tested CPT analogues strongly inhibited the Topo I activity in a cell-free system. Accordingly, we have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from ABCG2-overexpressing cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are good substrates for ABCG2 and therefore effectively extruded from cancer cells. Thus, hydrogen bond formation is considered to be involved in substrate recognition and/or transport processes of ABCG2. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Drug Design , Drug Resistance, Neoplasm/genetics , Neoplasm Proteins/genetics , Neoplasms/drug therapy , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Animals , Antineoplastic Agents, Phytogenic/chemical synthesis , Camptothecin/chemical synthesis , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/enzymology , Humans , Neoplasms/genetics , Topoisomerase I InhibitorsABSTRACT
Irinotecan (CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, the structure-activity-relationship (SAR) of 14 new camptothecin (CPT) analogues has been studied with respect to the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition. Based on the substrate specificity of ABCG2, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are recognized by ABCG2 and are thereby effectively extruded from cancer cells. To develop a platform for the molecular modeling to circumvent anticancer drug resistance, we have carried out quantum chemical calculations and neural network SAR analysis. Electrostatic potential iso-surfaces generated by ab initio MO calculations using restricted Hartree-Fock method have revealed that negative potential localized at positions 10 or 11 in the A ring is important for recognition by ABCG2.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/analogs & derivatives , Drug Resistance, Neoplasm/genetics , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Animals , Camptothecin/pharmacology , Drug Design , Humans , Models, Molecular , Neoplasms/geneticsABSTRACT
Irinotecan (CPT-11) is a widely-used potent anticancer drug that inhibits mammalian DNA topoisomerase I, however overexpression of the ATP-binding cassette transporter ABCG2 can confer cancer cells resistance to SN-38, the active form of CPT-11. In the present study, we have examined the contribution of three variant forms of ABCG2 to SN-38 resistance. Exogenous expression of the Arg482 (wild type), Gly482, and Thr482 variants of ABCG2 conferred HEK293 cells resistance to SN-38 by 15.0-, 5.0-, and 5.3-fold, respectively. In plasma membrane vesicles prepared from the ABCG2 variant cDNA-transfected HEK293 cells, [Arg482]ABCG2 transported SN-38 and its glucuronide conjugate in an ATP-dependent manner; however, only minimal transport activities were observed with the other variants (Gly482 and Thr482). In addition, we have screened natural flavonoids to find potent inhibitors of [Arg482]ABCG2. Quercetin was found to be the strongest inhibitor (Ki = 0.28 microM) among natural flavonoids tested in the plasma membrane system in this study. When [Arg482]ABCG2-transfected HEK293 cells were incubated with SN-38 in the presence of 20 microM quercetin, cellular resistance to SN-38 was partly reversed. In this context, certain flavonoid derivatives are considered to be good candidates for development of ABCG2 inhibitors.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents, Phytogenic/metabolism , Camptothecin/metabolism , Mutation/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Quercetin/pharmacology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Adenosine Triphosphate/metabolism , Biological Transport , Camptothecin/analogs & derivatives , Cell Membrane , Cells, Cultured , Drug Resistance , Glucuronides/metabolism , Humans , IrinotecanABSTRACT
Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin; CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN-38, the active form of CPT-11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2-overexpressing PC-6/SN2-5H cells transported SN-38 and its glucuronide conjugate in an ATP-dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827-32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell-free system, as did SN-38. Their antitumor activities in the SN-38-resistant PC-6/SN2-5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC-6/SN2-5H2 cells and ABCG2-transfected HEK-293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2-associated drug resistance, low-polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Camptothecin/analogs & derivatives , Camptothecin/toxicity , Drug Resistance, Neoplasm/drug effects , Neoplasm Proteins/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Adenosine Triphosphate/metabolism , Antineoplastic Agents/toxicity , Biological Transport , Camptothecin/pharmacokinetics , Carcinoma, Small Cell , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/physiology , Cloning, Molecular , Humans , Lung Neoplasms , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
A new poly(ethylene glycol) (PEG) conjugate of 10-amino-7-ethyl camptothecin, a potent antitumor analogue of camptothecin, has been synthesized and preliminary in vivo tests have been performed. Successful chemoselective N-acylation of 10-amino-7-ethyl camptothecin was accomplished using phenyl dichlorophosphate, a coupling reagent used in esterification of alcohols, while other coupling methods failed, due to the low nucleophilicity of the amino group in position 10. The conjugate was tested against P388 murine leukemia cell lines and resulted equipotent to CPT-11, a camptothecin analogue already in clinical use.
Subject(s)
Camptothecin/analogs & derivatives , Camptothecin/chemistry , Camptothecin/metabolism , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Acylation , StereoisomerismABSTRACT
Despite the high antitumor activity of camptothecins, few derivatives have been developed and tested for human treatment of solid tumors, due to unpredictable toxicity mainly connected to their poor water solubility. We report the conjugation of the antitumor agent 10-amino-7-hydroxy camptothecin (SN-392) to linear or branched poly(ethylene glycol)s (PEGs) of different loading capacity through a tri- or tetrapeptide spacer selectively cleaved by lysosomal enzymes (cathepsins). A synthetic strategy based on the chemoselective acylation of the aromatic amino group in the presence of the unprotected C20 tertiary alcohol allowed high overall yields. Two conjugates demonstrated good stability at physiological pH and in mouse plasma (nonspecific proteases) but slowly released the drug payload in the presence of the lysosomal enzyme cathepsin B1. Compound 3, selected for in vivo experiments, was very active against P388, P388/ADM leukaemia, and Meth A fibrosarcoma cell lines, scoring T/C% values comparable with the camptothecin derivative CPT-11. Pharmacokinetic studies indicated that 3 acts as a reservoir of 10-amino-7-ethylcamptothecin, as the mean residence time (MRT) is about 3-fold higher than that of the free drug.
Subject(s)
Antineoplastic Agents/chemical synthesis , Camptothecin/chemical synthesis , Oligopeptides/chemical synthesis , Polyethylene Glycols/chemistry , Prodrugs/chemical synthesis , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Camptothecin/analogs & derivatives , Camptothecin/pharmacokinetics , Camptothecin/pharmacology , Cathepsin B/chemistry , Cell Line, Tumor , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Hydrolysis , Male , Mice , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.