Osaka, JAPAN
Objectives: Response rates of 5-fluorouracil (5FU) and tegafur likely differ individually due to differences in the enzyme activities for anabolism and catabolism. Orotate phosphribosyl transferase (OPRT) is an essential enzyme for activation of 5FU. Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme for degradation of 5FU. To obtain a high percentage of response without side-effects, it would be important to evaluate the enzyme activities and to estimate the optimal duration of chemotherapy.
Patients and Methods: The study included 160 patients, whose colorectal cancer (Stage II to IV) were treated with uracil and tegafur (UFT) after surgery. (1) OPRT and DPD expressions were evaluated using immunohistochemistry. Relationships between their expressions and clinicopathological features. Survival curves were calculated using Kaplan-Meier method. (2) Using the collagen gel droplet embedded drug sensitivity test (CD-DST), we estimated the optimal duration of chemotherapy. Area under curve (AUC) was calculated with the inhibition rates assessed in variety of time and concentration in the tests. The AUC value for 50% inhibition (AUCIR50) was also calucated. The AUC24hr of 5FU infusion (250 mg/mm2/day), UFT (400 mg/m2/day) were about 1.7 and 1.4 (micro-gram x hr/mL). Based on these values, the duration to attain the AUCIR50 for 5FU and UFT were estimated.
Results: (1) OPRT expression showed a negative correlation with advances in cancer stage, though DPD expression showed positive correlations. The patients survival were better in those OPRT(+) than in those OPRT (-) (p=0.004). The patients survival were better in those DPD(-) than in those DPD(+) (p=0.008). In regard to the combination of these expression, the best survival curve was obtained for the OPRT(+) DPD(-) group and the worst was OPRT(-) DPD(+) group. (2) The AUCIR50 in 38% of the patients ranged < 100; 43% ranged 100-1000; 13% ranged 1000-10000; and 6% ranged > 10000 respectively. Therefore, the duration to attain the AUCIR50 by 5-FU infusion ranged < 10 weeks in 37% patients; 10-100 weeks in 44 %; and longer than 100 weeks in 19% respectively. The durations to attain the AUCIR50 by UFT ranged < 6 months in 55% patients; 6-12 months in 13 %; and longer than 12 months in 32% respectively.
Conclusions: OPRT expression related with better prognosis, although DPD expression were related with poor prognosis. The use of several determinants of response may identify a high percentage of responding patients. The optimal duration of chemotherapy with 5FU or tegafur may be estimated using CD-DST, though it differs in patients.
Developing a magic bullet against P-glycoprotein-mediated drug resistance by exploiting mechanism
TOMBLINE G1,3, HOLT JJ3, GANNON MK3, SAWADA G2, RAUB TJ2,
DONNELLY D3, Wetzel B3, Ye M3, Nygren CL3, Detty MR3
1Univ. of Rochester, Microb. & Immunol., Rochester NY, USA; 2Eli Lilly, Drug Disposition, Indianapolis IN, USA; 3Univ. at Buffalo, Chemistry, Buffalo NY, USA;.
Background: Multidrug resistance (MDR) is a major obstacle for the treatment of cancer as well as bacterial infections. Efflux pumps are often the root cause of MDR. The prototypical human multidrug resistance efflux pump P-glycoprotein (Pgp) couples drug export to ATP binding and hydrolysis. Details regarding drug trajectory, the molecular basis for coupling, and factors governing transport rate/efficiency remain unknown. Our goal is to reveal these details. Rhodamine dyes have been used as as drug mimics to assay Pgp-mediated transport. Herein, we employ novel synthetic analogues of Tetramethylrosamine (TMR) to probe Pgp mechanism. A unique advantage is our ability to tease apart the capacity of a drug to confer coupling to ATP catalytic sites versus transport rate/efficiency.
Methods: A library of TMR analogues was constructed and their effect on purified lipid-activated mouse MDR3 Pgp ATPase was determined. Coupling to ATP catalytic sites was also determined by their ability to promote ADP-Vi trapping, ATP occlusion in “catalytic carboxylate” mutant Pgp, as well as through inhibition of verapamil-dependent ATPase. Effect on drug transport was measured in vivo with Madin-Darby Canine Kidney monolayer cells that express Pgp (MDCK-MDR1).
Results: Nearly three orders of magintude variation in ATPase stimulation within the library was apparent. Importantly, the concentration of TMR required for coupling to the ATP site does not clearly corelate with turnover rate. In some cases, the ability to promote ATP occlusion is associated with robust turnover. Several other analogues stimulate ATP occlusion relatively well but displayed very slow turnover rates. This class also appeared relatively effective as Pgp inhibitors/modulators in vitro and in vivo.
Conclusions: Drug recognition may not be apparent from ATPase stimulation which may manifest as equivalent to (or below) basal values. Promotion of ATP occlusion appears to reveal the true affinity of a drug for Pgp and this does not corelate with transport rate. Molecules that promote ATP occlusion effectively yet confer slow turnover rates are good leads for development of competitive inhibitors.
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Bacterial resistance and influence on optimal choice of antibacterials
TOMIĆ Z, SABO A, HORVAT O, MILIJAŠEVIĆ B, VASOVIĆ V, VUKMIROVIĆ S, STILINOVIĆ N
Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine Novi Sad, Serbia
Background: Several studies have reported high rate of antimicrobial resistance among isolates from hospital. The aim of this study was review the pathogens associated with nosocomial infections at Clinic for abdominal surgery, Clinic for urology surgery, Clinic for Orthopedic surgery and Clinic for Resuscitation and Anesthesia Clinical Center of Vojvodina.
Methods: This investigation includes all bacterial strains isolated from patient’s material collected routinely from Clinic for abdominal surgery, Clinic for urology surgery, Clinic for Orthopedic surgery and Clinic for Resuscitation and Anesthesia Clinical Center of Vojvodina. The investigation was done in period of 01.01.2006. to 31.12.2006. year. The places from which isolates were collected are: surgery lesion, urine, patient’s blood.
Results: The results of analysis have shown that the collected data are insufficient for conclusions. The percentage of positive results was too low to enable to make recommendations. The list of antibiotics used for determination of sensitivity should include more first choice antibiotics according to international recommendations. The most frequently bacteria were tested to second and third choice antibiotics, to which the resistance rate is concerning high.
Conclusions: There is evidence for huge number of fecal contamination bacterias isolated from lesion and other places. Because of that there is need for urgent antisepsis and disinfection measures that we can lower contamination and need for antibiotics.
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Multiple Targets of Pyrvinium Pamoate in Mammalian and Parasite Mitochondria
Tomitsuka E1, Kita K 2, Esumi H 1
1Natl. Cancer Center Res. Inst. East, Chiba, Japan; 2Univ, Tokyo, Tokyo, Japan.
Background: Pyrvinium pamoate, an anthelmintic, interferes with absorption of glucose by the helminths. Recently, we have reported that pyrvinium pamoate inhibited cancer cell survival under glucose starved culture, shown as anti-cancer compound in mammalian. The differences in energy metabolisms between the mammalian and helminths have been thought to be a target of pyrvinium. One of the important metabolic systems in helminths, NADH-fumarate reductase system, which is the final step of the phosphoenolpyrvate carboxykinase (PEPCK)-succinate pathway, is plays and important role in the anaerobic energy metabolism. It has been thought that the effect of pyrvinium is from inhibition of the fumarate reductase activity, which appears to be the key enzyme in NADH-fumarate reductase system. This study examined which enzymes were related to these effects both of the mammalian and the parasite mitochondria.
Methods:Enzyme activities were measured in Ascaris sum, bovine heart and cancer cells’ mitochondria cultured with tumor-mimic conditions.
Results: Pyrvinium pamoate inhibited not only fumarate reductase (Complex II) but also NADH-quinine reductase (Complex I) in the anaerobic respiratory chain. Moreover, as same as in parasite, pyrvinium pamoate inhibited Complex I activity in mammalian mitochondria. However, compared with A. suum, pyrvinium showed different effect on mammalian Complex II, especially in cancer cells cultured with tumor-mimic conditions.
Conclusions: The species-specific, tumor-specific inhibitory effects by pyrvinium were caused by the different effects on Complex II activity in the mammalian and parasite mitochondria. These observations with pyrvinium pamoate emphasize the difference in metabolic systems between the parasite and the hosts, and the tumor in mammalian.
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Aminoglycoside Derivatives as Drug Transporters: Delivery Magic Bullets?
TOR Y
University of California, San Diego, Department of Chemistry and Biochemistry,La Jolla, California, U.S.A
Background: A major obstacle for the development of new therapeutic agents is drug delivery. Many drug candidates never make it to the clinic because of inappropriate pharmacokinetic characteristics and sluggish uptake. Delivery procedures based on passive diffusion across cell membranes encounter problems due to charged groups, and carriers that exploit endogenous membrane transporters limit the size of potential drug candidates. Low MW delivery vehicles that facilitate the uptake of diverse molecular cargos are highly desired. Guanidinoglycosides, a family of synthetic derivatives where all the ammonium groups of aminoglycosides have been converted into guanidinium groups, efficiently facilitate uptake of cargo into mammalian cells.
Methods: To quantitatively examine cellular uptake using flow cytometry, fluorescent conjugates of biotinylated guanidino-neomycin with streptavidinylated-phycoerythrin-cychrome were incubated with wild type and mutant CHO cells defective in glycosaminoglycan assembly. To examine the ability of guanidino-neomycin to deliver large proteins into cells and release them in active form, saporin was conjugated to guanidino-neomycin-biotin and cell killing was evaluated. This conjugated toxin was also used to examine the susceptibility of tumor cell lines to these carriers. Enzyme replacement therapy was explored in MPS fibroblasts with guanidinoneomycin conjugated to -gluocoronidase.
Results: (1) the cellular binding and uptake of guanidinoglycosides at low concentration depends exclusively on heparan sulfate (in contrast, the uptake of arginine-rich peptides follows multiple pathways); (2) guanidinoglycosides are capable of cellular delivery of high MW and bioactive cargo such as enzymes and protein toxins; (3) effective guanidinoglycoside-mediated delivery can be achieved at low transporter concentrations, with little or no cellular toxicity; and, preliminarily, (4) certain tumor cell lines (particularly breast cancer) show high susceptibility to guanidinoglycoside-linked toxins.
Conclusions: Guanidinoglycosides are promising low MW cellular transporters with high selectivity towards heparan sulfate. These carriers can be used as a screening tool to explore the surface glycobiology of tumor cell lines as well as for the targeted delivery of bioactive molecules (e.g., enzymes).
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Change in Immunisation Schedule and Sudden Infant Death Syndrome in Hungary
TÖRŐ K
Department of Forensic Medicine Semmelweis University, Budapest, Hungary
Infant mortality in Hungary was higher than in other European countries; however, the reported incidence of Sudden Infant Death Syndrome (SIDS) has been lower than those for Western Europe and the United States. Childhood immunisation has been reported to be a protective factor for SIDS. In Britain, the change to an earlier immunisation schedule for diphtheria, pertussis, and tetanus appeared to be associated with a shift in the age distribution of SIDS. In 1999, immunisation for Haemophilus influenzae type b (Hib) was introduced for Hungarian infants at the age of 2 months. Data for total infant mortality and SIDS in Hungary was analysed between 1990-2002. Infection was the major cause of death among Hungarian infants followed by SIDS. Following introduction of Hib immunisation, there was a decrease in deaths due to meningitis from an average of 3.5% of all infant deaths between 1990-1998 to an average of 1% of all infant deaths between 1999-2002 (p=0.00). There was also a significant decrease in the proportion of SIDS in the age range ³2 months from 48% in the earlier period to 39% after introduction of the vaccine (p=0.03). The decease in SIDS might be due to decrease in unrecognised Hib infections or to induction of antibodies by the tetanus toxoid to which the Hib polysaccharide is conjugated that are cross reactive with bacterial toxins implicated in SIDS.
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Generating C-reactive protein inhibitors for the treatment of cardiovascular disease
TORZEWSKI J
Department of Internal Medicine II-Cardiology, Robert Koch Str. 8, 89081 Ulm, Germany, Phone: + 49 731 500-45026, Fax: + 49 731 500-45005, jan.torzewski@uniklinik-ulm.de
In recent years, the role of C-reactive protein (CRP) in cardiovascular disease has been a lively topic of debate. The reasons for this interest can be summarized as follows. First, CRP has been identified as a powerful cardiovascular risk marker. Second, CRP may not only be a cardiovascular risk marker but also a risk factor, i.e., it may be causally involved in atherogenesis and its sequelae. Third, if it is indeed a cardiovascular risk factor, CRP may be a therapeutic target for primary or secondary prevention of cardiovascular disease. The medical and economic impact of these possibilities is evident. However, despite considerable research, the key questions concerning the role of CRP in cardiovascular disease remain unanswered. In this presentation we summarize the evidence for an active role of CRP in atherosclerosis and provide an update on the development of CRP-inhibitors for the treatment of cardiovascular disease.
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Use of Pharmacodynamic Modeling to Predict Efficacy of Doripenem, a Class 2 Carbapenem recently Licensed in Europe
TRAUTMANN M
Institute of Hospital Hygiene, Klinikum Stuttgart, Stuttgart, Germany
Background: Two class 2 carbapenems (meropenem and imipenem/cilastatin) and one class 1 carbapenem (ertapenem) have been licensed in Europe for a number of years. With the advent of the third class 2 carbapenem doripenem aspects of pharmacodynamics, dosage and clinical efficacy deserve consideration.
Methods: Experimental publications concerning the pharmacodynamics against relevant target pathogens under different dosing regimens are reviewed. Clinical results using these dosing regimens are presented.
Results: Experimental infection studies in small animals have shown that the time above MIC (t>MIC) is the relevant pharmacodynamic parameter predicting therapeutic response for ß lactam agents. Specifically, for carbapenems, it has been shown that t>MIC must reach a value of at least 30-40 % of the dosing interval. Interestingly, this value was not considerably different for application frequencies of 1 x , 2 x or 3 x daily. Relevant pharmacokinetic parameters in humans for a 500 mg i.v. dose of doripenem are: Cmax 46.9 7.4 mg/L, tmax 0.5 hrs, AUC 0-∞ 59,3 7.2 mg•h/L, Vss 11.0 1.7 L. Following a 500 mg dose infused over 0.5 h, mean serum levels exceeded MIC values of 16 mg/L for 1 h (t>MIC 12.7%), 4 mg/L for 3 hrs (t>MIC 37%), and 1 mg/L for 5.9 hrs (t>MIC 73%). Prolonging the duration of infusion to 4 hrs resulted in a 99% probability to exceed a t>MIC value of 40 % for pathogens with a MIC of 4 mg/L. For pathogens with higher MICs (8 mg/L), doses of 1000 mg with infusion periods of 4 hrs must be used to achieve the same 99% probability of target attainment.
Conclusions: On the basis of pharmacodynamic modeling and given the fact that most enterobacteria exibit MICs 1 mg/L, doses of 500 mg with an infusion time of 1 h are recommended for indications where P. aeruginosa is not considered. In clinical studies, this dosage regimen resulted in therapeutic response rates of 81.3 % (nosocomial pneumonia), or 85.9% (intraabdominal infection) of patients, respectively. For pathogens with higher MICs such as P. aeruginosa (typical MIC range 2-16 mg/L), doses must be increased or infusion periods prolonged. In general, for effective therapy of this organism, doses of 1000 mg given every 8 hrs for an infusion period of 4 hrs are recommended.
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mTOR Inhibitors for Hepatocellular Cancer (HCC) - A moving Target -
TREIBER G1
1Zollernalb Clinic, Dep Internal Medicine, Academic Teaching Hospital Tuebingen University, Balingen, Germany
Background: mTOR (mammalian target of rapamycin) is a central regulator of cell growth and angiogenesis.
Methods: A systematic review of the current literature
Results: The mTOR pathway is activated in 40-50% of patients with hepatocellular cancer (HCC), depending on the biomarker investigated. In different models (hepatoma cell lines, implanted HCC tumors in rats), mTOR inhibitors (mTORI) were effective in reducing cell growth and tumor vascularity. Synergistic effects were observed for mTORI and chemotherapeutic agents in these studies while other combinations involving mTORI and inhibitors of growth hormones and angiogenesis are awaiting further clinical testing.
A number of mTORI are already clinically available (sirolimus, temsirolimus, everolimus), sharing similiar pharmacokinetic parameters (except for absorption) and side effects. Clinical data are yet preliminary and are mainly derived from retrospective studies in patients who had received liver transplantation for HCC. Those patients had received sirolimus thereafter for immunosuppression and a much lower rate of tumor recurrence than with calcineurin inhibitors alone was noted.
Conclusions: Current prospective trials for treatment of advanced HCC include mTOR inhibitors alone or in combination with either transarterial chemoembolisation or other systemic drugs.
Authors’ disclosure statement: The author has received a research grant and consulting fees by Novartis Oncology, Nueremberg, Germany
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Liquid chromatography-tandem mass spectrometry assay for determination of raloxifene and its two glucuronide metabolites in human plasma and serum
TRONTELJ J, BOGATAJ M, MARC J, MRHAR A
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana SI- 1000, Slovenia
Background: Anti-osteoporotic agent raloxifene has recently attracted a lot of attention due to the newly discovered potent breast cancer preventing effects. Unconjugated raloxifene plasma concentrations are very low, cmaxss is only 1.36 ng/mL, while the majority of circulating raloxifene is in conjugated form with glucuronic acid. In addition, the raloxifene glucuronide standards were not commercially available. Therefore, the aim of our research was to develop a method for quantification of raloxifene (Ral) and its two glucuronides, raloxifene-6-glucuronide (M1) and raloxifene-4’-glucuronide (M2), in human plasma and serum samples.
Methods: Glucuronides (M1 and M2) were synthesized enzymatically with recombinant human UGT1A1, purified, characterized and used as authentic standards. The assay involves a solid phase extraction (SPE) procedure of 0.5 mL human plasma or serum spiked with haloperidol as internal standard on Strata X cartridges. After elution with 70% acetonitrile and 30% methanol containing 2% formic acid, the samples were dryed, reconstituted with 170 L of initial mobile phase and injected on a Luna C18(2) 50x2.0 3m column. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and in water (B) with a gradient from 10% to 100% A in 7 minutes. The flow was diverted to waste except from 3.9 to 6.3 min, when it entered the (+)ESI-QqQ. The following mass transitions were used for quantification [m/z +]: 650 474 for M1 and M2 and 474 112 and 376 165 for raloxifene and haloperidol, respectively.
Results: The calibration range was split into two overlapping regions with good linearity (r2 > 0.99) covering a wide range from 0.200 to 340 g/L, from 1.600 to 2720 g/L and from 0.088 to 60.00 g/L, for M1, M2 and Ral, respectively. The achieved limits of detection were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method was successfully applied to a pharmacogenetic study of UGT1A1*28 polymorphism on 57 postmenopausal women taking 60 mg Ral, where a significant effect of genotype on total raloxifene was observed.
Conclusion: The developed assay proved to be sensitive, accurate and precise. We expect this method can be applied with little modifications also to newer drugs from the SERM family, like bazedoxifene, arzoxifene and lasofoxifene.
Authors’ disclosure statement: The authors acknowledge financial support from the state budget through the Slovenian Research Agency (project “Pharmacogenetic study of raloxifene metabolism and transport.
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according to registration: Trost Jorgensen
Personalized Medicine - a Paradigm Shift in the Treatment of Cancer
JØRGENSEN J T
CMC Contrast AB, Lyngby, Denmark.
Background: The change from blockbuster medicine to personalized medicine will to a large extent influence the way that drugs are going to be developed, marketed and prescribed. In the future, it may be a pharmacodiagnostic test that decides which drug to choose for the individual cancer patient and to a less extent the average results from large randomized clinical trial combined with traditional tumor histology or the marketing efforts of the pharmaceutical companies [1].
Methods & Results: The implementation of personalized medicine will be a stepwise process where the stratification of patients, based on pharmacodiagnostic testing, into biological subgroups will be the first important step. Today, this is already the situation for several cancer diseases, e.g. breast cancer [2]. Development of pharmacodiagnostic tests are not only restricted to new drugs. As our knowledge about pathophysiology increases and the mechanisms of action of the drugs are explained, it will also be possible to develop such tests for drugs that are already on the market. One recent example is the predictive TOP2A FISH test for anthracyclin treatment in primary breast cancer. A number of clinical studies, including DBCG 89D, have shown that patients who have tumors with TOP2A gene aberrations, especially amplifications, have a significantly better effect of anthracycline-based chemotherapy than patients with a normal TOP2A gene status [2, 3].
Conclusions: Within cancer medicine, which has been on the forefront with respect to personalized or stratified medicine, it is expected that very few drugs, if any, will be prescribed without pharmacodiagnostic testing at the end of the next decade. With such development in mind other important challenges will arise such as education of health care professionals and a health care infrastructure that are able to cope with the more individualized treatments.
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