Development Of Highly Anti-HIV Active And Lowly Toxic 4’-C-Ethynyl-2’-Deoxy-2-Fluoroadenosine And A Proposal Of The Way To Develop Highly Active And Lowly Toxic Antiviral Modified Nucleosides
OHRUI H
Yokohama College of Pharmacy, Yokohama, Japan
Background: The existing antiretroviral therapy has several critical problems such as (i) the emergence of new drug-resistant HIV-1 mutants, (ii) the need to take large dosages of drugs, and (iii) drug side effects.
In order to solve these problems three working hypotheses, (i) the way to prevent the emergence of resistant HIV-1 mutants against nucleoside reverse transcriptase inhibitors, (ii) the way to decrease the toxicity of nuleosides, and (iii) the way to provide nucleosides with stability to both enzymatic and acidic glycolysis, were proposed in our study.
Methods: Based on the working hypotheses, many kinds of 4’-C-substituted nucleosides were synthesized and evaluated for their biological activity and stability to both enzymatic catabolism and acidic degradation.
Results: The scientific evidences obtained by our study proved the validity of the three working hypotheses and resulted in the development of 4’-C-ethynyl-2’-deoxy-2-fluoroadenosine(two positions modified 2’-deoxyadenosine derivative), which is highly potent against all HIV-1s, does not have acute mouse toxicity, is stable to intracellular enzymatic catabolism and acidic degradation, its triphosphate has a very long intracellular half-life, does not greatly inhibit mitochondria DNA polymerase γ.
Conclusions: 1) 4’-C-ethynyl-2’-deoxy-2-fluoroadenosine which is highly active against all HIV-1s and lowly toxic was developed. 2) The proposed three working hypotheses were proved to be valid. 3) The working hypotheses could be generally applied to the development of new highly active and lowly toxic antiviral modified nucleosides.
Authors’ disclosure statement:
Merck’s scientists developed a highly anti-HCV active and lowly toxic 2’-C-methyl-7-fluorotubercidine which is a two positions modified adenosine derivative.
Our study and Merck’s study showed that human DNA and RNA polymerases are more sophisticated than viral polymerases in the point that they scarcely accept the nucleosides modified at two or more positions of physiologic nucleosides into their active centers but viral polymerases do accept them, and further suggested that by taking advantage of the difference it will be possible to develop highly active and lowly toxic antivital modifie nucleosides, especially those against Flu-virus because Flu-virus uses RNA-dependent RNA polymerase like as HCV does.
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Blockade Of IL-6 Signalling With A Humanized Anti-IL-6 Receptor Antibody, Tocilizumab, For The Treatment Of Rheumatoid Arthritis
OHSUGI Y, MIHARA M
Chugai Pharmaceutical Co, Ltd., Tokyo, Japan
Background: IL-6 is a proinflammatory cytokine and is known to play important roles in the pathogenesis of rheumatoid arthritis (RA). Several clinical studies have demonstrated that blockade of IL-6 signalling by a humanized anti-IL-6 receptor antibody is highly effective in the treatment of patients with active RA who are inadequately responsive to traditional anti-rheumatic drugs. IL-6 has a variety of biological activities that match many of the symptoms observed in RA patients. The precise mechanisms of action of tocilizumab are still not fully understood, however.
Methods: We reviewed possible mechanisms of action of tocilizumab, based on recently published papers.
Results: The data from Phase III clinical studies confirmed that tocilizumab can improve symptoms (including the number of swollen joints, the number of tender joints, fever, fatigue, anaemia and anorexia) in moderate to severe active RA. It has also been reported that tocilizumab prevented the radiographic progression of joint destruction. Tocilizumab has generally been well tolerated. Interestingly and importantly, serum IL-6 levels gradually decreased during long-term treatment, even though tocilizumab does not directly inhibit the synthesis of IL-6. This might be explained by the finding, in an animal model, that blockade of IL-6 signalling suppressed the induction of Th17 cells, which play a pathogenic role in the development of autoimmune diseases. In addition, we recently found that tocilizumab inhibited IL-6-induced RANK ligand expression on synovial cells obtained from RA patients, resulting in the inhibition of osteoclast formation. Tocilizumab also inhibited the gene expression of vascular endothelial growth factor, which causes neovascularisation that increases the supply of oxygen and nutrition to growing synovial tissues.
Conclusion: Clinical studies have demonstrated that targeting the IL-6 signalling pathway with tocilizumab could be an attractive and innovative therapeutic option for RA. It is highlighted that high efficacy was achieved consistently in several studies, and this adds to the evidence for the deep involvement of IL-6 in the pathogenesis of RA.
In addition, blockade of IL-6 signalling inhibited the induction of Th17 cells and inhibited angiogenesis and bone destruction.
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Reconstruction Of In Vivo P-Glycoprotein Activity From In Vitro Information By Targeted Absolute Proteomics
OHTSUKI S, UCHIDA Y, TERASAKI T
Grad. Sch. of Pharm. Sci., Tohoku Univ., Sendai, Japan
Background: The reconstruction of P-glycoprotein (P-gp) function at human blood-brain barrier (BBB) from in vitro studies is a key issue for pharmaceutical industries, because P-gp plays a significant role to limit drug penetration across the BBB, influencing directly to pharmacological effect and toxicity in brain. The purpose of the present study was to reconstruct in vivo function of P-gp at BBB based on its absolute expression amount and in vitro transport activity in mouse.
Methods: In vitro P-gp transport activity was determined by the transcellular transport study using the monolayer of mouse P-gp transfected LLC-PK1 cells (L-mdr1a) and parental LLC-PK1 cells, which were kindly provided by Dr. Schinkel. P-gp absolute expression amount was determined according to the targeted absolute proteomics we developed (Kamiie et al., Pharm Res, 2008). Brain-to-plasma concentration ratio (Kp brain) was measured by intravenous constant infusion, and Kp brain ratio was calculated by dividing the Kp brain in P-gp knockout mice by that in wild-type mice.
Results: As a parameter of in vivo function of P-gp at mouse BBB, Kp brain ratio was reconstructed. First, intrinsic transport activities per P-gp molecule for 6 substrates were determined by dividing the in vitro P-gp transport activities by absolute expression amount of P-gp protein in L-mdr1a (15 fmol/μg protein). Then, by combining the obtained intrinsic transport activities with absolute expression amount of P-gp protein in mouse brain capillaries (14 fmol/μg protein), the Kp brain ratio was reconstructed (quinidine, 31; loperamide, 23; digoxin, 17; dexamethasone, 11; vinblastine, 12; cimetidine, 3.6). The reconstructed Kp brain ratio from in vitro studies corresponded well with the observed Kp brain ratio determined in mouse in vivo study (quinidine, 39; loperamide, 31; digoxin, 20; dexamethasone, 8.8; vinblastine, 8.0; cimetidine, 1.4) for all 6 substrates.
Conclusions: The present study demonstrated in mouse that absolute values of Kp brain ratio can be reconstructed by integrating P-gp absolute expression amount and in vitro P-gp transport activity. Applying this approach to human, it is possible to reconstruct P-gp function at human BBB from in vitro transport studies by using quantitative human transporter atlas of the BBB.
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Drug Screening Of Preserved Oral Fluid By Liquid Chromatography-Tandem Mass Spectrometry
ØIESTAD EL, JOHANSEN U, YTTREDAL B, CHRISTOPHERSEN AS
Norwegian Institute of Public Health, Oslo, Norway
Background: Oral fluid is an alternative matrix for drug analysis with potential applications in road-side drug screening, work-place drug testing, drug treatment programs, and epidemiological surveys.
Methods: We have developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for drug screening of preserved oral fluid, collected with either Intercept® (Øiestad et al. Clin. Chem. 53 (2) (2007), pp. 300–309).or Saliva Sampler® collection devices. Samples were prepared by liquid–liquid extraction with ethylacetate/heptane (4:1). LC-separation was achieved with an Atlantis dC18-column (2.1 X 50 mm, 3 µm particle). Mass detection was performed by positive ion mode electrospray LC-MS/MS and included the following drugs/metabolites: morphine, 6-monoacetylmorphine, codeine, buprenorphine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, cocaine, benzoylecgonine, Δ-9-tetrahydrocannabinol, lysergic acid diethylamide, alprazolam, bromazepam, clonazepam, 7-aminoclonazepam, diazepam, N-desmethyldiazepam, 3-OH-diazepam, fenazepam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, nitrazepam, 7-aminonitrazepam, oxazepam, zopiclone, zolpidem, carisoprodol, and meprobamat. The method has been used to analyse approx. 10800 samples from drivers in Norwegian traffic stopped at random (Gjerde et al, Accid. Anal. Prev., in press http://dx.doi.org/10.1016/j.aap.2008.06.015) and is currently being used for the DRUID project (http://www.druid-project.eu). Application of the method for drug screening in an opioid maintenance treatment program is currently under evaluation.
Results: Screening of 28-32 drugs was performed with a run time of 14 min. Within- and between-day relative CVs with the Intercept device varied from 2.0% to 31.8% and from 3.6% to 39.1%, respectively. Extraction recoveries were >50% except for morphine (30%) and benzoylecgonine (0.2%). Experiences with application of the method and differences in matrix effects and absorption to the sampling device for the two devices will be discussed.
Conclusions: The method allow rapid and sensitive oral fluid screening for the most commonly abused drugs in Norway and have been successfully applied to a large number of samples.
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Taxane-Based Tumor-Targeting Anticancer Agents
OJIMA I
Institute of Chemical Biology & Drug Discovery, State University of New York, Stony Brook, New York, U.S.A
Background: A long-standing problem in cancer chemotherapy is the lack of tumor-specific treatments. Therefore, the development of innovative and efficacious tumor specific drug delivery protocols or systems is urgently needed. Rapidly growing tumor cells overexpress many tumor-specific receptors, which can be used as targets to deliver cytotoxic agents into tumors.
Methods: We designed and developed new taxane-based anticancer agents possessing tumor-targeting ability and efficacy against various cancer types, especially drug-resistant tumors. These new anticancer agents are conjugates of the second-generation taxane anticancer agents with tumor-targeting modules through mechanism-based self-immolative disulfide linkers, which are specifically delivered to tumors, internalized into tumor cells, and the potent anticancer agents are released from the linker into the cytoplasm.
Results: We used monoclonal antibodies (for EGFR) and omega-3 polyunsaturated fatty acids, in particular DHA, as tumor-targeting molecules for drug conjugates, which exhibited remarkable efficacy against human tumor xenografts (e.g., A431, A121, DLD-1, CFPAC-1) in mouse models. Vitamin receptors are excellent biomarkers for cancers. Thus, biotin and folate were successfully employed as tumor-targeting molecules as well. In order to monitor and elucidate the mechanism of tumor-targeting, internalization and drug release, several fluorescent and fluorogenic probes were developed The use of functionalized single-wall carbon nanotube (SWNT) as a vehicle for drug conjugates bearing multiple guiding modules and warheads led to the development of novel nanopharmaceutical agents.
Conclusions: 1) DHA-SB-T-1214 provided highly promising results which identified this drug conjugate as the leading candidate for drug development. 2) We succeeded in monitoring the receptor-mediated endocytosis, drug release, and drug-binding to the target protein, microtubules, by means of confocal fluorescence microscopy. 3) We found that the mass drug delivery into the cytosol of the cancer cells using our novel drug delivery system (DDS) is superior than the simple exposure of the drug itself to the same cancer cells. These results strongly suggest that the functionalized SWNT-based DDS can serve as a highly promising drug delivery platform.
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Efficient Cancer Therapy By Use Of Neovascular-Targeted Liposomes
OKU N1, ASAI T1, SHIMIZU K1
1Sch. of Pharm. Sci., Univ. of Shizuoka, Shizuoka, Japan
Background: Since tumor angiogenesis is critical for tumor growth and blood borne metastasis, antiangiogenic therapy has been focused on for the tumor treatment. Furthermore, damaging angiogenic vessels would eradicate tumor due to cutoff the nutrients and oxygen. For this purpose we isolated a novel neovessel –targeted peptide, Ala-Pro-Arg-Pro-Gly (APRPG), from a phage displayed library, and liposomes were modified with APRPG-conjugate of distearoylphosphatidyl ethanolamine-polyethylene glycol (PEG-DSPE) for the specific delivery of antiangiogenic or anticancer agents to angiogenic endothelial cells.
Methods: In vivo distribution of radio-labeled APRPG-PEG-DSPE-modified liposomes (APRPG-PEG-Lip) and intratumoral distributions of APRPG-PEG-fluorescence-labeled APRPG-PEG-Lip were determined with radioactivity and LSM, respectively. Tumor growth suppression by the treatment of SU1498, an angiogenic inhibitor, or doxorubicin (DOX) encapsulated in APRPG-PEG-Lip was examined.
Results: APRPG-PEG-Lip accumulated in tumor similarly to control PEG-Lip, while intratumoral distribution was quite different: The former co-localized with angiogenic vessels and the latter accumulated interstitial tissues of the tumor around the vessels. SU1498 encapsulated in APRPG-PEG-Lip significantly suppressed tumor growth suggesting APRPG-PEG-Lip could deliver the antiangiogenic agent effectively to angiogenic endothelium. Moreover, DOX-encapsulated APRPG-PEG-Lip strongly induced apoptosis of tumor cells and suppressed tumor growth in tumor-bearing mice. This suggests that the damaging angiogenic endothelium by anticancer agents causes indirect and efficient eradication of tumor cells.
Conclusions: Tumor angiogenic endothelial cells are easily targeted by magic bullet, since the active-targeting drug carriers primarily interact with the cells after injection into bloodstream. Therefore, neovascular-targeted liposomes effectively deliver antiangiogenic or anticancer agents to neovessels and efficiently suppressed angiogenesis or damaged anigogenic vessels, respectively. Neovascular-targeted liposomes would be useful DDS carriers for the cancer treatment.
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Is The Induction Chemotherapy Response- And Recurrence Rate Depend On N0 Or N+ Stage In Oral Squamous Cell Cancer?
OLASZ L1, NYARADY Z1,2, SZALMA J1, ORSI E1
1University Pécs, Department of Oral and Maxillofacial Surgery, Hungary; 2University Szeged, Department of Dentistry, Oral and Maxillofacial Surgery, Hungary
Background: In Hungary the morbidity and mortality rate of head-neck cancer have increased in the last 30 years. One of the most important treatment failure is the high rate of primary recurrences after surgery. In this study the authors compare the regression and recurrence of N0 and N+ cases following chemotherapy.
Methods: During a 6 year period, 90 consecutive treated OSCC patients were entered into this retrospective study. From these patients 39 were N0 and 51 were N+ stages. Twenty-five patients received Bleomycin- Vincristrin- Methotrexate (BVM), twenty BVM+ Cisplatine and forty-five BVM+Mitolactol neoadjuvant chemotherapy. After three courses of chemotherapy the regression (CR,PR,NR) and side effect rate were determined. All of the patients were operated and observed the number and localisation of recurrences in three years follow-up time.
Results: The N0 cases came from T2-3, while N+ from T2-4. The regression were in N0 group CR:46%, PR:54%, but in N+ group CR:12%, PR:74%, NR:14%. The side effects were slight (grade I-II) and reversible. The recurrence rate for N0 group was 15%(lymph node metastasis), while for N+ group 59% (primary or metastasis). The recurrence rate was only 4% for complete responders. The three year tumor-free survival for N0 group 85%, N+ group 43%.
Conclusions: After neoadjuvant chemotherapy there was very good chance for tumor free survival in complete- coming from N0-N+ stages and partial responders coming from N0 stage. For these cases (45 patients) the recurrence rate was 13%. The chance was bad for partial- or no-responders coming from N+ stage (45 patients) as the recurrence rate was 70%.
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High Uptake Of Vaccines – A „Magic Bullet” In Control The Burden Of Hospitalisation Attributable To Childhood Mumps And Rotavirus Infections
OŁDAK E1, ROŻKIEWICZ D1, SZAFRAN D2, SULIK A1
1Medical University, Bialystok, Poland; 2 University Children’s Hospital, Białystok, Poland
Background: The Polish immunisation program consists of two vaccination systems; the routine (obligatory) and recommended (optional) vaccination. The routine vaccinations (mumps) are bought by Polish National Health Fund and recommended vaccinations (rotavirus [RV]) are paid by individuals. Aim: To assess the impact of different immunisation systems on the hospitalisation burden attributable to vaccine-preventable mumps and RV infections in children between 2003 and 2008, in north-eastern Poland.
Methods: A retrospective analysis of hospital discharge data obtained between January 9, 2003 and June 30, 2008, at University Children’s Hospital in Bialystok. Electronic hospital discharge data were reviewed to select records of children 0-18 years of age with ICD-10 codes for RV gastroenteritis (RVGE – A08.0) and for mumps (B26.0; B26.1; B26.3; B26.9). The nosocomial RV infections in 2008 were analysed based on the Hospital Infection Control Team database. The results were compared with mumps vaccines uptake (web database of the National Institute of Hygiene in Warsaw) and local RV vaccines uptake.
Results: Of 5213 children hospitalised during study period 2003-2007, 626 (12%) were discharged as RVGE, and 319 (6.1%) as mumps infection. The proportion of hospitalisations attributable to the mumps infection declined from 15.9% in 2003 to 0.2% in 2007 year, in line with increasing mumps vaccination uptake. In the year 2003, 39% of Polish children have been vaccinated against mumps whereas in 2007 – 97%. In the first 6 months of 2008 no case of mumps was hospitalised. The percentages of hospitalised cases attributable to RVGE increased during the study period from 7.7% in 2003 to 18.1% in 2007. In the first half of 2008, the percentage of hospitalised RVGE cases reached the highest level (39.7%). In 2008 year, the percentage of nosocomial RV cases among total RV cases was estimated to be 16.1%. In 2007, 89 infants under 6 months of age out of 1997 children born in our region were vaccinated against RV (vaccine coverage – 3%).
Conclusion: Routine vaccination system in line with high uptake of RV vaccines are needed to provide herd immunity and to control the burden of hospitalisation.
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