Ehrlich II –2nd World Conference on Magic Bullets

EHRLICH II –2^nd World Conference
on Magic Bullets

Celebrating the 100^thAnniversary of the

Celebrating the 100^th Anniversary of the

Nobel Prize Award to Paul Ehrlich



Nürnberg, Germany
October 3-5, 2008

Abstract Book

Combined targeting of growth-promoting genes with antisense oligonucleotides in human colorectal cancer cells: Chemo-sensitization potential ABAZA M-S I AND BAHMAN A M Department of Biological Sciences, Faculty of Science, Kuwait University, P. Box 5969, 13060 Safat, State of Kuwait. Background: Aberrant expression of growth-promoting genes contributes to the growth advantage of tumor cells, targeting such genes with phosphorothioated antisense oligonucleotides, AS[S]ODNs, might therefore be useful in controlling the abnormal proliferation of cancer cells. To explore the potential of combination antigene therapy in human colorectal cancer cells, we have examined the in vitro effects of AS[S]ODNs targeting c-myb, c-myc and cdc2 in human colorectal cancer cell lines. Method: Cancer cells were treated with c-myb-, c-myc- or cdc2-AS[S]ODNs individually or in combination. The effects of growth promoting-gene AS[S]ODNs on mRNA and protein expression were determined by RT-PCR and blot analysis. The effects of these combinations on cell growth, chemo-sensitization, apoptosis and genes controlling cell growth and apoptosis were monitored by MTT assay, DNA fragmentation and Real-time RT-PCR. Results: mRNA and protein expression were dramatically reduced after treatment with c-myb-, c-myc-, or cdc2-AS[S]ODNs. Combined targeting of c-myb/c-myc and c-myb/cdc2 had much higher dose and time dependent synergistic growth inhibitory effects; 5-100% and 5-95%, respectively; compared to single antigen therapy (5-55%). Combined targeting of c-myc / cdc2 also produced greater dose and time dependent additive or synergistic growth inhibitory effects (10-100%) compared to single antigene therapy (5-60%). The combined targeting of c-myb / c-myc / cdc2 exhibited much higher growth inhibitory effects (10-90%) compared to single antigene therapy (20-50%). Combined targeting of c-myb / c-myc / cdc2 produced marked inducing effects on both apoptosis and chemo-sensitization to taxol, 5FU, doxorubicin and vinblastine. Real-time RT-PCR indicated down-regulation of mRNAs of cdk1, cyclin B1, cdk2, cyclin E1, cdk4, cyclin D1, BcL2 and BclxL and up-regulation of mRNAs of p21, Bax and caspase. Conclusion: Our study suggests that combination antigene therapy targeting c-myb, c-myc and cdc2 can inhibit human colorectal cancer cell proliferation more effectively than monogene therapy by blocking the cell cycle and inducing apoptosis. Combination antigene therapy is thus a promising approach for cancer therapy.

Introduction of supercritical fluid extraction as a new sample-preparation procedure for isolation and identification of a pharmaceutical from biological fluids: Application to disposition kinetics ABD EL-ATY AM^1^,2, CHOI JH^3, KO MW^3, KHAY S^3, GOUDAH A^2, SHIN HC^1, SHIM JH^3 ^1Konkuk University, Seoul, Republic of Korea; ^2Cairo University, Giza, Egypt; ^3Chonnam National University, Gwangju, Republic of Korea Background: Since its commercial development in the early 1990s, supercritical fluid extraction (SFE) has attracted considerable attention as a sample-preparation procedure. However, other different sample preparation procedures, including precipitation, liquid- and/or solid-phase extraction in biological fluids, also remain in use. Aims: In this investigation, SFE was introduced to isolate and identify orbifloxacin (OBFX) from plasma and milk of lactating does. Methods: Four parameters, including the temperature and the pressure of supercritical fluid, modifier ratios, and dynamic extraction time, were evaluated and optimized to obtain the best yield of the analyte from the biological fluids. Determinations of the OBFX in the extracts were carried out using high-performance liquid chromatography coupled with fluorescence detector (HPLC-FLD). The linearity of the calibration curves as well as the instrument limit of detection /limit of quantitation (LOD/LOQ) were evaluated. Results: The optimum conditions of the extraction process that yielded the maximum analyte extraction efficiencies were 150°C vs. 60°C, 250 kg/cm^2, 30% vs. 35% methanol, and 40 min vs. 20 min, for plasma and milk, respectively. Good linearity (at least r^2 ≥ 0.999) of the calibration curves was obtained over the range from 0.2 to 0.01 µg/mL. The method showed good a recovery rate (74.2–127.73%) and precision (expressed as relative standard deviations (RSDs) 1.64–20%). The instrumental LOD and LOQ values were 0.004 µg/mLvs. 0.01 µg/mL or 0.006 µg/mLvs. 0.02 µg/ L, for plasma and milk, respectively. The method was successfully applied to estimate the pharmacokinetic variables of orbifloxacin in lactating does. Conclusions: To the best of our knowledge, this is the first time that SFE has been applied to isolate an antimicrobial agent from biological fluids. This method is promising for clinical applications and for pharmacokinetic studies of various pharmaceuticals in biological fluids.

Misoprostol and Postpartum Hemorrhage ABDEL-ALEEM H Department of Obstetrics and Gynecology, Faculty of Medicine ,Assiut University, Assiut , Egypt. Abstract: Every year more than half a million women lose their lives because of complications of pregnancy and childbirth. Postpartum hemorrhage is the leading cause of death especially in developing countries. Strategies have been developed to prevent and treat postpartum hemorrhage. One of these is the use of uterotonic drugs to prevent and treat atonic postpartum hemorrhage. Misoprostol (prostaglandin E1 analogue) has been emerged as an uterotonic drug with special advantages; being heat stable, can be used orally and rather cheap. So, it can be considered as a potential "Magic Bullet" in the management of postpartum hemorrhage. Because of this potentiality, World Health Organization has been conducting big multicentre randomized clinical trials to evaluate efficacy and safety of misoprostol in the prevention and treatment of postpartum hemorrhage. The findings and implications of these trials will be presented and discussed.

Magic bullets in prostate cancer ABDELBAKY A, HUSSAIN M, KAISARY AV Department of Urology, The Royal Free NHS Trust, London, U.K. It has been 100 years since Paul Ehrlich popularized the concept of a "magic bullet" during his 1908 Nobel prize lecture. Scientists have since keenly embarked on the search for these magic bullets. In the UK prostate cancer is the most common cancer diagnosed in men and the second leading cause of cancer death in men (1). Consequently a magic bullet is highly desired by patients and urologists alike. The first bullet comprises hormonal therapy resulting in androgen ablation, which represent the mainstay of treatment of metastatic prostate cancer. LHRH analogues can be implanted into patients where they cause a negative feedback effect, resulting in a decrease in LHRH production from the anterior pituitary and a subsequent reduction in testicular testosterone production. Degarelix, an LHRH antagonist has an immediate onset of action suppressing LHRH and testosterone, thus avoiding tumour flare. Studies suggest a PSA decrease by 97-98% after 1 year and the median time to 90% reduction in PSA was 8 weeks (2). Brachytherapy is ultrasound guided implanation of radioactive seeds into the prostate. Its popularity has increased as the use of transrectal ultrasound (TRUS) makes it easier to accurately direct the magic bullet. Patient selection is a key. In this scenario brachytherapy achieves a 12- year progression free survival of 66% (3). Heat effects are attractive. HIFU is a bullet where prostate tissue is heated to the point of coagulative necrosis using high-energy ultrasound allowing the selective destruction of tissues. Long-term results from clinical trials are awaited. Freezing or cryotherapy of the prostate cancer has increased over the past few years, especially with the improvement in cryotechnology. Multiple hollow core probes are placed percutaneously under TRUS guidance. This causes cell destruction at ^-20 - ^-40ºc, usually achieved by applying two cycles of freeze thaw. 96% of men with localised disease achieve a PSA of <0.2ng/ml within 6 months. Again long term results are awaited. Finally, Docetaxel, chemotherapy agent, is a magic bullet that may be used to treat androgen-independent prostate cancer, with promising results (4). 1) http://info.cancerresearchuk.org/cancerstats/types/prostate/ 2) Ragde H, Korb LJ, Elgamal AA et al.Modern prostate brachytherapy. Prostate specific antigen results in 219 patients with up to 12 years of observed follow-up.Cancer.2000: 89;135-41. 3) Tannock If Ronald de Wit, William R. Berry,et al Docetaxel plus Prednisone or Mitoxantrone plus Prednisone for Advanced Prostate Cancer. N Engl J Med. 2004 Oct 7;351(15):1502-12. 4) Van Poppel H, Tombal B, de la Rosette JJ,et al Degarelix: A Novel Gonadotropin-Releasing Hormone (GnRH) Receptor Blocker-Results from a 1-yr, Multicentre, Randomized, Phase 2 Dosage-Finding Study in the Treatment of Prostate Cancer. Eur Urol. 2008 May 8 [Epub ahead of print]

Non-Systemic Delivery of Ocular Brimonidine to the Brain: Extending the Therapeutic Benefits of Brimonidine to the CNS ABDULRAZIK M^1,2, TAMILVANAN S^1, BENITA S^1 ^1Pharmaceutics Department, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel, and ^2Department of Ophthalmology, Shaare Zedek Medical Center, Jerusalem, Israel. Background. Despite recent advancements in neuroscience, and ever-emerging novelties in the field of drug delivery, the blood brain barrier is still considered a major obstacle that critically limits the delivery to the brain of hydrophilic drugs or those with modest or weak hydrophobic character. Brimonidine, a selective alpha-2 agonist, is a widely used ocular hypotensive agent with promising merits as neuroprotectant. Aim. To demonstrate the efficient delivery to the brain of Brimonidine following ocular administration and to elucidate the route of non-systemic drug flow along the eye-brain axis. Methods. Brimonidine was used as a probe to study the eye and brain pharmacokinetics following topical ocular administration of one droplet of the radio-labeled drug. Animals were sacrificed at different time points following the administration of single 0.2% ^3H-Brimonidine droplet to the rabbit eye. Brain and eyes were dissected and selected brain and eye tissues, as well as systemic blood samples, were processed for detecting the concentration of radio-labeled Brimonidine. Fluorescent-labeled Dextran with molecular weight of 40 kDa was used as a probe to elucidate the route of the non-systemically mediated eye-to-brain drug flow following periocular administration. Results. Brimonidine accumulation in studied intracranial tissues was significant already at 5 minutes following single ocular administration while the detected Brimonidine levels in the systemic blood were very low at all time points. Evaluation by fluorescent microscopy showed that 40 kDa fluorescent-labeled Dextran that was administered to the periocular space didn’t permeate the ocular barriers and was flowing through veins that drain the eye and orbital tissues towards the intracranial cavernous sinus. Tissue sections that included the eye and its major vessels showed extensive accumulation in veins but with no signal of the fluorescented probe inside the ciliary artery, which corresponds to non-significant flow of the probe through the systemic circulation. Conclusion. Brimonidine, a non-toxic agent to the eye with well appreciated therapeutic merits, can accumulate in intracranial tissues at significant concentrations already at few minutes after simple ocular administration with very low systemic accumulation. Our results suggest an efficient method for the extension of Brimonidine therapeutic merits to the brain while avoiding systemic side effects.

A Role of Lysosomal Phospholipase A2 in Cationic Amphiphilic Drug-induced Phospholipidosis ABE A^1, KELLY R^1, KOLLMEYER J^1, HIRAOKA M^2, SHAYMAN JA^1 ^1University of Michigan, Ann Arbor, MI, USA, ^2Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, Japan Background: Many of cationic amphiphilic drugs (CADs) developed as therapeutic drugs, including those with anti-inflammatory, antineoplastic and antiangiogenic properties, and have been known to induce phospholipidosis in human and other mammalian tissues. Generally, CADs tend to accumulate in lysosomes and to inhibit lysosomal enzyme activities. Recently, it was found that the alveolar macrophages (AMs) prepared from lysosomal phospholipase A2 (LPLA2) deficient mice suffer from cellular phospholipid accumulation and phospholipidosis. This phenotype suggests a potential connection between CAD-induced phospholipidosis and LPLA2. The study on the reaction properties of LPLA2 could find a clue to understand the molecular mechanism of CAD-induced phospholipidosis. LPLA2 shows an increased activity towards zwitterionic phosphatidylcholine liposomes containing negatively charged lipids under acidic conditions. In the present study, the effect of negatively charged lipid on LPLA2 activity was investigated. Methods: Purified recombinant mouse LPLA2 was used as the source of LPLA2. Sulfatide, a lipid that is resistant to LPLA2, was chosen as a negatively charged lipid. Results: Sulfatide incorporated into 1, 2-dioleoyl-glycero-3-phosphocholine (DOPC) liposomes enhanced LPLA2 activity under acidic conditions. The enhancement by sulfatide was linear until 10% molar ratio of sulfatide to DOPC and weakened by the addition of NaCl in the reaction mixture. Amiodarone (AMD), a cationic amphiphilic drug that interacts with negatively charged lipids, reduced the LPLA2 activity in a concentration dependent manner. In addition, the co-sedimentation of LPLA2 with negatively charged liposomes occurred in centrifuge at 150,000 g and was markedly reduced by the addition of NaCl or AMD in the reaction mixture. These results indicate that LPLA2 adsorption to the negatively charged lipid membrane surface through an electrostatic attraction enhances the rate of phospholipid hydrolysis by LPLA2 at lipid-water interfaces. Conclusions: CAD-induced cellular phospholipidosis is linked to the impairment of phospholipid catabolism by inhibition of the binding of LPLA2 to the membrane treated with CDA.

Effect Of Flunixin On Enrofloxacin Clearance And Steady-State Serum Concentrations In Calves ABO-EL-SOOUD K^1, AL-ANATI L^2 ^1Pharmacology Department, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt; ^2Dept. of Veterinary Basic Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Jordan. Background: To evaluate whether the concomitant administration of flunixin may alter enrofloxacin pharmacokinetic parameters and the hepato-renal activities. Methods:: Ten clinically healthy, Freisian calves weighting 200-250 kg and 5-7 months old were used. They were divided up into two groups. First group was injected a single dose of enrofloxacin 2.5 mg/kg of body weight (BW) intravenously (IV). Second group was injected the same dose intramuscularly (IM). After 1 month washout period, each of the 10 animals was given flunixin (IM) at a dose 2.2 mg/kg of BW one-hour prior to with the injection of enrofloxacin in a dose of 2.5 mg/kg BW in calves of the first group IV. or the IM injection in the second group. Results: Co-administration of flunixin with IV injection of enrofloxacin reduced the volume of distribution at steady state Vd(SS) and total body clearance (Cltot) by 33.9% and 30% respectively. After IM injection of enrofloxacin, the elimination half-life (t1/2el) and mean residence time (MRT) were shorter in the flunixin-medicated calves. C^max: maximum plasma or milk concentration; T^max: time to peak concentration; AUC0-∞: area under the curve from zero to infinity by the trapezoidal integral; ^**P<0.01; ^***P<0.001 Conclusions: Concomitant administration of flunixin with enrofloxacin induced significant alterations in pharmacokinetic parameters and hepatic-renal functions in calves. Therefore, concurrent administration of flunixin with enrofloxacin should be avoided.

Selenium Derivatives as Cancer Preventive Agents ABOUL-FADL T Departments of Pharmaceutical Chemistry, King Saud University, Riyadh, Saudi Arabia. Background: The role of selenium in the prevention of cancer has been recently established by laboratory experiments, clinical trials, and epidemiological data. Most of the effects are related to the function of selenium in antioxidant enzyme systems. Animal data, epidemiological data, and intervention trials have shown a clear role for selenium derivatives in both prevention of specific cancers and antitumorigenic effects in postinitiation phases of cancer. Methods: Selenazolidine prodrugs (SCA) of selenocysteine were synthesized by the reaction of selenocysteine with the appropriate carbonyl derivative. Male CF1 mice were treated daily for 7 days with equi-selenium (1.25 mg Se/kg) doses of each agent, by either the intraperitoneal (ip) or intragastric (ig). route and the effects compared with those of selenocystine. Hepatic parameters were determined 24 hours after the last dose.The efficacy of SCA in reducing NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]-induced lung adenomas in female A/J mice, a model for tobacco-related lung tumorigenesis, has been investigated with selenazolidines in the diet for 1 month prior to carcinogen administration and during the subsequent 4 months of tumor development. Results: In general, few significant (p <0.05) changes were seen with ig as compared to ip administration. 2-butylSCA, 2-cyclohexylSCA, 2-phenylSCA and 2-oxoSCA were chemopreventive, significantly reducing mean lung tumor numbers from the 10.9 of unsupplemented controls to 4.7, 5.3, 2.8 and 4.7, respectively. When selenazolidine supplementation began three days after carcinogen administration (i.e., post-initiation), 2-butylSCA, 2-cyclohexylSCA, and 2-oxoSCA were chemopreventive. In both regimens, selenocystine was also chemopreventive. Both 2-butylSCA and 2-phenylSCA retained their chemopreventive activity (44% and 40% tumor number reduction, respectively), when the supplementation was shortened and restricted to a pre-initiation period (days −9 to−2). Conclusions: Although this study has not identified the mechanism, it firmly establishes that 2-substituted selenazolidine- 4(R)-carboxylic acids possess chemopreventive activity against NNK-induced lung tumors in a murine model. Dependent on the nature of the 2-substituent, the chemopreventive activity can arise from changes elicited in the post-initiation period, similar to selenocystine, or in the pre-initiation period.