Anti-Carbohydrate Specific Immune Response And Tumor Cell Lysis Correlate With Vaccination-Induced Systemic Release Of Stimulatory Cytokines KIRCHEIS R, SIEGL P, HALANEK N, NECHANSKY A
Vela Laboratories, Vienna, Austria
Background: Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn antigen, are frequently expressed on cancer cells and provide potential targets for vaccination. Carbohydrates, however, being T-cell independent antigens, are poorly immunogenic and fail to induce a memory response. To increase immunogenicity, SialylTn antigen was coupled to a highly immunogenic carrier, the murine monoclonal antibody mAb17-1A. An immunogenic formulation of SialylTn-mAb17-1A conjugate on alhydrogel (designated MB 402) with or w/o additional adjuvants was tested in Rhesus monkeys for tolerability and immunogenicity.
Methods: The SialylTn antigen was coupled to the mAb17-1A (mIgG2a) carrier. The coupling product was analyzed by SEC-HPLC, LDS-PAGE, Western blot, and IEF analysis. SialylTn-mAb17-1A conjugate was adsorbed onto aluminum hydroxide and co-formulated with QS-21.
Safety, tolerability and immunogenicity of multiple injections of MB 402 were evaluated in Rhesus monkeys vaccinated four times by s.c. injection and re-boosted on day 226. Blood samples were taken before and after immunization for serum analytic.
Immune response against mAb17-1A, SialylTn and Ovine submaxillary mucin, and SialylTn(+) tumor cells were measured. Cytokine release in serum was analyzed using xMAP Multiplex technology. NK lysis of tumor cells was measured using a 51Cr-release assay.
Results: Immunization induced a strong immune response against the carrier but only IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant, however, dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. Cell binding and ADCC of SialylTn positive tumor cells was induced. The kinetics of carbohydrate-specific IgG response correlated with a temporary release of cytokines in the serum. NK mediated cytotoxicity against tumor cells was found.
Conclusions: The present study demonstrates that synthetic vaccines eliciting specific immune response against defined target antigen(s) together with a synchronized cytokine release are promising candidates for cancer vaccines.
Pharmacogenetic diagnostics for optimization of psychotropic drug treatment KIRCHHEINER J Professor of Clinical Pharmacology
University of Ulm
Institute of Pharmacology of Natural Products & Clinical Pharmacology
Helmholtzstr. 20
D-89081 Ulm, Germany
Telephone: +49 (0731) 500-65603
Fax: +49 (0731) 500-65605
E-mail: julia.kirchheiner@uni-ulm.de For many drugs, pharmacogenetic polymorphisms are known affecting biotransformation and clinical outcome. The clinical importance of these variants depends on allele-frequency and the effect size of the clinical outcome parameters. Further, it depends on the therapeutic range of the drug which is affected, on predictability of drug response as well as on duration until onset of therapeutic efficacy. Consequences which arise from genotyping might be: adjustment of dose according to genotype, choice of therapeutic strategy or even choice of drug.
For many psychotropic drugs, pharmacogenetic polymorphisms are known affecting biotransformation and clinical outcome. In antidepressant drug treatment, most drugs are metabolized via the polymorphic cytochrome P450 enzymes CYP2D6 and CYP2C19. Huge differences in pharmacokinetic parameters have been consistently shown for many tricyclics, some SSRIs, and other antidepressant drugs. However, the effects on therapeutic efficacy and adverse events have been described controversially. Pharmacokinetic differences caused by genetic polymorphisms can be overcome by adapting the drug dosages and dosing intervals. Similar to bioequivalence studies, the aim to achieve similar plasma concentration time courses of antidepressants might help to reduce side effects and therapeutic failure.
In the field of antipsychotic drug treatment, genetic polymorphisms in drug metabolizing enzymes as well influence pharmacokinetic parameters to a large part. In these kinds of drug therapy, a more clear dose dependency of side effects such as extrapyramidal side effects exists, and the consideration of genetic polymorphisms might be more beneficial. Recent studies showed a relationship between the occurrence of adverse antipsychotic drug effects and CYP2D6 genotype. A prospective evaluation of the cost-benefit of genotyping in this field would be very helpful for the aim of introducing pharmacogenetic diagnostic into drug therapy.
Organ Independent Drug Elimination KISOR DF Ohio Northern University College of Pharmacy, Ada, Ohio, USA Background: Cisatracurium is a bisisoquinolinium nondepolarizing neuromuscular blocking agent used to facilitate tracheal intubation. Cisatracurium has been used for skeletal muscle relaxation during surgical procedures and/or during mechanical ventilator support. It has been shown that the major route of cisatracurium clearance is through the organ independent process of Hofmann elimination (pH and temperature dependent). Hofmann elimination takes place ubiquitously in plasma and tissue, and accounts for approximately 77% of the overall clearance of cisatracurium.1 Tight physiologic control of pH and temperature, maintaining Hoffmann elimination, results in low interpatient variability in the CL of cisatracurium (16 %).2 The aim of this study was to examine the relationships between pharmacokinetic parameters in the setting of organ independent elimination.
Methods: Geometric regression analysis was performed to look at the relationships between clearance and half-life, volume of distribution and half-life, and clearance and volume of distribution in 31 otherwise healthy patients who underwent minor surgical procedures requiring tracheal intubation.
Results: For cisatracurium, geometric regression analysis revealed that there is no relationship between clearance and half-life (n = 31; r2 = 0.01) nor is there a relationship between cisatracurium volume of distribution and half-life (n = 31; r2 = 0.01). However, cisatracurium clearance and volume of distribution were related (n = 31; r2 = 0.54; p < 0.001).
Conclusions: Cisatracurium being “removed” from the body mainly by the organ independent route of Hoffman elimination results in unique relationships among pharmacokinetic parameters. Conventional relationships between pharmacokinetic parameters, with the primary parameters of CL and Vd, being independent and the secondary parameter of t1/2 being dependent on CL and Vd, do not hold for cisatracurium as the half-life is essentially “fixed” by Hoffman elimination. Half-life is independent of the CL and Vd, while the CL and Vd are directly related. Other compounds that undergo organ independent elimination via a ubiquitous mechanism may also display these unique characteristics with respect to pharmacokinetic parameter relationships.
The Effect Of Some Endogenous Substances (Cl-, Oleate And Ca2+) On The Albumin Binding Of Triflupromazine (TFZ), Trifluoperazine (TFPZ) And Bendroflumethiazide (BFZ). An In Vitro Spectrometric Study KITAMURA K1, TAKEGAMI S1, OMRAN AA1,2, KITADE T1 1Kyoto Pharmaceutical University, Kyoto, Japan, 2Al-Azhar University, Assiut, Egypt
Background: Albumin, the most abundant protein in the blood, binds several endogenous and exogenous substances reversibly, and thus is a carrier of these substances in the body. It is well known that most of administered drugs are bound to albumin, and that the only unbound free drugs can move into the tissues. Long-chain fatty acids (LCFA), such as oleate, and inorganic ions exist abundantly in the blood, and as they bind to albumin, it must be studied whether their binding may affect the binding of drugs to change the unbound free drug concentration.
Methods: Human serum albumin (HSA, fatty acid free), and fluorine-containing these drugs studied were purchased from Sigma. Second-derivative (DS) ultraviolet spectra of the drugs in HSA buffer solutions (pH 7.40) were measured to calculate the binding constants (Ks). Structural study of the drug binding and the effects of the endogenous substances on it were performed by 19F NMR spectrometry.
Results: A single sharp 19F NMR signal of each drugs in buffer solutions was split and broadened by addition of albumin, revealing that the drugs bind at more than one site. From competitive 19F NMR experiments using known ligands, TFZ and TFPZ were found to bind to site I and another unknown site, and BFZ was to bind to both site I and II. Some of the 19F NMR signals of the bound drugs showed intensity reduction or increase upon addition of the endogenous substances. The K-values (TFZ, TFPZ) measured in the presence of Cl- (physiological concentration of 0.1 M) reduced to about 65% of the values measured without Cl-. Addition of oleate increased the K-values depending on the amount of oleate up to three times of that of albumin, however, further addition reduced them. Ca2+ induced concentration- dependent suppression effect on the drug binding (TFZ, BFZ).
Conclusions: 1) Buffers to be used in the determination of the drug-albumin biding constants should contain 0.1 M Cl-, otherwise, the binding constants may be over estimated. 2) The results of oleate suggested that the unbound free drug concentration may very depending on the amount of LCFA in the blood, which depends on physical conditions of the body. 3) Possible concentration fluctuation of Ca2+ in the blood may affect the free drug concentration.
KIT-Deficient Mouse And KIT-Targeted Drug KITAMURA Y Developmental Research Laboratories, Shionogi & Co., Ltd.
Mast cells were found by Paul Ehrlich and interstitial cells of Cajal (ICCs) by Ramon y Cajal, who also received the Novel Prize in 1906. We found deficiency of mast cells in W/W-v mutant mice, and thereafter the W locus was identified to encode the KIT receptor tyrosine kinase. Unexpectedly, W/W-v mutant mice lacked ICCs as well, indicating that the KIT signaling was essential for development of both mast cells and ICCs. Since we found gain-of-function mutations of the KIT gene in mouse and human mast cell tumors, we attempted to find tumors derived from ICCs. We identified the gastrointestinal stromal tumor (GIST) as such a tumor, and at the same time, gain-of-function mutations of the KIT gene in human GISTs. Unexpectedly again, a KIT-targeted drug, imatinib, was already present and has been successfully used for the treatment of GIST patients.
Genetic And Epigenetic Effects Of Pharmacological Doses Of Gamma-Hydroxybutyrate (GHB) In The Rat Brain KLEIN C, KEMMEL V, TALEB O, AUNIS D, MAITRE M
Department of Biochemistry and INSERM U-575, Faculty of Medicine, Strasbourg, France.
Background: Gamma-hydroxybutyrate (GHB) is at the same time an endogenous neuromodulator of some brain synapses at micromolar concentrations, a therapeutic substance used to induce deep sleep in narcoleptic patients or for anesthetic purposes, an agent that has been proposed to alleviate withdrawal symptoms in alcoholic, but also a substance of abuse which can induce addiction. In order to obtain therapeutic or recreative effects, large doses of GHB must be administered or absorbed. The principal targets of these pharmacological doses of GHB are primarily the brain endogenous GHB system and the GABAergic system. However, it can be predicted that millimolar brain concentrations of GHB will targets multiple proteins and finally adapts the expression of several genes.
Methods: The present study focuses on the transcriptome modifications and HDAC inhibition due to GHB overload in two brain regions, the hippocampus and frontal cortex of the rat, after acute administration of pharmacological doses of GHB. These modifications were explored by microarrays analysis and concern 248 genes which showed 1.5 or greater changes in expression in the two brain regions.
Results: If we exclude the large proportion of modified EST (54.9% of all probes set), numerous functional important genes were differentially regulated: These concern first neuronal signaling and metabolic processing, then regulation of DNA transcription, stress response and neuronal growth or structure. Several differences exist between hippocampus and frontal cortex, but some genes are also similarly affected in both structures. As it become evident that important epigenetic mechanisms are at the basis of modifications in gene expression and regulate drug addiction, we demonstrate that acute pharmacological doses of GHB increase histone H3 acetylation in both brain structures. These results were confirmed by quantitative immunocytochemical studies and by in vitro HDAC inhibition by GHB.
Conclusions: The present study completes the classical model which described pharmacological effects of GHB and shows that acute doses of this substance exerts a profound effect at the level of genes transcription, probably partly due to modifications of the accessibility of chromatin to regulatory factors.
Si Quantum Dots functionalized for siRNA delivery in Caco-2 cells KLEIN S1, KRYSCHI C1, NEUHUBER W2, SCHRÖDL F2, ZOLK O3 1Dept. Chemistry and Pharmacy, 2Chair of Clinical Pharmacology and Clinical Toxicology, 3Chair of Anatomy 1, University of Erlangen, Erlangen, Germany.
Background: Quantum dots are becoming popular as replacement for fluorescent dyes in biological fluorescence imaging because of their superior stability against photobleaching. The potential biocompatibility of silicon makes photoluminescent silicon quantum dots an ideal candidate for biological fluorescence imaging and versatile biomedical applications to human tissue. Quantum confinement of excitons in silicon nanoparticles smaller than 4.3 nm allows for tuning the luminescence wavelength in the visible range due to suitably adjusting the silicon quantum dot (Si qdot) size. One of the central challenges in silicon-based nanomedicine is to develop water-soluble Si qdots tailored for gene delivery, as image contrast agents and for diagnostic purposes.
Methods: For applications as image contrast agents and as transfection reagents for RNA interference (RNAi) the Si qdot surfaces were terminated with covalently linked ethenyl pyridine providing water solubility and positive charging. The surface chemistry and luminescence properties of the terminated Si qdots were characterized using FTIR and luminescence spectroscopy, respectively. The ability of the Si qdots to form complexes with siRNA was examined by gel electrophoresis. The Si qdot uptake by Caco-2 cells were investigated using TEM and confocal luminescence scanning microscopy. Downregulation of expression levels of MDR1 mRNA was determined by PCR.
Results: Ehthenyl-pyridine terminated Si qdots with mean sizes of 2.5 nm were observed to exhibit luminescence peaking at 520 nm. The uptake of ethenyl-pyridine terminated Si qdots by Caco-2 cells were shown by imaging cell slices and living cells with TEM and confocal luminescence scanning microscopy, respectively. The terminated, positively charged Si qdots were shown to bind negatively charged siRNA through their base function and were observed to enter Caco-2 cells via endocytosis. The siRNA molecules were released in the cytoplasm, where they participated in the RNAi pathway by targeting mRNA and therewith suppressing MDR1 gene expression. Complementary evidence for surpressed MDR1 gene expression were obtained from monitoring the reduced pharmacodynamic reponse of P-gycoprotein to rhodamine 123 with time-resolved fluorescence spectroscopy.
Cutaneous Microdialysis As A Useful Tool To Evaluate Skin Penetration Of Antiinfective Agents KLIMOWICZ A1, BIELECKA-GRZELA S2 1Division of Dermatopharmacotherapy, Dept. Dermatology, Pomeranian Medical University, Szczecin, Poland; 2Division of Aesthetic Dermatology, Dept. Dermatology, Pomeranian Medical University, Szczecin, Poland
Background: Treatment with antiinfective agents will be successful if the concentration of active drug and/or its metabolite in the target tissue will be above the minimum inhibitory concentration and below the toxic one. From the dermatological point of view concentrations of the drug in skin seems to be of importance. Aim of our study was to evaluate skin concentrations of two important drugs: antiviral – acyclovir as well as antibacterial and antiprotozoal – metronidazole following a single oral dose of the parent drug assessed by cutaneous microdialysis. Moreover, the penetration of each unchanged drug into skin was compared with its penetration into theoretically calculated peripheral compartment.
Methods: To evaluate skin concentrations after a single oral dose of 0.4 g acyclovir or 2.0 g metronidazole linear microdialysis probes with 2 kDa molecular-weight cut-off were inserted intradermally to 20 healthy drug-free volunteers divided into two groups. The probes were perfused with Ringer solution. Concentrations of the drugs were determined by HPLC with spectrophotometric detection
Results The average maximum concentrations of acyclovir in the plasma and skin were 3.16 and 0.94 µmol/L, respectively, and were found after about 1.6 and 2.4 h. The pharmacokinetic parameters differed significantly between skin and theoretical peripheral compartment.
The average maximum concentrations of metronidazole in the plasma and skin were 214 and 151 µmol/L, respectively, and were observed after about 2.1 and 2.8 h. In contrary to acyclovir, pharmacokinetic parameters of metronidazole did not differ significantly between skin and theoretical peripheral compartment.
Conclusions: In certain cases concentration of the drug in the skin should be determined instead of its plasma concentration. However, evaluation of skin concentrations cannot be replaced by their concentration in theoretical peripheral compartment.
Novel Generation of Antivirotics: Combination of Antimetabolic and Immunostimulatory Modes of Action KMONÍČKOVÁ E1, HOLÝ A2, ZÍDEK Z1 1Inst. Experimental Medicine, Acad. Sci., Prague, Czech Rep; 2Inst. Organic Chemistry and Biochemistry, Acad. Sci., Prague, Czech Rep.
Background: Acyclic nucleoside phoshonates (ANPs) are recognized class of novel compounds used in therapy of viral infections. The major mechanism of antiviral action is the inhibition of virus-induced DNA polymerases or of reverse transcriptases. The prodrugs of compounds tenofovir and adefovir were approved for treatment of AIDS and hepatitis B, respectively. The aim of the present study was to investigate possible immunobiological activity of ANPs, i.e. up-regulation of nitric oxide (NO) production and cytokine secretion and to compare the effects in cells of animal and human origin.
Methods: ANPs were synthesized in-house (Inst. Organic Chemistry and Biochemistry). Pooled peritoneal cells (PECs) were collected from female mice of the inbred strain C57BL/6 (4-8 mice/treatment). The sources of human peripheral blood mononuclear cells (PBMCs) were buffy coats from healthy donors. The animal PECs were cultured at final density of 2.0 x 106/ml, the human PBMCs at density of 1.0 x 106/ml in complete RPMI-1640 culture medium. The cells were cultured in presence of varying concentrations of ANPs (5-50 M). Levels of cytokines in supernatants of mouse and human cells were determined by ELISA. The length of culture was usually 16 h. The concentration of nitrites in supernatants of mouse cells was taken as a measure of NO production detected after the 24-h culture (a Griess reagent).
Results: Several ANPs have been found to stimulate secretion of TNF-, IL-10, RANTES and MIP-1 with good correlation between mouse PECs and human PBMCs: r = 0.969 (P < 0.0001) and r = 0.982 (P < 0.0001) for MIP-1 and RANTES. ANPs significantly augment production of NO primarily triggered in mouse peritoneal cells by IFN-. The effect is closely associated with their ability to stimulate secretion of cytokines. Highly significant correlation exists not only between the range of NO production and extent of cytokine stimulation in animal cells but also between NO in mouse and cytokines in human cells: r = 0.958 (P < 0.0001) and r = 0.969 (P < 0.0001) for MIP-1 and RANTES, respectively.
Conclusions: Acyclic nucleoside phosphonates are potent imunostimulators and thus represent new generation of antivirotics with a dual, i.e. antimetabolic and immunostimulatory modes of action.
Supported by grant: 1M0508
Discovery Of Dual-Targetting Ligands KNOX AJS, FLOOD CT, LLOYDD DG
Molecular Design Group, School of Biochemistry & Immunology, Trinity College Dublin, Dublin, Ireland
Background: A wealth of examples, both in literature and in the clinic currently exist whereby drugs have been shown to act via multiple targets, albeit these discoveries were generally retrospective. The intentional design of ligands that act at dual or multiple targets is becoming widely recognised as an approach to produce a single drug, in some cases with superior side-effect profiles and therapeutic effects, when compared with the combination of individual drugs. In general, methods such as generation of a conjugate ligand that incorporates functional groups key for binding to both targets are employed, with efficacy. However, this method often leads to the identification of higher MW compounds, as pharmacophores for both targets are not significantly overlapped.
We present a computational approach consisting of ‘Target Fishing’ concepts combined with Virtual Screening to converge on dual-targeting molecules with highly integrated pharmacophores for different unrelated protein families: Estrogen Receptor alpha/Tubulin and finally Hsp90/Tubulin.
Methods: Firstly, ‘Target-Fishing’ methods were employed to identify molecules with similar characteristics to known active ligands for the Estrogen Receptor for example, from a database containing a large number of molecules with known activities for certain biological targets (WOMBAT). Having identified the targets to which those molecules belong, we set about developing and optimizing Virtual Screening platforms combining cheminformatic, docking and pharmacophoric softwares to screen for molecules that ‘hit’ both targets.
Results: The first screen allowed identification of a molecule that binds potently (1.4nM) to the Estrogen Receptor and also to Tubulin. Importantly, we found that this compound is actually a naturally occurring compound contained in citrus fruits.
A second screen to identify molecules that modulate both Hsp90 and Tubulin was undertaken. A micromolar binding compound to both targets is revealed along with preliminary biological data on cell lines. Application of these methods should allow identification of more novel molecules targeting other dual or multiple proteins/enzymes in the future.
