Interactions Between Drug Target Binding Sites and the Remarkable Story of Dopamine D1/D2 Synergism LaHOSTE GJ University of New Orleans, New Orleans, LA, U.S.A.
Background: Biological effects elicited by concomitant binding of two or more drugs to distinct but interacting proteins identifies these binding networks as potential drug targets. For example, recent findings on ligand-activated receptor dimerization and receptor–G protein–accessory protein coupling suggest a potentially useful approach based on Boolean logic: “AND” operations lead to fewer possible outcomes than “OR” operations. Translation of these concepts to biological processes gives rise to the concept of “Magic Bullet Cocktails.” Twenty years ago, a remarkable discovery about signal transduction pathways in the brain was made, namely that the widespread effects of the neurotransmitter dopamine, with few exceptions, require concomitant agonist stimulation of both D1 and D2 receptor subtypes, a phenomenon referred to as “D1/D2 Synergism.” Even more remarkable is the fact that shortly after depletion of synaptic dopamine, there is a breakdown in synergism: all of the effects of dopamine can be elicited by drug stimulation of either D1 or D2 receptors which are now profoundly supersensitive to stimulation.
Methods: We have used receptor autoradiography, behavioral analysis, differential display, and gene knockout techniques to identify the mechanism(s) of D1/D2 synergism and its breakdown.
Results: First, we showed that increases in dopamine receptor number cannot account for these changes. In order to identify unknown candidate genes whose expression may contribute to these phenomena, we used Differential Display of mRNA, which led to the discovery of a novel transcript later identified as rhes, a gene encoding a Ras homolog that functions as a G protein accessory protein. We found that conditions that lead to a breakdown in synergism and profound supersensitivity consistently result in decreased expression of rhes mRNA and Rhes protein. Furthermore, Rhes knockout mice are supersensitive to D2 but not D1 receptor agonists. Thus Rhes may normally serve to inhibit D2-metiated signaling.
Conclusions: A Magic Bullet Cocktail consisting of a drug that facilitates the action of Rhes in combination with a D1 and a D2 antagonist should provide a novel treatment for schizophrenia that is superior to existing therapies.
Melanoma initiating cells: new perspectives for therapy MONZANI E, BAZZOTTI R, LA PORTA CAMa a Department of Biomolecular Science and Biotechnology, University of Milan, Italy
Keywords: melanoma, cancer stem cell, ABCG2, chemotherapy
Background: The model of cancer stem cells of tumor development states that tumor contain a subset of cells that both self renew and give rise to differentiated progeny. Recently our group identified a potential initiating/cancer stem cell subpopulation in melanoma which expresses ABCG2, an ABC-transporter involved in chemoresistance. Aims: 1) To identify and characterise melanoma CSCs, 2) To develop new therapeutic strategies for melanoma.
Methods: This study included human melanoma biopsies as well as human melanoma cell lines. In order to investigate the self-renewal capacity of these cells and the efficacy of new drugs, were carried out in vitro as well as in vivo studies.
Results: In human melanoma biopsy our group described a subpopulation expressing CD133 (Fig.1). Furthermore, a human melanoma cell line expressing high levels of CD133 was characterised and we described a subpopulation ABCG2 positive. CD133+/ABCG2+ cells were injected in NOD-SCID and, interestingly, the tumors expressed low levels of both markers accordingly to melanoma biopsies.
Fig. 1 Typical expression of CD133 in human melanoma biopsies (A) and tumour-initiating capability of CD133+ cells sorted and injected in NODSCID mice (B).
Conclusion: Considering that melanoma is one of the most aggressive forms of skin cancer and it is strongly resistant to conventional therapeutic agents, the molecular biology of CSCs opens interesting new perspectives from the pharmacological point of view.
The Many Lives of Hsp10: From Early Pregnancy Factor to Potential Antitumoral Agent. New Proteomic Data and a Review of the Literature Focusing on Its Immunologic Properties LA ROCCA G1, ANZALONE R1, CAPPELLO F1, CORRAO S1, TIMPERIO AM2, ZOLLA L2, CONWAY DE MACARIO E3, MACARIO AJ3, FARINA F1, ZUMMO G1.
1DIMES, University of Palermo, Italy; 2DISA, University of Viterbo, Italy; 3COMB, University of Maryland, Baltimore, USA.
Background: Hsp10 expression has been investigated in several cancer models, with contrasting results. It is homologue to early pregnancy factor (EPF), a secreted protein which modulates the immune response of the mother versus the fetus. The impact of cigarette smoke (a major risk factor for lung diseases) on Hsp10 expression by airway cells has not been characterized yet.
Methods: We studied the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of Hsp60 and Hsp10 in human lung cells. Proteomics was carried out by 2D-IPG, silver stain, western blotting, and mass-spectrometry (MS). Database searches and chaperonomics were used to identify the proteins and genes of interest.
Results: Following CSE cell exposure as compared with unstressed cells, significant variations in Hsp10 did occur, in both lung fibroblasts and epithelial cells. In unstressed cells, three isoelectric variants of Hsp10 were found, which have not been reported for any other system, yet. After CSE exposure, only the most basic isoform was still expressed. To characterize the three variants found in unstressed cells, we performed MS analyses. Digested spots were analysed by nano-RP-HPLC-ESI-MS/MS to determine the fragments’ amino acid sequences. Database searches showed that the most basic variant was human Hsp10 with 56% sequence coverage, and the other two isoforms had the same amino acid sequence, even if with a lower sequence coverage.
Conclusions: The data thus far indicate probably that Hsp10 protein variants are due to post-translational modifications. We recently showed the in vivo correlation between lung cancer development and downregulation of Hsp10 expression, and proposed a model for the antitumoral role for Hsp10, together with Hsp60. The precise role of Hsp10 in carcinogenesis is still unclear. The immunosuppressive activity of EPF/Hsp10 points towards a tumor-promoting role, mediating immune evasion and apoptosis resistance. On the other hand, the in vivo and in vitro evidences obtained in human lung models suggest that different Hsp10 isoforms may mediate diverse processes and should be differentially regulated.
Polypharmacy: A Major Risk In The Life Of The Elderly LABAULT-CABEZA NM Gerontology Program, Graduate School of Public Health, University of Puerto Rico
Addressing the high utilization of medications among the elderly, this study explored the degree of knowledge of polypharmacy and its consequences, as well as the pattern of utilization by a group of elders 65 years of age and over in the San Juan metropolitan area of Puerto Rico.
A dual technique was utilized: focus groups and a survey. Two focus groups were performed in order to explore issues pertaining the following domains: quality of life, doctor-patient and pharmacist-patient relationships, degree of knowledge of polypharmacy, and need of information. A questionnaire was administered to obtain information regarding their pattern of drug utilization.
Results of focus groups indicated that the elderly have learned how to cope with their chronic conditions and be able to continue with quality of life. Additionally, results suggested a need form the elderly to develop more assertiveness and closeness with their health care providers, both doctor and pharmacist; as well as a lack of sufficient knowledge of the serious implications that polypharmacy brings. Results from the survey revealed and confirmed their inappropriate utilization of medications, and suggest that this segment consults multiple physicians, all of which may bring potential problems of adverse drug interactions.
Further investigation is needed to examine fully the issue of polypharmacy, as well as the need of education that results in empowerment of patients, families and communities.
Biocompatible Nanoparticles, Carriers Of The Magic Bullet LACKO AG1, NAIR M1, PARANJAPE S1, MOOBERRY L1, McCONATHY WJ2 1University of North Texas Health Science Center, Fort Worth TX, USA .2Texas Tech University Health Science Center, Permian Basin, Odessa,USA.
Background: Current barriers to cancer chemotherapy include i) toxic side effects, ii) the limited accessibility of the drugs to tumor tissue, and iii) multi-drug resistance developed by malignant tumors during treatment. We developed a novel drug delivery system utilizing reconstituted high density lipoprotein (rHDL) nanoparticles with reduced toxicity to normal tissues and selective receptor-mediated uptake of anti-cancer drugs. The rHDL formulation is preferred over conventional drug delivery strategies because its small size and biocompatible components. A further advantage of the rHDL drug delivery model is the selective lipid uptake mechanism by which drugs are delivered to target cells via the scavenger receptor, class B, Type I (SR-BI).
Methods: The rHDL/paclitaxel (PTX) nanoparticles were characterized with regard to size, shape, stability and cytotoxicity against cancer cells using the MTT assay. Maximum tolerated dose studies were performed in C57Bl/6 female mice comparing rHDL/PTX with Taxol® and Abraxane®. In tumor suppression studies, the .
Results: The rHDL/PTX nanoparticles were found to have a diameter of 11.4 +/- 3.1 nm and 5-20 fold enhanced toxicity against cancer cells when compared to free PTX. the majority (82%) of the paclitaxel was taken up by cancer cells via a selectivel uptake mechanism, apparently via the SR-B1 receptor. Incubation of the cells with HDL3, the natural ligand of SR-BI, supressed paclitaxel uptake to 30.6% as compared to rHDL/Ptx alone (p<0.0001) supporting the specificity of the receptor uptake mechanism. During studies with mice a 2.3-fold and 1.4-fold higher dosage of rHDL/Ptx could were tolerated by mice, compared to Taxol® and Abraxane®, respectively. Recent tumor suppression studies with mice show that the rHDL delivery system is highly effective in reducing the tumor burden in mice carrying xenografts of human tumors.
Conclusions: Reconstituted high density lipoprotein (rHDL) provides a targeted delivery vehicle for the encapsulate paclitaxel via receptor mediated uptake of the drug by cancer cells and tumors. Encapsulation of chemotherapy drugs in rHDL enhances the tolerance of the drug while increases ist toxicity against cancer cells and tumors. The rHDL nanoparticles should thus reduce the toxic side effects seen with other formulations while enhancing the anti-tumor effectiveness of the encapsulated drug
Blood Choline Phospholipids As Preferential Sources Of Docosahexaenoic Acid To The Brain LAGARDE M Université de Lyon, UMR 870 Inserm / Insa-Lyon, 69621 Villeurbanne, France.
Docosahexaenoic acid (DHA) is the main polyunsaturated fatty acid (PUFA) of the brain phospholipids. It plays a major role in the brain development, learning activities and visual acuity. As a highly unsaturated molecule, it is supposed to be very sensitive to peroxidation, and it has been reported to be degraded, presumably in response to oxidative stress, in number of neurodegenerative diseases.
The accretion of DHA to the brain has been assumed to be done from the blood circulating pool associated to albumin, DHA being in its unesterified form which can cross the blood-brain barrier (BBB). Blood albumin also carries lysophospholipids, mainly lysophosphatidylcholine (LysoPC). We first found that the uptake of DHA by the brain was aound ten-fold more efficient when DHA was esterified in LysoPC compared with unesterified DHA (Thiès et al 1994). Then we found that DHA ingested either in triglycerides (oil) or phosphatidylcholines (PC) may circulate as DHA-containing LysoPC (LysoPC-DHA) in a way which is compatible with an efficient uptake of DHA under this form (Brossard et al 1996, 1997 & Lemaitre-Delaunay et al 1999). Furthermore, an in vitro reconstituted BBB allowed to find a preferential crossing of LysoPC-DHA over DHA (Bernoud-Hubac et al 1999). Also, a substantial amount of DHA has been found to circulate in the form of LysoPC-DHA with DHA at the sn-2 position (the usual position of PUFA in phospholipids), although rapidly isomerizing into sn-1-LysoPC-DHA (Croset et al 2000).
A recent work from Chen and Subbaiah (2007) has brought evidence for a preferential cleavage of DHA-containing PC by the so-called endothelial lipase which releases sn-2-Lyso-PC-DHA, making relevant this form of LysoPC-DHA for an efficient uptake by the brain.
As sn-2-Lyso-PC-DHA is rapidly isomerized into its sn-1 position isomer, we have set up a one step method (patent 2008) to produce 1-Acetyl,2-DHA-PC (AceDoPC) of which the structure is closely related to LysoPC-DHA. This method will be used to prepare 13C-labeled DHA-containing choline phospholipids in order to perform new human studies on the metabolic fate of DHA when ingested in those different forms. This work is in progress.
It is concluded that LysoPC-DHA or its stabilized form AceDoPC may be efficient carriers of DHA to the brain, and then could be used as a way to compensate for DHA-depleted brains, whatever the reason of such a depletion.
Desining Novel Antiinfective Concepts Combining Nanotechnology,
Bioplastics and Natural Products LAGARON JM1, TORRES-GINER S1, OCIO MJ1,2, BUSOLO M3, JACKSON J4, BURT H4, PLACKETT D5 1CSIC, Burjassot, Spain, 2Faculty of Pharmacy, University of Valencia, Valencia, Spain; 3Nanobiomatters S.L., Valencia, Spain; 4Faculty of Pharmaceutical Sciences, UBC, Canada; 5Risoe DTU, Roskilde, Denmark.
Abstract: This presentation reviews a number of research efforts carried out within our group where combining natural products, nanotechnology and biopolymers can be of value in the pharmaceutical and biomedical areas to design novel efficient antiinfective systems. The talk presents first our recent efforts to understand and optimize the antimicrobial properties of chitosan and electrospun nanofibers of chitosan and of other antimicrobial biomass derived biopolymers and blends (see Figure 1). It does later describe our most recent efforts to design antiinfective and bioactive bone replacement interphases and wound dressing systems based on nanostructured fiber mats of biopolymers carrying biocides and carried out within the EU FP6 project NEWBONE. Finally, the presentation describes the capacity of certain nanoclays to intercalate and control release biocide and bioactive plant extracts (see Figure 2) and, within a very recent collaboration with the University of British Columbia and Risoe DTU, of pharmaceutical antibiotics such as tetracycline.
Figure 2.Following the release of natural biocide thymol by FTIR spectroscopy from resorbable clay nanobiocomposites of PCL
Acknowledgements: The authors would like to acknowledge the EU FP6 project SUSTAINPACK, the CSIC overseas support program (A12008PM1), the Spanish research project MAT2006-10261-C03 and Nanobiomatters Ltd. for financial support.
Selection Of Cell Culture Substrate For Human Viral Vaccines LALOSEVIC D1, KNEZEVIC I2, LALOSEVIC V3, LAZAREVIC-IVANC LJ4 1Faculty of Medicine, Novi Sad, Serbia; 2World Health Organization, Geneva, Shwitzerland; 3Faculty for Agriculture, Novi Sad, Serbia; 4Pasteur Institute, Novi Sad, Serbia.
Background: Many cell cultures for human viral vaccines used up today, animal origin or human diploide cells (Wi-38), primary culture or continued cell lines like VERO. The rabies vaccine produced on BHK-21/C-13 (Baby Hamster Kidney) cell culture has been used for a long time in animals. Since the safety of the BHK cells for animals is not questionable, the possibility of their use for a human rabies vaccine has been discussed. Contamination by cellular DNA is not dangerous becouse the treatment with beta - propiolactone during virus inactivation completely inactivates the biological activity of DNA as well. This finding, which was accepted also by WHO, encourages the use of BHK cells as substrate for human vaccine production.
Methods: We multiplied L. Pasteur strain of rabies virus on the BHK-21 (C-13) cells and produced beta-propiolactone inactivated and aluminium-phosphate adsorbed rabies vaccine. A total of 300 adult subjects were vaccinated. A clinical testing was conducted with three doses of vaccine, intramuscularly in the deltoid region by the pre-exposition scheme 0-7-21 days. At 30th day post vaccination serum antibodies were measured by the RRFFIT (Rapid Rabies Focus Fluorescence Inhibition Test).
Results: In comparation with VERO cell line of monkey origin, we harvested one log more rabies virus from BHK cells and vaccine production is possible without virus concentration. Local reactions in few percents and no sistemic adverse reactions were registered. All vaccinees had antibody titer over acceptable mimimal (0,5 UI). We also adapted polio and measles viruses on BHK cells for possible vaccine production.
Conclusions: We conclude that this rabies vaccine is low cost, safe and effective for humans. The preliminary results with BHK/21 vaccine in volunteers confirmed its good tolerability and immunogenicity. This product designed is safe on the basis of experimental results that virus inactivation by beta-propiolactone destroyes contaminant DNA from cell culture.
Magic Bullets: Beynod Selective Targeting to Selective Killing Using Armed Antibodies LAMBERT JM ImmunoGen, Inc., Waltham, MA, USA.
Background: In recent years, there has been increasing interest in the use of highly toxic small molecules as the attached cell-killing agent. One reason for the heightened interest in the field is the increase in the number of companies pursuing antibody-based anticancer agents, since many tumor-targeting antibodies identified as a result of this effort lack meaningful anticancer activity of their own. Creating antibody-maytansinoid conjugate (AMC) compounds provides a means of achieving effective products from such antibodies.
Clinical Validation of Maytansinoid Technology: Currently eight AMC compounds are in clinical testing using ImmunoGen’s maytansinoid technology, with one more expected to enter the clinic in 2008. One of the compounds in the clinic, T-DM1, is a conjugate of the maytansinoid DM1 with the antibody trastuzumab. It is being evaluated in patients with HER2-expressing metastatic breast cancer that have progressed on treatment with a chemotherapy regimen that includes trastuzumab. The initial clinical data reported is encouraging, and includes a confirmed objective response rate of 44% (4/9) at a dose of 3.6 mg/kg q3weeks in a Phase I trial in such trastuzumab-nonresponsive patients.
Understanding the Mechanism of Cell Killing by AMCs:The maytansinoid molecules are linked to the antibody molecule at lysine residues via a disulfide or a non-reducible thioether link. The disulfide linkers are designed with steric hindrance at carbon atoms adjacent to the disulfide bond to maximize plasma stability of the conjugate and to facilitate the intracellular release of the maytansinoid by disulfide-reduction. The non-cleavable thioether link is designed to be stable in plasma, and the conjugate upon binding and internalization in the target cancer cell is lysosomally processed to release the thioether-linked maytansinoid attached to the lysine residue.
Broadening the Technology: Evaluation of the role of linkers in the effective intracellular release of the cytotoxic maytansinoid metabolites has led to the creation of novel hydrophilic linkers that are stable in plasma and yield even greater efficacy to the conjugates based on in vitro and in vivo pre-clinical studies. The hydrophilic linkers confer improved activity against multi-drug resistant (mdr) cancer cells, and also offer potential for agents against tumors that express the target antigen at low density.
Aerosolized Liposomal Antifungal Agents LAMBROS MP1, JOHNSON DL2, BOURNE DW 2 1Western University of Health Sciences, Pomona, CA, USA; University of Oklahoma HSC, Oklahoma City, OK, USA.
Background: In the past decade three classes of antifungal agents, polyenes, azoles, and echinocandins have been extensively studied and used. One of the most important antifungal agents, Amphotericin B (AmB), a polyene, has toxicity that limits the lung tissue doses that can be achieved through intravenous administration. Incorporation of AmB in liposomes reduces the toxicity and increases the therapeutic index for intravenous administration. Targeted delivery to lung tissues (those usually first colonized and infested by fungi) via inhaled liposomal AmB aerosol is an effective approach. Development of optimal aerosolized liposomal AmB therapies requires a better understanding of the effect that liposome surface charge has on lung clearance kinetics. In this work we evaluated the clearance kinetics and organ distribution of inhaled liposomal AmB in male Balb/C mice.
Methods: Mice were exposed via nose only to AmB-containing liposomal aerosols having positive, negative, or neutral surface charge characteristics. The formulations were aerosolized using a Collison nebulizer. Groups of animals were euthanized at predetermined times. The lungs and other organs were analyzed for AmB using an HPLC method. AmB was not detected in serum or other organs such as kidneys, liver, and brain.
Results: The disposition of neutral and positive liposomal amphotericin B in lungs followed biexponential kinetics. The alpha and beta phase half-lives for positive liposomes were 1.3 and 15.1 days, respectively, and 2.3 and 22 days for neutral liposomes. AmB delivered via negative liposomes exhibited monoexponential clearance with a half-life of 4.5 days.
Conclusions: These results suggest that toxic side effects in nontarget tissues are minimal and may indicate a potential for long term protection against fungal infections.
