Ehrlich II –2nd World Conference on Magic Bullets

A Study of Capecitabine and Cisplatin in the Treatment of Recurrent Carcinoma of the Uterine Cervix Benjapibal M, Thirapakawong C, Leelaphatanadit C, Therasakvichaya S, Inthasorn P Mahidol University, Bangkok, Thailand. Background: Platinum is the mainstay of treatment in advanced or recurrent cervical carcinoma, however, the duration of response is short lived as well as the median survival. Fluorouracil (5-FU) has been shown to be active in cervical carcinoma. Capecitabine, an oral fluoropyrimidine carbamate, is sequentially converted to 5-FU by thymidine phosphorylase (TP) which is found at higher concentrations in cervical carcinoma than normal tissue. In addition, cisplatin further upregulates TP. Capecitabine plus cisplatin has the potential to be an active treatment, which is more convenient than 5-FU-cisplatin. Methods: This study combines capecitabine and platinum in patients with recurrent cervical carcinoma with no potentially curative standard treatments. Sixteen patients (14 squamous cell carcinoma, and 2 adenocarcinoma) with a median disease-free interval of 11 months (range, 2-96) received cisplatin 50 mg/m^2 intravenously on day 1 and oral capecitabine 1000 mg/m^2 twice daily for two weeks with a one week rest period. Results: Median age was 50 years (range, 31-74). A total of 89 cycles were administered with a mean of 5.5 cycles (range, 3-6) per patient. Four of the sixteen patients had complete response (25%), 4 had partial response (25%), and 5 had disease stabilization (31%). Ten patients (63%) had recurrent disease outside the radiation field. The overall response rate in patients with recurrent disease within the previous irradiated field was 33% and 60% in patients with tumor outside the irradiated field. The median follow-up time was 29 months (range, 11-39). The median time to progression was 9 months (range, 5-37), with a median overall survival of 23 months (range, 5-37). The majority of adverse events were mild and there were no grade 4 adverse events. Hematological toxicity was the most frequent adverse event with grade 3 neutropenia in 19% of patients. Grade 2 and 3 hand-foot syndrome occurred in 38% and 6% of patients, respectively. There were no chemotherapy-related deaths. Conclusions: This active yet convenient combination of capecitabine and cisplatin shows a high response rate, long time to progression and survival with acceptable toxicities for patients with recurrent carcinoma of uterine cervix.

Girolline, a new antiplasmodial leader extracted from the sponge Cymbastela cantharella BENOIT-VICAL F^1,2, SALERY M^2, NJOMNANG SOH P^1,2, AHOND A^3, POUPAT C^3 ^1- Laboratoire de Chimie de Coordination du CNRS, UPR8241, 31077 Toulouse 4, France. ^2- Service de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Rangueil, 31059 Toulouse 9, France. ^3- Institut de Chimie des Substances Naturelles du CNRS, 91198 Gif-sur-Yvette cedex, France Background: Malaria is the most prevalent parasitic disease in the world today. In this context, there is clear demand to search for new antimalarial agents and research into new antimalarial drug candidates originating from natural sources has been actively pursued. Girolline (Figure1), a 2-aminoimidazole derivative extracted from Cymbastela cantharella (a New-Caledonian Sponge) already known for its antitumor activity, was tested against Plasmodium. Methods: We evaluated the effects of girolline and some of its analogues in vitro and in vivo against P. falciparum and P. vinckei petteri, respectively. We then evaluated its toxicity in vitro and in vivo. We have also determined the point of action of girolline in the erythrocytic life cycle of the malarial parasite and its synergistic action with chloroquine. Results: We have demonstrated that girolline presents a very promising activity against malaria both in vitro against four P. falciparum strains and in vivo on murine mdel. Girolline also showed a specific mode of action by inhibiting Plasmodium protein synthesis. Moreover, between girolline and chloroquine, a high synergistic effect was reported. Conclusions: Girolline is of a real interest as research basis for a new class of antimalarials. With such a biological profile, girolline could be considered as a model chemical structure for new candidates in the arsenal of new drugs and in particular of drugs able to fight malaria. Figure 1: Structure of Girolline

Natural Products (NP) - Microbial Metabolites (MM) - Antibiotics (AB): History, Facts and Problems, Where Now ? BÉRDY J Budapest, Hungary, e-mail: [e-mail] History: Great benefit and unpredictable negative consequences. The NP research and especially the discovery of new MM declined in the past years. Few new drugs were discovered. The reasons are: resistance-problem, less success with HTS and combinatorial chemistry, but the reasons are mainly economic and regulatory. In the last years the total synthetic efforts and the discovery of compounds from higher plants and marine organisms increasing, but sometimes no evident proof to the real producer species (endophytes, symbionts - taxol, patellamide). Facts: Close to half a million NP, including ~100 000 MM, ~20 000 microbial AB (~350 marketed), there are known. High percentage, (~40 %) of all drugs and about 70-80 % of all known AB drugs were derived from NP, as direct drugs, derivatives (semisynthetic and modified products), and other NP mimics (synthesized as NP analogue). The NP libraries have some advantages over random synthetic or combinatorial chemical libraries in several respects (e.g. complex structures hardly accessible by chemical methods). "Nature is the best combinatiorial chemist". They also meet the green chemistry. The bioproducts have inherent - but perhaps undiscovered - biological functions and drug-like structures, compatible to the host. The real function of MM is the communication with other microbes, higher organisms and the environment. They are the interface between microbes and the rest of world. The biosynthetic pathway of microbes, evolved in the millions of years under evolutionary pressure, ares mainly undiscovered (silent genes). Only ~1% of the existing bacteria are cultivable. Unlimited number of possible new structures exists in coded form in the metagenom. Where Now: Presently everything seems to be out of balance. Back to the pre-antibiotic era ? What can we do ? 1. Diversification of NP libraries. New ways to discover new leads to medicinal chemistry (inspiration for synthetic chemists). 2. New biochemical/genetic methods: mining and engineering of the biosynthesis (genom). 3. Discover new sources (marine species, endophytes, uncultivable, rare microbes). 4. New target-oriented effective screenings. 5.Chemical and biosynthetic post-evolution by combinatorial methods. 6. Better understanding and solving the problems of the drug-target-host interactions. The key of our co-evolution with microbes is the exact understanding of the life-cycle of microbes.

Possible Consequences of Transplacental Transfer of Viruses in Healthy Pregnants (Rewiev) BERENCSI GY, ÖRDÖG K, CSIRE M, KAPUSINSZKY B, YOUNES SA Division of Virology, National Center for Epidemiology, Gyáli Str. 2-6, H-1097 Budapest, Hungary Background: Viruses, which may cause illnesses of the fetus were shown to be transfered frequently through the placenta into fetal tissues without any clinical consequences. Rubella and rubella vaccine, measles and measles vaccine, Togaviruses, Flaviviruses, hepacivirus (HCV), Hepadnaviruses (HBV), , human cytomegalovirus (HCMV), human herpesvirus types 1 (HSV), 6 (HHV6), 7 (HHV7), and 8 (HHV8), human parvovirus B19 (HPV-B19), dependovirus (AAV), human adenovirus, Epstein-Barr virus (EBV), human papillomaviruses (HPaV), human polyomaviruses (HPyV), lentiviruses and the Anellovirus TTV were detected in the umbilical blood, amniotic fluid or fetal tissues at the end of healthy pregnancies. Mechanisms: IgG transport mechanisms, transported maternal cells and the lipid rafts were shown to be vehicles of this virus transport. Results: 1.) The first consequence of the contact of the developping fetal organism with latent viruses can be the life-long carriage of these viruses upon birth. 2.) The developping fetal immune system might create immunotolerance to certain viral antigens depending on the fact, whether the viruses replicate or are only latently present in the fetal cells. This partial immune tolerance may impair the post partum immune response facilitating tumour formation in the affected individuals. 3.) Herpesviruses may activate endogeneous retroviral genes in fetal cells, modifying their differentiation and surface properties. Alternatively the pathogenesis of autoimmune diseases might be initiated by these modifications. 4.) The viruses are coding for micro RNA molecules possibly influencing the replication and differentiation of the virus carrier cells. The genes of DNA viruses interfering with apoptotic mechanisms may also disturb the normal differentiation processes in the organs of the fetus. 5.) Finally the response to the mandatory antiviral vaccinations might be impaired by the transplacentally transcytosed viruses. Conclusion: The systematic testing of umbilical blood and urine of newborn babies would be of essential importance using molecular technics for the presence of the viruses in order to be prepared for yet unknown risks for the future life span.

Lacosamide is a Novel Antinociceptive and Antiepileptic Drug with a Dual Mode of Action BERKELS R^1, Beyreuther B^2 ,KREBSFAENGER N^2, Freitag J^2, Lees G^3, Errington A^3, STÖHR T^2 ^1UCB GmbH, Monheim, Germany; ^2SCHWARZ BioSciences GmbH, Monheim, Germany; ^3University of Otago, Dunedin, New Zealand; ^4UCB, Inc., Smyrna, GA, US Background: Lacosamide is an investigational drug that has demonstrated positive results in Phase III trials of neuropathic pain and epilepsy. Preclinical studies have shown neuroprotective effects of lacosamide both in animal models and in-vitro. Electrophysiology and proteomics experiments have identified two likely modes of action for lacosamide. Methods and Results: Electrophysiology experiments performed in mouse neuroblastoma cells indicate that lacosamide reduces sodium-channel availability by selectively enhancing slow-inactivation. Enhancing slow-inactivation is thought to raise channel-activation thresholds, reducing pathophysiological neuronal hyperexcitability. This mechanism is different from that of anesthetic and other antiepileptic agents, which non-selectively block the sodium channel pore and/or enhance fast- and slow-channel inactivation. A second mechanism of action may occur via the binding of lacosamide to collapsin-response mediator protein 2 (CRMP-2), a phosphoprotein that is involved in neuronal differentiation and axonal out-growth (processes that are maladaptive in the pathophysiology of pain and epilepsy). The interaction of lacosamide with CRMP-2 may underlie the apparent neuroprotective effects of lacosamide, since CRMP-2 appears to be important for mediating neuroprotection from excitotoxic insult and apoptosis. Conclusions: The dual mode of action of lacosamide represents two novel mechanisms for the treatment of neuropathic pain and epilepsy. Based on current studies, it is proposed that selective enhancement of slow-inactivation of sodium channels may underlie the immediate effects of lacosamide. Further characterization of the interaction with CRMP-2 may help to explain its role in lacosamide’s symptomatic and disease-modifying effects.

PK/PD Relationship of Antibiotics in Local Treatment of Prostethic Infections BERTAZZONI MINELLI E and BENINI A University of Verona, Verona, Italy Background. Infection is the most serious complication following orthopaedic surgery, and delivery of antibiotics to the surgical area is a way of reducing the infection frequency. Polymethylmethacrylate (PMMA) cements impregnated with antibiotics are currently utilised as local antibiotic carrier in orthopaedic surgical-site infection to treat prosthetic infections (hip, knee, etc) and are an adjunct to current therapy (surgical debridment and systemic antibiotic therapy). Cement intrinsic characteristics and capacity to release drug are essential for the final clinical outcome as well as antimicrobial drug pharmacodynamics at the site of infection. Methods. Several experimental models in vitro and in vivo have been developed to better understand the release kinetic of different antibiotic from cement and to optimise their use in clinical practice. Results. Aminoglycosides (A) and vancomycin (V) show good positive characteristics: bactericidal activity, adequate release, compatibility, mechanical resistance, and excellent tolerability. The release of A and V from PMMA cement seems prompt and effective, determining high local concentrations. The drug elution shows a bimodal profile, consisting of an initial high rapid release of drug followed by a much slower but sustained release. Initial drug concentration, cement surface area and porosity are important factors in determining the amount of drug release. A and V in combination show synergistic antimicrobial activity both in vitro and in vivo against multiresistant clinical isolates. A and V concentrations in drainage fluid following spacer implant are higher than those obtained with systemic administration; their kinetics is superimposable. These high local concentrations of the combination are effective against multiresistant pathogens responsible for prosthetic infections (high inhibitory bactericidal titre of drainage fluids). Moreover, the presence of A and V in PMMA specimens reduces the growth and bacterial adhesion of susceptible and intermediate-resistant Staphylococci. Their anti-adhesive effect depends on the characteristics of the microorganism and its capacity of adhering to antibiotic-loaded surfaces. Conclusions. PMMA antibiotic loaded cements improve local drug delivery at infection site and enhance pharmacodynamics of antibiotics for treatment of prosthetic orthopaedic infections.

Development of Unique Cisplatin Analogs for Site-Specific Treatment of Hormone-Dependent Female Cancers BÉRUBÉ G Univ. du Québec à Trois-Rivières, Trois-Rivières, Canada. Background: Chemotherapy remains, to this day, an effective treatment for several types of cancer. However, the severe side effects caused by the treatment limits its full potential for a cure. Thus, the development of site-specific anticancer therapy is a subject of intence research. Several strategies can be used to target cancer cells. For instance, the use of a carrier molecule being able to recongnise a specific receptor in the cell is a tactic of choice used by several research groups. We have developed several estradiol-platinum(II) (E2-Pt(II)) hybrid molecules using the following guiding principles a) potential for affinity towards the estrogen receptor b) potential for in vitro and in vivo selectivity on hormone-dependent female cancers c) ease of synthesis and, d) potential for large scale industrial production. Methods: The development of the E2-Pt(II) hybrid molecules will be initially discussed. Then, the most promising hybrid derivative, VP-128, is selected to examine its biological activity towards breast (MCF-7, ZR-75-1, MDA-MB-468, MDA-MB-231 and HS578-T) and ovarian (OVCAR-3, SKOV-3, A2780 and A2780-cp) cancer cells, in vitro (MTT assays) and in vivo (xenografts model) using ER positive or negative cells. Results: MTT assays revealed that VP-128 decreased the viability of breast and ovarian cancer cells more efficiently than cisplatin itself in vitro. Moreover, in the case of breast cancer the expression of ER sensitized the cells to the growth-suppressive effect of VP-128. Hoescht nuclear staining revealed an improved efficiency of VP-128 compared to cisplatin to induce apoptosis of breast cancer cells, which was enhanced in ER-positive cells. In cisplatin resistant A2780-cp cells, VP-128 was able to induce cell death indicating that the new drug might also be efficient to kill cisplatin resistant cancers. Finally, using human breast and ovarian cancer cell xenografts in nude mice, we found that VP-128 had stronger antitumour activity compared to cisplatin in vivo, and was more specific and selective towards hormone-dependent cancer cells. Conclusions: Experimental data show that VP-128 possesses enhanced anticancer activity compared to cisplatin and is able to specifically target hormone-dependent tumours in an in vivo model. Thus ultimately, VP-128 could provide new and/or alternative treatment modalities for breast and ovarian cancers.

Mediterranean spotted fever(MSF) in Oran (Algeria) BESTAOUI L, BENABDELLAH A, BENSADOUN F-Z, KOUIDED-BELKADI S-A, BENSAAD M Service des maladies infectieuses et tropicales, CHU.Oran, ORAN 31000, Algeria Abstract MSF due to rickettsia conori was thought ,for many years,to be the only tick-borne rickettsial disease prevalent in Algeria. However ,in recent years,other species within the spotted fever group of the genus Rickettsia have been described as emrging pathogens. Tick-borne agents include:Rickettsia aeschlimanii and rickettsia massiliae. Many rickettsia of unknown pathogenicity have also been detected from ticks and could represent potential emerging pathogens to be discovered in the future. Furthermore,a new spotted fever rickettsia,Rickettsia felis ,was found to be associated with cat flees and is an emerging human pathogen. Rickettsia felis is susceptible to doxycyclin,thiamphenicol,and fluoroquinolons but not to gentamicin,erythromycin ,amoxicillin or trimethoprim-sulfamethoxazole. The resistance of this new species to erythromycin is consistent with taxonomic position within the spotted fever group. We present an overview of these rickettsiodes,focusing on emerging diseases.

New Approaches for the Use of Old Drugs – Preclinical and Clinical Pharmacological Evaluation of the Pyrimidine Anti-neoplastics: 5-fluorouracil, zebularine, 5-fluoro-2’-deoxycytidine, gemcitabine, and THU. BEUMER JH^1, EISEMAN JL^1, NEWMAN EM^2, DOROSHOW JH^3, SYNOLD TW^2, ZEH HJ^1, BARTLETT DL^1, KUMMAR S^3, EGORIN MJ^1 ^1Univ. of Pittsburgh, Pittsburgh, USA; ^2City of Hope National Medical Center, Duarte, USA; ^3National Cancer Institute, Bethesda, USA. Background: Pyrimidines include some of the oldest anti-neoplastic agents. They are still an important part of many anticancer drug regimens, and continue to generate interest, as evidenced by recent studies of zebularine and 5-fluoro-2’-deoxycytidine (FdC), two DNA hypomethylating agents. Our aim is to develop innovative pyrimidine anticancer drugs and improve the use of existing ones. Methods: We developed chromatographic assays for zebularine and 3 metabolites (HPLC-radiodetection), FdC and 4 metabolites, gemcitabine (dFdC) and metabolite, 5-fluorouracil (5-FU), and tetrahydrouridine (THU) (all LC-MS/MS), and applied these to preclinical and clinical pharmacological studies. Results: Preclinical studies revealed that, in vivo, [^14C]-zebularine is quickly converted to uridine, producing hydrogen peroxide as a by-product. In mice, THU decreased metabolic degradation of FdC (>50-fold) and dFdC (5-fold) and substantially increased their oral bioavailability (25-fold and 4-fold, respectively). Clinical pharmacokinetics of intravenous FdC and THU were determined, and the oral route is being explored. Preclinical studies show that oral dFdC and THU is efficacious in a CFPAC-1 xenograft pancreatic tumor model (Treated/Control = 30%). The 5-FU LC-MS/MS assay was shown to correlate well with a newly developed 5-FU immunoassay, which will allow on-demand therapeutic drug monitoring. Conclusions: Pyrimidines are an important class of anticancer drugs, both in established regimens, and as agents in future treatments. Their metabolism is complex, but can be modulated to allow oral dosing, and different dosing schedules. New applications and targets of pyrimidine drugs are being discovered and should maintain interest in this class of compounds.

Novel Diastereoselective Synthetic Routes to Tamoxifen, Toremifene, and Droloxifene, Anti-Breast Cancer Agents via Organoboranes BHAT NG, CAVAZOS E, ORTIZ O, ELIZONDO D Department of Chemistry,The University of Texas-Pan American, 1201 West University Drive, Edinburg, Texas 78539-2999 USA; Background: Tamoxifen., toremifene, and drroloxifene are useful drugs used in the treatment of breast cancer. Several synthetic methods are known in literature to synthesize them. Some of the methods give a mixture of diastereomers. Organoborane reagents are known to achieve high selectivity in organic synthesis. Consequently, highly diastereoselective synthesis of tamoxifen, toremifene, and droloxifene is achieved using organoborane reagents. Our current methods are compared with the literature methods to prepare these drugs. Methods: This study included the reactions of alkynylborates with an electrophile such as trimethylchlorostannane followed by stepwise Stille coupling and Suzuki coupling. Since the chemicals used are highly air-sensitive, all of the reactions are carried out under nitrogen atmosphere. In the case of tamoxifen synthesis, phenylethynyllithium is reacted with triethylborane followed by Stille coupling with (N-{2-(4-bromophenoxy)ethyl}N,N-dimethylamine and Suzuki coupling with bromobenzene in the presence of tetrakis(triphenylphosphine)palladium. In the case of toremifene, 4-chloro-1-butynyllithium is reacted with triphenylborane followed by reaction with trimethylchlorostannane. The resulting intermediate is then subjected to palladium catalyzed Stille coupling with bromobenzene and palladium catalyzed Suzuki coupling with (N-{2-(4-bromophenoxy)ethyl}N,N-dimethylamine In a similar manner, droloxifene is synthesized by the reaction of 4-[2-(dimethylamino)ethoxy}-phenylethynyllithium with triphenylborane and chlorotrimethylstannane. The resulting intermediate is subjected to palladium catalyzed Stille and Suzuki coupling reactions with m-bromophenol and ethylbromide respectively. Results: Tamoxifen is obtained in 55% isolated yield. Toremifene and droloxifene are obtained in 48% and 50% isolated yields respectively. These compounds are purified by column chromatography.. The structures of these drugs are confirmed by nuclear magnetic resonance (NMR) spectroscopic methods. Conclusions: In summation, highly diastreoselective synthesis of tamoxifen, toremifene, and droloxifene is achieved using organoborane reagents, Stille, and Suzuki coupling reactions. The optimization of reaction yields of these drugs is currently in progress in our laboratory.

Mechanisms that Explain the Lower Incidence of Breast Cancer in Postmenopausal Women Treated with Conjugated Estrogens (CEE). Role of Estrogen Receptor  and . BHAVNANI BR^1,2 ^1,2Univ. of Toronto and The LKSKI of St. Michael Hospital, Toronto, Ontario, Canada. Background: Recent findings from the Women’s Health Estrogen Alone Trial (JAMA 2008), showed that long term treatment of hysterectomized postmenopausal women with CEE not only did not increase the incidence of breast cancer, but more importantly, may have reduced the risk in these women for this disease. These results raise some important questions as to the mechanisms involved and whether all types and of estrogen can impart this protection. In the present study, the relative binding affinities (RBAs) of 11 equine estrogens and their functional activities mediated via ER and ER were compared. Methods: The RBAs were estimated by competitive binding assays using ^3H-17 estradiol and unlabelled equine estrogens. The functional activity of the estrogens was measured in HepG2 cells transfected with human ER or ER or both and secreted-alkaline phosphalase (SEAP) gene and analyzed by a chemiluminescent assay. Results: In comparison to 17-estradiol (17-E2), the RBA’s of most ring B unsaturated estrogens were 2-8 fold lower for ER and ER, however, these unique estrogens had 2-4 times greater affinity for ER than for ER. The transcriptional activity of these 11 estrogens showed that all estrogens were functionally active. 17-estradiol induced the activity of SEAP by ER to a higher level than any other estrogen. Activity of other estrogens was 12 to 17% that of 17-E2. In contrast, 17-E2 stimulated the activity of ER to a 5 fold lower level than with ER. The activities of other estrogens mediated via ER were 66-290% that of 17-E2, with equilenin being the most active. Except for 17-E2, no correlation was observed between functional activities and the RBA’s for ERs. The activity of the ring B unsaturated estrogen components of CEE appear to be exerted predominantly through ER. To our knowledge, these are first such observations. Moreover, depending on the estrogen, ER can act as a dominant repressor or dominant activator of ER’s transcriptional activity. Conclusions: Taken together, these data indicate that all estrogens are not the same and have different pharmacology. Some ring B unsaturated estrogen components of CEE can via ER  inhibit the proliferative effects of 17-estradiol mediated via ER, thereby reduce the risk of breast cancer in women who are just taking equine estrogens alone.

Nanodevices for targeted delivery: An evaluation of toxicological models BHOGAL N FRAME, Russell & Burch House, 96-98 North Sherwood Street, Nottingham, UK Background: Engineered nanoparticles and liposomal delivery systems (nanodevices) are increasingly utilised and investigated as a means of overcoming problems with the bioavailability, stability and toxicity of therapeutics. Such systems promise to revolutionise clinical management and to obviate the need for needle-based self-administration. Methods: Some of the major challenges are described and the prospects for simplifying and stratifying toxicological assessment of nanodevices is considered. The importance of selecting the most appropriate toxicological models for biocompatibility testing is highlighted by the surge of new nanodevice-medicine combinations currently in the pipeline. Results: Nanomaterials display unique reactivities that are dependent on physicochemical properties, surface chemistry and biophysical and biological interactions. These reactivities are often very different from those of bulk materials and can be unpredictable owing to material heterogeneity. The estimation of the rate of drug release from nanodelivery devices, issues pertaining to bioretention are problematic. Differences in anatomical features between test species and human, including accessibility via natural barriers, targeting dependent on physical characteristics of tissue vasculature, immune cell density and lymphatic system characteristics present interesting challenges. For instance, it is clear that transdermal delivery is likely to depend on follicular transport. Yet animal models, with their vastly different follicle diameters are unlikely to give an indication of absorption through human skin. Similarly, it is clear that human skin is much more densely innervated and supplied with immune cells that most species. Thus, the relevance of animal models to assess the biocompatibility and as well bioavailability via this route of delivery is highly questionable. Whether human skin equivalents or cadaveric human skin can successfully replace animal studies is dubious, particular given that most organotypic models lack follicles, paracrine function and full barrier function. Conclusions: The application of existing animal and in silico approaches to resolving species differences and informing biocompatibility testing strategies of nanoparticles is questionable, calling for a review of methods for nanomaterial testing. Authors’ disclosure statement: FRAME is a scientific charity that conducts research into scientifically sound alternatives to animal testing that afford the highest standards of human health protection.

New derivatives of BM 212 with improved antimycobacterial activity. Biava M^1*, Porretta GC^1,Poce G^1,Pompei R^2, De Logu A^2, Manetti F^3, Botta M^3 ^1Università "La Sapienza", Rome, Italy. ^2Università degli Studi di Cagliari, Cagliari, Italy; ^3Università degli Studi di Siena, Siena, Italy. Background: According to the report 2007, compiled by the World Health Organization (WHO), the total number of new cases of Tuberculosis (TB) worldwide in 2005 had risen to approximately 8.8 million and there were approximately 1.6 million TB deaths during the same year. Increased infection with the M. avium complex (MAC) is also contributing to the morbidity and mortality in AIDS patients. The most urgent goal of chemotherapy of tuberculosis infections should be the development of highly active and low-cost drugs, which should be used not only in industrialized countries but also in developing ones in which both these infections are now rapidly increasing. As active molecules already introduced in therapy very soon generate resistance, scientists have focused their attention on the development of new antimycobacterial compounds acting with a mode of action without cross-resistance. Methods: Many pyrrole derivatives, analogues of BM 212, have been synthesized on the basis of previous results and molecular modelling considerations based on the pharmacophore model previously identified for them. All the derivatives were tested for their cytotoxicity and in vitro activity against many strains of M. tuberculosis, atypical mycobacteria, drug-resistant mycobacteria of clinical origin and intracellular mycobacteria. Protection Index (PI) was calculated and for the most active of them the bioavailability was also evaluated. The in vivo tests and the study of the mode of action are currently under study. Results: Some of the synthesized compounds revealed more active than BM 212 against mycobacteria. In particular the PI for many of them was comparable to that of reference compounds, Isoniazid, Streptomycin and Rifampin. Many of the synthesized compounds revealed also to be active against intracellular mycobacteria and they showed to inhibit drug-resistant mycobacteria of clinical origin. Conclusions: On the basis of our previous studies and molecular modelling considerations, many new derivatives of BM 212 were identified. In particular some of them revealed very active and low toxic so that they can be considered very promising for future studies.

The Insulin-Stem Cell Connection: What Insulin Does To Stem Cells And How We Get It From Them BIEBERICH E^1 ^1Medical College of Georgia, GA, U.S.A. Background: Replacement of pancreatic β-cells by stem cell-derived insulin producing cells (IPCs) is a promising strategy to treat diabetes. Therefore, generating IPCs has been a major goal of academic and industrial efforts to harness the differentiation potential of embryonic or adult stem (ES or AS) cells. To date, most differentiation protocols yield only a small proportion (<5%) of cells that genuinely produce sufficient amount of insulin in response to high glucose levels. A major obstacle in identifying true IPCs is that serum-free culture conditions require supplements with insulin to ensure survival of the differentiating stem cells. This exogenous insulin is taken up by the cells and confounds the determination of cell-derived insulin using antibody-based techniques. Methods: Our laboratory has developed, for the first time, a differentiation protocol replacing insulin with a recombinant peptide analog of insulin-like growth factor-1 (IGF-1). This IGF-1 analog-supplemented, serum-free medium (NeoITS medium) allows for the identification of genuine IPCs. Results: Using chemically defined NeoITS medium instead of insulin/transferrin/selenite (ITS) or serum supplement, we derived nestin(+)/C-peptide(+) and nestin(-)/C-peptide(+) cells at proportions of 40% or 25%, respectively. These IPCs increased insulin secretion by 20-fold when exposed to 20 mM glucose. The determination of secreted insulin was not possible when ES cells were differentiated in the presence of conventional ITS or serum. Conclusions: Insulin or IGF-1 are essential cell survival factors for differentiating stem cells. A recombinant peptide analog of IGF-1 in NeoITS can efficiently replace IGF-1 or insulin and allows for the determination of secreted insulin. Using NeoITS will tremendously facilitate the identification of early IPCs when differentiated from ES or AS cells (including induced pluripotent stem cells or iPS cells). We will now determine the potential of these IPCs to normalize glucose levels in hyperglycemic mice.

Liqiud Chromatography and Capillary Electrophoresis as a Tools to Study Ligand-Receptor Interactions BIELEJEWSKA A Institute of Physical Chemistry PAS, Kasprzaka 44/52, Warsaw 01-224, Poland The phenomenon responsible for enantioseparation in chromatographic and electrophoretic methods is the same; it is the enantioselective interaction between the enantiomers and a chiral selector. The principal difference between these two techniques arises from different separation process mechanisms and sometimes different environments of complexation The remarkable capacity of cyclodextrin (CD) for enantioseparation has been used to advantage in many chromatographic and electrophoretic applications The chromatographic and electrophoretic methods which are very sensitive to structure, size, shape and dynamics of the analytes have been used not only in separation science but also in the study of molecular recognition proceses. The enantiomeric separation of basic chiral pharmaceuticals such as pheniramine, brompheniramine, metoxyphenamine, cyclopentolate, doxylamine, and ketamine was investigated in capillary electrophoresis (CE) and liquid chromatography (HPLC) using negatively charged sulfated--cyclodextrin (s--CD) and neutral cyclodextrins (CDs). The apparent stability constants for the model compounds with cyclodextrins in both techniques were estimated. Both methods seem to be complementary for the study of complexation phenomena. It can be seen that brompheniramine forms stronger complexes with -CD than pheniramine and doxylamine. Complexation of pheniramine and doxylamine by -CD is very similar. The weakest complexes -CD forms with metoxyphenamine. For the studied compounds, TM--CD forms very weak complexes. The stability constant for DM--CD is very similar to that obtained for -CD. From the native CDs the best chiral selectors for the studied compounds are -CD and HP--CD. For the studied compounds the best recognition between enantiomers was obtained for cyclopentolate (K1/K2=1.32, K1/K2 =1.45 and K1/K2 =1.26 for -CD,for HP--CD and TM--CD, respectively) As the CE is the more efficient method, chiral recognition is better visible in this method than in HPLC. Conclusions: The obtained results shows that chromatographic and electrophoretic methods may be used as additional tools for studying weak interactions responsible for molecular recognition between ligand and receptor

Treatment Oucome And Management Of Acromegaly Before And After The Introduction Of Somatostatin Analogs A long term follow-up study BIERMASZ NR, PEREIRA AM, ROMIJN JA Leiden University Medical Center, Dept. of Endocrinology and Metabolism, Leiden, The Netherlands. Background: Acromegaly is a rare disease resulting from GH overproduction. The available treatments for acromegaly are surgery, radiotherapy and medical treatment. Somatostatin analogs (SMS) were the first effective drugs available sinds 1980s, first as three daily injection and from 1998 as monthly depotpreparation. This drug changed the treatment strategy of acromegaly, and is now used as the first option after unsuccessful surgery, but also as primary in stad of surgery in some patients. Methods: Long-term biochemical and clinical data are available of 164 patients diagnosed with acromegaly in the Leiden University Medical Center from 1978 onwards. We evaluated the long-term outcome of surgery, irradiation and SMS treatment in this cohort. Biochemical remission and clinical outcome, i.e. mortality, co-morbidity and quality of life parameters were evaluated. We also considered the effect of different treatment on detailed hormone secretion profile. We reviewed the effect of the introduction of SMS on the used treatment modalities in our patients, and in literature. Results: The surgical remission rate is 66 % in short term, but there was a recurrence rate of 15 % resulting in a final remission rate by surgery only of 54% after a mean of 12 years. Radiotherapy was associated with a long-term remission rate of 75 %, however, the duration until normalization of GH excess was long and there was a high incidence of hypopituitarism. Postoperative SMS treatment was well-tolerated and resulted in a remission rate of 60 %. An iv dose of octreotide well-predicted the long-term outcome of medical therapy. Detailed growth hormone secretion was normalized following successful surgery, but not after radiotherapy and during SMS. Mortality near-normalized, but quality of life remained impaired. Conclusions: The introduction of SMS analogs has changed the treatment algorithm of acromegaly. It is the preferred treatment in case of unsuccessful surgery and in this cohort of patients treated by surgery, radiotherapy and SMS analogs about 95% of patients achieve remission. At present equal biochemical remission rates are achieved with surgery and medical therapy only. Further study is required to assess the clinical outcome of this new treatment regimen.

Mannitol, a Key Probe Molecule in the Assessment of Small Intestinal Permeability? BIJLSMA PB^1, TAMINIAU JAJM^1, JODAL M^2 ^1Academic Medical Center, Amsterdam, The Netherlands; ^2University of Göteborg, Göteborg, Sweden. Background: The urinary recovery of orally ingested solutions containing Mannitol (mol.radius 4 Å) and Lactulose or Cr-EDTA (mol. radii 5 Å) is a widely used clinical test for small intestinal barrier function. In humans, recovery of Lactulose is  0.5 %, of Mannitol  20 %, giving a L/M recovery ratio of 0.025, which is very low compared to theorethically expected L/M diffusion ratios of 0.8 through aqueous pores Aims: To investigate the underlying mechanisms of this discrepancy by comparison of in vivo and in vitro probe permeability. Methods: 1) Small intestinal sheets from rodents (rats, guinea pigs, rabbits) and human intestinal biopsies were mounted in Ussing chambers and mucosa-to-serosa fluxes of L/M were determined (n = 4-8). Urinary recovery of orally applied probes was measured in rodents, cats and humans (n = 2-5). 2) Absorption of Cr-EDTA, Mannitol and water was studied in in situ perfused jejunal loops in anaestetized cats (n = 8), using four isotonic perfusion solutions with varying contents of NaCl and glucose. Results: L/M flux ratios in vitro were about 0.8 in all tested species (0.68 to 0.89). Urinary recovery L/M ratios in rodents ranged from 0.60 to 0.28. L/M ratios in cats and humans were 0.03 and 0.02, due to high mannitol recovery, resp. 29 and 22 %. In in situ perfused cat jejunum there was a strong positive correlation between water absorption and mannitol clearance (r = 0.99, p < 0.003), no correlation between water absorption and Cr-EDTA clearance (r = 0.05, p = 0.95). Likewise, there was a strong negative correlation between water absorption and Cr-EDTA/Mannitol ratios (r = 0.98, p < 0.02). Conclusions: Interspecies variation in urinary recovery of mannitol is caused by differences specific for the intact small intestine in vivo. Hyperosmolality of villus tips in vivo varies, being highest in humans and cats as a result of efficient vascular countercurrent multiplication because of their villus vascular anatomical structure. Thus we hypothesize that the high mannitol recovery in both species is caused by solvent drag through pores that allow the passage of Mannitol but not of Lactulose. The positive correlation between water absorption and Mannitol clearance in cat jejunum perfused with varying solutions which differentially affect the capability of the countercurrent multiplier mechanism confirms this hypothesis.

Safe and Effective Delivery of Paclitaxel through Amphiphilic Beta-Cyclodextrin Nanoparticles BILENSOY E^1 ^1Univ. Hacettepe, Fac. of Pharmacy, Dept. Pharmaceutical Technology, Ankara, Turkey Background: Paclitaxel is a potent anticancer drug associated with severe side effects due to the use of co-solvent Cremophor in its commercial injectable formulation. It is known that nanoparticulate drug delivery systems can provide an alternative for the effective delivery of anticancer agents due to the EPR effect facilitating the targeting of the encapsulated anticancer drug to tumor cells instead of healthy tissues. Objective of this study was to develop a nanoparticulate carrier system for paclitaxel with high encapsulation efficiency, controlled drug release property which may be more advantageous than cremophor vehicle in terms of safety and efficacy. Methods: Nanoparticles of amphiphiphilic ß-cyclodextrins loaded with paclitaxel were prepared using nanoprecipitation technique. Particle size distribution and zeta potential of the nanoparticles were measured by QELS technique. Encapsulation efficiency of amphiphilic cyclodextrin nanoparticles were measured as well in vitro release profiles for paclitaxel with HPLC assay. Safety of the nanoparticles (nanocapsules and nanospheres) were assessed in terms of both hemolysis and cytotoxicity to L929 cells in comparison to commercial injectable formulation. Anticancer efficacy of paclitaxel loaded nanospheres and nanocapsules were evaluated by MTT assay against human breast cancer cell line MCF7 with MTT assay. Results: Size for nanocapsules and nanospheres were found to be around 350 nm and 180 nm respectively and was found to be stable for a storage period of 12-months as seen in Figure 1. Encapsulation efficiency was increased by 2 to 3 fold by incorporating paclitaxel into cyclodextrin nanoparticles. The drug was released within 6 hours. Hemolytic order was found to be Cremophor vehicle>nanocapsules>nanospheres. Erythrocytes were imaged by SEM after treatment with paclitaxel loaded formulations. Cytotoxicity of blank nanoparticles were signficantly lower than cremophor commercial vehicle as paclitaxel demonstrated equal anticancer efficacy in nanoparticles. Figure 1. AFM photomicrograph of paclitaxel loaded amphiphilic cyclodextrin nanoparticles Conclusions: Amphiphilic cyclodextrin nanoparticles can be considered as an alternative dosage form for injectable paclitaxel in terms of safety and efficacy. Authors’ disclosure statement: Author would like to acknowledge Hacettepe University Research Fund Grant 0202301005 for financial support of this study.

Two Passengers on the Cancer Road: Clomipramine and Lithium Chloride BİLİR A ^1, ERGUVEN M ^2, YAZIHAN N ^3 Istanbul University, Istanbul, Turkey ^1Istanbul Faculty of Medicine, ^2 Istanbul Faculty of Medicine and Faculty of Pharmacy; ^3Ankara University, Faculty of Medicine, Ankara, Turkey. Background: The aim was to investigate whether a tricyclic anti-depressant clomipramine (CIM) and an anti-psychorotic lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) at SHSH-5Y human neuroblastoma cells in vitro besides their conventional mode of action. Methods: The IC50 values of VNR, CIM and LiCl were determined as 8 , 14.23 and 200 μM. Four sets of experiments were performed for 96 hrs both for monolayer and three dimensional (spheroid) cultures of SHSH-5Y cells. These were i) Control group, cells treated with ii) Singly applied VNR, CIM and LiCl, iii) VNR with CIM, and iv) VNR with LiCl.Their effects on monolayer cultures were determined by the evaluation of cell proliferation, the percentage of cells in S-phase by BrDU-LI, apoptosis by Annexin V-FITC/PI staining, and cAMP levels by RIA; on spheroid cultures by evaluation of the percentage of cells in S phase, spheroid size, and the ultrastructure by TEM. Results: In comparison to the control group, single and combination drug medications significantly reduced the proliferation index (PI) for 96 hrs. The most potent reduction of PI was observed at VNR with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective to reduce BrDU-LI of both monolayer cell and spheroid cultures, spheroids size, cAMP levels. VNR with LiCl increased apoptosis potently at 24 hrs, however VNR with CIM increased apoptosis at 96 hrs. In ultrastructural evaluation of spheroids, increased presumably autophagic vacuoles, mitochondria damage and debris of lytic cells in extracellular area were observed at VNR with CIM. Striking data were catched at VNR with LiCl applied spheroids. The VNR applied spheroids revealed intact nuclear and cellular membranes. LiCl led to nuclear membrane breakdown at nearly all cells of spheroids. When VRL was used with LiCl, in addition to nuclear membrane breakdown, the cellular membranes inside spheroids disappeared and the cellular membranes were appeared as an unique membrane constructions only in spheroid surface. As a result of this, one spheroid resembled one giant cell. Conclusions: 1) Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity with few exceptions. 2) Interestingly, VNR with LiCl led to selective nuclear and cellular membranes destruction. This effect could be due to the premature activation of cyclin dependent kinase 2.

Analysis of drug-receptor interaction at equilibrium BINDSLEV N  Synagics lab  Panum Institute  University of Copenhagen  Denmark Aim: We want to analyze our dose-response data at equilibrium by models. We may have an idea about a possible physical interpretation of obtained response curves and use either mathematical or mechanistic descriptions. Background: From 1901 to 1910, receptors as separate entities were recognized by Elliott (1904) and Langley (1905) in Cambridge and Ehrlich (Frankfurt 1907). Deviation from a simple Langmuirian hyperbole (1918) was documented by C. Bohr (Copenhagen 1904) for oxygen-hemoglobin binding. Mono-ligand systems: For mono-ligand systems with dose-responses deviating from a simple hyperbole, Hill=s equation can give you a quantitative measure of co-operativity; but without assessing possible interactions between binding sites. Therefore, co-operative values from a Hill scheme most likely has no physical correlate. For a physical description of systems deviating from simple hyperbolism, I suggest the homotropic two-state model, HOTSM (2004). This model can handle both positive and negative co-operativity as well as bell-shaped and reverse bell-shaped relationships. Two-ligand systems: Models for two ligands may again be based on either a Anon-interaction@ scheme such as the non-competitive inhibition scheme without two-states of the un-liganded receptor or on an extended Monod-Wyman-Changeux scheme (Monod et al 1965; Rubin & Changeux 1966) with two-states. Both these approaches assume no interaction between binding sites, i.e., upon binding of a ligand there is no change in the binding constant for a second drug. Instead, for combinatorial drug effects with assumed site interaction, analysis of data may be performed by the ternary complex model, TCM, a one-state scheme for two ligands with site interaction (Ehlert 1988), or alternatively by the allosteric two-state model, ATSM, with site interaction (Hall 2000). ATSM implementation: As one of the first, Jäger et al have implemented the ATSM (2007). Thus, physical effects are quantified for naphmethonium on acetylcholine binding and on pilocarpine activation of an M2 receptor subtype. Lately, Ehlert-Griffin (2008) have extended the ATSM with an additional ligand. A tri-ligand two-state model. Conclusion: Depending on the synagic system to be analyzed, i.e., the actual dose-response relationships at equilibrium, we may choose between Hill, Hall or other models. A critical choice is crucial for our extraction and use of relevant mechanistic parameters.