Ehrlich II –2nd World Conference on Magic Bullets

Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli ALI FAKHIMI AND AHMAD REZA SHAHVERDI Department of Pharmaceutical Biotechnology and Medical Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155/6451, Tehran, Iran Abstract Silver nanoparticles have been known to have inhibitory and bactericidal effects. Resistance to antimicrobial agents by pathogenic bacteria has emerged in recent years and is a major health problem. The combination effects of silver nanoparticles with the antibacterial activity of antibiotics have not been studied. Here, we report on the synthesis of metallic nanoparticles of silver using a reduction of aqueous Ag+ ion with the culture supernatants of Klebsiella pneumonia. Also in this investigation, these nanoparticles were evaluated for their part in increasing the antimicrobial activities of different antibiotics against Staphylococcus aureus and Escherichia coli. The antibacterial activities of penicillin G, amoxicillin, erythromycin, clindamycin and vancomycin were increased in the presence of silver nanoparticles against both test strains. The highest enhancing effects were observed for vancomycin, amoxicillin and penicillin G against Staphylococcus aureus.

Effect of gentamicin on serum digoxin level in patients with congestive heart failure. ALKADI HO, MAJED AN, YAHIA AR Univ. Sana'a , Sana'a, Yemen Background: Gentamicin is frequently used to treat infectious diseases in patients receiving digitalis therapy. The aim of this study is to evaluate the effect of gentamicin on serum digoxin level. Methods: Twenty-four diabetic patients and patients with congestive heart failure and twelve normal healthy volunteers were enrolled in this study. The patients received digoxin treatment 0.25 mg/day. Gentamicin in a dose of 80 mg i.m. twice a day for 7 days was prescribed for these patients to treat chest infection. Serum digoxin and creatinine levels were determined before and after gentamicin administration. Results: Gentamicin induced a significant increase in serum digoxin level of diabetic patients and patients with congestive heart failure. Serum creatinine level increased significantly before and after i.m. injection of gentamicin. Conclusions: The present study indicated that increase serum digoxin level when combined with gentamicin should be considered a risk factor for digitalis toxicity.

Controlling of Systemic Absorption of Gliclazide through Incorporation into Alginate Beads. AL-KASSAS RS^1, AL-GOHARY OM^2, AL-FAADHEL MM^2 ^1Univ. AUK, Auckland, New Zealand; ^2Univ. KS, Riyadh, Saudi Arabia Background: Gliclazide is an oral hypoglycaemic second generation sulfonyl urea drug which is useful for a long term treatment of non insulin dependent diabetes mellitus. However, the absorption rate of gliclazide from gastrointestinal tract is slow and varied among the subjects. Several studies on healthy volunteers and diabetic patients revealed that the time to reach plasma concentration (tmax) ranged from 2 hours to 8 hours following a single oral administration of 80 mg of gliclazide tablet. Aims: (1) to develop controlled release formulations of gliclazide using alginate beads. (2) to study the effect of processing conditions on the physical characteristics of the prepared beads and the in vitro release rate of the drug (3) to investigate the hypoglycaemic effect of the prepared gliclazide loaded alginate beads on the diabetic rabbits and to compare the developed systems with marketed conventional gliclazide tablets. Methods: Gliclazide loaded Ca-alginate beads were prepared using ionotropic gelation method. The variation in polymers concentrations, stirring speed, internal phase volume and the type of surfactants in external phase were examined systemically for their effects on particle size, incorporation efficiency, flow properties of the beads and in vitro drug release rates. The in vivo studies involved measuring blood glucose level of 18 diabetic male New Zealand white rabbits treated (6 rabbits per treatment) with the following formulations (1) normal saline solution (2) marketed conventional gliclazide tablet (Gliclazide ®) (3) gliclazide loaded alginate beads. Results: The average mean diameters of gliclazide beads decreased with decreasing polymer concentration, increasing speed of stirring, and increasing the internal phase volume. All prepared beads possessed excellent flowability. The swelling behaviour was strongly dependent on polymer concentration in the formulation and the pH of the medium. The in vitro release experiments revealed that the swelling is the main parameter controlling the release rate of gliclazide from the beads. In vivo studies on diabetic rabbits showed that the hypoglycaemic effect induced by gliclazide loaded alginate beads was significantly greater and more prolonged than that induced by the marketed conventional gliclazide tablet (gliclazide ®). Conclusions: Alginate beads can control, improve and prolong the systemic absorption of the gliclazide through their mucoadhesive properties. This effect results in maintaining tight blood glucose level and improved patient compliance.

Pharmaceutical Analysis using Sequential Injection Analysis (SIA): A review of present Applications and future possibilities Al-KINDY SMZ, SULIMAN FO Sultan Qaboos University, Muscat, Oman Background: The need for automation in pharmaceutical analysis has lead to the development of rapid and sensitive detection methods. Our aims are to develop robust and simple sequential injection analysis (SI) methods for the essay of pharmaceutical samples and to investigate various luminescence detection modes in combination with SIA system. The approaches compared will be spectrophotomety, micellar enhanced fluorescence, and lanthanide enhanced luminescence and chemiluminescence (CL). Method: The development of analytical protocols for monitoring of various drugs is presented. The chemical system is developed based on the structural properties of the given drug. When compounds lack efficient chromophores the use of a metal ion or organic derivatizing agents that can form highly absorbing or fluorescent products is necessary. On the other hand lanthanides such as Eu(III), and Tb(III) are used to sensitize the luminescence of drug samples offering excellent analytical characteristics. CL is also emerging as an efficient tool when coupled to flow techniques. Ru(III) complexes were used to develop simple and robust assay for group of common drugs such as chlorpheniramine (CPA) and ephedrine(EP) and some fluoroquinolone antibiotics. Several types of SI designs were employed. The development of the analytical system thereafter hinges on the selection of the most suitable environmental factors that result in an enhanced signal-to-noise ratio. Therefore a systematic optimization protocol must be used for this purpose. Then the methods developed are validated and compared to standard and official methods. Results: using spectrophotometric techniques penicillamine (PA) was complexed with Fe(II) ions in acidic media forming blue complex that absorbs strongly at 600 nm. A linear dynamic range for the determination of PA of 25-300 ppm was obtained with sampling frequency of 50 h ^-1. PA and ephedrine were determined using tris(bipyridyl)ruthenium(II) as a CL reagent and potassium peroxydisulfate as an oxidant in the presence of light.. Derivatization of PA and EP with aldehydes produced a significant enhancement of the CL emission, leading to detection limits (LOD) of 0.1ppm for PA and 0.03 ppm for EP. Optimum conditions for the determination of BRZ in pharmaceutical formulation were 0.6 % tergitol surfactant in the presence of 0.1 M lactose. Piroxicam (PX) and ibuprofen (IB) were assayed using lanthanide sensitized luminescence. Eu(III) when complexed to PX resulted in a huge enhancement in the emission of the EU(III) bands.. which allow the determination of 100–1000 ppb of PX with LOD of 29 ppb. Recoveries of PX in pharmaceutical formulations and in urine samples were 100.87±1.7% and 97.57±2.0%. IB was determined after complexation with Tb (III) ions giving a detection limit of 1.0 × 10^−7 mol/L. Fluoroquoinolone antibiotics eg levefloxacin ( LV), were determined using Ce(IV) ions as oxidant and tris(1,10-phenanthroline)ruthenium(III) as the CL reagent giving characteristic orange emission with LOD of 0.02 ppm. Conclusions: SIA is a powerful tool when coupled to sensitive luminescence methods for the determination of drugs in the pharmaceutical formulations and in biological fluid. This combination results in an increased sampling frequency and an enhanced sensitivity. The use of micelles has lead to an increase in the solubilisation of BRZ and an improvement in the sensitivity of the method. Lanthanide sensitized luminescence lead to a sensitize detection of the drugs with emission in the longer wavelength of the lanthanide ion that achieve a better selectivity of the methods. Future work include the development of methodology for the determination of drugs in various environmental samples such as round water and sewage, in order to cope with the increase problem of pollution brought about by the continuous charging of drugs into the environment.

Clinical Pharmacokinetics of Gentamicin: Estimation of Initial Dosing Parameters in Hospitalized Patients at Kuwaiti Hospital. AL-LANQAWI Y^1, CAPPS P^2, ABUDLMALEK K^3, AL-ANEZI K^4, THUSU A^5, AND SHARMA P ^1, 3, ^4, ^5AL-Amiri Hospital, Ministry of Health, Kuwait, and ^2,6Faculty of Pharmacy - Kuwait University, Kuwait. Background: Gentamicin has a narrow range between its therapeutic and toxic blood levels. This has prompted the development and wide use of pharmacokinetic dosing equations in order to maximize drug safety and efficacy. Dosing equations commonly rely on estimating gentamicin clearance (Clgent) and volume of distribution (Vd). These parameters are subject to considerable variability’s. The objective of this study was to: (a) develop equations for estimating Clgent and Vd based on Kuwaiti population and (b) evaluate these equations by comparison with other methods in their predictive ability to estimate Clgent and Vd. Methods: Clgent and Vd were calculated in 47 patients (group 1) using the Sawchuk-Zaske method. Regression analysis was used to derive a correlation between creatinine clearance (Clcr) and Clgent, Vd and actual body weight (ABW). Based on actual Clgent and Vd values, the predictive ability of the estimated parameters from the regression equations was validated and compared with 4 methods, using mean error (ME) (bias), mean squared error and root mean squared error (MSE and RMSE, respectively) (precision). All equations were also evaluated in an independent second group (group 2) of 23 patients. Results: (a) The mean (±SD) for Clgent and Vd, was 4.0 (±1.8) L.h^-1 and 16.8 (±6.7) L, respectively. (b) The derived equations were: Clgent = (0.760) (Clcr)+1.117 (r = 0.701) and Vd = (0.165)(ABW)+5.604 (r = 0.532). In comparison to the four published methods, the derived equations were found to be less biased (ME=0.00), and more precise (MSE=1.68, RMSE=1.02) in predicting Clgent (p<0.05), and less biased (ME=-0.01) with no difference in precision (MSE=36.22, RMSE=4.59) in predicting Vd, (p>0.05). This precision was confirmed in the second group of 23 patients, where the derived equations were less biased (ME=-0.1) and more precise (MSE=3.22, RMSE=1.48) in predicting Clgent (p<0.05) and no differences were found for prediction of Vd (p>0.05). Conclusion: The equations developed in this study provided a reliable estimation of Clgent and Vd. It is planned to use them at Kuwait Hospitals help provide more individualized patient dosing information to physicians.

Complexation of Itraconazole with Cyclodextrins for Enhanced Solubility, Dissolution and Bioavailability AL-MARZOUQI AH^1, JOBE B^1, SHEHATTA I^2, HASSAN HA^1, HAMZA AA^1 ^1UAE University, Al-Ain, United Arab Emirates; ^2Mansoura University, Mansoura, Egypt. Background: Itraconazole is an antifungal agent whose poor aqueous solubility restricts its use for the treatment of oropharyngeal candidiasis, which is the first symptom of HIV infection. Therefore, the aim of this study was to prepare itraconazole-cyclodextrin inclusion complexes in solid state by different methods in order to enhance the solubility, dissolution and bioavailability of itraconazole. Methods: The formation of inclusion complexes between itraconazole and α-, β-, γ-, and hydroxylpropyl-β-cyclodextrin (HP-β-CD) were assessed using phase solubility techniques. Solid state inclusion complexes between itraconazole and cyclodextrins were prepared using supercritical (SC) CO2 and conventional methods. The physico-chemical properties of the products were characterized by UV, DSC, FTIR, PXRD and SEM. Dissolution amounts of the products obtained by different methods were measured in gastric fluid. Finally, pharmacokinetic studies of the inclusion complexes were conducted in blood, liver and kidney of male Wistar rats to assess the bioavailability of the prepared complexes. Results: The aqueous solubility of itraconazole increased linearly as a function of cyclodextrin concentration according to the rank order: HP-β-CD > β-CD > γ-CD > α-CD. Inclusion formation was influenced by the preparation technique. Products obtained by the SC CO2 method were among the ones showing the highest interaction between itraconazole and the CDs, leading to about three times higher dissolution amounts than pure itraconazole. Temperature, pressure and drug:CD ratio had significant effects on the inclusion yield. In vivo drug pharmacokinetic studies showed that the itraconazole-β-CD product prepared using SC CO2 results in higher bioavailability of itraconazole than those obtained by physical mixing or coprecipitation methods. Conclusions: Cyclodextrins significantly improved the solubility of itraconazole in aqueous solutions, which should improve the therapeutic effects of itraconazole against antifungal infections. SC CO2 method proved to be an effective technique for preparing solid complexes between cyclodextrins and itraconazole. Since SC CO2 method has no toxic solvent residue, products obtained by this method should provide minimal side effects in humans.

Pathogenesis of Osteoarthritis: possible target molecules for new therapeutic strategies. ALMONTE-BECERRIL M, CRUZ R, GONZALEZ EC, MIRANDA M, ROJAS-ORTEGA M, KOURI JB Investigation center and advanced studies of National Polytechnic Institute (CINVESTAV-IPN); México, DF. Background: Changes in the morphology and function of the chondrocytes during the Osteoarthritis (OA) progression can be related with the expression of molecules involved in the inflammatory process. Some of these molecules could be an important target to avoid, retard or even stop the progression of OA. Aims: 1) To describe the chondrocytes phenotype during early OA. 2) To identify possible target molecules involved in phenotype changes in OA chondrocytes. 3) To identify programmed cell death of OA chondrocytes. Methods: The experimentally OA-induced model was accomplished by unilateral knee menisectomy and post-surgery training; normal rats were used as a control. Right femoral condyles were removed and processed for either electron microscopy (EM) or Immunohistochemistry (IHQ). Samples from rats with 1 to 5 training days (td) were used to evaluate ultrastructural changes by EM. Moreover, cartilage from rats with 3, 6 and 10 td were analyzed by IHQ for IL-1, IL-10, TGF-1 and TNF-. Finally, programmed cell death was identified with TUNEL, caspasa 3 and LC3II at 20 and 45 td. Fluorescent signals were analyzed by confocal microscopy. Results: EM revealed changes in the superficial zone chondrocytes, where cell phenotype changed from elongated to a rounded shape, in addition, the cells developed prominent endoplasmic reticulum (ER) with dilated cisterns and enhanced Golgi membranes. Expression of the proinflammatory cytokines IL-1β and TNF-α and began at 6 td, in the superficial zone and reached their highest levels at 10 td in all the cartilage zones. TGF-1 showed diminished expression at 6 and 10 td. IL-10 was constant in all samples. Finally the cell death analysis, showed a co-expression of autophagic and apoptotic mechanisms. Conclusions: Our results suggest that in early OA, chondrocytes changes its phenotype in order to synthesize proteins required for extracellular matrix (ECM) repairment. However, when its capacity is overwhelmed, chondrocytes begin the synthesis of catabolic molecules like IL-1β and TNF-α that stimulate an inflammatory process. Furthermore, the decrease of anti-inflammatory molecules could be involved in the beginning of the OA. Finally, the chondrocytes execute its own process of cell death, that include both autophagy and apoptosis.

Molecular Dynamics Simulations of human membrane transport proteins from the Major Facilitator Superfamily. ALMQVIST J (A), HUANG Y (B), HOVMÖLLER S (A), LAAKSONEN A (C) AND WANG D-N (D). (a) Division of Structural Chemistry, Arrhenius Laboratory, Stockholm University, Sweden. (b) Department of Molecular Biology, Uppsala Biomedical Center, Swedish University of Agricultural Sciences, Sweden. (c) Division of Physical Chemistry, Arrhenius Laboratory, Stockholm University, Sweden. (d) Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, USA. The Major Facilitator Superfamily (MFS) constitutes the largest group of secondary active transporter proteins, with members ubiquitous in all kingdoms of life. Recent crystal structures of bacterial MFS proteins - including the Oxalate transporter, Glycerol-3-phosphate transporter, Lactose permease and the multidrug resistance protein D - has revealed a common structural architecture. This MFS fold features two transmembrane domains composed of six helices each, connected with a cytosolic linker region of approximately 30 amino acids. Human MFS transporter structures, however, have not yet been obtained. From a pharmaceutical standpoint, the human MFS transporters are of significant interest. This talk will present our studies on two human MFS proteins; (1) the glucose-6-phosphate transporter (G6PT), directly involved in the glycogen storage disease type 1b and (2) the vesicular glutamate transporter (VGLUT1) from the membranes of synaptic vesicles located in excitatory neural cells in the mammalian central nervous system. In the absence of structural data for these important human proteins, we have modelled atomic structures of G6PT and VGLUT1 from high-resolution crystal structures of bacterial homologues, and carried out extensive Molecular Dynamics simulations to better understand fundamental questions related to substrate binding, inhibition and conformational change of human MFS proteins.

Verapamil Reverts Acute Renal Functional Impairment Induced by Angiotensin II Converting Enzyme Inhibitors ALVAREZ GREGORI JA^1,2, BOUCHOUTROUCH Y ^2, MACIAS NUÑEZ JF ^2 ^1 CS. Casto Prieto. UDMFYC. SACYL. Salamanca; ^2Faculty of Medicine. University of Salamanca Background: Antihypertensive agents have been found effective in arresting glomerulosclerosis. Initially, it was thought that the healthy effect of these drugs was exclusively due to their hemodynamic effects. However, it has become clear that nonhemodynamic actions of these agents are an important component of their beneficial effects. Among thepharmac ological agents that may have a favorable influence in the course of renal failure, angiotensin converting enzyme (ACE) inhibitors, and calcium channel blockers (CCB) have generated the most interest. Angiotensin-converting enzyme inhibitors (ACEI) have proven to be effective drugs for the treatment of hypertension and represent a major therapeutic breakthrough in the management of hypertension and renal function preservation in diabetic and nondiabetic nephropathies. In some patients, ACEI may induce a rapid deterioration of renal function, assessed as an increase in serum creatinine (SCr), which can be reversed by withdrawing the drug.In these cases, maintaining the renal protection due to ACEI, instead of withdrawing these drugs could be desirable. Calcium antagonists are a heterogeneous group of agents with diverse effects in terms of nephroprotection. Some of these differences relate to their effects on renal microcirculation. Dihydropyridine agents appear to act only on the afferent arteriole, increasing intraglomerular pressure, and albumin excretion rate. In contrast, nondihydropyridine agents like verapamil, may dilate efferent arterioles in addition to afferent arterioles and with normalization of the systemic blood pressure, verapamil may reduce intraglomerular pressure, and proteinuria. However, some other non hemodynamic protective effects of CCB could be explained by its capacity to inhibit the extracellular calcium influx, an important signal for the proliferative effect of mesangial cells mitogens, its influence in the decrease in mesangial entrapment of macromolecules and, possibly, its effect as free radical scavenger Münter et al. describe that nephroprotective effects of ACEI/CCB combination can occur at doses, which do not significantly alter systemic blood pressure in the stroke-prone SHR. We have described that the combined therapy with these agents provide in the remnant kidney model a synergistic effect in preventing renal injury, independently of their effects on blood pressure.In a preliminary study, we have demonstrated in a small group of patients that nondihydropiridin (nonDHP) CCB are able to revert renal function reduction associated to ACEI treatment. The main purpose of this study was to assess the efficacy and safety of low doses of verapamil (180 mg/day) added to the previous ACEI treatment for reverting decreased glomerular filtration rate observed in patients treated with ACEI. A secondary purpose was to test the ability of the fixed combination Verapamil-SR 180 mg plus Trandolapril 2 mg in attaining BP control and maintaining there renal function throughout the study. Methods: This was a multicenter, nonrandomized, prospective, open study developed in five Spanish Hospitals. All patients were referred from the outpatients Departments of Internal Medicine and Nephrology. The Institutional Review Boards approved the study protocol and each patient entering the study signed a written informed consent. Eligible patients presented a previous diagnosis of hypertension and an increase in SCr of 20% or 45 mmol/L from last values, in the course of ACEI treatment for more than four weeks. Exclusion criteria were renal insufficiency, defined as SCr >354 mmol/L (4 mg/dL), stroke, AMI in the last three months, unstable angina, cardiac failure, other causes of renal hypo-perfusion, or low volume syndrome such as dehydration, vomiting, diarrhea, laxative, and known hypersensitivity to verapamil. In the selected group, a clinical evaluation was carried out, including clinical history, physical examination, BP measurement, ECG, and biochemistry (Visit 1). Patients fulfilling the inclusion/exclusion criteria were enrolled in the study. Three to ten days later a new BP measurement, serum, and urine creatinine and 24 h proteinuria were also measured (Visit 0 or Baseline) and creatinine clearance (CrCl) calculated. If the SCr was not higher than 10% respect to Visit 1, the patients were included in the study and Verapamil (180 mg/day) was added. Patients returned to the hospital after four weeks for new measurement of SCr and BP (Visit 1). If an increase on SCr superior to 10% respect to Visit 0 or BP was _140/90mm Hg was detected, ACEI was discontinued, and the patient withdrawn from the study. If not, treatment was continued for eight weeks. At this moment (Visit 2) patients were evaluated again. If SCr had increased more than 20% with respect to baseline, or BP was 140/90mmHg, the patients were withdrawn. If not, the patients were placed on the Trandolapril (2mg)-Verapamil (180 mg) association for eight more weeks. Patients were followed until 20 weeks of follow-up were completed. All the patients were recommended to limit sodium intake. At each study visit, blood pressure was measured three times at 2 min intervals after 10 min rest in the sitting position, using calibrated mercury sphygmomanometers. Blood pressure values were estimated as the mean of the three readings. The mean value obtained in Visit 0 was accepted as BP basal level. Biochemistry and SCr (Jaffe´ reaction) were determined in an automatic analyzer (Hitachi 747). Creatinine clearance was calculated as: urinary creatinine (mg/L) urine volume (mL)/serum creatinine (mg/L)/1.73m2.

Anti-tumour Vaccination in Advanced Malignancy with Class I & II hTERT Peptide-pulsed Dendritic Cells (DCs) Generates Suboptimal Antigen Specific CD8+Cytotoxic T Cell (CTL) Responses and Induces Regulatory T Cells in the Circulation ALOYSIUS MM^1, VERMA C^2, ROBINS RA^2, EREMIN JM^3, SREENIVASAN T^3, FARZANEH F^4, HARDWICK N^4, HABIB N^5, KECHNIE AMC^1, HARRISON PR^6, EL-SHEEMY M^4, AND EREMIN O^1, 4 ^1Section of Surgery and ^2Institute of Infection and immunity, School of Molecular Medical Sciences, Nottingham University Hospitals, University of Nottingham, NG7 2RD, UK. ^3Lincoln Oncology Centre and ^4Research and Development Department, Lincoln County Hospital, Lincoln, UK. ^4Department of Haematological & Molecular Medicine, Rayne Institute, King’s College, 123 Coldharbour Lane, London, SE5 9NU. ^5Surgery Section, Department of Surgical Oncology and Technology, Imperial College London, Du Cane Road, London, W12 0NN, UK. ^6The Liver Unit, King's College Hospital, Denmark Hill, London SE5 9RS, UK. Purpose: Human telomerase reverse transcriptase (hTERT) is expressed in >85% of human cancers and is becoming an increasingly popular molecular target for anticancer immunotherapy. We have previously shown that autologous DCs pulsed with class I epitopes of hTERT are capable of generating hTERT tetramer+, CD8+ CTLs in circulation of advanced cancer patients, albeit short-lived. By including CD4+cognate T cell help in the vaccination, through pulsing autologous monocyte-derived DCs with class II promiscuous hTERT peptides, we aimed to optimize the hTERT+, CD8+ CTL response in magnitude and duration. Experimental Design: A Phase I clinical trial in advanced cancer patients was performed to evaluate the immunological and clinical impact of vaccinating (6 occasions, 2 to 3 weekly) advanced cancer patients with the HLA-A2-restricted class I hTERT epitopes I540 & I865 (n=10), with or without class II epitopes 766 & 675 (n=5), pulsed on autologous monocyte-derived ex vivo generated DCs. Results: Peptide/MHC tetramer-CD8+ CTLs and CD4+CD25+ foxp3+ T cells were tracked sequentially through the vaccinations. Four patients with advanced cancer (prostate, renal, head & neck and colon) received an average (range) of 8.5 (6-12) vaccinations. . hTERT tetramer+, CD8+ CTLs were induced in 13/15 patients, with circulating tumour marker level reductions in 4/6 patients with prostate cancer. These were only short-lived and declined despite continuing vaccination. T regulatory cells (CD4+CD25+foxp3) tended to increased in the circulation during vaccination and showed a negative correlation with tetramer+ CD8+ CTL levels. Conclusions: These results demonstrate that vaccination with autologous monocyte-derived DCs pulsed with hTERT class I & II peptides was unable to induce an optimal and sustained tetramer+, CD8+ CTL response but generated T regulatory suppressor cells in the circulation.

All the basal variables were analyzed descriptively. Data are given as Mean ± standard deviation. Adverse events are codified according to the WHO Adverse Reaction Terminology List (WHO-ARTL). Repeated measurements ANOVA was used with an exploratory approach in order to test the following null hypotheses: changes of creatinine, systolic, and diastolic blood pressure, uric acid and potassium over time. The Wilcoxon test (two tailed) for paired samples was used to test the following hypotheses: changes of creatinine clearance and albuminuria between inclusion and week 12. Changes in creatinine between Visit 1 and inclusion, inclusion and week 12, inclusion and week 20, week 12, and week 20, historic control, and week 20. All the p-values are exploratory as type I error has not been adjusted for multiplicity. Results: Forty-six patients, 23 female and 23 male, with a mean age of 54.9±7.3 years and BMI of 27.3±2.2 kg/m2, were included. Five of them (10.9%) had suffered from cerebrovascular disease, eight (17.4%) coronary heart disease, 24 (52%) had left ventricular hypertrophy on ECG or echocardiography, seven (15%) lower limbs peripheral vascular disease, and 35 (76%) some degree of retinopathy. Fourteen patients had been diagnosed from diabetes 4.5±8.0 years earlier (a median of 2.6 years): one was treated with diet, 12 with oral anti-diabetics drugs, and one with insulin. They were known hypertensive for a median of 4.5 years and all of them had been treated with ACEI for at least four weeks previous to SCr deterioration. Previously used ACEI were: enalapril in 12 patients, Lisinopril in nine, Cilazapril in seven, Perindopril in five, Quinapril in five, Ramipril in four, Trandolapril in two, and Captopril in two. Nine patients were withdrawn from the study, three at week four and six at week 12. Eight were withdrawn due to no BP control and no treatment compliance in one patient. In one patient, the reason was no BP control plus SCr increase>20%. A total number of 37 patients finalized the study. The evolution of the main clinical and biochemical parameters is shown in the following Table. All the 46 patients were considered for the safety analysis. Throughout the study, 13 patients reported 19 adverse events. Symptoms were mild/moderate in intensity in all cases. Any patient interrupted the study because of adverse events . Conclusions: The major finding of this study is the possibility of reverting renal functional impairment induced by ACEI treatment in hypertensive patients by adding verapamil without further modification in ACEI therapeutical regime. These patients had normal serum creatinine levels before ACEI treatment. ACEI have been demonstrated to reduce morbidity and mortality in patients with heart failure. Renal impairment is the most important factor associated with prescription of lower-than-recommended doses. A recent review of 12 randomized clinical trials evaluating renal disease progression and ACEI-based therapy, shows that an acute increase of serum creatinine in the first two months usually follows the ACEI therapy in patients with preexisting renal failure. This is not our case, as the patients in our study had normal values of plasma creatinine before ACEI treatment. The review also shows that a limited elevation, of up to 30%, is strongly associated with long-term preservation of renal function when ACEI therapy is continued. In our study, we have observed increases in plasma creatinine up to 50% respect to pretreatment levels. These increases were completely reverted by adding verapamil to the ACEI treatment. Some calcium antagonists seem to offer additional benefit in hypertensive patients when renal function is impaired, and may reduce proteinuria in hypertensive diabetic patients. The deterioration of renal function in patients receiving ACE-inhibitors seems to be due to a decrease in intraglomerular pressure, and, subsequently in the filtration net pressure. Thus, as angiotensin II has a predominant vasoconstrictor effect in the efferent arteriole, the decrease in angiotensin II due to ACE inhibition leads to an efferent arteriolar vasodilatation, and subsequently to a decrease in glomerular capillary pressure. This is one of the major beneficial effects of ACE inhibitors, as increased capillary pressure is a major cause of the progression of renal failure, but if the decrease on glomerular pressure is below normal values, glomerular filtration rate will be also decreased. However, it is difficult to accept this as the only cause of filtration rate decrease after ACEI treatment, because the autoregulation threshold is not always reached. If it is overpassed, an immediate deterioration in renal function must occurs at least in absence of decreased effective arterial volume, by far the commonest cause of acute rise in serum creatinine. The additive effect of verapamil and ACEIs could be owing to that the two drug types protect the kidney by different mechanisms. Dworkin et al. have reported that either enalapril or nifedipine reduce renal injury in the remnant kidney model by different mechanisms: nifedipine reduces glomerular hypertrophy whereas enalapril reduces glomerular hypertension. Verapamil is able to diminish renal vasoconstriction mediated by BK receptor activation, prevent intrarenal vasoconstriction mediated by adenosine, and prevent from mesangial cells contraction and proliferation induced by several agents. Verapamil is also able to stimulate NO release. The addition of verapamil to trandolapril treatment might promote other beneficial effects on renal structure and function. Thus, CCB are able to inhibit platelet aggregation, and this inhibition has been reported to ameliorate glomerular injury in rats with reduced renal mass. In addition, Harris et al. have shown that verapamil lessened oxygen consumption in the isolated, perfused remnant kidney and, therefore, it might ameliorate glomerular injury by reducing damaging oxygen radicals production rate. The results of this study confirm our previous preliminary data suggesting a therapeutical approach, which allows revert the renal failure induced by ACEI treatment in hypertensive patients with previous normal renal function, maintaining this therapy, and adding verapamil 180 mg/day to the ACEI treatment. These findings are clinically relevant and will be of paramount interest for the patients in which ACE illicit a doubtless protection of target organs, even in nonhypertensive patients. The trandolapril-verapamil combination allows maintain an excellent BP control decreasing further the levels of serum creatinine.

Hydrogels for Sustained and Selective Release of Diclofenac ALVAREZ-LORENZO C Univ. Santiago de Compostela, Santiago de Compostela, Spain. Background: The development of hydrophilic systems able to load hydrophobic drugs and to control the site and rate at which they are delivered is a target issue in pharmaceutical technology. The aim of our work was to prepare functionalized hydrogels based on semi-synthetic cellulose derivatives, acrylic copolymers or cyclodextrins, with high affinity for diclofenac for improving the loading and the release performance and even able to deliver the drug in response to certain stimuli. Methods: The hydrogels were i) cross-linked cationic cellulose ethers with ethyleneglycol diglycidylethers (EGDE); ii) cross-linked cellulose ethers/ cyclodextrins with EGDE; iii) copolymerized hydroxyethylmethacrylate (HEMA) with aminopropyl methacrylamide (AMPA) or vinylpyridine (VP); and iii) interpenetrated networks of N-isopropylacrylamide (NIPA) and chitosan. Results: Cross-linked cationic cellulose networks loaded 250 mg diclofenac per gram and selectively released the drug at pH>7, being useful for specialized intestinal delivery. A novel cross-linking method to prepare cyclodextrin hydrogels led to systems that hosted the drug in the cyclodextrin cavities (100 mg/g) and sustained the delivery for 8 hours. Poly(HEMA-co-APMA) and poly(HEMA-co-VP) loaded 15 mg/g and did not release the drug in water but in the presence of ions; the release being sustained for several days. Such hydrogels display features particularly adequate for the development of medicated soft contact lenses. Interpenetrating NIPA/chitosan networks showed temperature- and pH-sensitive loading and release behavior. The IPNs had a notably greater affinity for diclofenac (18 mg/g) than the pure PNIPA hydrogel and were able to sustain the drug release for more than 8 h in 0.9% NaCl solutions or pH 8 phosphate buffer. The IPNs with low chitosan postcross-linking degree showed high temperature-sensitive release patterns. Conclusions: Loading of diclofenac and its release pattern can be tunned designing tailored hydrogels in which functional elements performing as high affinity binding points and environment-responsive sensors are combined. These functionalized hydrogels may be particularly useful for site-specific drug delivery. Authors disclosure statement: Some information described in this abstract is the subject of patent applications filed by the University of Santiago de Compostela (WO 2006/089993; ES 200802364).