Discovery of Small Molecule Renal Outer Medullary Potassium (ROMK) Channel Inhibitors: A Brief History of Medicinal Chemistry Approaches To Develop Novel Diuretic Therapeutics.

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir, Kir1.1) channels. It is primarily expressed in two regions of the kidney, the cortical collecting duct (CCD) and the thick ascending loop of Henle (TALH). At the CCD it tightly regulates potassium secretion while controlling potassium recycling in TALH. As loss-of-function mutations lead to salt wasting and low blood pressure, it has been surmised that inhibitors of ROMK would represent a target for new and improved diuretics for the treatment of hypertension and heart failure. In this review, we discuss and provide an overview of the medicinal chemistry approaches toward the development of small molecule ROMK inhibitors over the past decade.

Preparation and characterization of bee venom-loaded PLGA particles for sustained release.

Bee venom-loaded poly(lactic-co-glycolic acid) (PLGA) particles were prepared by double emulsion-solvent evaporation, and characterized for a sustained-release system. Factors such as the type of organic solvent, the amount of bee venom and PLGA, the type of PLGA, the type of polyvinyl alcohol, and the emulsification method were considered. Physicochemical properties, including the encapsulation efficiency, drug loading, particle size, zeta-potential and surface morphology were examined by Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction (XRD). The size of the bee venom-loaded PLGA particles was 500 nm (measured using sonication). Zeta-potentials of the bee venom-loaded PLGA particles were negative owing to the PLGA. FT-IR results demonstrated that the bee venom was completely encapsulated in the PLGA particles, indicated by the disappearance of the amine and amide peaks. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the bee venom in the bee venom-loaded PLGA particles was intact. In vitro release of the bee venom from the bee venom-loaded PLGA particles showed a sustained-release profile over 1 month. Bee venom-loaded PLGA particles can help improve patients' quality of life by reducing the number of injections required.

PLGA microspheres containing bee venom proteins for preventive immunotherapy.

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections.

Seguridad de inmunoterapia con veneno de himenópteros en pauta agrupada. Perspectiva de enfermería / Safety of hymenopteran venoms immunotherapy in a cluster schedule. A nursing perspective

Fundamento. La gravedad inherente a la hipersensibilidadIgE mediada a veneno de himenópteros impone la necesidadde alcanzar, en el menor plazo de tiempo posible, ladosis de mantenimiento en la inmunoterapia con venenos.El objetivo del presente trabajo es valorar la seguridad deuna pauta agrupada (cluster) de inmunoterapia subcutáneacon veneno de himenópteros, que reduce de 12 a 3 semanasel tiempo necesario para llegar a la dosis de mantenimiento.Material y métodos. El estudio fue realizado en 30 pacientes,24 varones y 6 mujeres con una media de edad de 46,06años, que habían sido diagnosticados de hipersensibilidada veneno de himenópteros y se les había indicado tratamientocon inmunoterapia. Los pacientes recibieron inmunoterapiafrente a veneno de himenópteros Pharmalgen®(ALK Abelló), 13 fueron de Apis mellífera, 12 de Véspula spp,y 5 de Polistes spp, con una pauta agrupada que consistióen: día 1 (4μg + 6μg), día 8 (10μg + 30μg) y día 15 (40μg +60μg). Se valoraron las reacciones ocurridas durante la fasede inicio entre abril de 2005 y febrero de 2008.Resultados. De los 30 pacientes vacunados 2 presentaronreacción local exagerada, otros síntomas inespecíficos endos ocasiones y 2 más reacción sistémica. Uno tras administrarla dosis de 40μg, presentó reacción sistémica grado III,y el otro tras recibir la dosis de 60 μg, presentó una reacciónsistémica grado II. Ambos pasaron a una pauta convencionalde administración de inmunoterapia y tuvieron nuevasreacciones de grado III por lo que se les mantiene la inmunoterapiacon premedicación con antihistamínicos orales.Discusión. El estudio confirma que la pauta utilizada es seguracon una baja incidencia de reacciones adversas, 1,67%presentó reacción local exagerada, 1,11% tuvo reaccióninespecífica y 1,11% reacción sistémica(AU)

Background. The inherent seriousness of IgE mediated hypersensitivityto hymenopteran venoms makes it necessary toreach, in the shortest period of time, the maintenance dosein immunotherapy with poisons. The aim of this article is toevaluate the safety of a cluster schedule of subcutaneous Hymenopteranvenoms immunotherapy, which reduces the timeneeded to reach the maintenance dose from 12 to 3 weeks.Material and methods. Thirty patients, 24 men and 6 womenwith an average age of 46.06 years, who had beendiagnosed with hypersensitivity to the poison of hymenopteransand for whom immunotherapy had been prescribedparticipated in the study. The patients received Pharmalgen® (ALK Abelló) immunotherapy against hymenopteranvenoms, 13 Apis mellífera, 12 Véspula spp, and 5 Polistes spp,with a cluster schedule that consisted of: day 1 (4μg + 6μg),day 8 (10μg + 30μg) and day 15 (40μg + 60μg). The reactionsoccurring during the starting phase between April 2005 andFebruary 2008 were evaluated.Results. Of the 30 vaccinated patients 2 presented an exaggeratedlocal reaction, there were other non-specific symptomson two occasions, and another 2 presented systemicreaction. One following administration of the dose of 40μgpresented a grade III systemic reaction, and the other afterreceiving the dose of 60μg presented a grade II systemicreaction. Both passed to a conventional model of immunotherapyadministration and had new grade III reactions, andwere therefore kept on immunotherapy with premedicationwith oral antihistamines.Discussion. The study confirms that the model employedis safe with a low incidence of adverse reactions, 1.67%presented an exaggerated local reaction, 1.11% had an nonspecificreaction and 1.11% a systemic reaction(AU)

Kinetics and dynamic evaluation of specific immunotherapy.

UNLABELLED: Specific immunotherapy (SIT) is frequently used in the treatment of allergic diseases. However, the mechanisms by which SIT achieves clinical improvement remained unclear. We decided to study the in vivo kinetics of this therapy, using a nuclear medicine approach (leukocytes labelled with 99mTc-HMPAO) in patients on maintenance doses of specific immunotherapy with confirmed clinical efficacy. MATERIAL AND METHODS: We studied 13 allergic patients grouped according to different treatment schedules: subcutaneous aqueous allergenic extract (3 latex and 2 hymenoptera venom), subcutaneous depot extract (2 house dust mite and 2 pollens), subcutaneous modified allergens (2 pollens), sublingual extract (2 house dust mites). The control group included two allergic patients submitted to subcutaneous injections of bacterial extract (1 patient--positive control), and aqueous solution (1 patient). At the same time that the therapeutic allergen was administered subcutaneously, the autologous labelled white cells were injected intravenously in a peripheral vein in the contralateral arm. A thoracic dynamic acquisition of 60 mins, 64x64 matrix, 2 frame/min, in anterior view was performed. Static acquisition for 256x256 matrix, during 5 mins each at 60, 90, 120, 180, 240, 300 and 360 mins after the administration of the radiolabelled leukocytes, in thoracic (anterior and posterior), and abdominal view were performed. During the examination, the local erythema was monitored. A similar procedure was undertaken for Sublingual administration of immunotherapy. RESULTS: The inflammatory activity at the site of SIT injection (aqueous depot extract) started in the first hour and the increase was time related. For modified allergen extract and sublingual SIT the activity was present since the beginning of the administration. The ascendant lymphatic drainage, which was directed to the homolateral axillary region, to the lymphoid tissue of the upper mediastinum and to the anterior region of the neck began earlier. Thoracic focalisations were present for all the patients, whereas bowel focalisations were only observed for the subcutaneous route of administration. Sublingual SIT did not induce axillary or intestinal inflammatory focalisations, even though the patients had swallowed the allergenic extract. The uptake coefficient in individualized areas corrected to the uptake coefficient background was also studied. CONCLUSIONS: For the subcutaneous route of administration, except for glutaraldehyde-modified allergen, the local inflammatory activity at the allergenic injection site was significantly higher in depth and was time dependent, maintaining activity even after complete disappearance of the erythema and/or wheal. These results express a prompt inflammatory involvement of the immune system with this allergenic therapy, which was unexpected until now. We also observed differences concerning allergic diseases, the type of allergenic extracts and routes of administration.

Oral colon-specific drug delivery for bee venom peptide: development of a coated calcium alginate gel beads-entrapped liposome.

Colon-specific drug delivery systems (CDDSs) can be used to improve the bioavailability of protein and peptide drugs through the oral route. A novel formulation for oral administration using coated calcium alginate gel beads-entrapped liposome and bee venom peptide as a model drug has been investigated for colon-specific drug delivery in vitro. Drug release studies under conditions mimicking stomach to colon transit have shown that the drug was protected from being released completely in the physiological environment of the stomach and small intestine. The release rate of bee venom from the coated calcium alginate gel beads-entrapped liposome was dependent on the concentration of calcium and sodium alginate, the amount of bee venom in the liposome, as well as the coating. Furthermore, a human gamma-scintigraphy technique was used in vivo to determine drug delivery more precisely. The colonic arrival time of the tablets was found to be 4-5 h. The results clearly demonstrated that the coated calcium alginate gel beads-entrapped liposome is a potential system for colon-specific drug delivery.

A sensitive enzyme-linked immunosorbent assay for evaluating the concentration of bee venom in rat plasma.

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) was developed to determine the concentration of bee venom in rat plasma. The intra- and inter-assay coefficients of variation for the ELISA were less then 3% between 0.1 and 1,000 ng mL(-1) venom, and the sensitivity of the detection was 0.1 ng mL(-1). Total recovery of the bee venom added to rat plasma was determined. Using this ELISA, serum levels of bee venom were easily determined. The rats were administered a single intravenous injection or oral dose of bee venom (1 mg kg(-1) of body weight). The bioavailability of the bee venom under the two administrations was compared using pharmacokinetic parameters. Results showed that intravenous administration of bee venom produced high plasma concentrations with a short half-life. The area under the curve for oral administration was 10 times lower than for intravenous administration. This loss of bee venom may be due to the degradation that occurs in the enzymatic and acidic environment of the gastrointestinal tract.

Rate and quantity of delivery of venom from honeybee stings.

To determine the rate and completeness of delivery of venom from honeybee stings, European bees were collected at the entrance of a hive and studied with the use of two laboratory models. In one model bees were induced to sting the shaved skin of anesthetized rabbits. The stings were removed from the skin at various time intervals after autotomization, and residual venom was assayed with a hemolytic method. In the other model the bees were induced to sting preweighed filter paper disks, which were weighed again after removal of the sting at various intervals. Results of both experiments were in agreement, showing that at least 90% of the venom sac contents were delivered within 20 seconds and that venom delivery was complete within 1 minute. The data suggest that a bee sting must be removed within a few seconds after autotomization to prevent anaphylaxis in an allergic person. The extensive variation found in the amount of venom delivered at each time point may explain inconsistencies in relationships among reactions to field stings, sting challenge testing, venom skin tests and RAST.