Drug repurposing from the perspective of pharmaceutical companies.

Drug repurposing holds the potential to bring medications with known safety profiles to new patient populations. Numerous examples exist for the identification of new indications for existing molecules, most stemming from serendipitous findings or focused recent efforts specifically limited to the mode of action of a specific drug. In recent years, the need for new approaches to drug research and development, combined with the advent of big data repositories and associated analytical methods, has generated interest in developing systematic approaches to drug repurposing. A variety of innovative computational methods to enable systematic repurposing screens, experimental as well as through in silico approaches, have emerged. An efficient drug repurposing pipeline requires the combination of access to molecular data, appropriate analytical expertise to enable robust insights, expertise and experimental set-up for validation and clinical development know-how. In this review, we describe some of the main approaches to systematic repurposing and discuss the various players in this field and the need for strategic collaborations to increase the likelihood of success in bringing existing molecules to new indications, as well as the current advantages, considerations and challenges in repurposing as a drug development strategy pursued by pharmaceutical companies. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Contenido en algunos nutrientes del alga marina comestible Monostroma undulatum Wittrock de la costa patagónica argentina / Chemical composition and seasonal fluctuations of the edible green seaweed, Monostroma undulatum, Wittrock, from the Southern Argentina coast

The chemical composition of green seaweed, Monostroma undulatum, Wittrock, growing in the Southern Argentina coast, was studied. Samples were collected in Puerto Deseado, province of Santa Cruz (47 degrees 45'L.S., 65 degrees 55'L.W.), from October to December 1999 and 2000. It has been analyzed six sample during this period. Algae were washed with sea water and dried at room temperature for 24 hs. Moisture, nitrogen, lipids and ashes were determined according to AOAC; fiber (total, soluble and insoluble), according to Lahaye. After mineralization with nitric acid, sodium and potassium were determined by flame photometry, calcium by complexometric method, and phosphorus by Gomori's method. The ranges expressed per 100 g dry algae were: protein (Nx6.25): 12.89-21.85; ashes (g): 33.92-40.05; lipid (g): 0.32-1.47; total fiber (g): 14.36-19.6; digestible carbohydrates (calculated by difference) (g): 20.86-32.48; sodium (g): 7.39-13.11; potassium (g): 1.38-3.18; calcium (mg): 149-226; phosphorus (mg): 190-447; Vitamin C (mg): 159-455. These results show that this green seaweed is an important source for protein, fiber, macronutrients minerals and vitamin C, during the macroscopic period. There was an important fluctuation that must be taken into account to consider the commercial collection to use it in human nutrition.

Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression.

Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT after release. To explore protein kinase C-mediated SERT regulation, we generated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hSERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and antidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-cell currents. In these cells, the protein kinase C activator beta-PMA caused a time-dependent reduction in 5-HT uptake capacity (Vmax) after acute application and a reduction in SERT-mediated currents. Effects of beta-PMA were mimicked by the phorbol ester beta-PDBu, were not observed with the inactive alpha-isomers, and could be blocked by treatment of cells with the protein kinase C inhibitor staurosporine. Biotinylation/immunoblot analyses showed that activity reductions are paralleled by a staurosporine-sensitive loss of surface SERT protein. These data indicate that altered surface abundance, rather than reduced catalytic transport efficiency, mediates acute PKC-dependent modulation of 5-HT uptake.

Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells.

1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced currents (approximately -100 pA at -60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1-mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Na+ into the cell, which then leads to electrogenic efflux through GAT1 at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr approximately 10(9) s-1 under nominal conditions (V = -60 mV, 30 microM GABA, 130 mM Na+ and room temperature). This indicates either very high levels of expression (approximately 10(4) microns-2), assuming published turnover rates (approximately 10 s-1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Na+ and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed-stoichiometry transporter mode of conduction.

[Tuberculosis of the spine in children. Personal experience]. / Tubercolosi della colonna vertebrale in età pediatrica. Nostra esperienza.

We are currently witnessing a worldwide return of tuberculosis. An extremely rare form is tuberculosis of the spine which is reported above all in extra-European studies. The authors report a case of Pott's disease in a child aged 3 years and 3 months who was referred to their attention due to the appearance of left inguinal swelling, fever and anemia. Diagnostic tests (ETG, CT, MR) showed an abscess involving the L5-S1 intersomatic space, the intervertebral disc and osteolytic lesions of S1, with impairment of the left psoas muscle and diffusion as far as the inguinal region. Chemotherapy was commenced using isoniazid, ethambutol, rifampicin, and streptomycin and lasted 24 months associated with drainage of the ileopsoas abscess. Conservative orthopedic treatment lasting for one year initially took the form of decubitus in bed with hyperdistension of the vertebral column, followed by the creation of a plaster-cast cot on the back and lastly a glass-reinforced resin orthopedic jacket. The follow-up of 2 years and 10 months showed recovery with reconstruction of the vertebral elements and the preservation of intervertebral space.

Detection of HIV-1 sequences in children using radioactive and colorimetric polymerase chain reactions.

The detection of HIV-1 proviral DNA in children born to seropositive mothers was studied using the polymerase chain reaction with either a radioactive electrophoretic method or a noval procedure that employs colorimetric microwell visualization. Peripheral blood mononuclear cell lysates from 18 HIV-1 infected children and 28 uninfected subjects were assayed for a 142 bp fragment of DNA from the gag region of HIV-1 using the primer pair SK145-431. Detection of amplified DNA was carried out by hybridization with a radiolabeled SK102 probe, or with a tagged SK102 probe permitting colorimetric detection. The radioactive detection procedure demonstrated 100% specificity and correlated with the serological results. The assay was more sensitive than the p24 antigen test, but two false negative results were obtained. One was from a sample taken at 2 weeks, an age at which undetectable provirus levels were reported in almost all HIV-1 infected newborns. The second was probably due to a low copy number of proviral DNA, as positive results were obtained in all other (6) samples from this child. Comparative analysis in a limited number of specimens of radioactive and colorimetric detection following PCR revealed 100% specificity and comparable sensitivity with 4 discordant results. The results show that PCR is the best method for early diagnosis of HIV-1 infection in pediatric subjects. The study also demonstrated the value of a colorimetric detection method for PCR products. This colorimetric microwell plate procedure may prove a useful technique in routine diagnosis of HIV-1 infection in children.

Ca channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

The ability of Ca ions to inhibit Ca channels presents one of the most intriguing problems in membrane biophysics. Because of this negative feedback, Ca channels can regulate the current that flows through them. The kinetics of the channels depend on voltage, and, because the voltage controls the current, a strong interaction exists between voltage dependence and Ca dependence. In addition to this interaction, the proximity of pores and the local concentration of ions also determine how effectively the Ca ions influence channel kinetics. The present article proposes a model that incorporates voltage-dependent kinetics, current-dependent kinetics, and channel clustering. We have based the model on previous voltage-clamp data and on Ca and Ba action currents measured during the action potential in beating heart cells. In general we observe that great variability exists in channel kinetics from patch to patch: Ba or Ca currents have low or high amplitudes and slow or fast kinetics during essentially the same voltage regime, either applied step-protocols or spontaneous cell action potentials. To explain this variability, we have postulated that Ca channels interact through shared ions. The model we propose expands on our previous model for Ba currents. We use the same voltage-dependent rate constants for the Ca currents that we did for the Ba currents. However, we vary the current-dependent rate constants according to the species of the conducting ion. The model reproduces the main features of our data, and we use it to predict Ca channel kinetics under physiological conditions. Preliminary reports of this work have appeared (DeFelice et al., 1991, Biophys. J. 59:551a; Risso et al., 1992, Biophys. J. 61:248a).