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ABCG2/BCRP is an ABC transporter that plays an important role in tissue protection by exporting endogenous substrates and xenobiotics. ABCG2 is of major interest due to its ...involvement in multidrug resistance (MDR), and understanding its complex efflux mechanism is essential to preventing MDR and drug-drug interactions (DDI). ABCG2 export is characterized by two major conformational transitions between inward- and outward-facing states, the structures of which have been resolved. Yet, the entire transport cycle has not been characterized to date. Our study bridges the gap between the two extreme conformations by studying connecting pathways. We developed an innovative approach to enhance molecular dynamics simulations, ‘kinetically excited targeted molecular dynamics’, and successfully simulated the transitions between inward- and outward-facing states in both directions and the transport of the endogenous substrate estrone 3-sulfate. We discovered an additional pocket between the two substrate-binding cavities and found that the presence of the substrate in the first cavity is essential to couple the movements between the nucleotide-binding and transmembrane domains. Our study shed new light on the complex efflux mechanism, and we provided transition pathways that can help to identify novel substrates and inhibitors of ABCG2 and probe new drug candidates for MDR and DDI.
The theranostic emerging radionuclide 47Sc is under the spotlight of the scientific community due to its medically favorable decay characteristics (t1/2 =3.3492 d, Eγ=159.381 keV, Eβ−, mean=162.0 ...keV) for both the diagnostic and treatment procedures. In the framework of the LaRAMED (LAboratory of RAdionuclides for MEDicine) program at INFN-LNL (National Institute of Nuclear Physics-Legnaro National Laboratories), the investigation of the possible 47Sc production routes using a 70 MeV proton beam is one of the goals of the REMIX (Research on Emerging Medical radIonuclides from the X-sections) project, funded by INFN for the years 2021-2023. Since the LARAMED bunkers are currently under completion, experiments are performed in collaboration with the GIP ARRONAX facility (Nantes, France), where a 70 MeV multi-particle cyclotron is operational. In this work, the cross-section measurements using enriched 48,49Ti targets are reported and the results presented in comparison to the previous literature data, when available. The 47Sc excitation functions are analyzed in relation to the contaminants’ ones since the co-produced radionuclides can affect the dose delivered to a patient. 46Sc cross-section curves are mainly taken into account since 46Sc cannot be chemically separated from 47Sc and its half-life (t1/2=83.79 d) is longer than the 47Sc one (t1/2=3.3492 d).
Background:
The rate of entry of cocaine into the brain is a critical factor that influences neuronal plasticity and the development of cocaine addiction. Until now, passive diffusion has been ...considered the unique mechanism known by which cocaine crosses the blood-brain barrier.
Methods:
We reassessed mechanisms of transport of cocaine at the blood-brain barrier using a human cerebral capillary endothelial cell line (hCMEC/D3) and in situ mouse carotid perfusion.
Results:
Both in vivo and in vitro cocaine transport studies demonstrated the coexistence of a carrier-mediated process with passive diffusion. At pharmacological exposure level, passive diffusion of cocaine accounted for only 22.5% of the total cocaine influx in mice and 5.9% in hCMEC/D3 cells, whereas the carrier-mediated influx rate was 3.4 times greater than its passive diffusion rate in vivo. The functional identification of this carrier-mediated transport demonstrated the involvement of a proton antiporter that shared the properties of the previously characterized clonidine and nicotine transporter. The functionnal characterization suggests that the solute carrier (SLC) transporters Oct (Slc22a1-3), Mate (Slc47a1) and Octn (Slc22a4-5) are not involved in the cocaine transport in vivo and in vitro. Diphenhydramine, heroin, tramadol, cocaethylene, and norcocaine all strongly inhibited cocaine transport, unlike benzoylecgonine. Trans-stimulation studies indicated that diphenhydramine, nicotine, 3,4-methylenedioxyamphetamine (ecstasy) and the cathinone compound 3,4-methylenedioxypyrovalerone (MDPV) were also substrates of the cocaine transporter.
Conclusions:
Cocaine transport at the BBB involves a proton-antiporter flux that is quantitatively much more important than its passive diffusion. The molecular identification and characterization of this transporter will provide new tools to understand its role in addictive mechanisms.
LARAMED project aims to set up an advanced science and technology facility to develop new and efficient methods to produce medical radioisotopes at Legnaro National laboratories of National Institute ...of Nuclear Physics. Waiting for the facility full operation, LARAMED group has already started working on the cyclotron production of several conventional and emerging radionuclides. Suitable target preparation is one of the most critical aspects in cyclotron production of radioisotopes. LARAMED group has investigated a set of non-classical techniques for metallic target preparation. Magnetron sputtering technique developed for ultra-thick film deposition was applied for the preparation of the Mo solid targets for 99mTc production, and Y ones for 89Zr production. Spark plasma sintering method provides efficient sintering of powders and good bonding of metallic pellet to a backing. Like magnetron sputtering, it was tested for natl00Mo and natY targets, as well as for natCr targets preparation aimed at 52Mn production. The High energy Vibration Powders Plating technique was instead applied for natural (Mo, Ti) and enriched 48Ti metallic powders with >95% deposition efficiency. All three techniques tested provided the cyclotron solid targets with high thermomechanical performance under the beam (IkW/cm2).
Zirconium-89 (89Zr) is an emerging radionuclide for positron emission tomography (PET), with nuclear properties suitable for imaging slow biological processes in cellular targets. The 89Y(p,n)89Zr ...nuclear reaction is commonly exploited as the main production route with medical cyclotrons accelerating low-energy (< 20 MeV) and low-current (< 100 μA) proton beams. Usually, natural yttrium solid targets manufactured by different methods, including yttrium electrodeposition, yttrium sputtering, compressed yttrium powders, and foils, were employed. In this study, the Spark Plasma Sintering (SPS) technique has been investigated, for the first time, to manufacture yttrium solid targets for an efficient 89Zr radionuclide yield. The natural yttrium disc was bonded to a niobium backing plate using a commercial SPS apparatus and a prototype machine assembled at the University of Pavia. The resulting targets were irradiated in a TR19 cyclotron with a 12 MeV proton beam at 50 μA. A dedicated dissolution module, obtained from a commercial system, was used to develop an automated process for the purification and recovery of the produced 89Zr radionuclide. The production yield and recovery efficiency were measured and compared to 89Zr produced by irradiating standard yttrium foils. SPS manufactured targets withstand an average heat power density of approximately 650 W∙cm−2 for continuous irradiation up to 5 h without visible damage. A saturation yield of 14.12 ± 0.38 MBq/μAh was measured. The results showed that the obtained 89Zr production yield and quality were comparable to similar data obtained using standard yttrium foil targets. In conclusion, the present work demonstrates that the SPS technique might be a suitable technical manufacturing solution aimed at high-yield 89Zr radioisotope production.
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The REMIX project is focused on the cyclotron-based production of 47Sc, 149Tb, 152Tb, 155Tb and 161Tb radionuclides, whose decay characteristics make them suitable for medical applications. This work ...will outline the main results achieved withing the REMIX collaboration, that is organized in the following Work Packages (WP): WP1. Target manufacturing (49Ti, 50Ti and 155Gd2O3) and characterization; WP2. Nuclear cross section (XS) measurements with 49Ti and 50Ti targets for 47Sc production; WP3. Nuclear XS measurements with natDy, 159Tb and natEu targets for xxTb production; WP4. Nuclear XS modeling for 47Sc and 155Tb production; WP5. Dosimetric calculations for 47Sc- and xxTb-labelled radiopharmaceuticals; WP6. 155Tb Thick Target Yield (TTY) measurements; WP7. Apparatus design and realization for irradiation tests with the LARAMED beamline. Since the LARAMED bunkers and ancillary laboratories are currently under completion at the INFN-LNL, the nuclear XS experiments are carried out in collaboration with the GIP ARRONAX facility (Saint-Herblain, France) and the Thick Target Yield (TTY) measurements are performed at the Sacro Cuore Don Calabria hospital (SCDCh, Negrar, Verona, Italy).
Background: Opioids are widely used in pain management, acting via opioid receptors
and/or Toll-like receptors (TLR) present at the central nervous system (CNS). At the blood-brain
barrier (BBB), ...several influx and efflux transporters, such as the ATP-binding cassette (ABC)
P-glycoprotein (P-gp, ABCB1), Breast Cancer Resistance Protein (BCRP, ABCG2) and multidrug
resistance-associated proteins (MRP, ABCC) transporters, and solute carrier transporters (SLC), are
responsible for the transport of xenobiotics from the brain into the bloodstream or vice versa.
Objective: ABC transporters export several clinically employed opioids, altering their neuropharmacokinetics
and CNS effects. In this review, we explore the interactions between opioids
and ABC transporters, and decipher the molecular mechanisms by which opioids can modify their
expression at the BBB.
Results: P-gp is largely implicated in the brain-to-blood efflux of opioids, namely morphine and
oxycodone. Long-term exposure to morphine and oxycodone has proven to up-regulate the expression
of ABC transporters, such as P-gp, BCRP and MRPs, at the BBB, which may lead to increased
tolerance to the antinociceptive effects of such drugs. Recent studies uncover two mechanisms by
which morphine may up-regulate P-gp and BCRP at the BBB: 1) via a glutamate, NMDA-receptor
and COX-2 signaling cascade, and 2) via TLR4 activation, subsequent development of neuroinflammation,
and activation of NF-kB, presumably via glial cells.
Conclusion: The BBB-opioid interaction can culminate in bilateral consequences, since ABC
transporters condition the brain disposition of opioids, while opioids also affect the expression of
ABC transporters at the BBB, which may result in increased CNS drug pharmacoresistance.
To determine concentration-dependent P-gp-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in rats.
The transport of radiolabeled colchicine and ...vinblastine across the rat BBB was measured with or without PSC833, a well known P-gp inhibitor, and within a wide range of colchicine and vinblastine concentration by an in situ brain perfusion. Thus, the difference of brain transport achieved with or without PSC833 gives the P-gp-mediated efflux component of the compound transported through the rat BBB. Cerebral vascular volume was determined by coperfusion with labeled sucrose in all experiments.
Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. P-gp limited the uptake of both colchicine and vinblastine, but the compounds differ in that vinblastine inhibited its own transport. Vinblastine transport was well fitted by a Hill equation giving IC50 at approximately 71 microM, a Hill coefficient (n) approximately 2, and a maximal efflux velocity Jmax of approximately 9 pmol s(-1) g(-1) of brain.
P-gp at the rat BBB may carry out both capacity-limited and capacity-unlimited transport, depending on the substrate, with pharmacotoxicologic significance for drug brain disposition and risk of drug-drug interactions.
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