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Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model.

Vaquer S, Cuyàs E, Rabadán A, González A, Fenollosa F, de la Torre R - F1000Res (2014)

Bottom Line: Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases.MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples.Further research is needed to provide a conclusive answer in this regard.

View Article: PubMed Central - PubMed

Affiliation: Departament de Farmacologia Humana, Institut Municipal d'Investigació Mèdica de Barcelona (IMIM), Barcelona, 08003, Spain ; Corporació Sanitària i Universitària Parc Taulí, Sabadell, 08208, Spain.

ABSTRACT
Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and interesting research lines in biology and human physiology with the potential for significant benefits for both space and terrestrial medicine.

No MeSH data available.


Related in: MedlinePlus

Final prototype configuration.This picture shows the final configuration of the experiment on board the parabolic aircraft. The three racks presented were, from left to right: storage rack, main rack, fluids and control rack. All principal electric components were placed in a sealed fire-proof cabinet underneath the main rack to protect the equipment from water contact in case of failure of the containment system.
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f2: Final prototype configuration.This picture shows the final configuration of the experiment on board the parabolic aircraft. The three racks presented were, from left to right: storage rack, main rack, fluids and control rack. All principal electric components were placed in a sealed fire-proof cabinet underneath the main rack to protect the equipment from water contact in case of failure of the containment system.

Mentions: An electro-mechanical engineering system was especially designed to undertake the required procedures in microgravity following the design and safety specifications for parabolic flights (Figure 1 &Figure 2). Four experimentation units composed the prototype, one per each specific group of samples. Accurate fluid displacement was achieved implementing high precision component manufacturing and accurate linear engines (Schneider Electric, France). These engines presented 0.05 mm of displacement error, which represented a calculated 16 μl fluid displacement error (< 10% in our experimental setup). Sample temperature was strictly controlled using a dedicated thermal control system, which consisted of four preheating chambers to raise the sample temperature from 2–6°C to 37°C and four reaction chambers to maintain the temperature at 37.3°C during the reaction. In order to protect the vesicle integrity, 45°C was established as the maximum permissible temperature at syringe wall in preheating chambers given syringe radius and its thermal conductivity. Several temperature probes feedback a central computer, which maintained the temperatures at the required levels (Minco, Minneapolis, USA; TC-Direct, Madrid, Spain). This system also enabled cold storage of ATP and washing mix at 2–6°C in two certified isothermal boxes (Tecnisample, Barcelona, Spain). All procedures were controlled by an electrical and control system, which provided electrical power, actuator control, synchronization, monitoring, logging and a user-friendly interface (Telemecanique, France) (see supplementary materialAppendix 2. List of Components for a more detailed description of equipment and components used).


Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model.

Vaquer S, Cuyàs E, Rabadán A, González A, Fenollosa F, de la Torre R - F1000Res (2014)

Final prototype configuration.This picture shows the final configuration of the experiment on board the parabolic aircraft. The three racks presented were, from left to right: storage rack, main rack, fluids and control rack. All principal electric components were placed in a sealed fire-proof cabinet underneath the main rack to protect the equipment from water contact in case of failure of the containment system.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4264636&req=5

f2: Final prototype configuration.This picture shows the final configuration of the experiment on board the parabolic aircraft. The three racks presented were, from left to right: storage rack, main rack, fluids and control rack. All principal electric components were placed in a sealed fire-proof cabinet underneath the main rack to protect the equipment from water contact in case of failure of the containment system.
Mentions: An electro-mechanical engineering system was especially designed to undertake the required procedures in microgravity following the design and safety specifications for parabolic flights (Figure 1 &Figure 2). Four experimentation units composed the prototype, one per each specific group of samples. Accurate fluid displacement was achieved implementing high precision component manufacturing and accurate linear engines (Schneider Electric, France). These engines presented 0.05 mm of displacement error, which represented a calculated 16 μl fluid displacement error (< 10% in our experimental setup). Sample temperature was strictly controlled using a dedicated thermal control system, which consisted of four preheating chambers to raise the sample temperature from 2–6°C to 37°C and four reaction chambers to maintain the temperature at 37.3°C during the reaction. In order to protect the vesicle integrity, 45°C was established as the maximum permissible temperature at syringe wall in preheating chambers given syringe radius and its thermal conductivity. Several temperature probes feedback a central computer, which maintained the temperatures at the required levels (Minco, Minneapolis, USA; TC-Direct, Madrid, Spain). This system also enabled cold storage of ATP and washing mix at 2–6°C in two certified isothermal boxes (Tecnisample, Barcelona, Spain). All procedures were controlled by an electrical and control system, which provided electrical power, actuator control, synchronization, monitoring, logging and a user-friendly interface (Telemecanique, France) (see supplementary materialAppendix 2. List of Components for a more detailed description of equipment and components used).

Bottom Line: Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases.MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples.Further research is needed to provide a conclusive answer in this regard.

View Article: PubMed Central - PubMed

Affiliation: Departament de Farmacologia Humana, Institut Municipal d'Investigació Mèdica de Barcelona (IMIM), Barcelona, 08003, Spain ; Corporació Sanitària i Universitària Parc Taulí, Sabadell, 08208, Spain.

ABSTRACT
Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and interesting research lines in biology and human physiology with the potential for significant benefits for both space and terrestrial medicine.

No MeSH data available.


Related in: MedlinePlus