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Fly Your Thesis! 2009 – Final Report 1

Fly your Thesis! 2009

Final Report

1. Executive Summary ABC transporter family is one of the main active trans-membrane transport systems in human cells. Their study in microgravity is a modern, innovating and original idea, which aims to establish a base for future research in drug behaviour in space. This project aims to determine whether microgravity can modify ABC transporter mediated trans-membrane drug transport in human model cells. Changes in ABC transporter activity can have implications in space physiology and physiopathology, space pharmacology and health care systems in current and future space missions. Additionally this research is expected to enhance current knowledge of the ABC protein family, its intricate mechanism of action and provide new information for improving Earth treatments. Developed to fly on the 51st ESA Parabolic Flight Campaign and under the auspices of the “Fly your thesis” project a tailored electro-mechanical prototype was designed to perform specific advanced laboratory procedures for ABC transporter analysis in microgravity. This system enabled researchers to perform required fluid mixing, within exact timeframes, in exact quantities and at desired temperatures. MRP2 (Multi-drug Resistance Protein 2) and estradiol-17-beta-glucuronide were chosen to determine transport speed modifications in microgravity. High MRP2 content insect-derived membrane vesicles were obtained from Solvo Biotechnology TM and were used to perform a vesicular transport assay, in which weightlessness MRP2 activity was determined by measuring estradiol-17-beta-glucuronide content after exposing the transporter to this drug and ATP (Adenosine tri-phosphate) at human body temperature during microgravity phases of parabolic flights. Benzbromarone, a known ABC transporter pharmacological inhibitor was also used in order to assess transport inhibition capacity in microgravity. At the moment of delivery of this report 50% of samples had been analyzed, being insufficient to provide a reliable answer to whether microgravity can affect ABC transporter mediated drug transport in human cell models. However, results showed internal coherence demonstrating reliability of experiments performed and good performance of equipment. Complete analysis of samples is required as well as in-depth statistical evaluation of results before withdrawing any definitive and valid conclusions.

2. Student Team Description

Sergi Vaquer Araujo M.D. (P.I.) Medical Doctor by Universitat Autònoma de Barcelona, currently 2nd year resident in Intensive Care Medicine (Hospital Parc Taulí, Sabadell). He has experience in applied research in the field of human embryology, human physiology, as well as in areas of pharmacology and biomedicine. Intern of the Crew Medical Support Offcie at the European Astronaut Center (September 2007 – March 2008). Delegate of the International Astronautical Congress 2006 in Valencia and the Space Generation Congress 2006. Member of the International Astronautical Federation, Space Generation Network and Laboratory for Experimentation in Space and Microgravity (LEEM). He is the originator of the project and acts as a general coordinator.

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Arnau Rabadán Barea. (Chief Engineer) Mechanical Engineer by Escola Universitària d'Enginyeria Tècnica Industrial de Barcelona (EUETIB) from the Polytechnic University of Catalonia, where he majored in mechanical engineering. This project represents his final university project. At Fundació CIM he has developed several projects in industrial laboratories, manufacture of prototypes and he is a certified teacher on various computer aided design courses. His main task has been to conceive and develop the experimental prototype following all scientific requirements and safety constraints. During the extension of the parabolic flight campaign he was doing a research fellow at the Fundació CIM.

Albert González Manresa (Electrical and Control Engineer) Electrical Engineer (Bsc) by Escola Universitària d'Enginyeria Tècnica Industrial de Barcelona (EUETIB) of the Polytechnic University of Catalonia, specialization and intensification in electrical power systems and automated systems. Has supported various projects in the company RUBATEC SA, which include the maintenance of Barcelona�s Harbour premises. This project is his graduation project. His main task is to design and build all electrical systems, including automatic drivers, heating system, harness, monitoring system and logging.

Elisabet Cuyàs Navarro (Biochemistry Head) Graduated Biochemicist by Universitat Autònoma de Barcelona, with intensifications in Biochemistry and Molecular Biology. Currently developing her research activity for her doctorate under the Biochemistry and Molecular Biology Program in the Pharmacology Department of the Institut Municipal d�Investigació Mèdica. Elisabet has a wide background and expertise on biochemistry research and plays a primordial role in the ABCtr team as the biochemistry group director, a team which has the task of adapting high precision terrestrial biochemical research to microgravity environments for space application and further analysis of samples.

Felip Fenollosa Artés (Endorsing Professor) Director of Research and Development of the CIM Foundation and associate professor of Mechanical Engineering at the Polytechnic University of Catalunya. From his position directs and coordinates the development of scientific teams in Fundació CIM. He directs and teaches a Master in Computer Integrated Manufacturing and Engineering. Author of several patents, scientific articles and engineering papers at conferences. As Fields of technological interest he is an expert in product development, with a specialization in rapid prototyping technologies, always in touch with the industrial environment of SMEs. It is also expert in continuous transmission systems aimed at both the automotive sector and the electricity generation.

3. Project Description

ABC transporters in microgravity

3.1. The ABC transporters family ABC (ATP Binding Cassette) transporters are trans-membrane proteins located in all cells of the human body. Their main function is to act as toxic cleaners and to detect dangerous substances pumping them to the exterior of the cell. These products range from metabolism-derived waste products to commonly used drugs (Galvinas, Krajcsi, & Sarkadi, 2004). At a higher organisational level, these transporters give the organism the ability to excrete to the outer environment many kinds of dangerous substances (Beringer & Slaughter, 2005).

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These transporters have been chosen for their analysis in microgravity due to their known relevance in pharmacokinetics of numerous pharmacological agents. ABC transporters regulate absorption and excretion of drugs affecting their dose, semi-live time, side effects, drug-drug interactions and treatment resistance. Given the later, these proteins not only modify the effect of drugs in healthy cells but also cause resistance to treatment of cancer therapies, cleaning tumoral cell interior of antineoplasic drugs providing immunity to current treatments (Galvinas, Krajcsi, & Sarkadi, 2004). This is the reason why research on ABC transporters is focused on acquiring the capacity of selectively modulate their action to affect those located in ill cells without harming healthy cells of the organism. Nevertheless, and despite all efforts made until the moment, ABC transporters and their mechanism of action remains unknown, which makes it harder to develop new and more specific drugs (Choudhuri & Klaassen, 2006). However, recent research suggests that interaction between drug and transporter, and cell membrane is the key to explain the transport of drugs and toxic waste in human cells (Choudhuri & Klaassen, 2006).

3.2. Enzymes & Microgravity Despite common sense would suggest that gravity (weak force at a molecular level) should not be able to modify biochemical reactions in which stronger intermolecular forces play the main role, there are signs that gravity might certainly have an effect in biochemical reactions not yet considered. It has been observed that numerous enzymes, amongst which are drug metabolism related proteins, see their effect modified in animal models exposed to microgravity. The most relevant are activity changes observed in various key enzymes of the organism like HMG-CoA reductase, cytochrome P-450 depending enzymes aniline hydroxilase and etilmorfine N-desaminase, phosphofructokinase and succinate dehydrogenase (Graebe, Schuck, & Derendorf, 2004). Further research demonstrated that several basic enzymatic processes are modified in microgravity, like phosphorilation of certain urchin sperm proteins (Tash & Bracho, 1999). However, research developed by Maccarrone M. and Giachetti E. deserves special attention. Each of them performed their research tests during the same parabolic flight. Prof. Giachetti had obtained similar results in an experiment performed onboard a sounding rocket. These experiments demonstrated that distinct enzymes behave differently under microgravity conditions. Soybean Lipooxigenase-1, substrates of which are free fatty acids, seems to increase its affinity for them by fourfold (Maccarrone, Bari, Battista, & Finazzi-Agrò, 2001). However, isocitrate lyase which cleaves isocitrate to succinate and glioxilate does not modify its function (Giachetti, Ranaldi, & Fiusco, 1999). It has been suggested that a possible explanation to this phenomena would rely in water-lipid interactions which might be modified in microgravity (Macarrone & Finazzi-Agró, 2001). These results have risen the interest of the scientific community and require a deeper and more accurate study (Giachetti, Ranaldi, & Vanni, 2001). Trans-membrane ion channels also show alterations due to the lack of gravity, reducing significantly their open state probability (Goldermann & Hanke, 2001). Authors suggest this phenomenon could be explained by the interaction between lipidic part of the cell membrane and ion channels sensible to gravitational vector changes. It has been considered that this could be the method some marine species orientate themselves under water (Goldermann & Hanke, 2001) Given the uncertainty about drug behaviour in space and scarce medical resources onboard current spaceships, big and ambitious clinical trials on orbit have not been considered so far, since they could endanger the safety and health of the astronauts. However, there are efforts focused to discover indirect means by which drug safety and efficiency can be demonstrated. Amongst others, research developed by Dr. Gandia is quite remarkable and showed unexpected phenomena when blood drug levels of Paracetamol (Acetaminophen) were measured in astronauts during a spaceflight. Thereafter new research projects focused on splitting drug metabolism processes into its different levels are under development to ascertain which of them has a bigger relevance to explain strange behaviour of drugs observed in space.

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3.3. Objectives The project “ABC transporters in microgravity” aims to demonstrate the ability to perform reliable and reproducible biochemical experiments with ABC transporters in microgravity, to find out whether gravity plays a significant role in its mechanism of action. With this project we would like to call attention upon the need for greater effort in studying effects of microgravity on chemical reactions level and to facilitate future research of other cellular components involved in the metabolism of drugs in microgravity. Since there are no publications so far that analyse these cellular elements in microgravity, background on which to base solid hypothesis is scarce. Nor are there adequate systems to develop this type of research in microgravity. Nevertheless, based on current knowledge about the effects of gravity on enzymes and components of cell membranes, the project aims to ascertain:

Whether changes in gravity are capable of affecting ABC transporters mediated transport across human cell model membranes.

Validate electromechanical equipment that allows automated enzymology and biochemical experiments in microgravity environments. With accurate timing and exact substrate quantities.

In addition, the study of these elements in conditions of weightlessness can provide new perspectives and concepts that help clarify the current uncertainties about the functioning mechanism of these transporters in healthy and ill subjects on Earth. ABC transporters are related to pharmacokinetics of multiple pharmacological agents and their interactions, as well as effectiveness of anticancer therapies. Although with this project it will not be possible to give a totally certain answer about whether a change in the activity of ABC transporters in microgravity is relevant or not for space pharmacology, it is expected that it will serve to foster future research initiatives of these elements in microgravity and more generally to study the general molecular level effects of microgravity.

3.4. Materials & Methods

Parabolic flights as the best microgravity platform Parabolic flights were selected for conducting this study due to the time required for completion of each experiment and the cost of different microgravity platforms. Although drop towers offered microgravity at low cost, drop periods were too short for developing our experiment, this could have been a handicap due to the need of unaffordable number of repetitions to get statistically significant results. On the other hand the International Space Station represented the ideal platform, however its high operational costs requires previous equipment and protocol validation. However, it is certainly a future goal for this project, if expected results are obtained. Sounding rockets offered appropriate time and costs for our project, however impossibility of monitoring the experiment during the flight, inability of modifying tests once launched if an anomaly is observed, a single chance to perform a successful battery of tests, lack of quick reproducibility opportunities, time lapse between selection and flight, ruled out this option for our project. Furthermore, there still are uncertainties on high vibration and high G force effect on used biotechnological materials. We have exposed vesicles to low frequency vibration for mixing ensuring that a moderate vibration can be tolerated, for instance during a parabolic flight takeoff, however high vibration amplitudes destroy vesicles. Other structures providing pseudo microgravity were not suitable for this project. Vesicles have not been designed for clinostat usage, turbulent currents produced by rotating compartments could destroy or fuse together vesicles.

Biotechnology ABC transporter MRP2 (Multidrug Resistance Protein 2) and its major substrate transport estradiol-17-beta-glucuronide were chosen as a model to determine microgravity transport

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activity. Regular protocols provided by MRP2 producer (Solvo Biotechnology) were optimized to be able to conduct experiments with adjusted volumes and in short time periods. ABC transporters are obtained through biotechnology techniques and genetic engineering. By using a viral vector insect cell is transfected with human transporter gene (Galvin, Krajcsi, & Sarkadi, 2004). Cells carrying a human gene synthesize the selected transporter abundantly and place it on the membrane. Cells are destroyed and membrane fragments are rebuilt into vesicles with 10-15% ABC transporter percentage. Transporters are reoriented out-side-in on the membrane thus transporting towards the interior substrates from and assay mix. Once reaction is initiated by exposition of vesicles to a drug rich medium and ATP (Adenosine Tri-phosphate), drug is introduced at high rates into the vesicles. The addition of a washing mix, which drastically reduces temperature, stops the reaction. Substrate is then recoverable from inside the vesicles by millipore fibreglass filter filtration, which isolates vesicles from the medium. Once isolated, vesicles are destroyed and drug is recovered by elution. Final estradiol concentration is measured by gas chromatography separation with coupled mass spectrometry detection (GS/MS). MRP2 mediated transport was measured by determination of estradiol levels contained inside vesicles compared to MRP2 defective (MRP transporter with induced mutation that causes total incapacitation) activity in equal transport conditions. Additionally two other controls were designed to provide information on pharmacological inhibition capacity in microgravity, using a known ABC transporter inihibitor (Benzbromarone), and spontaneous not ATP-dependent MRP2 transport, often observed in these transport systems.

Engineering A tailored electro-mechanical prototype was designed to perform specific advanced laboratory procedures for ABC transporter analysis in microgravity. This system enabled researchers to perform required fluid mixing, within exact timeframes, in exact quantities and at desired temperatures. Schematically, 4 experimentation units composed the prototype, each of them performing one specific control simultaneously. These experimentation units were commanded by a central computer or PLC, which received feedback from several sensors enabling constant optimization of experimental conditions as well as keeping all parameters inside safety limits. Human interaction was required for feeding the system with new samples while replacing already used ones. Prototype’s general structure can be observed in figures 1, 2 & 3 and was composed by the following subsystems:

Structure (Figure 3) The ABCtr prototype was designed according to parabolic flight safety minimums for experimental rack design established by Novespace. All components were oversized in order obtain a minimum safety factor of 1.5 in shear force, traction force, bending moment and linear load.

Fluid system (Figure 4) The fluid system had the objective of ensuring exact fluid distribution within its compartments and very precise fluid quantity displacement on each phase of the experiment. Increased level of accuracy was obtained by the combination of high precision machining of components and the implementation of Schneider Electric’s high precision linear engines. These engines were certified for 0.05 mm displacement error, which represented a calculated 16L fluid displacement error, <10% in current experimental setup. Once fully prepared for an experimental round, prior to microgravity phase, each of the four experimentation units and its fluid systems were composed by one sample syringe, one ATP/Assay syringe and a number of Washing Mix syringes. These syringes and a filter case were interconnected by two three way valves and plastic hoses. The filter case was also connected to three waste syringes

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for disposable fluid collection. In all connections where in-flight removal of components was required, diaphragm valves were installed to avoid fluid leaks. Additionally a second sealed sample-containing syringe was allocated in a pre-heating chamber one parabola prior to its utilisation and underwent a pre-warming protocol in order to stabilize its temperature at 37ºC. Syringes, valves and waste syringes were contained into three different watertight containers ensuring 2 or 3 watertight levels from the cabin and electrical core components. For ATP and washing mix syringes, these containers were also isothermal boxes for temperature control.

Electrical & Control systems (Figure 5)

The Electrical system was designed to provide electrical power, control and monitoring capability of all processes performed during parabolic flight, as well as to provide a user-friendly interface by which researchers could monitor/interact with ongoing automated activities. The system also performed a continuous log of events and sensor feedback that served as a posterior quality control of the experiments. This system was designed to provide 220V AC and 24VDC power lines to a control unit (PLC), heating system, four linear engines, eight stepper motors, a control panel and a touch screen. Eight temperature probes and inductive position sensors were constantly providing feedback to the PLC. Following strict parabolic flight regulations, all electrical core systems were contained into an inflammable certified electrical cabinet and were protected against direct and indirect shortcuts, for over voltage and intensity oscillations. Harness was designed to be watertight, however additional watertight hoses were used for its insulation. Harting connection systems were used for connecting the electrical cabinet to all systems inside the main watertight box, ensuring fluid containment.

Heating system (Figures 6 & 7) The main role of the heating system was to maintain reaction chambers at a given temperature (37ºC in this experiment) and to perform a pre-heating protocol for target temperature acquisition in sample syringes. PLC commanded a total of 8 warming resistances, 4 of them performed a warm up procedure of samples in a specialised pre-heating chamber while 4 additional resistances maintained each sample at constant temperature during experiment rounds. A Total of 8 temperature probes provided feedback to the PLC with constant temperature monitoring of 8 heating resistances. Information could be displayed on real time on the touch screen and laptop screen. The PLC selectively delivered power to a specific resistance according to its temperature. For resistances on pre-heating chambers the system would deliver power until 46ºC were reached lately maintaining 37ºC. This temperature peak was needed to quickly raise sample temperature from 2ºC to 37ºC minimizing warm up time. Previous computer based simulations using COMSOL Multyphisics software and real experimental demonstrations were performed in order to establish proper temperature curve that was able to warm up samples to 37ºC without exceeding 45ºC at any point of the inner wall of sample syringes, so biotechnological material was not destroyed. On the other hand, for heating resistances on reaction chambers, in which constant 37º C were required, reaction chambers contained a cylindrical compartment filled with thermal oil that helped distributing temperature along syringe’s surface. The whole system was protected by several active and passive safety systems in order to avoid overheating, shortcutting and ignition. Such systems comprised:

o Constant automatic temperature monitoring to avoid temperature rising above a given limit.

o Maximum continuous power delivery time to four minutes in case of temperature probe malfunction.

o Thermal fuses limited to 72ºC and 84ºC for reaction chamber and preheating chamber respectively protected all heating resistances.

o Prototype was designed so critical elements were contained into an inflammable polycarbonate sealed box, held by TeflonTM attachments.

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Additionally to temperature control, PLC performed a continuous logging of temperature values in each reaction chamber of the system. Latter analysis of this log showed temperature oscillation ranging from 37ºC to 45ºC on preheating chambers inner wall (outer syringe wall). This log demonstrated no peaks above critical temperature in any of the flights. On reaction chambers, temperature ranged from 36,7ºC to 37,8ºC during the whole 3 flights, which is considered optimal for this project.

Cooling system (Image 8, Figure 9)

The cooling system was designed to maintain samples, ATP and washing mix at a stable temperature between 2-6ºC during the whole parabolic flight. Two certified isothermal boxes with cold accumulators, both allocated at the external part of the experimentation rack composed the system. ATP and Washing mix syringes were connected to the experimentation rack using double watertight hoses with three levels of thermal coating to minimize cold looses during fluid displacement.

Containment system (Figure 9) Free-floating fluids are commonly considered as very dangerous threat that may imply a catastrophic risk in any microgravity environment. Thus all fluids used in this experiment during microgravity phases were isolated from electrical components and the airplane cabin with a minimum of two watertight insulation levels. This protection was reduced to one level during inter-parabola 1g phases for sample exchange. For critical locations inside the experimental rack, like the reaction chamber and waste deposit, an additional watertight insulation level was installed. The experimental rack itself was a watertight box, shielded with polycarbonate foils. All joints, angles and holes were covered with glued silicon. Storage boxes used were IP67 certified and connections between them and the main rack were double coated with plastic hoses and watertight joins.

3.5. Inflight Experimental Procedure Fifty experiments each of them consisting on four different sample syringes were to be performed in microgravity during the first two flights, twenty five experiments per flight, one hundred fifty samples in two flights. Twenty experimental rounds had to be performed in microgravity phases whereas five would be performed during 4’, 5’, 8’ breaks. These experimental rounds were used as a positive control in exact research conditions except from microgravity, providing key information about the state of vesicles after transport and their performance in aircraft’s vibration, pressure and temperature environment. However, due to a combination of man-induced issues and some system faults certain number of samples were lost on each flight, detailed report on final available samples can be found hereinafter. For the last flight, reaction time was extended to 4 minutes disregarding micro/hypergravity phases. These samples reacted until reaching known transport steady state, thus should provide information on whether microgravity alone on gravitational stresses modify transport rates when compared to microgravity alone and 1g samples. The following chart outlines main procedures performed on a regular 20s parabola. For other previously mentioned experimental scenarios (Flight 3) time was increased without changing any other parameter.

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Phase Step Action

Before parabola (1g)

1- Sample Syringe insertion (360 µl) (manually)

Previous sample syringe (Jx) is manually reallocated from the pre-heating slot to the sealed fluid heating system (reaction chamber) becoming J1.


2- Sample Syringe (Jx) insertion to preheating chamber (360 µl) (manually)

A new sample syringe (Jx) is manually inserted on the pre-heating slot to undergo a pre-heating process for temperature acquisition and stabilisation, which will last the whole parabolic maneouver


3- Filter installation (manually)

Filter FLT is manually installed in the filter slot at the final section of the fluid system

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Before parabola (1g and 2g injection phase)

4- Heating (automatically / manually, Button “ARM”)

Heating of Jx and J1. Valve V1 rotation in preparation for ATP injection. Linear actuator approaches J1 plunger (plunger lock must be manually attached to actuator) enabling automatic plunger displacement.

At the beginning of microgravity phase

5 - Injection 250 µL ATP + 20s of microgravity (automatically, Button “START”)

ATP 250 µl injection into reaction chamber. The time for injection is included into total time calculations to fit inside each parabola. After injection valves V1 and V2 rotate in preparation for washing mix injection.

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At the end of microgravity phase

6- Washing Mix injection (1400 µL) – Reaction Stop (automatically)

Washing mix injection into reaction chamber. Reaction Stops.

After microgravity phase (2g)

7- Filter (automatically)

Valves V1 and V2 rotate liquid displacement towards micropore filter. Vesicles are captured and isolated from reaction medium.

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After microgravity phase (2g)

8- Wash out – Washing Mix 2000 µL (automatically)

Valves V1 and V2 rotate to allow Washing Mix injection into reaction chamber. Then valves re-orientate and plunger displaces liquid toward the filter. Wash out of the filter and conductions occur.

After parabola (1g)

9 – Finalisation (manually)

Syringe J1 extraction and disposal, filter extraction and cold stowage. (Jx sample syringe continues pre-heating process) Filters are retrieved, tapped and stored into the used-filters watertight box in rack 2.

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3.6. Pre and postflight tasks during the PFC

Before each flight cold accumulators had to be retrieved from freezers and installed inside isothermal boxes to maintain their temperature within 2-6ºC. ATP, Assay Mix and Wash Buffer syringes had also to be retrieved from freezers, unfrozen, and connected to specific conductions. These conductions had also to be cleaned and purged in preparation for first parabola. Sample syringes, properly labelled with four colours were transported frozen directly into the flying isothermal box, which had to be installed inside the watertight box allocated in rack 2. This procedure was aimed to maintain the cold chain as much as possible. Clean filters had also to be loaded into their specific case before each flight. After each flight cold accumulators had to be brought back to freezers for the next day, so did Washing Mix and ATP / Assay mix syringes. Used filters had to be eluded with 3 ml of ethanol which minimized estradiol degradation, a preliminary phase of later sample analysis in IMIM laboratories. Filter cases must be cleaned and dried for next day experiments.

4. Parabolic Flight Campaign

4.1. Before the flights One week prior to 51st ESA Parabolic Flight Campaign, the ABC transporters in microgravity prototype rack 1 was shipped in its shipment configuration and arrived one week later at Novespace premises. Racks 2 & 3 and other materials were transported by the team. Biotechnological samples arrived in cold storage during the first week of the campaign and were properly stored in cold facilities in Novespace. Some concerns arose at the moment of reception of these materials since there was a critical low amount of dry ice inside the shipment box, thus cold chain was suspected to have broken. However, samples were still frozen at the moment of reception so it was considered that, although optimal temperature had not been maintained during the transport, it was kept within minimum margins preventing major damage of samples. During preliminary safety assessment performed by Novespace personnel it was determined that the weight of the prototype was higher than the expected, stated on the ESDP, thus center of gravity calculations were wrong and above limits for bending moment. It was decided to take a direct measurement of CG rather than using calculations, which resulted on a lower CG than expected, therefore mechanical characteristics of the prototype fit within Novespace safety requirements. Watertightness test were performed demonstrating sealing of critical compartments and insulation of electrical components. A general breaker switch test was performed and the superimunized differential breaker switch, which had been installed was not suitable and had to be replaced. Superimunized breakers had been recommended by Schneider ElectricTM since they can bare current oscillations produced by motors used for this experiment. In spite of the change, no spontaneous power cuts were observed when using the motors and regular differential switch. Power up was performed without other contingencies. All systems were checked and performed as expected, ready for flight. However, it was recommended to ad two additional steal profiles to the electrical cabinet in order to enhance structural strength towards the X+ direction of the plane. The team finalised padding the racks, and prepared filters and samples for the first flight.

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In order to enter the aircraft a tilting tool was mounted to main rack so it could trespass the aircraft door in a 90º-tilted position. Once inside the aircraft rack fixation was performed as expected.

4.2. During the flights Flight One Major issues during this flight consisted on a fluid leak on Unit 3 (no ATP). All water-tightness barriers performed as expected and no fluid got in contact with any electrical component neither did it disperse into the cabin. Unit 3 was forced to a self-sealing position ensuring water-tightness for the rest of the flight. At the end of the flight a total of 21 samples from the initial 100 (21%) had been lost, 13 (13%) due to Unit 3 malfunction, the remaining 12 (12%) due to other human-related errors. In conclusion this flight was successful from the fluid containment and control system point of view, but its outcome in means of scientific production was impaired due to the amount of samples lost. It was latter determined that a misalignment while replacing a filter case on Unit 3 bended an anti-leak diaphragm valve so homogeneous contact between the valve and filter cases was not possible, causing a constant leak when fluids where displaced through this conduction. Fortunately, with a soft pull of the plastic diaphragm it returned back to its original position. Water-tightness tests were performed to ensure correct valve performance. Since no leak was observed any further repairs were performed. The whole team was instructed on a new and more cautious method of filter case allocation, which can prevent this problem to occur in the future. Flight two With an initial load of 100 samples major issues in this flight involved malfunction of the fluid containment system. In this flight it was quickly determined that Unit 2 (MRP2 Defective) had a major leak due to a diaphragm valve total destruction. Water-tightness barriers performed as expected, containing fluids inside the main rack. However, Unit 2 had to be stopped and configured into a self-sealing position. MRP2 Def. control lost implied a major impairment to scientific outcome of this flight, thus it was decided to disregard Benzbromarone control samples and use their experimentation unit for running MRP2 Def. control samples. Team’s decision and reconfiguration times were quick enough so sample lost was minimized. At the conclusion of this second flight only 4 (4%) MRP2 Def. samples had been lost, but 11 (11%) Benzbromarone samples were lost in return. A little amount of other samples were lost, 6 (6%), due to miscellaneous problems. Nevertheless, during this flight it was observed that several filter cases presented important leaks on their screwing parts. This problem had been already observed but in a very reduced portion of filter cases during the previous flight. It was a latter on-ground evaluation and testing of filter cases that showed a fabrication flaw on some filter cases in which screws did not provide sufficient sealing, permitting water to escape when high pressures were applied. Due to a lack of enough spare filter cases the available ones were reinforced with Teflon coating for the next flight. Tests showed an outstanding performance of reinforced filter cases. Flight Three In this flight experimental reaction time was extended to 4’, therefore only 60 samples were loaded on board (15 per each control). Only 8 samples (13%) were lost during this flight. No major issues arose and all systems performed flawlessly.

5. Scientific Results CONFIDENTIAL 6. Conclusions

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The ABC transporters in microgravity project aims to ascertain whether microgravity is able to modify ABC transporter mediated active trans-membrane drug transport. A positive answer to this question has a series of very relevant scientific implications, which range from microgravity molecular biology to microgravity cellular physiology, space pharmacology and can even affect how do we understand some human physiological responses observed in weightlessness. It can also have an important impact on future space missions health care systems if further evaluations demonstrate ABC transporter’s altered function in microgravity can represent a real modification in drug distribution, thus impairing drug efficiency or enhancing their side effects. Furthermore, results may extend to physics domain. Thereafter, more detailed analysis should be performed in the future to disguise how microgravity is able of modifying processes in which stronger intermolecular forces are implied. Understanding all previously mentioned implications is of prime importance when deciding to withdraw a definitive conclusion from obtained data. Therefore, given the high dispersion of results obtained so far it is not yet possible to provide a conclusive answer to whether microgravity is able of affecting ABC transporters activity in microgravity. The contrary would be irresponsible and lacking of scientific rigor. Nevertheless, this project already demonstrated it is feasible to perform reliable and reproducible biochemical evaluations in microgravity where trans-membrane cell transporters are implied, which fosters a new and exciting area of knowledge into microgravity research. Work on this project will continue until all samples have been analyzed. Additional samples will be obtained in our lab in similar conditions to parabolic flight in order to have a solid gold standard set of values to compare with in 1g. Nevertheless, given the complexity of the procedures required for sample processing, results may not be available in a short period of time. Once all data has been gathered, in-depth statistical analysis will be performed to withdraw a definitive conclusion. It is foreseen that a final official paper will be developed for its publication in a scientific journal.

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7. Figures / Attachments

Figure 1. Schematic of main rack. One experimentation unit comprising one liner engine, reaction chamber and waste deposit is highlighted.

Figure 2. Airplane distribution of racks. Note that the electrical cabinet (dark blue) was allocated underneath main rack.

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Figure 3. Main Rack Structure.

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Figure 4. Fluid System

Figure 5. Electrical & Control Systems

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Figure 6. Heating and Pre-Heating Chambers

Figure 7. Heating system schematic.

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First insulation layer: nylon tape Second insulation layer: foam

hose Third insulation layer: aluminium tape

Image 8. Cooling system conductions, insulation.

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Figure 9. Containment system. New filters box, new syringes box, used syringes box, used filters box and ATP/WB box were IP67 certified watertight boxes.

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8. References Beringer, P. M., & Slaughter, R. L. (2005). Transporters and their impact on drug disposition. The Annals of Parmacotherapy , 39, 1097-1108. European Astronaut Centre - EAC. (2007). International Space Station Medical Checklist & Medical Operations. Cologne, Germany. Evetts, S. N. (2007). Space Medicine. European Space Agency, European Astronaut Centre, Cologne. Galvinas, H., Krajcsi, P., & Sarkadi, B. (2004). The role of ABC transporters in Drug Resistance, Metabolism and Toxicity. Current Drug Delivery , 1, 27-42. Gandia, P., Saivin, S., & Houin, G. (2005). The influence of weightlessness on pharmacokinetics. Fundamental & Clinical Pharmacology (19), 625-636. Giachetti, E., Ranaldi, F., & Fiusco, A. (1999). Enzyme kinetic parameters are not altered by microgravity. Microgravity Science Technology , 36-40. Giachetti, E., Ranaldi, F., & Vanni, P. (2001). Enzyme catalysis in microgravity: an intricate problem to be solved. Federation of European Ciochemical Societies , 78-79. Goldermann, M., & Hanke, W. (2001). Ion channel are sensitive to gravity changes. Microgravity Science & Technology , 13 (1), 35-38. Graebe, A., Schuck, E., & Derendorf, H. (2004). Physiological, Pharmacokinetic and Pharmacodynamic Changes in Space. The Journal of Clinical Pharmacology , 44, 837-853. Integrated Medical Group (IMG). (n.d.). International Space Station Medical Checklist. Maccarrone, M., Bari, M., Battista, N., & Finazzi-Agrò, A. (2001). The catalytic efficiency of soybean lipoxygenase-1 is enhanced at low gravity. Biophysical Chemistry , 90, 97-101. Macarrone, M., & Finazzi-Agró, A. (2001). Enzyme activity in microgravity: a problem of catalysis at the water-lipid interface? Federation of European Biochemical Societies , 504, 80. Ranaldi, F., Vanni, P., & Giachetti, E. (2003). Enzime catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction. Biophysical Chemistry , 103, 169-177. Robinson, J. A. (2006). NASA utilization of the International Space Station and the vision for space exploration. International Astronautical Congress. Tash, J. S., & Bracho, G. E. (1999). Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility. FASEB , 13 (Suplement), 43-54.

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