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

Introduction

Critical medical situations can occur in space missions¹. In such situations, adequate treatment may require the use of drugs with potential severe side effects. Pharmacokinetics and pharmacodynamics can be modified under weightlessness conditions by many factors^2,3, which may lead to undesired pharmacological effects. These parameters should be assessed carefully in order to ensure safe use of medications in space. However, insufficient information exists in this regard in microgravity^4,5 and proper empirical evaluation is not feasible due to the significant associated risks.

ABC (ATP-Binding Cassette) transporters are a large family of trans-membrane proteins widely present in many life forms, from bacteria to mammals. In humans, they play important roles in many physiological processes and in drug pharmacokinetics, pharmacodynamics, and drug-to-drug interactions of many currently used medications in oncology, emergency medicine, critical care and general practice^6,7. Inhibition of these transporters has been correlated with clinically relevant metabolic disorders, drug overdosing, and drug toxicity⁷. On the other hand, their overexpression can lead to treatment failure due to enhanced drug cell clearance and increased drug excretion from the body⁷. Multi-drug resistance-associated protein 2 (MRP2) is a well-studied representative of the ABC transporters family, by which transport of glucuronide, sulphate and gluthatione-conjugated drugs is made against a concentration gradient requiring adenosine-tri-phosphate (ATP) hydrolysis⁸.

Genetic expression of ABC transporters has been found to change in real and simulated microgravity conditions. Two independent studies evaluated gene expression pattern in two models of medically relevant species of microorganisms (Salmonella sp. and Candida sp.) and found upregulation of certain ABC transporters genes during short duration spaceflight missions^9,10. Similarly, results from a microgravity simulation study showed that significant variation of certain ABC transporter levels could be found in the liver and kidneys of a murine model¹¹. These changes could lead to a modified antimicrobial susceptibility in microgravity and to different pharmacokinetic characteristics of medications during spaceflight. Furthermore, recent reports suggest that microgravity can modify the activity of certain important cell components^12–14, especially transmembrane proteins and ion channels^15–17 with structural and functional similarities to ABC transporters. Changes in ABC transporters activity could lead to clinically significant metabolic disorders and potentially dangerous undesired pharmacological effects during spaceflight. However, there is no current technology that permits a functional evaluation of these cellular components in microgravity. Therefore, we developed a new approach combining the use of a currently available commercial assay, a high precision electromechanical system and an enhanced drug detection protocol to evaluate ABC transporters activity in microgravity conditions. A validation study of this new biotechnological approach is presented.

Glossary

Microgravity: A condition in which there is very little net gravitational force, as of a free-falling object, an orbit, or interstellar space. In this text microgravity, weightlessness and zero gravity (0g) are considered equivalents.

Parabolic flight/parabolic flight campaign: To create a weightless environment, an especially prepared airplane flies in a long parabolic arc, in which two 2g periods are experienced while ascending (injection phase) and recovering (pull out phase) from the manoeuvre. In between these two periods, the aircraft enters into a free falling state, lasting approximately 22s, in which microgravity is obtained inside the cabin. A regular parabolic flight consists of 30 parabolas with a number of breaks. A parabolic flight campaign consists of three flights.

Methods

This study was performed during the 51^st European Space Agency (ESA) parabolic flight campaign, under the auspices of the “Fly your thesis” programme, and was developed following the specifications and experimental requirements of ESA/Novespace parabolic flight campaigns¹⁸.

Equipment

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 material Appendix 2. List of Components for a more detailed description of equipment and components used).

Figure 1. Main experimental rack.

The designed electromechanical system was composed of three racks. In this figure a scheme of the main rack is presented, containing the four experimentation units. One of the four experimentation units is highlighted with a dashed line. Several electrical holes permitted connection to all electrical components in the main rack (sensors, actuators, linear engines, resistances). One ATP-WB hole permitted access to liquid conductions transporting ATP and Wash Buffer to each experimentation units. Lengths are expressed in mm.

Figure 2. 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.

Results

All four different assays presented expected E17βG signal profile in microgravity and on ground 1g conditions (Table 1), being MRP2+ samples the most active and MRPdef the less active group of samples in all gravitational conditions. The ANOVA test revealed statistically significant signal differences between sample groups in each gravitational condition (on ground F = 4.95, p = 0.005 and microgravity F = 13.45, p < 0.001) and post-hoc Dunnett t test confirmed statistically significant differences between MRP2+ and MRPdef in both microgravity and 1g conditions (Table 2). These results confirmed activation of the transporter in MRP2+ samples. MRP2 net transport activity of 1g on ground samples laid within commercial transport standards (4.61 ng in a range of 3.7 – 7.4 ng of E17βG in our experimental configuration), conversely microgravity samples suffered a 19-fold activity reduction with statistically significant differences of E17βG transported in all cases. The Mean activity differences between microgravity and 1g on ground samples were 10.3 ng of E17βG in MRP2+ samples (Mann-Witney U < 0.001; p < 0.001; CI 95% = 7.4 – 13.2 ng E17βG), 5.95 ng of E17βG in MRPdef samples (Mann-Witney U < 0.001; p < 0.001; CI 95% = 4 – 7.9 ng E17βG), 7.65 ng of E17βG in noATP samples (Mann-Witney U < 0.001; p < 0.001; CI 95% = 6.14 – 9.16 ng E17βG) and 9.12 ng of E17βG in BZM samples (Mann-Witney U < 0.001; p < 0.001; CI 95% = 7.9 – 10.3 ng E17βG).

The use of consecutive 2g periods in order to simulate the effects of hypergravity present during a parabolic flight did not show differences in shape, size or number of vesicles between centrifuged vesicles and control samples. Aggregation of vesicles was not observed, neither macroscopically nor microscopically during this simulation. The thermal control system implemented in our prototype maintained the temperature of reaction chambers at 37.25°C (CI 95%: 37.14 – 37.34°C) during the whole duration of experiments and kept the sample syringe wall temperature below 45°C in preheating chambers in all cases.

Discussion

To our knowledge, this is the first validation study of a biotechnological approach aimed at evaluating human ABC transporters-mediated active transmembrane transport capabilities in microgravity. Despite the high signal variability expected, the precision attained by our electromechanical system and the enhanced sensibility of the detection protocol used, enabled us to detect net MRP2 transport activity in the short microgravity periods provided by parabolic flights. Furthermore, activation profile and signal levels detected in 1g on ground samples laid within expected commercial standards, confirming the validity of the equipment and procedures used.

The results obtained in microgravity samples showed a significant reduction in MRP2 transport activity, when compared to on ground 1g controls, but presented a comparable activation profile. In order to ensure reliability of these findings, vesicle stability during a parabolic flight was assessed on ground. Simulations revealed that vesicle microscopic structure could remain stable during parabolic manoeuvres, in spite of repeated hypergravity periods. Provided that materials, equipment, procedures and transportation means were equal for all samples in all gravitational conditions, we hypothesized that gravitational stress during parabolic flights may have exerted an effect upon the vesicle membrane or the transporter, preventing proper transport or retention of E17βG. However, such modification should occur at an ultra-structural level, out of reach of our microscopic evaluation. Unfortunately, given the lack of similar research precedents, an ultra-structural evaluation of samples was not planned and was not possible after analysis, since all microgravity samples had been eluted to retrieve E17βG. Therefore, the results of the present evaluation cannot provide a definitive explanation of the effect of microgravity upon ABC transporters-mediated active transmembrane transport. Notwithstanding the aforementioned limitation, we tried to elaborate a plausible hypothesis to explain the present observations.

Compelling evidence indicates that gravity is able to affect cellular and even molecular interactions in numerous life forms, such as in certain protozoa^12,14, bacteria¹⁵, plant cells^13,16,17 or mammals^21–23. Enzymes from the cell membrane²⁴, cytoskeleton²⁵, cytosol²⁶ and nucleus²⁴ have been found to be sensible to gravitational changes. In principle, gravity is a weaker force compared to intermolecular bonds, polar-apolar interactions and van der Waals forces, therefore it is not clear yet how gravity could affect enzymatic reactions. However, changes in polar-apolar interactions and conformational changes of lipid structures occurring in microgravity could explain some observations^24,25. Structures such as ion channels and trans-membrane signalling systems can be significantly affected by changes in physicochemical properties of the lipid bi-layer cell membrane. Similarly, the mechanism of action of ABC transporters, although still not fully unveiled, is highly dependent on transporter-membrane interactions²⁸, which play a principal role in transporter stabilization and substrate recognition. Interestingly, we were able to demonstrate a reduction of the signal detected in MRPdef controls, where only simple diffusion of substrate was possible. This finding could be explained by an impeded E17βG diffusion into vesicles, or by an increase in membrane permeability in microgravity, which would lead to increased E17βG leakage. In any case, the present observations are indicative of important structural changes in the lipid bi-layer in microgravity, which could be induced by modified polar-apolar interactions of lipid structures in microgravity, as suggested elsewhere²⁸. In this context, the interaction of ABC transporters with the membrane, substrate recognition capabilities and transporter activation could become significantly affected with the potential for relevant biological, physiological and medical consequences in microgravity. However, additional research is required to confirm the present hypothesis.

There are many potential applications of the presented technology, which can benefit both space and terrestrial medicine. Insufficient information exists on drug effects in space^4,5 and is often based in very reduced observational studies³⁰ or in animal models³¹. While clinical experience throughout spaceflight history has shown no critical metabolic alterations in astronauts, few medications have been studied from a pharmacological point of view in space. A reduction of transmembrane drug transport capabilities in microgravity could lead to drug accumulation and potentially toxic effects during spaceflight. However an increase in membrane permeability would lead to an enhanced drug clearance, increased absorption and significant changes in pharmacological proprieties of drugs. Although not conclusive, our results warrant additional and thorough evaluation of this and other human drug transportation systems in a more stable microgravity platform. To this end, this study describes a new method, based on in vitro analysis of human drug transmembrane transport capabilities, by which an approximation to drug effects in microgravity can be made. Similarly, reduced activity of certain ABC transporters has been associated with a number of serious medical conditions on Earth. On the other hand, enhanced activity of ABC transporters can decrease intestinal drug absorption, facilitate hepatic and renal drug excretion, modify drug distribution, and limit drug penetration to certain body tissues. These transporters are known to be responsible for a significant portion of the variability in treatment response to certain drugs used in emergency and critical care medicine^6–8 and can cause multidrug resistance in a number of solid and hematological neoplasias, where overexpression of certain ABC transporters is present^6,7. Further research derived from this study may help elucidate the intricate mechanism of action of ABC transporters and provide new information for developing more effective treatments for oncology and other medical specialities based on microgravity effects upon these transporters.

There are several limitations in this study. First, we designed a novel electromechanical system to allow the required biochemical reactions to be undertaken precisely and automatically in parabolic flights. This equipment had never been tested before in such conditions. However, results from 1g on ground control samples laid within commercial transport standards, proving functionality and validity of the research equipment and protocols used. Additionally, MRP2 net E17βG transport activity in microgravity fell within the detection range of our enhanced detection method, which confirmed that the required sensitivity was reached. Second, as previously addressed by Macarrone et al.²⁸, “microgravity is likely to favour the dispersion in water of less dense molecules such as lipids and the opposite is observed in hypergravity”. The contact surface available for ABC transporter exposition to drug-rich medium would be reduced, and transport capability would consequently be diminished, if vesicles precipitated during 2g phases in parabolic flights. Furthermore, vesicle structural integrity has never been evaluated in variable gravitational conditions. Nevertheless, no macroscopic precipitation of samples was observed in-flight due to combined effects of aircraft vibration and the ATP turbulent injection jet. On-ground hypergravity simulation data suggested that vesicles remained stable and did not aggregate after repeated 2g periods. However ultrastructural alterations affecting the membrane or the transporter could still be possible and were not evaluated. Further research should focus on performing a detailed ultra-structural evaluation of vesicles and transporters in a more stable microgravity platform and use whole human cells to provide a more conclusive answer on the potential physiological and clinical effects of our findings. Other factors were also considered as possible disruptors of results and were evaluated. The temperature was strictly maintained within a narrow margin during experiments but aircraft pressure was reduced in flight (800 hPa in flight). However, pressure variations were not expected to cause any modification in sample composition nor in transport activity in our experimental setup.

In conclusion, we validated a new methodology for evaluating ABC transporters activity in microgravity during parabolic flights. Our results demonstrated that despite time constrains, the combination of an adapted commercial assay, a highly accurate electromechanical system and an enhanced drug detection protocol can provide enough accuracy, reproducibility and sensitivity to detect transport activity in microgravity. A significant decrease in transport activity of fully activated samples and a reduction of signal detected in negative controls could be observed in microgravity. While the present results are insufficient for drawing conclusive explanations to these observations, we hypothesize that altered polar-apolar interactions induced by gravitational stress during parabolic manoeuvres affected vesicle’s lipid bi-layer membrane, eventually changing its physicochemical properties, limited simple diffusion phenomena and impaired transporter - membrane interaction. However, alternative explanations are possible. Therefore, additional studies will be required to assess membrane and transporter ultra-structures in microgravity, and to confirm these results. The novel methodological approach presented here opens new and interesting research lines in biology, microbiology and human physiology with the potential for significant benefits for both space and terrestrial medicine.

Data availability

Dataset 1. Data of active transmembrane drug transport in microgravity, 10.5256/f1000research.4909.d34169³²