Viola
Hähnel
a,
Isabell
Weber
b,
Simon
Tuemmler
b,
Bernhard
Graf
b,
Michael
Gruber
*b,
Ralph
Burkhardt
a and
Norbert
Ahrens
a
aInstitute of Clinical Chemistry and Laboratory Medicine, Transfusion Medicine, University Hospital Regensburg, Regensburg, Germany
bDepartment of Anesthesiology, University Hospital Regensburg, Regensburg, Germany. E-mail: michael.gruber@ukr.de; Tel: +49 (0) 941/944-7870
First published on 8th July 2020
Background: Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy for various diseases. Autologous leukocytes are collected, photoactivated with a photosensitizer (8-methoxypsoralen, 8-MOP) and UVA light, and subsequently reinfused back to the patient. Leukapheresis and UVA irradiation systems can be integrated into one device (inline) or handled by two separate devices (offline). ECP works via intercalation of 8-MOP into DNA helices and UVA-based interactions to inhibit DNA replication. 8-MOP is known to adhere to plastic materials, which might reduce its availability for intercalation. In the present study we examined the bioavailability of 8-MOP when different plastic materials and solvents are used as matrices. Methods: Varying amounts of shredded ethylene vinyl acetate (EVA) and polyvinylchloride (PVC) from the MacoGenic irradiation bag (EVA1), UVA PIT irradiation bag (EVA2), UVA PIT recirculation bag (PVC A) and UVA PIT tubing (PVC B) by MacoPharma and PIT Medical Systems, respectively, were incubated with 200 ng mL−1 8-MOP dissolved in diisopropyl ether (DIPE) plus toluene 90/10 vol%, deionized water or plasma. After 2 h 8-MOP concentrations were determined by GC-MS. Results: After incubation, 8-MOP concentrations varied depending on the amount and type of plastic (PVC > EVA) and solvent (water > plasma > DIPE/toluene). Absorption to 200 mg EVA1 or EVA2 resulted in 8-MOP concentrations of 57% or 32% in water, 91% or 80% in plasma, and 93% or 92% in DIPE/toluene, while 200 mg PVC A and PVC B yielded recovery rates of 26% and 10% in water, 76% and 75% in plasma, and 55% and 30% in DIPE/toluene, respectively. Remaining 8-MOP differed significantly between container materials (EVA vs. PVC; p < 0.022) but not manufacturers (MacoPharma vs. PIT Medical Systems). Conclusion: Absorption loss of 8-MOP depends on the type of plastic and solvent and is more pronounced with water than with plasma. As the DNA binding effect of 8-MOP is dose-dependent, ECP starting doses should be adjusted to ensure that a sufficient concentration of free bioavailable 8-MOP is present during UV irradiation.
8-MOP intercalates into DNA and, following exposure to UVA, forms monoadducts and DNA interstrand cross-links,4–7 resulting in the inhibition of DNA replication, white blood cell apoptosis and, ultimately, immunosuppression.8,9 After administration, the unbound fraction of 8-MOP is rapidly metabolized and excreted in the urine.10
8-MOP binds to DNA of cells, to protein and membrane structures and may absorb to plastic materials due to its lipophilic structure.11 The resulting loss of the drug could be dependent on the type of plastic container and solvent (liquid matrix) in which the treated cells are suspended (e.g., plasma or physiological saline). As the binding of 8-MOP to DNA is dose-dependent,12 the starting dose would need to be adjusted to ensure that sufficient quantities of 8-MOP are present in the cell suspension during reinfusion.
Several methods have been described for psoralen detection, including gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS).13–18 In this study, we applied a GC-MS method to examine the adsorption-dependent drug bioavailability of 8-MOP utilizing various commonly used plastic materials and solvents in the liquid and solid matrix.
Absorption was tested with zero, 80, 100, 120, 150, 180 and 200 mg of sample material (5–6 each) that was incubated for 2 hours with 200 ng 8-MOP from stock solution dissolved in 1000 μL of liquid matrix—diisopropyl ether (DIPE; Honeywell) plus toluene 90/10 v/v, deionized water or plasma (BRK Munich), respectively. 8-MOP stock solutions (2000 ng mL−1) were prepared by toluene extraction of 8-MOP (Uvadex, Therakos, Pennsylvania, USA) in the case of DIPE/toluene, and by 1:
10 dilution of 8-MOP in deionized water in the case of plasma and water.
Aliquots of 200 μL supernatant were transferred to glass tubes. We used 5-MOP (Sigma Aldrich, Germany) as internal standard in a concentration of 20.8 ng μL−1. In triplicate experiments, the psoralens were extracted from deionized water or plasma with toluene (200 μL). The combined toluene fractions were then vaporized at 40 °C and subsequently re-dissolved in 300 μL toluene before analysis. With the DIPE/toluene matrix, the samples were dried in a gentle stream of N2 at 40 °C and directly re-dissolved in 300 μL toluene. A blank sample containing 200 μL solvent alone was always co-tested.
For recovery from absorption (elution), the plastic samples were surface-cleaned with 1000 μL toluene after the above mentioned incubation, transferred to separate tubes and subsequently incubated in 1 mL toluene for 24 hours. 290 μL supernatant and 10 μL internal standard were mixed and analyzed by GC-MS.
The solid to liquid partition coefficients (k) were estimated by the following equation to avoid density inaccuracies:
With DIPE/toluene (organic solution) as the liquid matrix, remaining 8-MOP decreased with sample weight from 62% (80 mg) to 30% (200 mg) for PVC A and from 72% (80 mg) to 55% (200 mg) for PVC B (Fig. 1b) compared to decreases from 97% (80 mg) to 92% (200 mg) for EVA2 and 93% (200 mg) for EVA 1.
Compared to the other liquid matrices, plasma resulted in less loss of 8-MOP with remaining 72% (PVC A) and 76% (PVC B, Fig. 1c), and 80% (EVA1) and 91% (EVA2, all 200 mg each).
(a) P value | (b) P value | |
---|---|---|
a: EVA 1 (MacoGenic irradiation bag) versus PVC A (UVA PIT recirculation bag); b: EVA 1 (MacoGenic irradiation bag) versus EVA 2 (UVA PIT irradiation bag); n.s., p ≥ 0.05. | ||
Deionized water | <0.01 | n.s. |
DIPE/toluene | <0.01 | 0.014 |
Plasma | 0.022 | n.s. |
Samples of EVA1 and EVA2 absorbed less of the photochemical, as reflected by recovery rates of 34% and 22% (DIPE/toluene), 59% and 78% (water) and 11% and 19% (plasma), respectively.
In general, the higher affinity of 8-MOP to PVC, especially in water, is illustrated by the solid to liquid partition coefficients (Fig. 3). Partition coefficients for EVA were 7.7, 0.8, and 1.2 in water, plasma, and DIPE/toluene, while those for PVC were 29.2, 2.3, and 5.5, respectively.
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Fig. 3 Partition coefficients of 8-MOP [g mL−1] dependent on the type of plastic material and solvent. |
This study was conducted to simulate the influence of different solvents and container materials on the bioavailability of 8-MOP during ECP. Irradiation bags usually consist of EVA, a plastic material which should have less affinity to 8-MOP than others. Nevertheless, about 30% of free bioavailable 8-MOP is lost due to adsorption to EVA.20
Loss of 8-MOP from the liquid matrix into the solid matrix varied dependent on the type and surface area of plastic material in the solid matrix as well as on the type of solvent in the liquid matrix (Table 2). Our test results indicated that 8-MOP was more likely absorbed into the plastic matrix rather than just being adsorbed onto its surface. PVC absorbed more 8-MOP than EVA. Incubation with increasing amounts of plastic material resulted in decreasing concentrations of free 8-MOP in solvent (recovery). Absorption loss was more pronounced in water than in plasma.
Deionized water | Plasma | DIPE/toluene | |
---|---|---|---|
Symbols: weak (↑), moderate (↑↑), strong (↑↑↑), unchanged (↔). | |||
EVA | ↑↑ | ↔ | ↔ |
PVC | ↑↑↑ | ↑ | ↑↑ |
Similarly, Laulhé et al. observed that 8-MOP adsorption was significantly higher in NaCl (79%) than in plasma (94%), resulting in the inhibition of lymphocyte proliferation and an increase in T-cell apoptosis.21 Hence, plasma seems to be associated with lower bioavailability of 8-MOP even though adsorption is less pronounced. This could be caused by protein binding of 8-MOP in plasma.
In conclusion, the type of liquid matrix in which leukocytes are suspended influences the availability of 8-MOP. To ensure that free, bioavailable drug is present during ECP treatment, the starting dose should be considered accordingly.
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