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. 2023 May 23:3:1113493.
doi: 10.3389/fddev.2023.1113493.

Identification of transport systems involved in eflornithine delivery across the blood-brain barrier

Christopher P Watson  1 , Gayathri Nair Sekhar  1 , Sarah A Thomas  1
Affiliations

Identification of transport systems involved in eflornithine delivery across the blood-brain barrier

Christopher P Watson et al. Front Drug Deliv. .

Abstract

Human African Trypanosomiasis (HAT) is a neglected parasitic disease that continues to persist in sub-Saharan Africa. It is fatal if untreated. The first stage of the disease is associated with the presence of the parasite in the periphery and the second stage with the presence of the parasites in the CNS. The treatment of CNS stage HAT requires the drugs to cross the blood-brain barrier (BBB). Eflornithine is an amino acid analogue that is used to treat second stage HAT gambiense both alone and in combination with nifurtimox. Recent studies have identified that accumulation of eflornithine into the parasites (trypanosomes) involves the amino acid transporter (Trypanosoma brucei AAT6). In this study we tested the hypothesis that eflornithine uses a cationic amino acid transport system to cross the BBB. We particularly focused on system-y+ and system-B0,+. To do this we utilized specialist databases to compare the physicochemical characteristics of relevant molecules and an in vitro model of the BBB to explore the mechanisms of eflornithine delivery into the CNS. Our results confirmed that eflornithine is related to the endogenous amino acid, ornithine. At pH 7.4, eflornithine is predominately (92.39%) a zwitterionic (dipolar) amino acid and ornithine is predominately (99.08%) a cationic (tripolar) amino acid. In addition, the gross charge distribution at pH 7.4 of eflornithine is much smaller (+0.073) than that of ornithine (+0.99). Further results indicated that eflornithine utilized a saturable transport mechanism(s) to cross the hCMEC/D3 cell membranes and that transport was inhibited by the presence of other amino acids including ornithine. Eflornithine transport was also sodium-independent and sensitive to a y+-system inhibitor, but not a B0,+-system inhibitor. Eflornithine transport was also inhibited by pentamidine, suggestive of transport by organic cation transporters (OCT) which are expressed in this cell line. We confirmed expression of the y+-system protein, CAT1, and the B0,+-system protein, ATB0,+, in the hCMEC/D3 cells. We conclude that eflornithine uses the cationic amino acid transporter, system y+, and OCT to cross the BBB. This research highlights the potential of system-y+ to deliver drugs, including eflornithine, across the BBB to treat brain diseases.

Keywords: OCT; amino acids; blood-brain barrier; eflornithine; transporter; trypanosomiasis; y+-system.

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Conflict of interest statement

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The percentage distribution and chemical structures of the four eflornithine microspecies found at physiological pH.
FIGURE 2
FIGURE 2
The effect of unlabelled eflornithine and ornithine on the accumulation of [3H]eflornithine in hCMEC/D3 cells. All data expressed as mean ± S.E.M, n = 3–5 (plates), with 6 replicates (wells) per plate. The [3H]eflornithine Vd has been corrected for [14C]sucrose Vd. Data were analysed using two-way ANOVA with SigmaPlot 13. Significant differences compared to control (i.e., [3H]eflornithine alone) were observed in hCMEC/D3 cells—*p < 0.05, **p < 0.01.
FIGURE 3
FIGURE 3
The effect of unlabelled L-lysine, L-arginine, ADMA and leucine on [3H]eflornithine accumulation in hCMEC/D3 cells. The [3 H]eflornithine Vd has been corrected for [14C]sucrose Vd. Significant differences compared to control were observed in hCMEC/D3 cells—*p < 0.05, **p < 0.01. All data expressed as mean ± S.E.M, n = 3–4 (plates), with 6 replicates (wells) per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.
FIGURE 4
FIGURE 4
[3H]eflornithine accumulation in hCMEC/D3s in the absence and presence of L-homoarginine, BCH or Na+Cl− free buffer. The [3H]eflornithine Vd has been corrected for D-[14C]sucrose Vd. All data expressed as mean ± S.E.M, n = 3 (plates), with 6 replicates (wells) per plate. Data were analysed using two-way ANOVA with SigmaPlot 13. Significant differences compared to control were observed with L-homoarginine at all time-points—***p < 0.001.
FIGURE 5
FIGURE 5
The effect of anti-HAT drugs on radiolabelled eflornithine accumulation in hCMEC/D3 in the presence of DMSO. The [3H]eflornithine. Vd has been corrected for [14C]sucrose Vd. All data expressed as mean ± S.E.M, n = 3 (plates), with 6 replicates (wells) per plate. Data were analysed using two-way ANOVA with SigmaPlot 13. Significant differences were observed with pentamidine in hCMEC/D3 cells -**p < 0.01. The control and test groups were performed in the presence of 0.05% DMSO.
FIGURE 6
FIGURE 6
The effect of suramin on [3H]eflornithine accumulation in hCMEC/D3s. The [3H]eflornithine Vd has been corrected for [14C]sucrose Vd. All data expressed as mean ± S.E.M, n = 4 plates, with 6 replicates (wells) per plate. Data were analysed using two-way ANOVA with SigmaPlot 13. No differences compared to control were observed.
FIGURE 7
FIGURE 7
Potential cytotoxic effects of test conditions were assessed by an MTT assay in hCMEC/D3 over 30 min. No significant effects were observed except for the positive control 1% Triton X-100 (***p < 0.001). All data expressed as mean ± S.E.M, n = 4 plates, with 6 replicates (wells) per plate. Data were analysed using one-way ANOVA with SigmaPlot.
FIGURE 8
FIGURE 8
Expression of CAT-1 in hCMEC/D3 cells. HepG2 cells was used as a positive control. A band at 68 kD was observed in all lanes and assumed to be CAT-1. Lane 1- hCMEC/D3 passage 28, Lane 2- hCMEC/D3 passage 33, Lane 3- control HepG2 cells. Lanes 1, 2 and 3 present on same blot.
FIGURE 9
FIGURE 9
Expression of ATB0,+ in hCMEC/D3 cells. (A) An example Western blot. Following deglycosylation with PNGase F, SDS-PAGE and WB analysis revealed ATB 0,+ expression in hCMEC/D3 (passage 28) and wild type MCF7 whole cell lysate lysed in TGN lysis buffer. Bands from 70 to 55kD were observed. Lane 1 –MCF7 without PNGase, Lane 2-MCF7 with PNGase, Lane 3- hCMEC/D3 passage 28 without PNGase, Lane 4-hCMEC/D3 passage 28 with PNGase. (B) ATB 0,+ expression was also demonstrated by immunofluorescence performed on hCMEC/D3 cells (passage 30) grown on rat tail collagen type-1-coated coverslips, fixed with 4% formaldehyde and stained for the primary and secondary antibody and viewed at 63x with oil emersion using a Zeiss LSM710 confocal microscope and image analysis software Zen 2009. Scale bar 10 μm. Cell nuclei were counterstained with 1 mg/mL DAPI. For negative staining, cells were stained with secondary antibody only along with DAPI (inset figures).
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