A pilot clinical phase II trial MemSID: Acute and durable changes of red blood cells of sickle cell disease patients on memantine treatment

Abstract An increase in abundance and activity of N‐methyl D‐aspartate receptors (NMDAR) was previously reported for red blood cells (RBCs) of sickle cell disease (SCD) patients. Increased Ca2+ uptake through the receptor supported dehydration and RBC damage. In a pilot phase IIa‐b clinical trial MemSID, memantine, a blocker of NMDAR, was used for treatment of four patients for 12 months. Two more patients that have enrolled into the study did not finish it. One of them had psychotic event following the involuntary overdose of the drug, whereas the other had vertigo and could not comply to the trial visits schedule. Acute and durable responses of RBCs of SCD patients to daily oral administration of memantine were monitored. Markers of RBC turnover, changes in cell density, and alterations in ion handling and RBC morphology were assessed. Acute transient shifts in intracellular Ca2+, volume and density, and reduction in plasma lactate dehydrogenate activity were observed already within the first month of treatment. Durable effects of memantine included (a) decrease in reticulocyte counts, (b) reduction in reticulocyte hemoglobinization, (c) advanced membrane maturation and its stabilization as follows from reduction in the number of NMDAR per cell and reduction in hemolysis, and (iv) rehydration and decrease in K+ leakage from patients’ RBC. Memantine therapy resulted in reduction in number of cells with sickle morphology that was sustained at least over 2 months after therapy was stopped indicating an improvement in RBC longevity.

serum albumin (BSA). RBCs were re-suspended in the same solution to Hb levels of 90-100 g/l, incubated in a thermoshaker at 37 o C and under continuous shaking for 6 hours. Each hour extracellular K + concentration was measured, and kinetics of its accumulation plotted against time and normailsed per Hb content.

Flow cytometry for detection of CD71+ RBC and intracellular free Ca 2+ content
The number of RBCs positive for CD71 (reticulocytes) was assessed using Gallios Flow Cytometer (Becton Dickenson AG, Allschwil, Switzerland) for 100 000 cells at medium flow rate.

RBC density measured using separation on Percoll gradients
RBCs were fractionated into low-, medium-and high-density fractions on Percoll density gradient as described elsewhere 22 . One ml of whole blood was layered on top of 13 ml of the 90% isotonic Percoll solution and centrifuged at 48 000×g at 34-36•C for 15 min to achieve separation of cells into fraction of low (L), medium (M) and high (H) densities. Image of the distribution of RBCs within the gradient was taken against the homogeneous light source and analyzed using ImageJ software (see Suppl Fig 2). In addition, cells forming L, M and H fraction were then collected and the number of cells within the fractions in % was calculated using capillary hematocrit measurements for whole blood and for reach fraction.

Assessment of the number of NMDARs per cell using [ 3 H]MK-801 binding assay
The radiolabeled NMDA receptor antagonist ([ 3 H]MK-801) binding assay was used to detect the number of receptor copies in RBCs forming M density fraction as described elsewhere 25 . Briefly, RBCs were washed with plasma like solution and resuspended to a hematocrit of 40-50%. 5 μl of  2D projected images were then binarized by setting in first place a threshold on the image pixel intensity value followed by a typical binarization routine. The identified objects (sRBCs) were set to pixel value 1 (white) and the background to pixel value 0 (black). The coordinates of cell borders were defined as the positions of the interface between the objects and the background. The number of pixels inside the cell border was defined as the cell projected area, A. From the covariance matrix obtained by a fitting of a multivariate normal distribution to the cell border coordinates, the major and the minor axis, a and b respectively, of an ellipse that fits to the cell contour and its eccentricity  were obtained. "sickle", considered as so according to Corbett (Corbett, et al 1995) description of cells which bearer single or multicentral HbF crystalls; 3) "others", to include additional shapes (e.g. teardrop cells), and deformed cells without a clear typical shape observed in sickle cell disease. The classification procedure was repeated tree times with an error of 2%. Eccentricity was considered as a reference value to distinguish discocytes from ellyptocytes: cells with ε ≥ 0.7 were classified as ellyptocytes and therefore included in the group "others". Changes in the prevalences of those shape groups after the memantine treatment are show on the Supplementary Figure 3C.

Supplementary table 1
Effect of memantine therapy on the white blood cell counts in SCD patients Average of the values at the start of the MemSID trial and the up-dosing phase (base) are compared to the average of the last 3 months of treatments (20 mg/day) and a down-dose phase (end). Stars denote significance (p<0.05) between the "base" and the "end" datasets. (B) Automated and manual analysis of fixed RBC shapes. Fixed RBC shape to projected area distributions based on the manual classification and eccentricity values before and after the treatment and by the end of the follow-up phase. As mentioned in the extended methods section, projected area was defined as number of pixels inside the cell border using Matlab R2017b software routine and re-calculated to µm 2 thereafter.
(C) Impact of memantine treatment on the solidity and eccentricity of RBCs of individual patients compared to three healthy donors (S1-S3, grey color). Samples were collected at prescreening (pre, blue color), by the end of down-dosing phase (end, black color) and at the end of the follow-up phase (post, red color). Paired t-tests were used to assess the significance of changes for individual patients with memantine treatment and its interruption.

Supplementary literature
Corbett