Page 110 - Haematologica March 2020

J.G. McArthur et al.
Hydroxyurea (HU) was the first approved disease mod- ifying therapy for SCD.20-24 HU was originally developed as a chemotherapeutic agent, and is believed to mitigate disease pathology and organ damage sequelae by increas- ing RBC expression of fetal hemoglobin (HbF) and reduc- ing WBC counts.8,23,25-27 HU has been proposed to stimulate soluble guanyl cyclase, resulting in the elevation of cellular cGMP levels and activation of protein kinase G, which ultimately induces HbF expression.26 HU may also indi- rectly affect NO biology as a result of these activities, or directly increase NO levels. Despite its activity on multi- ple pathways that can improve SCD pathophysiology, HU is under-used in patients with SCD and often under- dosed.28,29 Use of HU is challenged by responder effects and the careful safety monitoring required due to its myelosuppressive properties, and by concerns about toxi- cities, including HU impact on fertility and long-term car- cinogenic potential.30-35 As a result of these risks, female and male patients are advised to discontinue HU therapy when trying to conceive or during pregnancy.
The cGMP specific phosphodiesterase 9 (PDE9) enzyme degrades cGMP and therefore PDE9 inhibitors (PDE9i) increase intracellular cGMP levels recapitulating the HbF induction mechanism of HU.36-38 PDE9 is highly expressed in erythropoietic cells, and is further elevated in neu- trophils and reticulocytes from patients with SCD.39 A PDE9i originally developed for the treatment of neurode- generative diseases (BAY73-6691) has been shown to increase HbF transcripts in K562 cells.38 BAY73-6691 also reduced WBC adhesion to endothelial cells, the adhesion of patient-derived neutrophils to immobilized fibronectin, leukocyte recruitment to the microvasculature, and, in conjunction with HU, it reduced the lethality of TNF-α induced vaso-occlusion in a mouse model of SCD.38,40
We describe here a novel, potent, and selective phospho- diesterase 9A inhibitor (IMR-687) that induced cGMP and HbF in the erythroid cell line K562 and increased HbF expression in erythroid cells derived from multiple SCD patients. In murine SCD models, IMR-687 increased plasma cGMP levels and HbF expression in RBC and impacted a number of disease-relevant features of SCD, including reducing lung inflammation, RBC sickling, and occlusion of micro-vessels. Furthermore, unlike PDE9i that was devel- oped for neurodegenerative diseases, IMR-687 did not alter cognition in mice and, unlike HU, did not induce myelosup- pression. In summary, IMR-687 demonstrated disease-rele- vant improvements in several aspects of SCD with compa- rable efficacy to HU.
Methods
Phosphodiesterase enzyme inhibition
Phosphodiesterase enzyme (PDE) inhibition IC50 values were determined for IMR-687 using recombinant human PDE enzymes in a radiometric assay.41
K562 and UT-7 erythroid cells
Human erythroleukemic K562 and UT-7 cells (American Type Culture Collection) were cultured as described in the Online Supplementary Methods. Terminal cell viability was determined by use of a trypan-blue exclusion technique (Thermo Fisher Scientific, France), ATP-based assays (Cell-Titer Glo, Promega), or automated cell counts (Countess Automated Cell Counter, Life Technologies). Apoptosis was assessed by Annexin V FACS assay (Biolegend).
Fetal hemoglobin quantification
K562 cells (5x106) supernatants were assayed using an ELISA kit for HbF (Cloud Clone Corp, CEA996Hu) (see Online Supplementary Methods). Permeabolized cells were stained with PE-mouse anti-human HbF and the percentage of HbF+ cells (% HbF) and the HbF levels (MFI) determined by flow cytometry (see Online Supplementary Methods).
Sickle cell disease patient cells
Blood was collected from five adult patients with severe SCD, aged 19-33 years (median age 32 years), admitted to the Biotherapy Department of Necker Hospital for an exchange transfusion. All samples used in this study were obtained from patients who signed informed consent forms approved by the ethical committee of Necker Hospital on 11th September 2015 (study IMNIS2015-01). CD34+ cells were were cultured in the presence of 15% BIT 9500 [mixture of bovine serum albumin (BSA) + insulin + transferrin from Stem Cell Technologies], 100 U/mL penicillin-streptomycin, 2 mM L-glutamine, 10 ng/mL recombinant human (rh) IL-3 (Peprotech), 100 ng/mL rhIL-6 (Peprotech), and 100 ng/mL rhSCF (Peprotech) for seven days and then CD36+ cells, isolated and cultured in media containing 100 ng/mL rhSCF, 10 ng/mL rhIL-3 and 2 UI/mL erythropoetin (Cilag, France) supplemented with dimethyl sulfoxide (DMSO), 30 mM HU or 10 mM IMR-687 for five days, at which point the HbF+ erythroid cells (LD–/GPA+/Band3+) was determined by FACS.
Animals
Townes model. HbSS-Townes mice42 on a 129/B6 background (Jackson Laboratory, Bar Harbor, ME, USA; 10-12 weeks old, n=7 per group) were dosed daily by gavage with vehicle (poly- ethylene glycol in water 1:3), 50 or 25 mg/kg of HU, or 30 mg/kg of IMR-687. On day 30, mice were anesthetized and blood counts, spleen weights, and plasma bilirubin, LDH, nitrite, HbF and free Hb determined (see Online Supplementary Methods).
Table 1. Phosphodiesterase enzyme (PDE) selectivity of IMR-687.
Enzyme IC50 (mM) Enzyme
PDE1A3 88.4 PDE4D4
PDE1B 8.48 PDE4D5 PDE1C 12.2 PDE4D7 PDE2A3 NI PDE5A1 PDE3A NI PDE5A2 PDE3B NI PDE5A3
IC50 (mM)
NI
NI
NI >100 81.9 >100 NI NI NI >100 NI NI 0.008 0.010 >100 NI
PDE4A1 PDE4A4 PDEA10 PDE4B1 PDE4B2 PDE4B3 PDE4C2 PDE4D1 PDE4D2 PDE4D3
>100 PDE6AB >100 PDE6C NI PDE7A NI PDE7B >100 PDE8A1 >100 PDE8B
NI PDE9A1 >100 PDE9A2 >100 PDE10A2
NI PDE11A1
Enzyme inhibition by IMR-687 on human recombinant PDE was demonstrated to be more selective to PDE9 by 1,000-10,000-fold over PDE1A3, 1B, 1C, and PDE5A2 iso- forms.No measurable inhibition was observed in PDE2A3,3A,3B,4A1,4A4,4A10,4B1, 4B2, 4B3, 4C2, 4D1, 4D2, 4D3, 4D4, 4D5, 4D7, 5A1, 5A3, 6AB, 6C, 7A, 7B, 8A1, 8B, 10A1, 10A2 or 11A1 at doses up to 100 uM. NI: no inhibition detected.
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