Page 114 - Haematologica March 2020

J.G. McArthur et al.
strated significant changes in all measures of reticulocyto- sis including a 36% reduction in reticulocyte counts (Figure 2H), a 27% increase in mature RBC (Figure 2I), a 10% increase in hematocrit (Figure 2J), and a 1.5g/dL increase in Hb (Figure 2K). HU at a dose of 100 mg/kg pro-
Figure 4. Treatment with phosphodiesterase 9A inhibitor (IMR-687) reduces vessel-occlusion in the Townes-HbSS sickle cell disease model. Townes-HbSS mice were dosed orally for 30 days with IMR-687 at 30 or 10 mg/kg or hydrox- yurea (HU) at 100 mg/kg or 30 mg/kg IMR-687 in combination with 100 mg/kg HU. After ten days of treatment, animals were exposed to hypoxic conditions for quantification of microvessel occlusion via dorsal skin-fold chambers implanted on day 7 of treatment. On day 10 of treatment, 20-23 flowing venules in the chamber window were selected and mapped. Mice were then exposed to 1 h of hypoxia (7% O2) and then returned to room air. The same venules were re-exam- ined at 1 h (A) and 4 h (B) post hypoxia for blood flow, and static (no flow) venules were counted and the data expressed as percent stasis. Data are pre- sented as means±Standard Deviations. Statistical significance was calculated for each agent and dose compared to a vehicle-treated control. *P<0.05; **P<0.01; ns: not significant (P>0.05). Data are presented as means±Standard Errors.
duced smaller changes in reticulocyte counts, RBC, hema- tocrit and Hb; the changes in Hb were not significant. At HU doses of 25 and 50 mg/kg, only the change in reticu- locyte counts was significant.
Townes mice have elevated circulating WBC counts, the major component of which are neutrophils. WBC were 36% lower in IMR-687 (24.1x109/L vs. 38.2x109/L; P<0.05) and 21% lower in 100 mg/kg HU treatment groups (30.4x109/L vs. 38.2x109/L) (Figure 3A). While the reduc- tion in WBC with HU treatment can result from the myelotoxicity of HU, the IMR-687 reduction in peripheral WBC was not due to myelotoxicity as demonstrated in long-term toxicology studies conducted in normal rats (data not shown) and dogs (Figure 3B) treated with IMR-687 for up to 6 and 9 months, respectively. In these studies, super-physiological doses of IMR-687 did not result in any reduction in peripheral WBC counts. Furthermore, a histo- logical examination of bone marrow smears from IMR- 687-treated rats and dogs did not demonstrate any myelo- toxicity (data not shown). This reduction in WBC counts with IMR-687 treatment likely reflects reduced WBC acti- vation or mobilization in this sickle cell model.
Not only were peripheral WBC counts increased in Townes mice, but soluble WBC-derived factors were ele- vated, including lung-associated myeloperoxidase (MPO), which is released by activated neutrophils, reduces plasma NO, and contributes to vascular damage.48 MPO levels were elevated over 5-fold in HbSS-Townes mice com- pared to control mice (3.1 mU/L vs. 0.57 mU/L in control mice; P<0.0001) (Figure 3C). MPO levels were reduced 2.3-fold in IMR-687-treated mice and 2.1-fold in 100 mg/kg HU-treated mice (1.3 mU/L and 1.5 mU/L, respec- tively; P<0.0001). Lower doses of HU also reduced MPO levels in the lungs.
Reduced vaso-occlusion in hemoglobin S-Townes mice
Occlusion of vessels by sickled RBC and adhesive WBC
Figure 5. IMR-687 increases F-cells in patient-derived sickle cell disease (SCD) CD36+ cells. CD36+ cells derived from CD34+ adult SCD peripheral blood cells were cultured as described in the Methods section. Increase in the percentage of F-cells for each treatment is shown. Statistical significance was calculated for each agent compared to a vehicle-treated control (n=9). **P<0.01; ns: not sig- nificant (P>0.05). Errors are presented as Standard Error.
B
A
628
haematologica | 2020; 105(3)