Optimized lactoferrin as a highly promising treatment for intracerebral hemorrhage: Pre-clinical experience

Abstract

Intracerebral hemorrhage (ICH) is the deadliest form of stroke for which there is no effective treatment, despite an endless number of pre-clinical studies and clinical trials. The obvious therapeutic target is the neutralization of toxic products of red blood cell (RBC) lysis that lead to cytotoxicity, inflammation, and oxidative damage. We used rigorous approaches and translationally relevant experimental ICH models to show that lactoferrin-(LTF)-based monotherapy is uniquely robust in reducing brain damage after ICH. Specifically, we designed, produced, and pharmacokinetically/toxicologically characterized an optimized LTF, a fusion of human LTF and the Fc domain of human IgG (FcLTF) that has a 5.8-fold longer half-life in the circulation than native LTF. Following dose-optimization studies, we showed that FcLTF reduces neurological injury caused by ICH in aged male/female mice, and in young male Sprague Dawley (SD) and spontaneously hypertensive rats (SHR). FcLTF showed a remarkably long 24-h therapeutic window. In tissue culture systems, FcLTF protected neurons from the toxic effects of RBCs and promoted microglia toward phagocytosis of RBCs and dead neurons, documenting its pleotropic effect. Our findings indicate that FcLTF is safe and effective in reducing ICH-induced damage in animal models used in this study.

Keywords: Intracerebral hemorrhage; cytoprotection; lactoferrin; neuroprotection; phagocytosis.

Figure 1.

(a, b) FcLTF reduces the neurotoxicity of RBC lysates in vitro. Cultured SD rat cortical neurons at 12d of age were exposed to RBC lysates in the presence or absence of FcLTF, and viability was assessed using LDH release (a) and MTT assay (b). (c, d) FcLTF increased phagocytosis in vitro. Bar graphs quantifying the number of phagocytosed dead neurons (DNs) (c) or RBCs (d) per single rat microglial cell. FcLTF was given at 0–5 µg/ml. The n = 50 or 30 indicate the number of individual microglial cells that were inspected for internalized DN or RBC. (e) Representative confocal image of microglial cells (green) that engulfed DN (red). Nuclei were counterstained with DAPI (blue).

Figure 2.

Comparison of efficacy of FcLTF vs. rhLTF in reducing neurological deficits (NDS) in three  month male mice subjected to ICH. Treatment was initiated 24 h after ICH (10 mg/kg in 100 µL, i.v.) and then at 1 mg/kg in 100 µL by oral administration on d2 and d3. The NDS on d3 was measured after the last treatment. Bar graphs shows grand NDS (a combination score of NDS measured by using postural flexing, forward placing, foot faults and cylinder test) and performance of individual tests on d1 after ICH (immediately before treatment) and on d3, two days after treatment with saline, FcLTF, or rhLTF. *p ≤ 0.05, vs. saline group at the same time point (d3 after ICH). #p ≤ 0.05 between FcLTF and rhLTF groups. Data are presented as mean ± SD, n = 9 mice per group.

Figure 3.

FcLTF dose response in NDS and brain edema in mice after ICH. (a) Grand NDS (A composite score of all 4 tests) on d3 and d10, and (b) individual test (postural flexing, forward placing, foot-faults, and wire test) scores on d10 after ICH. n = 10 animals per group. The FcLTF (0.1–5 mg/kg) was first administered 3 h after ICH i.v. and then i.p. on d1 and d2. *p ≤ 0.05, vs. control. (c) Brain edema = brain water content–([wet-dry]/wet weight) in the ICH-affected (Ipsi) and contralateral (Contra) striatum on day 3 after ICH. Data are presented as mean ± SD, n = 5/group. *p ≤ 0.05 vs. indicated group.

Figure 4.

The effect of FcLTF treatment regimen on neurological deficit (NDS) in mice after ICH. (a) Mice received FcLTF (5 mg/kg/injection) starting at either 3, 6, 24, or 48 h after ICH onset. Some of the animals received FcLTF daily for either two or five days as indicated in the figure. The first treatment was given by i.v. infusion, and subsequent treatments were i.p. injection. Grand NDS (a composite score encompassing the Postural Flexing, Forward Placing, Footfaults and Wire tests) was determined on d1, d3, d7, and d10 after ICH; Values are mean ± SD, n = 10–12 mice per group. *p ≤ 0.05, vs. vehicle control. (b) Performance on each individual behavior test on d10 after ICH. *p ≤ 0.05, vs. indicated group; ^#p ≤ 0.05 vs. all remaining groups. A repeated two-way ANOVA was used to analyze the data.

Figure 5.

FcLTF reduces neurological deficit (NDS) in aged mice after ICH. Postural flexing, forward placing, corner, foot-faults, and wire tests were used to generate a composite grand neurological deficit (NDS) score for d3, d7, d14, and d21 after ICH. FcLTF at 1 or 5 mg/kg or saline (vehicle) was first given 6 h after ICH (i.v.) and then daily for an additional six days (i.p.). n indicates number of animals per group. *p ≤ 0.05, vs. vehicle control group at the same time point.

Figure 6.

FcLTF reduces neurological deficits (NDS) in male SHR rats after ICH. Postural flexing, foot faults, corner, and tape removal test results were used to calculate a grand NDS on d3, d7, d14, and d21 after ICH. FcLTF (1 mg/kg in 100 µL, i.v.) or saline (vehicle) was given first at 6 h after ICH and then daily at 1 mg/kg i.p. for an additional six days. n = 10/group *p ≤ 0.05, vs. the vehicle control group at the same time point.