Drug regimen for treatment of cerebral ischemia

Abstract

Treatment of subjects experiencing cerebral ischemia is improved when the treatment employs a thrombolytic and an inhibitor against vascular endothelial growth factor receptor signal transduction (VEGF-RST) at a reduced, low dosage compared to that used to treat cancer patients. The treatment is also improved to permit point-of-care use by formulating protein drugs for long term stability at room temperature, providing doses appropriate for the method, and by combining the therapeutic agents with a point-of-care diagnostic for blood brain barrier integrity.

Claims

1. A method of treatment to reduce neuronal damage caused by a cerebral ischemic event in a human patient said method comprising administering to said patient an effective amount of a thrombolytic agent or a thrombolytic intervention and an effective amount of an inhibitor of vascular endothelial growth factor (VEGF) receptor-mediated signal transduction (VEGF-RST) wherein the effective amount of said inhibitor provides inhibition of >50% of said VEGF-RST activity over 24 hours and wherein the serum concentration level of said inhibitor is reduced below this pharmacologically active level at least by 48 hours after administration, wherein the inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to VEGF or binds to VEGF-R and is antagonistic thereto selected from the group consisting of ranibizumab, aflibercept, or bevacizumab, or is an inhibitor of a kinase associated with VEGF-R signaling selected from the group consisting of Tivozanib, Apatinib, Lenvatinib, Axitinib, Imtinib, Motesanib, Fruquitinib, Brivanib, Cediranib, Regorafenib, Sulfatinib, and Sunitinib.

2. The method of claim 1, where the antibody or antigen-binding fragment thereof that specifically binds to VEGF is ranibizumab, aflibercept, or bevacizumab.

3. The method of claim 1, wherein the kinase inhibitor is selected from the group consisting of Tivozanib, Apatinib, Lenvatinib, Axitinib, Imtinib, Motesanib, Fruquitinib, Brivanib, Cediranib, Regorafenib, Sulfatinib, and Sunitinib.

4. The method of claim 1, wherein said thrombolytic agent comprises tissue plasminogen activator (tPA), urokinase, streptokinase, desmoteplase, single chain urokinase-type plasminogen activator (uPA).

5. The method of claim 1, wherein the thrombolytic agent and the inhibitor of VEGF-RST are formulated and/or packaged for long term stability at room temperature.

6. The method of claim 1, wherein the thrombolytic intervention is mechanical disruption.

7. The method of claim 1, which further includes conducting an assay for blood-brain barrier (BBB) integrity loss resulting from said ischemic event.

8. The method of claim 7, wherein the assay comprises measuring total S100B and/or S100BB homodimer in the blood.

9. A method of treatment to reduce neuronal damage caused by a cerebral ischemic event in a human patient said method comprising mechanically removing a blood clot associated with said ischemia event and administering in association with said removing a measured dose of an inhibitor of VEGF-RST effective to inhibit >50% of signal transduction over 24 hours and wherein the serum concentration level of said inhibitor is reduced below this pharmacologically active level at least by 48 hours after administration, and wherein the inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to VEGF or binds to VEGF-R and is antagonistic thereto selected from the group consisting of ranibizumab, aflibercept, or bevacizumab, or is an inhibitor of a kinase associated with VEGF-R signaling selected from the group consisting of Tivozanib, Apatinib, Lenvatinib, Axitinib, Imtinib, Motesanib, Fruquitinib, Brivanib, Cediranib, Regorafenib, Sulfatinib, and Sunitinib.

10. The method of claim 9, wherein the antibody or antigen-binding fragment thereof that specifically binds to VEGF is ranibizumab, aflibercept, or bevacizumab.

11. The method of claim 9, wherein the inhibitor is selected from the group consisting of Tivozanib, Apatinib, Lenvatinib, Axitinib, Imtinib, Motesanib, Fruquitinib, Brivanib, Cediranib, Regorafenib, Sulfatinib, and Sunitinib.

12. The method of claim 9, which is performed by a device comprising a catheter for said mechanical removal and wherein said inhibitor is administered by means of a catheter that is part of a device for said mechanical removal.

Description

EXAMPLE 1

Assay to Evaluate Combined Administration of tPA and Anti-VEGF Antibody as a Function of Treatment Window

(1) A cerebral infarction rat model is disclosed in the above-referenced WO2011/013668 and U.S. Pat. Nos. 8,652,476 and 9,439,961. Briefly, a thrombus is formed by coagulating autologous blood from rats and thrombin as a gel in a polyethylene tube catheter. This is allowed to stand overnight and cut to have a length of 1 mm. The thrombus is injected from the external carotid artery into the middle cerebral artery of the rat model under anesthesia with halothane. Cerebral blood flow is measured before and 30 minutes or 24 hours after injection of the thrombus. Animals exhibiting a cerebral blood flow lower than 50% of that measured before injection of the thrombus are used as models in the experiments.

(2) After injection of the thrombus, BBB status is assessed by comparing the serum S100B level as a function of time as compared to the S100B level measured prior to injection of the thrombus. The anti-VEGF treatment markedly reduces the S100B level normalized against the level in the same individual rat prior to the induced stroke. The efficacy is seen at both time points (3-9 hours and 24 hours following the induced stroke with 4 out of 5 rats showing decreased S100B in the anti-VEGF group compared to only one in the control group.

EXAMPLE 2

Measurement of VEGF and S100B in Human Stroke Thrombus Samples

(3) A review of multiple S100B studies concluded that it is not suitable as a marker of stroke in general, but is useful as a surrogate marker for cerebral damage (Dassan, P., et al., Cerebrovasc. Dis. (2009) 27:295-302). The use of tPA combined with an agent to ameliorate tPA toxicity by blocking VEGF signaling is most appropriate for patients with more severe cerebral damage, for which S100B is a useful marker for selecting patients to receive this combination therapy.

(4) Currently, tPA is the only pharmacological intervention widely used to dissolve intra-arterial clots, helping to restore cerebral blood flow. Other interventional strategies include the use of tools that mechanically disrupt and remove intra-arterial clots, notably including: MERCI® or Trevo® (Stryker; Kalamazoo, Mich.); Solitaire™ (Medtronic; Minneapolis, Minn.); Apollo™ (Penumbra; Alameda, Calif.). To test whether the same local elevation of VEGF observed in the rat model was present in human brain after an embolic stroke event, 39 patients were enrolled for this study. Of these, 18 received tPA intravenously before interventional thrombectomy was initiated. All patients underwent endovascular recanalization procedure with 20 cases classified as hemorrhagic by clinical and radiologic criteria. Extent of stroke by Mill was measured as diffusion weighted imaging (DWI) and quantified by a modified Alberta Stroke Program Early CT score (ASPECTS). European Cooperative Acute Stroke Study (ECASS) criteria were used to define the nature of the hemorrhage. The extracted thrombus and aspirate are normally discarded, but in this study were saved for analysis. Full recanalization was achieved in 50% of cases, partial success was achieved in 25% of interventions. The values of S100B and VEGF were not statistically correlated with the success or extent of recanalization. Peripheral blood samples were also collected and assayed for VEGF or S100B by ELISA assay. Analyte levels did not depend on the mechanism of stroke etiology (atherosclerotic, 7%; cardioembolic, 56%; large vessel occlusion, 15%; cryptogenic, 22%).

(5) When values of circulating VEGF were compared to levels of tPA in clot, there was a statistically significant correlation between tPA levels and VEGF (P<0.006). After partitioning the patients as VEGF.sub.Clot>VEGF.sub.Peripheral, it was observed that only 42% of patients with low VEGF experienced a hemorrhage (determined by CT scans), while 72% of patients with elevated brain VEGF were affected by hemorrhage (difference between groups P=0.05). The relationship between peripheral S100B and presence of hemorrhage was significant (P<0.05) and did not depend on the method used (CT or MRI). When all patients were analyzed together, S100B and VEGF in clot correlated well with radiologic endpoints; clot VEGF and peripheral S100B were specifically correlated with poor outcome (Discharge NIH stroke scale (NIHSS)).

(6) Based on the human data above, a further experiment in the rat experimentally induced intra-arterial thrombotic model was performed to examine S100B after treatment with a rabbit polyclonal anti-VEGF antibody. In untreated animals, 80% showed increased S100B in serum at 8 and 24 hours following ischemic stroke (normalized to previous day baseline level). By contrast, 80% of the anti-VEGF treated animals had marked reductions in S100B at both time points.

EXAMPLE 3

Dose Adjustment for Antibody Against VEGF

(7) The effective amount of an inhibitor of VEGF-RST is determined from available sources as follows: Studies establishing the utility of an antibody against VEGF for ameliorating the hemorrhagic activity of tPA used a polyclonal rabbit serum designated RB-222 (Thermo Fisher): Kanazawa, M., et al., J. Cerebral Blood Flow and Metabolism (2011) 31:1461-1474. The dose given to rats was 30 or 100 ug/rat; in Zhang, H-T., et al., Mol Med Rep. (2017) 15:57-64, a dose of RB-222 of 10 ug/rat was more efficacious than 5 ug/rat. With a weight of 500-1000 g, these results imply an effective dose was ˜75 ug/kg. Applied to humans, with an average weight of 70 kg, this corresponds to providing ˜5 mg dose to a person. A standard commercial single dose vial of the anti-VEGF antibody Avastin® (bevacizumab, Genentech) contains 100 mg with multiple vials used to achieve the recommended 10-15 mg/kg total dose. While this dose is appropriate for cancer treatment, the foregoing Kanazawa and Zhang data demonstrate an effective dose for combination with thrombolysis in treatment of stroke is only ˜5 mg per patient if a polyclonal such as RB-222 is employed. For the stroke indication described here, the appropriate dose for the combination treatment is ˜1% of the dose used for cancer.

(8) The EC.sub.50 of Avastinx binding to VEGF in an in vitro ELISA assay is 0.1 ug/mL. The vendor's recommended concentration of RB-222 for the closely related immunohistochemistry application is 10 ug/mL. That is, the monoclonal antibody is 100-fold more potent than the polyclonal serum in vitro. The equivalent human dose for the acute stroke indication by this comparison is thus 1% of the dose used for cancer.

EXAMPLE 4

Dose Adjustment Based on Pharmacokinetics

(9) A PK model was constructed using published data on low dose Avastin® arising from spillage into systemic circulation from the antibody given intra-ocularly to treat macular degeneration Avery R. L., Br. J. Ophthalmol. (2014) 98:1636-41. Assay of VEGF in an extracted clot following thrombectomy showed that the peak concentration at that site is ˜10 nM. The published Kd of Avastin® for VEGF is 2.2 nM, and the model predicts that Avastin® at ˜1 nM (0.15 μg/ml) will be sufficient to sequester the peak VEGF level in the clot.

(10) This concentration of antibody (˜1 nM) across 5 L of blood is achieved with a dose of 0.01 mg/kg, or 0.7 mg for a 70 kg person. This is 0.1% of the standard cancer dose. At that dose, the normal clearance reduces the antibody concentration to <10% of its Kd within 48 hours after which it will have negligible pharmacological activity.

(11) In a murine model, this is borne out. A dosage range of 2.67×10.sup.−3 mg/kg to 8.8×10.sup.−3 mg/kg Avastin® IV gave peak blood levels 1.5×10.sup.−4-9×10.sup.−4 μg/ml or 6-18 nM. This was sufficient to depress VEGF levels in serum from 22 pg/ml to 10 pg/ml.