Anti-amyloid β activity of intravenous immunoglobulin (IVIG) in vitro
View Patent ↗The present invention relates to a cell-based in vitro screening assay for identifying and selecting therapeutic agents that inhibit amyloid β-induced cytotoxicity.
1. A method of preparing a pharmaceutical composition comprising IVIG in a therapeutically effective dose for treatment of Alzheimer's Disease, the method comprising:
a) contacting a first test cell culture with a cytotoxic Aβ aggregate and a first sample from a first IVIG lot,
b) contacting a second test cell culture with a cytotoxic Aβ aggregate and a second sample from a second IVIG lot,
c) detecting the level of cytotoxicity in the first and second test cell cultures, and
d) comparing the level of cytotoxicity in the first test culture with the level of cytotoxicity in the second test cell culture,
e) preparing a pharmaceutical composition comprising IVIG in a therapeutically effective dose for treatment of Alzheimer's Disease only from the IVIG lot having a higher level of inhibition of cytoxicity.
2. The method of claim 1 , further comprising the step of determining the level of cytotoxicity in a control cell culture.
3. The method of claim 2 , wherein the control cell culture further comprises a cytotoxic Aβ aggregate.
4. The method of claim 3 , wherein the control cell culture further comprises a control agent known to inhibit Aβ-induced cytotoxicity.
5. The method of claim 1 , wherein the first and second samples are contacted with the first and second test cell cultures before the cytotoxic Aβ aggregates.
6. The method of claim 1 , wherein the cytotoxic Aβ aggregates are contacted with the first and second test cell cultures before the first and second samples.
7. The method of claim 1 , wherein the first and second samples and the cytotoxic Aβ aggregates are pre-mixed before contacting the first and second test cell cultures.
8. The method of claim 2 , wherein the first and second test cell cultures and the control cell culture are grown under serum-free conditions.
9. The method of claim 3 , wherein the first and second test cell cultures and the control cell culture comprise PC-12 cells.
10. The method of claim 4 , wherein the control agent is an anti-Aβ antibody.
11. The method of claim 1 , wherein the Aβ aggregates are comprised of Aβ-40 or Aβ1-42.
12. The method of claim 1 , wherein the cytotoxic aggregates are comprised of at least an Aβ dimer.
13. The method of claim 12 , wherein a fibril content of the cytotoxic Aβ aggregate is quantitated using a thioflavin T fluorescence assay.
14. The method of claim 1 , wherein the cytotoxic Aβ aggregates are present in a concentration range from about 2.5 μM to about 25 μM.
15. The method of claim 1 , wherein the cytotoxic Aβ aggregates are present in a concentration range from about 5 μM to about 30 μM.
16. The method of claim 1 , wherein the cytotoxic Aβ aggregates are present in a concentration range from about 10 μM to about 15 μM.
17. The method of claim 4 , wherein the first sample in the first test cell culture, the second sample in the second test cell culture, and the control agent in the control cell culture are all present in equal molar concentrations.
18. The method of claim 17 , wherein the first sample in the first test cell culture, the second sample in the second test cell culture, and the control agent in the control cell culture are all present in a range of molar concentration ratios with the cytotoxic AP aggregate from about 30:1 to about 1:30.
19. The method of claim 1 , wherein the level of cytotoxicity is determined by measuring the concentration of lactate dehydrogenase in the culture media.
20. The method of claim 1 , wherein the level of cytotoxicity is determined by measuring the level of apoptosis in the cell culture.