SELECTIVE TARGETING AGENTS FOR MITCOCHONDRIA

Abstract

The present invention relates to compositions and methods for providing mitochondria-selective targeting agents covalently linked to desired cargo such as radical scavenging agents. Compositions and methods are disclosed for treating an illness that is caused or associated with cellular damage or dysfunction which is caused by excessive mitochondrial production of reaction oxygen species (ROS). Compositions which act as mitochondria-selective targeting agents using specific structural signaling features recognizable by cells as mitochondrial targeting sequences are discussed. A method for delivering these agents effectively into cells and mitochondria where they act as electron scavengers by way of certain targeting sequences is also disclosed. Mitochondria and cell death by way of apoptosis is inhibited as a result of the ROS-scavenging activity, thereby increasing the survival rate of the patient. In a preferred embodiment, the compositions and methods may be administered therapeutically in the field to patients with profound hemorrhagic shock so that survival could be prolonged until it is feasible to obtain surgical control of the bleeding vessels. In further preferred embodiments, the composition for scavenging radicals in a mitochondrial membrane includes a radical scavenging agent and a membrane active compound having a high affinity with said mitochondrial membrane and associated methods. In another embodiment, the cargo transported by mitochondrial-selective targeting agents may include an inhibitor of nitrous oxide system (NOS) enzyme activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of TEMPOL and the seven hemigramicidin derivative compounds.

FIG. 1A shows TEMPOL.

FIG. 1B shows a dipeptidic TEMPO analog—XJB-5-208.

FIG. 1C shows a hemigramicidin-TEMPO conjugate—XJB-5-125.

FIG. 1D shows a hemigramicidin compound that does not have the TEMPO moiety—XJB-5-127.

FIG. 1E shows a hemigramicidin-TEMPO conjugate—XJB-5-131.

FIG. 1F shows a hemigramicidin compound that does not have the TEMPO moiety—XJB-5-133.

FIG. 1G shows a hemigramicidin-TEMPO conjugate—XJB-5-197.

FIG. 1H shows a hemigramicidin compound that does not have the TEMPO moiety—XJB-5-194.

FIG. 2 depicts an example of a synthetic pathway for the TEMPO-hemigramicidin conjugates.

FIG. 3 shows an EPR-based analysis of integration and reduction of nitroxide Gramicidin S peptidyl-TEMPO conjugates in MECs.

FIG. 4 shows an flourescein isothiocyanate-dextran (FD4) read-out which reflects the effect of Gramicidin-S TEMPO conjugates on rat ileal mucosal permeability following profound hemorrhagic shock. Data are expressed as a percentage of the change permeability relative to that observed in simultaneously assayed control segments loaded during shock with normal saline solution.

FIG. 4A shows an FD4 read-out of TEMPOL which is used as a “positive control” for the gut mucosal protection assay.

FIG. 4B shows an FD4 read-out of TEMPO conjugate XJB-5-208 reflecting gut mucosal protection.

FIG. 4C shows an FD4 read-out of XJB-5-125 which has the TEMPO payload, but fails to provide protection against gut barrier dysfunction induced by hemorrhage.

FIG. 4D shows an FD4 read-out of XJB-5-127 which lacks the TEMPO payload and fails to provide protection against gut barrier dysfunction induced by hemorrhage.

FIG. 4E shows an FD4 read-out of TEMPO conjugate XJB-5-131 reflecting gut mucosal protection.

FIG. 4F shows an FD4 read-out of XJB-5-133 which lacks the TEMPO payload even though it possesses the same hemigramicidin mitochondria targeting moiety as the most active compound, XJB-5-131.

FIG. 4G shows an FD4 read-out of XJB-5-197 which has the TEMPO payload, but fails to provide protection against gut barrier dysfunction induced by hemorrhage.

FIG. 4H shows an FD4 read-out of XJB-5-194 which lacks the TEMPO payload and fails to provide protection against gut barrier dysfunction induced by hemorrhage.

FIG. 5 shows graphical representations of the effect of nitroxide conjugates on ActD-induced apoptosis.

FIG. 5A is a graphical representation of superoxide production based upon mean fluorescence intensity from 10,000 ileal cells.

FIG. 5B is a graphical representation of phosphatidylserine (PS) externalization as indicated by the percentage of annexin V-positive cells.

FIG. 5C is a graphical representation of caspase-3 activity as indicated by amount of its specific substrate present, Z-DVED-AMC, in nmol/mg protein.

FIG. 5D is a graphical representation of DNA fragmentation as indicated by propidium iodide fluorescence.

FIG. 5E is a graphical representation of PS externalization at different concentrations of the compound 5a.

FIG. 5F is a graphical representation of adenosine triphosphate (ATP) levels in mitochondria in the presence or absence of 5a or 2-deoxyglucose.

FIG. 6 illustrates the effects of intraluminal XJB-5-131 on hemorrhage-induced peroxidation of phospholipids in intestinal mucosa.

FIG. 6A is a graphical representation of the peroxidation of phosphatidylcholine (PC).

FIG. 6B is a graphical representation of peroxidation activity with respect to phosphatidylethanolamine (PE).

FIG. 6C is a graphical representation of peroxidation activity with respect to phosphatidylserine (PS).

FIG. 6D is a graphical representation of peroxidation activity with respect to cardiolipin (CL).

FIG. 7 is a graphical representation of caspase 3 and 7 activity that illustrates the effects of intraluminal XJB-5-131.

FIG. 8 is a graphical representation of permeability of XJB-5-131 with respect to Caco-2_BBe human enterocyte-like monolayers subjected to oxidative stress. The permeability of the monolayers is expressed as a clearance (pL·h⁻¹·cm⁻²).

FIG. 9A is a graphical representation of the effects of intravenous treatment with XJB-5-131 on MAP (mean arterial pressure, mm Hg) of rates subjected to volume controlled hemorrhagic shock.

FIG. 9B is a graphical representation of the effects of intravenous treatment with XJB-5-131 on survival probability of rates subjected to volume controlled hemorrhagic shock.

Claims

1. A composition for scavenging the radicals in a mitochondrial membrane comprising: a. a radical scavenging agent; andb. a membrane active compound having a high affinity with the mitochondria.

2. The composition of claim 1 wherein said membrane active compound has antibiotic properties.

3. The composition of claim 1 wherein said membrane active compound has radioprotective properties.

4. The composition of claim 1 wherein said membrane active compound has therapeutic protective properties.

5. The composition of claim 1 wherein said membrane active compound is a peptidyl fragment with properties selected from the group of antibiotic, radioprotective, protective therapeutic and combinations thereof.

6. The composition of claim 1 wherein said membrane active compound are peptidyl fragments with properties selected from the group of antibiotic, radioprotective, protective therapeutic and combinations thereof.

7. The composition of claim 1 wherein said membrane active compound is selected from the group consisting of bacitracins, gramicidins, valinomycins, enniatins, alamethicins, beauvericin, serratomolide, sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinum peptides.

8. The composition of claim 1 wherein said radical scavenging agent is selected from the group consisting of XJB-5-234, XJB-5-133, XJB-5-241, and XJB-5-127.

9. The composition of claim 1 wherein said composition is characterized by the property of resisting oxidative damage.

10. The composition of claim 1 wherein said composition is characterized by the property of resisting nitrosative damage.

11. The composition of claim 1 wherein said radical scavenging agent is a ubiquinone analog.

12. The composition of claim 11 wherein said radical scavenging agent is a ubiquinone analog fragment moiety.

13. The composition of claim 11 wherein said radical scavenging agent is a ubiquinone analog fragment moiety lacking a hydrophilic tail.

14. The composition of claim 1 wherein said radical scavenging agent is a superoxide dismutase mimetic.

15. The composition of claim 1 wherein said radical scavenging agent is a superoxide dismutase biomimetic.

16. The composition of claim 1 wherein said radical scavenging agent is a salen-manganese compound.

17. A method for delivering a composition to mitochondria comprising transporting to said mitochondria a radical scavenging agent and a membrane active compound having a high affinity with the mitochondrial membrane.

18. The method of claim 17 wherein said membrane active compound is selected from the group consisting of bacitracins, gramicidins, valinomycins, enniatins, alamethicins, beauvericin, serratomolide, sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinum peptides.

19. The method of claim 17 wherein said radical scavenging agent is selected from the group consisting of XJB-5-234, XJB-5-133, XJB-5-241, and XJB-5-127.

20. The method of claim 17 wherein said composition is characterized by the property of resisting oxidative damage.

21. The method of claim 17 wherein said composition is characterized by the property of resisting nitrosative damage.

22. The method of claim 17 wherein said radical scavenging agent is a ubiquinone analog.

23. The method of claim 22 wherein said radical scavenging agent is a ubiquinone analog fragment moiety.

24. The method of claim 23 wherein said radical scavenging agent is a ubiquinone analog fragment moiety lacking a hydrophilic tail.

25. The method of claim 17 wherein said radical scavenging agent is a superoxide dismutase mimetic.

26. The method of claim 17 wherein said radical scavenging agent is a superoxide dismutase biomimetic.

27. The method of claim 17 wherein said radical scavenging agent is a salen-manganese compound.

28. A composition for delivering cargo in a mitochondrial membrane comprising: a. cargo; andb. a membrane active compound having a high affinity with the mitochondria.

29. The composition of claim 28 wherein said membrane active compound has antibiotic properties.

30. The composition of claim 28 wherein said membrane active compound has radioprotective properties.

31. The composition of claim 28 wherein said membrane active compound has therapeutic protective properties.

32. The composition of claim 28 wherein said membrane active compound is a peptidyl fragment with properties selected from the group of antibiotic, radioprotective, protective therapeutic and combinations thereof.

33. The composition of claim 28 wherein said membrane active compound are peptidyl fragments with properties selected from the group of antibiotic, radioprotective, protective therapeutic and combinations thereof.

34. The composition of claim 28 wherein said membrane active compound is covalently bonded to said cargo.

35. The composition of claim 28 wherein said cargo is an inhibitor of nitrous oxide system (NOS) enzyme activity.

36. A method for delivering a composition to mitochondria comprising transporting to said mitochondria cargo and a membrane active compound having a high affinity with the mitochondrial membrane.

37. The method of claim 36 wherein said membrane active compound has antibiotic properties.

38. The method of claim 36 wherein said membrane active compound has radioprotective properties.

39. The method of claim 36 wherein said membrane active compound has therapeutic protective properties.

40. The method of claim 36 wherein said membrane active compound is a peptidyl fragment with properties selected from the group of antibiotic, radioprotective, protective therapeutic and combinations thereof.

41. The method of claim 36 wherein said membrane active compound are peptidyl fragments with properties selected from the group of antibiotic, radioprotective, protective therapeutic and combinations thereof.

42. The method of claim 36 wherein said membrane active compound is covalently bonded to said cargo.

43. The method of claim 36 wherein said cargo is an inhibitor of nitrous oxide system (NOS) enzyme activity.

44. The method of claim 36 employing compounds with antibiotic properties whose mechanism of action includes bacterial wall targets.