Composition for collecting and preserving placental stem cells and methods of using the composition

• US 20070190042A1
• Filed: 12/28/2006
• Published: 08/16/2007
• Est. Priority Date: 12/29/2005
• Status: Active Grant

• Current Assignee: Celularity, Inc.
• Original Assignee: Clarity Acquisition II LLC
• Inventors: Hofgartner, Wolfgang, Edinger, James, Heidaran, Mohamad
• Granted Patent: US 9,598,669 B2
• Time in Patent Office: Days
• US Class Current: 424/94.63
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly

Abstract

The present invention provides improved compositions and methods for the collection of stem cells from an organ, e.g., placenta. The invention provides a stem cell collection composition comprising an apoptosis inhibitor and, optionally, an enzyme such as a protease or mucolytic enzyme, vasodilator, necrosis inhibitor, oxygen-carrying perfluorocarbon, or an organ preserving compound. The invention provides methods of using the stem cell collection composition to collect stem cells and to preserve populations of stem cells.

Claims

1. A composition comprising, in a physiologically-acceptable solution, an inhibitor of apoptosis and a protease,

wherein said composition, when contacted with population of stem cells, reduces or prevents apoptosis in said population of stem cells as compared to a population of stem cells not contacted with the composition, and wherein said composition is not a naturally-occurring composition.

2. The composition of claim 1, wherein said inhibitor of apoptosis is a caspase inhibitor.

3. The composition of claim 1, wherein said inhibitor of apoptosis is a JNK inhibitor.

4. The composition of claim 1, wherein said JNK inhibitor does not modulate differentiation or proliferation of said stem cells.

5. The composition of claim 1, wherein said protease is present in an amount sufficient to detectably dissociate the cells of a tissue from which said stem cells may be derived.

6. The composition of claim 1, additionally comprising an inhibitor of necrosis.

7. The composition of claim 6, wherein said inhibitor of necrosis is 2-(1H-Indol-3-yl)-3-pentylamino-maleimide.

8. The composition of claim 1, additionally comprising an oxygen-carrying perfluorocarbon.

9. The composition of claim 1 wherein said physiologically-acceptable solution is a saline solution or culture medium.

10. The composition of claim 9 wherein said saline solution is 0.9% NaCl solution or phosphate buffered saline.

11. The composition of claim 1, wherein said protease is a matrix metalloprotease or a neutral protease.

12. The composition of claim 11, wherein said matrix metalloprotease is collagenase.

13. The composition of claim 11, wherein said neutral protease is thermolysin or dispase.

14. The composition of claim 11 additionally comprising a mucolytic enzyme.

15. The composition of claim 14, wherein said mucolytic enzyme is hyaluronidase.

16. The composition of claim 1, additionally comprising hydroxyethyl starch, lactobionic acid and raffinose.

17. The composition of claim 1, additionally comprising UW solution.

18. The composition of claim 1, wherein said JNK inhibitor is an indazole.

19. The composition of claim 1, wherein said JNK inhibitor has the structure

wherein:

A is a direct bond, —

(CH₂)_a—

, —

(CH₂)_bCH═

CH(CH₂)_c—

, or —

(CH₂)_bC≡

C(CH₂)_n—

;

R₁ is aryl, heteroaryl or heterocycle fused to phenyl, each being optionally substituted with one to four substituents independently selected from R₃;

R₂ is —

R₃, —

R₄, —

(CH₂)_bC(═

O)R₅, —

(CH₂)_bC(═

O)OR⁵, —

(CH₂)_bC(═

O)NR₅R₆, —

(CH₂)_bC(═

O)NR₅(CH₂)_cC(═

O)R₆, —

(CH₂)_bNR₅C(═

O)R₆, —

(CH₂)_bNR₅C(═

O)NR₆R₇, —

(CH₂)_bNR₅R₆, —

(CH₂)_bOR₅, —

(CH₂)_bSO_dR₅ or —

(CH₂)_bSO₂NR₅R₆;

a is 1, 2, 3, 4, 5 or 6;

b and c are the same or different and at each occurrence independently selected from 0, 1, 2, 3 or 4;

d is at each occurrence 0, 1 or 2;

R₃ is at each occurrence independently halogen, hydroxy, carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —

C(═

O)OR₈, —

OC(═

O)R₈, —

C(═

O)NR₈R₉, —

C(═

O)NR₈OR₉, —

SO₂NR₈R₉, —

NR₈SO₂R₉, —

CN, —

NO₂, —

NR₈R₉, —

NR₉C(═

O)R₉, —

NR₈C(═

O)(CH₂)_bOR₉, —

NR₉C(═

O)(CH₂)_bR₉, —

O(CH₂)_bNR₉R₉, or heterocycle fused to phenyl;

R₄ is alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, each being optionally substituted with one to four substituents independently selected from R₃, or R₄ is halogen or hydroxy;

R₅, R₆ and R₇ are the same or different and at each occurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, wherein each of R₅, R₆ and

R₇ are optionally substituted with one to four substituents independently selected from R₃; and

R₈ and R₉ are the same or different and at each occurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle, or heterocycloalkyl, or R₈ and R₉ taken together with the atom or atoms to which they are bonded form a heterocycle, wherein each of R₅, R₉, and R⁸ and R₉ taken together to form a heterocycle are optionally substituted with one to four substituents independently selected from R₃;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

20. The composition of claim 1, wherein said JNK inhibitor has the structure

wherein:

R₁ is aryl or heteroaryl optionally substituted with one to four substituents independently selected from R₇;

R₂ is hydrogen;

R₃ is hydrogen or lower alkyl;

R₄ represents one to four optional substituents, wherein each substituent is the same or different and independently selected from halogen, hydroxy, lower alkyl and lower alkoxy;

R₅ and R₆ are the same or different and independently —

R₈, —

(CH₂)_aC(═

O)R₉, —

(CH₂)_aC(═

O)OR₉, (CH₂)_aC(═

O)NR₉R₁₀, (CH₂)_aC(═

O)NR₉(CH₂)_bC(═

O)R₁₀, —

(CH₂)_aNR₉C(═

O)R₁₀, —

(CH₂)_aNR₁₁C(═

O)NR₉R₁₀, —

(CH₂)_aNR₉R₁₀, —

(CH₂)_aOR₉, —

(CH₂)_aSO_cR₉ or —

(CH₂)_aSO₂NR₉R₁₀;

or R₅ and R₆ taken together with the nitrogen atom to which they are attached to form a heterocycle or substituted heterocycle;

R₇ is at each occurrence independently halogen, hydroxy, cyano, nitro, carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, substituted heterocycle, heterocycloalkyl, —

C(═

O)OR₈, —

OC(═

O)R₈, —

C(═

O)NR₈R₉, —

C(═

O)NR₈OR₉, —

SO_cR₈, —

SO_cNR₈R₉, —

NR₈SO_cR₉, —

NR₈R₉, —

NR₈C(═

O)R₉, —

NR₉C(═

O)(CH₂)_bOR₉, —

NR₈C(═

O)(CH₂)_bR₉, —

O(CH₂)_bNR₈R₉, or heterocycle fused to phenyl;

R₈, R₉, R₁₀ and R₁₁ are the same or different and at each occurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl;

or R₈ and R₉ taken together with the atom or atoms to which they are attached to form a heterocycle;

a and b are the same or different and at each occurrence independently selected from 0, 1, 2, 3 or 4; and

c is at each occurrence 0, 1 or 2;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

21. The composition of claim 1, wherein said JNK inhibitor has the structure

wherein R₀ is —

O—

, —

S—

, —

S(O)—

, —

S(O)₂—

, NH or —

CH₂—

;

the compound of structure (III) being;

(i) unsubstituted, (ii) monosubstituted and having a first substituent, or (iii) disubstituted and having a first substituent and a second substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8, 9, or 10 position, wherein the first and second substituent, when present, are independently alkyl, hydroxy, halogen, nitro, trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a group represented by structure (a), (b), (c), (d), (e), or (f);

wherein R₃ and R₄ are taken together and represent alkylidene or a heteroatom-containing cyclic alkylidene or R₃ and R₄ are independently hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, or di-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino, di-alkylamino, arylamino, arylalkylamino, cycloalkylamino, cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, or di-alkylaminoalkyl;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof

22. The composition of claim 1, additionally comprising an immunomodulatory compound.

23. The composition of claim 22, wherein said immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;

3-(4′

aminoisolindoline-1′

-onw)-1-piperidine-2,6-dione;

4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;

or α

-(3-aminophthalimido) glutarimide.

24. The composition of claim 22, wherein said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═

O, the other of X and Y is C═

O or CH₂, and R² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof

25. The composition of claim 22, wherein said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═

O and the other is CH₂ or C═

O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴, (C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³, C(S)NHR³, C(O)NR³R³, C(S)NR³R³ or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^3′

are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-C₂-C₅)heteroaryl, (C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, (C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or (C₀-C₄)alkyl-C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or (C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or (C₀-C₈)alkyl-C(O)O—

R⁵ or the R⁶ groups can join to form a heterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof

26. The composition of claim 22, wherein said immunomodulatory compound is a compound having the structure

wherein:

one of X and Y is C═

O and the other is CH₂ or C═

O;

R is H or CH₂OCOR′

;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R¹, R², R³, or R⁴ is nitro or —

NHR⁵ and the remaining of R¹, R², R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′

is R⁷—

CHR¹⁰—

N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —

(C_nH_2n)—

in which n has a value of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene, pentamethylene, hexamethylene, or —

CH₂CH₂X₁CH₂CH₂—

in which X₁ is —

O—

, —

S—

, or —

NH—

;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

27. The composition of claim 1, wherein said perfusion solution additionally comprises a vasodilator.

28. The composition of claim 27, wherein said vasodilator is an antihypertensive drug.

29. The composition of claim 27, wherein said vasodilator activates guanylyl cyclase, ADP-ribosyl transferase or cyclooxygenase, or inhibits lipoxygenase.

30. The composition of claim 27, wherein said vasodilator is atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate.

31. The composition of claim 30, wherein said hydralazine is present in a concentration of from about 0.1 mM to about 10 mM.

32. The composition of claim 30, wherein said adenosine is present at a concentration of about 0.001 mM to about 10.0 mM.

33. The composition of claim 30, wherein said adenosine triphosphate is present at a concentration of about 0.1 mM to about 1000 mM.

34. The composition of claim 30, wherein said indomethacin is present at a concentration of about 1 mg/kg to about 20 mg/kg, wherein “

kg”

is the weight of the placenta.

35. The composition of claim 30, wherein said magnesium sulfate is present at a concentration of about 0.1 mM to about 20 mM.