Stabilization of Metabolically-Active Cells in a Blood Sample at Ambient Temperatures
The present invention relates to the stabilization of one or more metabolically-active cell in a blood sample at ambient temperatures. In particular, formulations, compositions, articles of manufacture, kits and methods for substantially stable storage of one or more metabolically-active cell in a blood sample at ambient temperatures are provided.
1 . A formulation for substantially stable storage of one or more metabolically-active cell in a blood sample at ambient temperatures, wherein the one or more cell remains metabolically-active after storage at room temperature for a period of at least three days.
2 . The formulation of claim 1 , wherein at least 80% of the substantially stored cells remain metabolically-active after storage at room temperature for a period of at least three days.
3 . The formulation of claim 1 or claim 2 , wherein at least 80% of the cells remain metabolically-active at room temperature for a period of at least 18 days.
4 . The formulation of any one of claims 1 - 3 , comprising:
(i) a pH buffer;
(ii) a chelating agent; and
(iii) a peptide.
5 . The formulation of claim 4 , wherein the pH buffer is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), and a combination thereof.
6 . The formulation of any one of claims 4 - 5 , wherein the peptide is a di-peptide or a tri-peptide.
7 . The formulation of any one of claims 4 - 6 , wherein the di-peptide sequence is Ala-Gln or Gly-Gly andthe tri-peptide sequence is Gly-Gly-Gly.
8 . The formulation of any one of claims 4 - 7 , wherein the chelating agent is EDTA.
9 . The formulation of any one of claims 1 - 8 , wherein the one or more metabolically-active cell is selected from the group consisting of a leukocyte, an erythrocyte, a circulating tumor cell, and a combination thereof.
10 . The formulation of any one of claims 1 - 3 , comprising:
(i) a pH buffer;
(ii) a chelating agent;
(iii) a phosphatase inhibitor; and
(iv) a purine.
11 . The formulation of claim 10 , wherein the phosphatase inhibitor is a serine-threonine phosphatase inhibitor.
12 . The formulation of claim 11 , wherein the serine-threonine phosphatase inhibitor is 2-glycerol phosphate.
13 . The formulation of claim 10 , wherein the purine is adenine or guanine.
14 . The formulation of claim 13 , wherein the purine is adenine.
15 . The formulation of claim 13 , wherein the purine is guanine.
16 . The formulation of any one of claims 10 - 15 , further comprising a chelating agent, and at least one compound selected from the group consisting of a cationic compound, a zwitterionic compound, and a peptide.
17 . The formulation of claim 16 , wherein the chelating agent is sodium gluconate.
18 . The formulation of any one of claims 16 - 17 , wherein the zwitterionic compound is a compound of formula (I):
wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH 2 , CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), wherein A is an unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and Z is CO 2 —, SO 3 — or OPO 3 —.
19 . The formulation of any one of claims 16 - 17 , wherein the cationic compound is selected from the group consisting of:
(a) a compound of formula (II):
wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH 2 , CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is an unsubstituted or substituted alkyl, aryl, arylalkyl or any side chain typically found in one of the 20 naturally occurring amino acids; Z is CO 2 A; and X is a pharmaceutically acceptable anion;
(b) a compound of formula (III):
wherein R1, R2, R3, and R4 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH 2 , CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is an unsubstituted or substituted alkyl, aryl, arylalkyl or any side chain typically found in one of the 20 naturally occurring amino acids; and X is a pharmaceutically acceptable anion; and
(c) a compound of formula (IV):
wherein R1 and R2 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl; and X is a pharmaceutically acceptable anion.
20 . The formulation of claim 18 or claim 19 , wherein R1 and R2 of a compound of formula (I), formula (II), or formula (III) form a morpholino ring, pyrrolidinium ring, a piperidinium ring, or an oxazinium ring.
21 . The formulation of claim 16 , wherein the zwitterionic compound is selected from the zwitterionic compounds set forth in Table 1.
22 . The formulation of claim 16 , wherein the cationic compound is selected from the cationic compounds set forth in Table 1.
23 . The formulation of claim 16 , wherein the zwitterionic compound is a quaternary inner salt.
24 . The formulation of claim 23 , wherein the quaternary inner salt is selected from the group consisting N,N-dimethyl-N-(2-hydroxyethyl)-3-ammonium-proprionate or N-ethyl-piperidinium-4-butylsulfonate.
25 . The formulation of any one of claims 16 - 24 , wherein the peptide has the amino acid sequence of Ala-Gln, Gly-Gly, or Gly-Gly-Gly.
26 . The formulation of any one of claims 10 - 25 , further comprising an acetate buffer, sucralose, glucose, potassium chloride, myo-inositol, N-methylglucamine, magnesium chloride, or a combination thereof.
27 . The formulation of any one of claims 10 - 26 , wherein the one or more metabolically-active cell is selected from the group consisting of a leukocyte, an erythrocyte, a circulating tumor cell, and a combination thereof.
28 . The formulation of any one of claims 1 - 3 , wherein the formulation is selected from the formulations set forth in Table 2.
29 . A composition comprising a substantially, stably stored one or more purified, metabolically-active leukocyte admixed with the formulation any one of claims 1 - 8 and 10 - 28 .
30 . A composition comprising a substantially, stably stored one or more purified, metabolically-active erythrocyte admixed with the formulation any one of claims 1 - 8 and 10 - 28 .
31 . A composition comprising a substantially, stably stored one or more purified, metabolically-active circulating tumor cell admixed with the formulation any one of claims 1 - 8 and 10 - 28 .
32 . An article of manufacture, comprising the formulation of any one of claims 1 - 28 contained within a blood collection tube.
33 . The article of manufacture of claim 32 , wherein the blood collection tube is an evacuated blood collection tube.
34 . A kit, comprising any one of the articles of manufacture of claim 32 or 33 and a package insert.
35 . A method for substantially stable storage of one or more metabolically-active cell in a blood sample at ambient temperatures, comprising: admixing a sample of collected blood from a subject with the formulations any one of claims 1 - 28 , wherein the one or more cell remain metabolically-active at room temperature for a period of at least three days.
36 . The method of claim 35 , wherein the cell remains metabolically-active at room temperature for a period of at least 18 days.
37 . The method of claim 35 or claim 36 , wherein the cell is a leukocyte, an erythrocyte a circulating tumor cell, or a combination thereof.
38 . The method of any one of claims 35 - 37 , wherein the subject is an animal.
39 . The method of any one of claims 35 - 37 , wherein the subject is a mammal.
40 . The method of any one of claims 35 - 37 , wherein the subject is a human.