U.S. patent application number 16/106504 was filed with the patent office on 2019-01-31 for formulation of mk2 inhibitor peptides.
This patent application is currently assigned to MOERAE MATRIX, INC.. The applicant listed for this patent is MOERAE MATRIX, INC.. Invention is credited to Colleen Brophy, Cynthia Lander, Caryn Peterson.
Application Number | 20190031730 16/106504 |
Document ID | / |
Family ID | 56356475 |
Filed Date | 2019-01-31 |
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United States Patent
Application |
20190031730 |
Kind Code |
A1 |
Lander; Cynthia ; et
al. |
January 31, 2019 |
FORMULATION OF MK2 INHIBITOR PEPTIDES
Abstract
The described invention provides pharmaceutical formulations
comprising a polypeptide of amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent
thereof with improved stability and bioavailability.
Inventors: |
Lander; Cynthia; (Mendham,
NJ) ; Brophy; Colleen; (Nashville, TN) ;
Peterson; Caryn; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MOERAE MATRIX, INC. |
Morristown |
NJ |
US |
|
|
Assignee: |
MOERAE MATRIX, INC.
Morristown
NJ
|
Family ID: |
56356475 |
Appl. No.: |
16/106504 |
Filed: |
August 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14991531 |
Jan 8, 2016 |
10087225 |
|
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16106504 |
|
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62101190 |
Jan 8, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0019 20130101;
C07K 14/4703 20130101; A61K 9/0078 20130101; A61P 43/00 20180101;
A61K 47/26 20130101; A61P 9/00 20180101; A61K 9/145 20130101; A61K
38/00 20130101; A61K 38/1709 20130101; A61K 9/0075 20130101; A61K
47/02 20130101; A61K 47/32 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 47/02 20060101 A61K047/02; A61K 47/26 20060101
A61K047/26; A61K 47/32 20060101 A61K047/32; A61K 38/17 20060101
A61K038/17; A61K 9/00 20060101 A61K009/00; A61K 9/14 20060101
A61K009/14 |
Claims
1. A pharmaceutical formulation for delivery by inhalation
comprising a therapeutic amount of an MK2i polypeptide of amino
acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional
equivalent thereof, up to 5% w/w solids before drying, up to 7.3%
glycerin, and a pharmaceutically acceptable carrier, wherein the
formulation: (a) is isosmotic and non-buffered; (b) has a stable pH
of about 7.0 for at least 2 weeks at 60.degree. C.; and (c) is
effective to preserve at least about 93% stability of physical,
chemical, microbiological, therapeutic, and toxicological
specifications of the MK2i polypeptide for at least 2 weeks at
60.degree. C., and bioavailability of the MK2i polypeptide.
2. The pharmaceutical formulation according to claim 1, wherein the
pharmaceutical formulation is a particulate pharmaceutical
formulation.
3. The pharmaceutical formulation according to claim 1, wherein the
pharmaceutical formulation is an aerosolized pharmaceutical
formulation.
4. The pharmaceutical formulation according to claim 1, wherein the
formulation is prepared by a process of spray drying.
5. The pharmaceutical formulation according to claim 2, wherein the
pharmaceutical formulation comprises 1% w/w solids before
drying.
6. The pharmaceutical formulation according to claim 2, wherein the
pharmaceutical formulation comprises 5% w/w solids before
drying.
7. The pharmaceutical formulation according to claim 1 further
comprising trehalose.
8. The pharmaceutical formulation according to claim 1, wherein the
functional equivalent is made from a fusion between a first
polypeptide that is a protein transduction domain (PTD) and a
second polypeptide that is a therapeutic domain (TD).
9. The pharmaceutical formulation according to claim 8, wherein the
protein transduction domain (PTD) is selected from the group
consisting of a polypeptide of amino acid sequence YARAAARQARA (SEQ
ID NO: 11), FAKLAARLYR (SEQ ID NO: 16), and KAFAKLAARLYR (SEQ ID
NO: 17), and a second polypeptide that is a therapeutic domain (TD)
of amino acid sequence KALARQLGVAA (SEQ ID NO: 2).
10. The pharmaceutical formulation according to claim 1, wherein
the pharmaceutical formulation is delivered to a subject via a dry
powder inhalation device (DPI).
11. The pharmaceutical formulation according to claim 3 further
comprising saline before drying.
12. The pharmaceutical formulation according to claim 11, wherein
the saline is NaCl.
13. The pharmaceutical formulation according to claim 12, wherein
the polypeptide of amino acid sequence YARAAARQARAKALARQLGVAA (SEQ
ID NO: 1) or the functional equivalent thereof is at a
concentration of 0.7 mg/mL.
14. The pharmaceutical formulation according to claim 12, wherein
the polypeptide of amino acid sequence YARAAARQARAKALARQLGVAA (SEQ
ID NO: 1) or the functional equivalent thereof is at a
concentration of 7.0 mg/mL.
15. The pharmaceutical formulation according to claim 3, wherein
the pharmaceutical formulation is formulated to be used via a
nebulizer.
16. The pharmaceutical formulation according to claim 1, comprising
an ionic complex of the MK2i polypeptide of amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent
thereof and a nano-polyplex polymer, wherein the ionic complex
dissociates in intracellular compartments selected by intracellular
pH conditions such that bioactivity and stability of the peptide is
preserved.
17. The pharmaceutical formulation according to claim 16, wherein
the nano-polyplex polymer is anionic and endosomolytic.
18. The pharmaceutical formulation according to claim 17, wherein
the nano-polyplex polymer is poly(propylacrylic acid) (PPAA).
19. The pharmaceutical formulation according to claim 16, wherein
the pharmaceutical formulation comprises a charge ratio (CR) of the
MK2i polypeptide of amino acid sequence YARAAARQARAKALARQLGVAA (SEQ
ID NO: 1) or a functional equivalent thereof to PPAA selected from
the group consisting of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,
2:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and
1:10.
20. The pharmaceutical formulation according to claim 19, wherein
the charge ratio (CR) is 1:3.
21. The pharmaceutical formulation according to claim 16, wherein
the functional equivalent is made from a fusion between a first
polypeptide that is a protein transduction domain (PTD) and a
second polypeptide that is a therapeutic domain (TD).
22. The pharmaceutical formulation according to claim 21, wherein
the protein transduction domain (PTD) is selected from the group
consisting of a polypeptide of amino acid sequence YARAAARQARA (SEQ
ID NO: 11), FAKLAARLYR (SEQ ID NO: 16), and KAFAKLAARLYR (SEQ ID
NO: 17), and a second polypeptide that is a therapeutic domain (TD)
of amino acid sequence KALARQLGVAA (SEQ ID NO: 2).
23. The pharmaceutical formulation according to claim 16, wherein
the pharmaceutical formulation is delivered to a subject via an
implantation device.
24. The pharmaceutical formulation according to claim 16, wherein
the pharmaceutical formulation is delivered to a subject
topically.
25. The pharmaceutical formulation according to claim 16, wherein
the pharmaceutical formulation is delivered to a subject
parenterally.
26. A method for treating a vascular graft-induced intimal
hyperplasia in a subject in need of such treatment, the method
comprising administering the pharmaceutical formulation of claim 16
comprising a therapeutic amount of a polypeptide of amino sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent
thereof, and a nano-polyplex polymer, wherein the therapeutic
amount is effective to inhibit MK2; and to treat a vascular
graft-induced intimal hyperplasia.
27. The method according to claim 26, wherein the nano-polyplex
polymer is anionic and endosomolytic.
28. The method according to claim 27, wherein the nano-polyplex
polymer is poly(propylacrylic acid) (PPAA).
29. The method according to claim 26, wherein the pharmaceutical
formulation comprises a charge ratio (CR) of the polypeptide of
amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a
functional equivalent thereof to PPAA selected from the group
consisting of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1,
1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10.
30. The method according to claim 29, wherein the charge ratio (CR)
is 1:3.
31. The method according to claim 29, wherein the nano-polyplex
polymer is poly(acrylic acid) (PAA).
32. The method according to claim 26, wherein the functional
equivalent is made from a fusion between a first polypeptide that
is a protein transduction domain (PTD) and a second polypeptide
that is a therapeutic domain (TD).
33. The method according to claim 32, wherein the protein
transduction domain (PTD) is selected from the group consisting of
a polypeptide of amino acid sequence YARAAARQARA (SEQ ID NO: 11),
FAKLAARLYR (SEQ ID NO: 16), and KAFAKLAARLYR (SEQ ID NO: 17), and a
second polypeptide that is a therapeutic domain (TD) of amino acid
sequence KALARQLGVAA (SEQ ID NO: 2).
34. The method according to claim 26, wherein the administering is
by an implantation device.
35. The method according to claim 26, wherein the administering is
by topical administration.
36. The method according to claim 26, wherein the administering is
by parenteral administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
provisional patent application Ser. No. 62/101,190, filed Jan. 8,
2015, entitiled "FORMULATION OF MK2 INHIBITOR PEPTIDES", the
content of which is incorporated by reference herein in its
entirety.
FIELD OF INVENTION
[0002] The described invention relates to the fields of cell and
molecular biology, polypeptides, pharmaceutical formulations and
therapeutic methods of use.
BACKGROUND
Kinases
[0003] Kinases are a ubiquitous group of enzymes that catalyze the
phosphoryl transfer reaction from a phosphate donor (usually
adenosine-5'-triphosphate (ATP)) to a receptor substrate. Although
all kinases catalyze essentially the same phosphoryl transfer
reaction, they display remarkable diversity in their substrate
specificity, structure, and the pathways in which they participate.
A recent classification of all available kinase sequences
(approximately 60,000 sequences) indicates kinases can be grouped
into 25 families of homologous (meaning derived from a common
ancestor) proteins. These kinase families are assembled into 12
fold groups based on similarity of structural fold. Further, 22 of
the 25 families (approximately 98.8% of all sequences) belong to 10
fold groups for which the structural fold is known. Of the other 3
families, polyphosphate kinase forms a distinct fold group, and the
2 remaining families are both integral membrane kinases and
comprise the final fold group. These fold groups not only include
some of the most widely spread protein folds, such as Rossmann-like
fold (three or more parallel .beta. strands linked by two .alpha.
helices in the topological order
.beta.-.alpha.-.beta.-.alpha.-.beta.), ferredoxin-like fold (a
common .alpha.+.beta. protein fold with a signature
.beta..alpha..beta..beta..alpha..beta. secondary structure along
its backbone), TIM-barrel fold (meaning a conserved protein fold
consisting of eight .alpha.-helices and eight parallel
.beta.-strands that alternate along the peptide backbone), and
antiparallel .beta.-barrel fold (a beta barrel is a large
beta-sheet that twists and coils to form a closed structure in
which the first strand is hydrogen bonded to the last), but also
all major classes (all .alpha., all .beta., .alpha.+.beta.,
.alpha./.beta.) of protein structures. Within a fold group, the
core of the nucleotide-binding domain of each family has the same
architecture, and the topology of the protein core is either
identical or related by circular permutation. Homology between the
families within a fold group is not implied.
[0004] Group I (23,124 sequences) kinases incorporate protein S/T-Y
kinase, atypical protein kinase, lipid kinase, and ATP grasp
enzymes and further comprise the protein S/T-Y kinase, and atypical
protein kinase family (22,074 sequences). These kinases include:
choline kinase (EC 2.7.1.32); protein kinase (EC 2.7.137);
phosphorylase kinase (EC 2.7.1.38); homoserine kinase (EC
2.7.1.39); I-phosphatidylinositol 4-kinase (EC 2.7.1.67);
streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC
2.7.1.82); streptomycin 3'-kinase (EC 2.7.1.87); kanamycin kinase
(EC 2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin
kinase (EC 2.7.1.103); [hydroxymethylglutaryl-CoA reductase
(NADPH2)] kinase (EC 2.7.1.109); protein-tyrosine kinase (EC
2.7.1.112); [isocitrate dehydrogenase (NADP+)] kinase (EC
2.7.1.116); [myosin light-chain] kinase (EC 2.7.1.117);
hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin-dependent
protein kinase (EC 2.7.1.123); rhodopsin kinase (EC 2.7.1.125);
[beta-adrenergic-receptor] kinase (EC 2.7.1.126); [myosin
heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC
2.7.1.135); macrolide 2'-kinase (EC 2.7.1.136);
I-phosphatidylinositol 3-kinase (EC 2.7.1.137);
[RNA-polymerase]-subunit kinase (EC 2.7.1.141);
phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); and
phosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group I
further comprises the lipid kinase family (321 sequences). These
kinases include: I-phosphatidylinositol-4-phosphate 5-kinase (EC
2.7.1.68); I D-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127);
inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140);
I-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149);
I-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150);
inositol-polyphosphate multikinase (EC 2.7.1.151); and
inositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further
comprises the ATP-grasp kinases (729 sequences) which include
inositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate,
phosphate dikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC
2.7.9.2).
[0005] Group II (17,071 sequences) kinases incorporate the
Rossman-like kinases. Group II comprises the P-loop kinase family
(7,732 sequences). These include gluconokinase (EC 2.7.1.12);
phosphoribulokinase (EC 2.7.1.19); thymidine kinase (EC 2.7.1.21);
ribosylnicotinamide kinase (EC 2.7.1.22); dephospho-CoA kinase (EC
2.7.1.24); adenylylsulfate kinase (EC 2.7.1.25); pantothenate
kinase (EC 2.7.1.33); protein kinase (bacterial) (EC 2.7.1.37);
uridine kinase (EC 2.7.1.48); shikimate kinase (EC 2.7.1.71);
deoxycytidine kinase (EC 2.7.1.74); deoxyadenosine kinase (EC
2.7.1.76); polynucleotide 5'-hydroxyl-kinase (EC 2.7.1.78);
6-phosphofructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC
2.7.1.113); tetraacyldisaccharide 4'-kinase (EC 2.7.1.130);
deoxynucleoside kinase (EC 2.7.1.145); adenosylcobinamide kinase
(EC 2.7.1.156); polyphosphate kinase (EC 2.7.4.1);
phosphomevalonate kinase (EC 2.7.4.2); adenylate kinase (EC
2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4.4); guanylate
kinase (EC 2.7.4.8); thymidylate kinase (EC 2.7.4.9);
nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10);
(deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase
(EC 2.7.4.14); and uridylate kinase (EC 2.7.4.22). Group II further
comprises the phosphoenolpyruvate carboxykinase family (815
sequences). These enzymes include protein kinase (HPr
kinase/phosphatase) (EC 2.7.1.37); phosphoenolpyruvate
carboxykinase (GTP) (EC 4.1.1.32); and phosphoenolpyruvate
carboxykinase (ATP) (EC 4.1.1.49). Group II further comprises the
phosphoglycerate kinase (1,351 sequences) family. These enzymes
include phosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate
kinase (GTP) (EC 2.7.2.10). Group II further comprises the
aspartokinase family (2,171 sequences). These enzymes include
carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4);
acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC
2.7.2.1) and uridylate kinase (EC 2.7.4.). Group II further
comprises the phosphofructokinase-like kinase family (1,998
sequences). These enzymes include 6-phosphofrutokinase (EC
2.7.1.11); NAD(+) kinase (EC 2.7.1.23); I-phosphofructokinase (EC
2.7.1.56); diphosphate-fructose-6-phosphate I-phosphotransferase
(EC 2.7.1.90); sphinganine kinase (EC 2.7.1.91); diacylglycerol
kinase (EC 2.7.1.107); and ceramide kinase (EC 2.7.1.138). Group II
further comprises the ribokinase-like family (2,722 sequences).
These enzymes include: glucokinase (EC 2.7.1.2); ketohexokinase (EC
2.7.1.3); fructokinase (EC 2.7.1.4); 6-phosphofructokinase (EC
2.7.1.11); ribokinase (EC 2.7.1.15); adenosine kinase (EC
2.7.1.20); pyridoxal kinase (EC 2.7.1.35);
2-dehydro-3-deoxygluconokinase (EC 2.7.1.45);
hydroxymethylpyrimidine kinase (EC 2.7.1.49); hydroxyethylthiazole
kinase (EC 2.7.1.50); I-phosphofructokinase (EC 2.7.1.56); inosine
kinase (EC 2.7.1.73); 5-dehydro-2-deoxygluconokinase (EC 2.7.1.92);
tagatose-6-phosphate kinase (EC 2.7.1.144); ADP-dependent
phosphofructokinase (EC 2.7.1.146); ADP-dependent glucokinase (EC
2.7.1.147); and phosphomethylpyrimidine kinase (EC 2.7.4.7). Group
II further comprises the thiamin pyrophosphokinase family (175
sequences) which includes thiamin pyrophosphokinase (EC 2.7.6.2).
Group II further comprises the glycerate kinase family (107
sequences) which includes glycerate kinase (EC 2.7.1.31).
[0006] Group III kinases (10,973 sequences) comprise the
ferredoxin-like fold kinases. Group III further comprises the
nucleoside-diphosphate kinase family (923 sequences). These enzymes
include nucleoside-diphosphate kinase (EC 2.7.4.6). Group III
further comprises the HPPK kinase family (609 sequences). These
enzymes include 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
pyrophosphokinase (EC 2.7.6.3). Group III further comprises the
guanido kinase family (324 sequences). These enzymes include
guanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7.3.2);
arginine kinase (EC 2.7.3.3); and lombricine kinase (EC 2.7.3.5).
Group III further comprises the histidine kinase family (9,117
sequences). These enzymes include protein kinase (histidine kinase)
(EC 2.7.1.37); [pyruvate dehydrogenase (lipoamide)] kinase (EC
2.7.1.99); and [3-methyl-2-oxybutanoate dehydrogenase(lipoamide)]
kinase (EC 2.7.1.115).
[0007] Group IV kinases (2,768 sequences) incorporate ribonuclease
H-like kinases. These enzymes include hexokinase (EC 2.7.1.1);
glucokinase (EC 2.7.1.2); fructokinase (EC 2.7.1.4); rhamnulokinase
(EC 2.7.1.5); mannokinase (EC 2.7.1.7); gluconokinase (EC
2.7.1.12); L-ribulokinase (EC 2.7.1.16); xylulokinase (EC
2.7.1.17); erythritol kinase (EC 2.7.1.27); glycerol kinase (EC
2.7.1.30); pantothenate kinase (EC 2.7.1.33); D-ribulokinase (EC
2.7.1.47); L-fucolokinase (EC 2.7.1.51); L-xylulokinase (EC
2.7.1.53); allose kinase (EC 2.7.1.55);
2-dehydro-3-deoxygalactonokinase (EC 2.7.1.58); N-acetylglucosamine
kinase (EC 2.7.1.59); N-acylmannosamine kinase (EC 2.7.1.60);
polyphosphate-glucose phosphotransferase (EC 2.7.1.63);
beta-glucoside kinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1);
butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid kinase (EC
2.7.2.14); and propionate kinase (EC 2.7.2.15).
[0008] Group V kinases (1,119 sequences) incorporate TIM
.beta.-barrel kinases. These enzymes include pyruvate kinase (EC
2.7.1.40).
[0009] Group VI kinases (885 sequences) incorporate GHMP kinases.
These enzymes include galactokinase (EC 2.7.1.6); mevalonate kinase
(EC 2.7.1.36); homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC
2.7.1.46); fucokinase (EC 2.7.1.52); shikimate kinase (EC
2.7.1.71); 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythriol kinase
(EC 2.7.1.148); and phosphomevalonate kinase (EC 2.7.4.2).
[0010] Group VII kinases (1,843 sequences) incorporate AIR
synthetase-like kinases. These enzymes include thiamine-phosphate
kinase (EC 2.7.4.16) and selenide, water dikinase (EC 2.7.9.3).
[0011] Group VIII kinases (565 sequences) incorporate riboflavin
kinases (565 sequences). These enzymes include riboflavin kinase
(EC 2.7.1.26).
[0012] Group IX kinases (197 sequences) incorporate
dihydroxyacetone kinases. These enzymes include glycerone kinase
(EC 2.7.1.29).
[0013] Group X kinases (148 sequences) incorporate putative
glycerate kinases. These enzymes include glycerate kinase (EC
2.7.1.31).
[0014] Group XI kinases (446 sequences) incorporate polyphosphate
kinases. These enzymes include polyphosphate kinases (EC
2.7.4.1).
[0015] Group XII kinases (263 sequences) incorporate integral
membrane kinases. Group XII comprises the dolichol kinase family.
These enzymes include dolichol kinases (EC 2.7.1.108). Group XII
further comprises the undecaprenol kinase family. These enzymes
include undecaprenol kinases (EC 2.7.1.66).
[0016] Kinases play indispensable roles in numerous cellular
metabolic and signaling pathways, and are among the best-studied
enzymes at the structural, biochemical, and cellular level. Despite
the fact that all kinases use the same phosphate donor (in most
cases, ATP) and catalyze apparently the same phosphoryl transfer
reaction, they display remarkable diversity in their structural
folds and substrate recognition mechanisms. This probably is due
largely to the diverse nature of the structures and properties of
their substrates.
[0017] Mitogen-Activated Protein Kinase (MAPK)-Activated Protein
Kinases (MK2 and MK3)
[0018] Different groups of MAPK-activated protein kinases
(MAP-KAPKs) have been defined downstream of mitogen-activated
protein kinases (MAPKs). These enzymes transduce signals to target
proteins that are not direct substrates of the MAPKs and,
therefore, serve to relay phosphorylation-dependent signaling with
MAPK cascades to diverse cellular functions. One of these groups is
formed by the three MAPKAPKs: MK2, MK3 (also known as 3pK), and MK5
(also designated PRAK). Mitogen-activated protein kinase-activated
protein kinase 2 (also referred to as "MAPKAPK2", "MAPKAP-K2",
"MK2") is a kinase of the serine/threonine (Ser/Thr) protein kinase
family. MK2 is highly homologous to MK3 (approximately 75% amino
acid identity). The kinase domains of MK2 and MK3 are most similar
(approximately 35% to 40% identity) to calcium/calmodulin-dependent
protein kinase (CaMK), phosphorylase b kinase, and the C-terminal
kinase domain (CTKD) of the ribosomal S6 kinase (RSK) isoforms. The
MK2 gene encodes two alternatively spliced transcripts of 370 amino
acids (MK2A) and 400 amino acids (MK2B). The MK3 gene encodes one
transcript of 382 amino acids. The MK2- and MK3 proteins are highly
homologous, yet MK2A possesses a shorter C-terminal region. The
C-terminus of MK2B contains a functional bipartite nuclear
localization sequence (NLS)
(Lys-Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Arg-Arg-Lys-Lys;
SEQ ID NO: 21) that is not present in the shorter MK2A isoform,
indicating that alternative splicing determines the cellular
localization of the MK2 isoforms. MK3 possesses a similar nuclear
localization sequence. The nuclear localization sequence found in
both MK2B and MK3 encompasses a D domain
(Leu-Leu-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 22), which was shown to
mediate the specific interaction of MK2B and MK3 with p38.alpha.
and p38.beta.. MK2B and MK3 also possess a functional nuclear
export signal (NES) located N-terminal to the NLS and D domain. The
NES in MK2B is sufficient to trigger nuclear export following
stimulation, a process which may be inhibited by leptomycin B. The
sequence N-terminal to the catalytic domain in MK2 and MK3 is
proline rich and contains one (MK3) or two (MK2) putative Src
homology 3 (SH3) domain-binding sites, which studies have shown,
for MK2, to mediate binding to the SH3 domain of c-Abl in vitro.
Recent studies suggest that this domain is involved in MK2-mediated
cell migration.
[0019] MK2B and MK3 are located predominantly in the nucleus of
quiescent cells while MK2A is present in the cytoplasm. Both MK2B
and MK3 are rapidly exported to the cytoplasm via a chromosome
region maintenance protein (CRM1)-dependent mechanism upon stress
stimulation. Nuclear export of MK2B appears to be mediated by
kinase activation, as phosphomimetic mutation of Thr334 within the
activation loop of the kinase enhances the cytoplasmic localization
of MK2B. Without being limited by theory, it is thought that MK2B
and MK3 may contain a constitutively active nuclear localization
signal (NLS) and a phosphorylation-regulated nuclear export signal
(NES).
[0020] MK2 and MK3 appear to be expressed ubiquitously, with
increased relative expression in the heart, lungs, kidney,
reproductive organs (mammary and testis), skin and skeletal muscle
tissues, as well as in immune-related cells such as white blood
cells/leukocytes and dendritic cells.
[0021] Activation
[0022] Various activators of p38.alpha. and p38.beta. potently
stimulate MK2 and MK3 activity. p38 mediates the in vitro and in
vivo phosphorylation of MK2 on four proline-directed sites: Thr25,
Thr222, Ser272, and Thr334. Of these sites, only Thr25 is not
conserved in MK3. Without being limited by theory, while the
function of phosphorylated Thr25 is unknown, its location between
the two SH3 domain-binding sites suggests that it may regulate
protein-protein interactions. Thr222 in MK2 (Thr201 in MK3) is
located in the activation loop of the kinase domain and has been
shown to be essential for MK2 and MK3 kinase activity. Thr334 in
MK2 (Thr313 in MK3) is located C-terminal to the catalytic domain
and is essential for kinase activity. The crystal structure of MK2
has been resolved and, without being limited by theory, suggests
that Thr334 phosphorylation may serve as a switch for MK2 nuclear
import and export. Phosphorylation of Thr334 also may weaken or
interrupt binding of the C terminus of MK2 to the catalytic domain,
exposing the NES and promoting nuclear export.
[0023] Studies have shown that while p38 is capable of activating
MK2 and MK3 in the nucleus, experimental evidence suggests that
activation and nuclear export of MK2 and MK3 are coupled by a
phosphorylation-dependent conformational switch that also dictates
p38 stabilization and localization, and the cellular location of
p38 itself is controlled by MK2 and possibly MK3. Additional
studies have shown that nuclear p38 is exported to the cytoplasm in
a complex with MK2 following phosphorylation and activation of MK2.
The interaction between p38 and MK2 may be important for p38
stabilization since studies indicate that p38 levels are low in
MK2-deficient cells and expression of a catalytically inactive MK2
protein restores p38 levels.
[0024] Substrates and Functions
[0025] MK2 shares many substrates with MK3. Both enzymes have
comparable substrate preferences and phosphorylate peptide
substrates with similar kinetic constants. The minimum sequence
required for efficient phosphorylation by MK2 was found to be
Hyd-Xaa-Arg-Xaa-Xaa-pSer/pThr (SEQ ID NO: 22), where Hyd is a
bulky, hydrophobic residue.
[0026] Accumulating studies have shown that MK2 phophorylates a
variety of proteins, which include, but are not limited to,
5-Lipooxygenase (ALOX5), Cell Division Cycle 25 Homolog B (CDC25B),
Cell Division Cycle 25 Homolog C (CDC25C), Embryonic Lethal,
Abnormal Vision, Drosophila-Like 1 (ELAVL1), Heterogeneous Nuclear
Ribonucleoprotein A0 (HNRNPAO), Heat Shock Factor protein 1 (HSF1),
Heat Shock Protein Beta-1 (HSPB1), Keratin 18 (KRT18), Keratin 20
(KRT20), LIM domain kinase 1 (LIMK1), Lymphocyte-specific protein 1
(LSP1), Polyadenylate-Binding Protein 1 (PABPC1), Poly(A)-specific
Ribonuclease (PARN), CAMP-specific 3',5'-cyclic Phosphodiesterase
4A (PDE4A), RCSD domain containing 1 (RCSD1), Ribosomal protein S6
kinase, 90 kDa, polypeptide 3 (RPS6KA3), TGF-beta activated kinase
1/MAP3K7 binding protein 3 (TAB3), and Tristetraprolin
(TTP/ZFP36).
[0027] Heat-Shock Protein Beta-1 (also termed HSPB1 or HSP27) is a
stress-inducible cytosolic protein that is ubiquitously present in
normal cells and is a member of the small heat-shock protein
family. The synthesis of HSPB1 is induced by heat shock and other
environmental or pathophysiologic stresses, such as UV radiation,
hypoxia and ischemia. Besides its putative role in
thermoresistance, HSPB1 is involved in the survival and recovery of
cells exposed to stressful conditions.
[0028] Experimental evidence supports a role for p38 in the
regulation of cytokine biosynthesis and cell migration. The
targeted deletion of the mk2 gene in mice suggested that although
p38 mediates the activation of many similar kinases, MK2 seems to
be the key kinase responsible for these p38-dependent biological
processes. Loss of MK2 leads (i) to a defect in lipopolysaccharide
(LPS)-induced synthesis of cytokines such as tumor necrosis factor
alpha (TNF-.alpha.), interleukin-6 (IL-6), and gamma interferon
(IFN-.gamma.) and (ii) to changes in the migration of mouse
embryonic fibroblasts, smooth muscle cells, and neutrophils.
[0029] Consistent with a role for MK2 in inflammatory and immune
responses, MK2-deficient mice showed increased susceptibility to
Listeria monocytogenes infection and reduced inflammation-mediated
neuronal death following focal ischemia. Since the levels of p38
protein also are reduced significantly in MK2-deficient cells, it
was necessary to distinguish whether these phenotypes were due
solely to the loss of MK2. To achieve this, MK2 mutants were
expressed in MK2-deficient cells, and the results indicated that
the catalytic activity of MK2 was not necessary to restore p38
levels but was required to regulate cytokine biosynthesis.
[0030] Knockout or knockdown studies of MK2 provide strong support
that activated MK2 enhances stability of IL-6 mRNA through
phosphorylation of proteins interacting with the AU-rich 3'
untranslated region of IL-6 mRNA. In particular, it has been shown
that MK2 is principally responsible for phosphorylation of hnRNPAO,
an mRNA-binding protein that stabilizes IL-6 RNA. In addition,
several additional studies investigating diverse inflammatory
diseases have found that levels of pro-inflammatory cytokines, such
as IL-6, IL-1.beta., TNF-.alpha. and IL-8, are increased in induced
sputum from patients with stable chronic obstructive pulmonary
disease (COPD) or from the alveolar macrophages of cigarette
smokers (Keatings V. et al, Am J Resp Crit Care Med, 1996,
153:530-534; Lim, S. et al., J Respir Crit Care Med, 2000,
162:1355-1360).
[0031] Regulation of mRNA Translation.
[0032] Previous studies using MK2 knockout mice or MK2-deficient
cells have shown that MK2 increases the production of inflammatory
cytokines, including TNF-.alpha., IL-1, and IL-6, by increasing the
rate of translation of its mRNA. No significant reductions in the
transcription, processing, and shedding of TNF-.alpha. could be
detected in MK2-deficient mice. The p38 pathway is known to play an
important role in regulating mRNA stability, and MK2 represents a
likely target by which p38 mediates this function. Studies
utilizing MK2-deficient mice indicated that the catalytic activity
of MK2 is necessary for its effects on cytokine production and
migration, suggesting that, without being limited by theory, MK2
phosphorylates targets involved in mRNA stability. Consistent with
this, MK2 has been shown to bind and/or phosphorylate the
heterogeneous nuclear ribonucleoprotein (hnRNP) A0, tristetraprolin
(TTP), the poly(A)-binding protein PABP1, and HuR, a ubiquitously
expressed member of the ELAV (Embryonic-Lethal Abnormal Visual in
Drosophila melanogaster) family of RNA-binding protein. These
substrates are known to bind or copurify with mRNAs that contain
AU-rich elements in the 3' untranslated region, suggesting that MK2
may regulate the stability of AU-rich mRNAs such as TNF-.alpha.. It
currently is unknown whether MK3 plays a similar role, but LPS
treatment of MK2-deficient fibroblasts completely abolished hnRNP
A0 phosphorylation, suggesting that MK3 is not able to compensate
for the loss of MK2.
[0033] MK3 participates with MK2 in phosphorylation of the
eukaryotic elongation factor 2 (eEF2) kinase. eEF2 kinase
phosphorylates and inactivates eEF2. eEF2 activity is critical for
the elongation of mRNA during translation, and phosphorylation of
eEF2 on Thr56 results in the termination of mRNA translation. MK2
and MK3 phosphorylation of eEF2 kinase on Ser377 suggests that
these enzymes may modulate eEF2 kinase activity and thereby
regulate mRNA translation elongation.
[0034] Transcriptional Regulation by MK2 and MK3
[0035] Nuclear MK2, similar to many MKs, contributes to the
phosphorylation of cAMP response element binding (CREB), Activating
Transcription Factor-1 (ATF-1), serum response factor (SRF), and
transcription factor ER81. Comparison of wild-type and
MK2-deficient cells revealed that MK2 is the major SRF kinase
induced by stress, suggesting a role for MK2 in the stress-mediated
immediate-early response. Both MK2 and MK3 interact with basic
helix-loop-helix transcription factor E47 in vivo and phosphorylate
E47 in vitro. MK2-mediated phosphorylation of E47 was found to
repress the transcriptional activity of E47 and thereby inhibit
E47-dependent gene expression, suggesting that MK2 and MK3 may
regulate tissue-specific gene expression and cell
differentiation.
[0036] Other Targets of MK2 and MK3
[0037] Several other MK2 and MK3 substrates also have been
identified, reflective of the diverse functions of MK2 and MK3 in
several biological processes. The scaffolding protein 14-3-3.zeta.
is a physiological MK2 substrate. Studies indicate that
14-3-3.zeta. interacts with a number of components of cell
signaling pathways, including protein kinases, phosphatases, and
transcription factors. Additional studies have shown that
MK2-mediated phosphorylation of 14-3-3.zeta. on Ser58 compromises
its binding activity, suggesting that MK2 may affect the regulation
of several signaling molecules normally regulated by
14-3-3.zeta..
[0038] Additional studies have shown that MK2 also interacts with
and phosphorylates the p16 subunit of the seven-member Arp2 and
Arp3 complex (p16-Arc) on Ser77. p16-Arc has roles in regulating
the actin cytoskeleton, suggesting that MK2 may be involved in this
process. Further studies have shown that the small heat shock
protein HSPB1, lymphocyte-specific protein LSP-1, and vimentin are
phosphorylated by MK2. HSPB1 is of particular interest because it
forms large oligomers which may act as molecular chaperones and
protect cells from heat shock and oxidative stress. Upon
phosphorylation, HSPB1 loses its ability to form large oligomers
and is unable to block actin polymerization, suggesting that
MK2-mediated phosphorylation of HSPB1 serves a homeostatic function
aimed at regulating actin dynamics that otherwise would be
destabilized during stress. MK3 also was shown to phosphorylate
HSPB1 in vitro and in vivo, but its role during stressful
conditions has not yet been elucidated.
[0039] It was also shown that HSPB1 binds to polyubiquitin chains
and to the 26S proteasome in vitro and in vivo. The
ubiquitin-proteasome pathway is involved in the activation of
transcription factor NF-kappa B (NF-.kappa.B) by degrading its main
inhibitor, I kappa B-alpha (I.kappa.B-alpha), and it was shown that
overexpresion of HSPB1 increases NF-kappaB (NF-.kappa.B) nuclear
relocalization, DNA binding, and transcriptional activity induced
by etoposide, TNF-alpha, and Interleukin-1 beta (IL-1.beta.).
Additionally, previous studies have suggested that HSPB1, under
stress conditions, favors the degradation of ubiquitinated
proteins, such as phosphorylated I kappa B-alpha (I.kappa.B-alpha);
and that this function of HSPB1 accounts for its anti-apoptotic
properties through the enhancement of NF-kappa B (NF-.kappa.B)
activity (Parcellier, A. et al., Mol Cell Biol, 23(16): 5790-5802,
2003).
[0040] MK2 and MK3 also may phosphorylate 5-lipoxygenase.
5-lipoxygenase catalyzes the initial steps in the formation of the
inflammatory mediators, leukotrienes. Tyrosine hydroxylase,
glycogen synthase, and Akt also were shown to be phosphorylated by
MK2. Finally, MK2 phosphorylates the tumor suppressor protein
tuberin on Ser1210, creating a docking site for 14-3-3.zeta..
Tuberin and hamartin normally form a functional complex that
negatively regulates cell growth by antagonizing mTOR-dependent
signaling, suggesting that p38-mediated activation of MK2 may
regulate cell growth by increasing 14-3-3.zeta. binding to
tuberin.
[0041] Accumulating studies have suggested that the reciprocal
crosstalk between the p38 MAPK-pathway and signal transducer and
activator of transcription 3 (STAT3)-mediated signal-transduction
forms a critical axis successively activated in lipopolysaccharide
(LPS) challenge models. It was shown that the balanced activation
of this axis is essential for both induction and propagation of the
inflammatory macrophage response as well as for the control of the
resolution phase, which is largely driven by IL-10 and sustained
STAT3 activation (Bode, J. et al., Cellular Signalling, 24:
1185-1194, 2012). In addition, another study has shown that MK2
controls LPS-inducible IFN.beta. gene expression and subsequent
IFN.beta.-mediated activation of STAT3 by neutralizing negative
regulatory effects of MK3 on LPS-induced p65 and IRF3-mediated
signaling. The study further showed that in mk2/3 knockout
macrophages, IFN.beta.-dependent STAT3 activation occurs
independently from IL-10, because, in contrast to IFN.beta.-,
impaired IL-10 expression is not restored upon additional deletion
of MK3 in mk2/3 knockout macrophages (Ehlting, C. et al., J. Biol.
Chem., 285(27): 24113-24124).
[0042] Kinase Inhibition
[0043] The eukaryotic protein kinases constitute one of the largest
superfamilies of homologous proteins that are related by virtue of
their catalytic domains. Most related protein kinases are specific
for either serine/threonine or tyrosine phosphorylation. Protein
kinases play an integral role in the cellular response to
extracellular stimuli. Thus, stimulation of protein kinases is
considered to be one of the most common activation mechanisms in
signal transduction systems. Many substrates are known to undergo
phosphorylation by multiple protein kinases, and a considerable
amount of information on primary sequence of the catalytic domains
of various protein kinases has been published. These sequences
share a large number of residues involved in ATP binding,
catalysis, and maintenance of structural integrity. Most protein
kinases possess a well conserved 30-32 kDa catalytic domain.
[0044] Studies have attempted to identify and utilize regulatory
elements of protein kinases. These regulatory elements include
inhibitors, antibodies, and blocking peptides.
[0045] Inhibitors
[0046] Enzyme inhibitors are molecules that bind to enzymes thereby
decreasing enzyme activity. The binding of an inhibitor may stop a
substrate from entering the active site of the enzyme and/or hinder
the enzyme from catalyzing its reaction (as in inhibitors directed
at the ATP biding site of the kinase). Inhibitor binding is either
reversible or irreversible. Irreversible inhibitors usually react
with the enzyme and change it chemically (e.g., by modifying key
amino acid residues needed for enzymatic activity) so that it no
longer is capable of catalyzing its reaction. In contrast,
reversible inhibitors bind non-covalently and different types of
inhibition are produced depending on whether these inhibitors bind
the enzyme, the enzyme-substrate complex, or both.
[0047] Enzyme inhibitors often are evaluated by their specificity
and potency. The term "specificity" as used in this context refers
to the selective attachment of an inhibitor or its lack of binding
to other proteins. The term "potency" as used herein refers to an
inhibitor's dissociation constant, which indicates the
concentration of inhibitor needed to inhibit an enzyme.
[0048] Inhibitors of protein kinases have been studied for use as a
tool in protein kinase activity regulation. Inhibitors have been
studied for use with, for example, cyclin-dependent (Cdk) kinase,
MAP kinase, serine/threonine kinase, Src Family protein tyrosine
kinase, tyrosine kinase, calmodulin (CaM) kinase, casein kinase,
checkpoint kinase (Chkl), glycogen synthase kinase 3 (GSK-3), c-Jun
N-terminal kinase (JNK), mitogen-activated protein kinase 1 (MEK),
myosin light chain kinase (MLCK), protein kinase A, Akt (protein
kinase B), protein kinase C, protein kinase G, protein tyrosine
kinase, Raf kinase, and Rho kinase.
[0049] Small-Molecule MK2 Inhibitors
[0050] While individual inhibitors that target MK2 with at least
modest selectivity with respect to other kinases have been
designed, it has been difficult to create compounds with favorable
solubility and permeability. As a result, there are relatively few
biochemically efficient MK2 inhibitors that have advanced to in
vivo pre-clinical studies (Edmunds, J. and Talanian, MAPKAP Kinase
2 (MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin,
J and Laufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175,
the Royal Society of Chemistry, 2012; incorporated by reference in
its entirety).
[0051] The majority of disclosed MK2 inhibitors are classical type
I inhibitors as revealed by crystallographic or biochemical
studies. As such, they bind to the ATP site of the kinase and thus
compete with intra-cellular ATP (estimated concentration 1 mM-5 mM)
to inhibit phosphorylation and activation of the kinase.
Representative examples of small-molecule MK2 inhibitors include,
but are not limited to,
##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005##
Blocking Peptides
[0052] A peptide is a chemical compound that is composed of a chain
of two or more amino acids whereby the carboxyl group of one amino
acid in the chain is linked to the amino group of the other via a
peptide bond. Peptides have been used inter alia in the study of
protein structure and function. Synthetic peptides may be used
inter alia as probes to see where protein-peptide interactions
occur. Inhibitory peptides may be used inter alia in clinical
research to examine the effects of peptides on the inhibition of
protein kinases, cancer proteins and other disorders.
[0053] The use of several blocking peptides has been studied. For
example, extracellular signal-regulated kinase (ERK), a MAPK
protein kinase, is essential for cellular proliferation and
differentiation. The activation of MAPKs requires a cascade
mechanism whereby MAPK is phosphorylated by an upstream MAPKK (MEK)
which then, in turn, is phosphorylated by a third kinase MAPKKK
(MEKK). The ERK inhibitory peptide functions as a MEK decoy by
binding to ERK.
[0054] Other blocking peptides include autocamtide-2 related
inhibitory peptide (AIP). This synthetic peptide is a highly
specific and potent inhibitor of Ca.sup.2+/calmodulin-dependent
protein kinase II (CaMKII). AIP is a non-phosphorylatable analog of
autocamtide-2, a highly selective peptide substrate for CaMKII. AIP
inhibits CaMKII with an IC50 of 100 nM (IC50 is the concentration
of an inhibitor required to obtain 50% inhibition). The AIP
inhibition is non-competitive with respect to syntide-2 (CaMKII
peptide substrate) and ATP but competitive with respect to
autocamtide-2. The inhibition is unaffected by the presence or
absence of Ca.sup.2+/calmodulin. CaMKII activity is inhibited
completely by AIP (1 .mu.M) while PKA, PKC and CaMKIV are not
affected.
[0055] Other blocking peptides include cell division protein kinase
5 (Cdk5) inhibitory peptide (CIP). Cdk5 phosphorylates the
microtubule protein tau at Alzheimer's Disease-specific
phospho-epitopes when it associates with p25. p25 is a truncated
activator, which is produced from the physiological Cdk5 activator
p35 upon exposure to amyloid .beta. peptides. Upon neuronal
infections with CIP, CIPs selectively inhibit p25/Cdk5 activity and
suppress the aberrant tau phosphorylation in cortical neurons. The
reasons for the specificity demonstrated by CIP are not fully
understood.
[0056] Additional blocking peptides have been studied for
extracellular-regulated kinase 2 (ERK2), ERK3, p38/HOG1, protein
kinase C, casein kinase II, Ca.sup.2.+-./calmodulin kinase IV,
casein kinase II, Cdk4, Cdk5, DNA-dependent protein kinase
(DNA-PK), serine/threonine-protein kinase PAK3, phosphoinositide
(PI)-3 kinase, PI-5 kinase, PSTAIRE (the cdk highly conserved
sequence), ribosomal S6 kinase, GSK-4, germinal center kinase
(GCK), SAPK (stress-activated protein kinase), SEK1 (stress
signaling kinase), and focal adhesion kinase (FAK).
Protein Substrate-Competitive Inhibitors
[0057] Most of the protein kinase inhibitors developed to date are
ATP competitors. This type of molecule competes for the ATP binding
site of the kinase and often shows off-target effects due to
serious limitations in its specificity. The low specificity of
these inhibitors is due to the fact that the ATP binding site is
highly conserved among diverse protein kinases. Non-ATP competitive
inhibitors, on the other hand, such as substrate competitive
inhibitors, are expected to be more specific as the substrate
binding sites have a certain degree of variability among the
various protein kinases.
[0058] Although substrate competitive inhibitors usually have a
weak binding interaction with the target enzyme in vitro, studies
have shown that chemical modifications can improve the specific
biding affinity and the in vivo efficacy of substrate inhibitors
(Eldar-Finkelman, H. et al., Biochim, Biophys. Acta,
1804(3):598-603, 2010). In addition, substrate competitive
inhibitors show better efficacy in cells than in cell-free
conditions in many cases (van Es, J. et al., Curr. Opin. Gent. Dev.
13:28-33, 2003).
[0059] In an effort to enhance specificity and potency in protein
kinase inhibition, bisubstrate inhibitors also have been developed.
Bisubstrate inhibitors, which consist of two conjugated fragments,
each targeted to a different binding site of a bisubstrate enzyme,
form a special group of protein kinase inhibitors that mimic two
natural substrates/ligands and that simultaneously associate with
two regions of given kinases. The principle advantage of
bisubstrate inhibitors is their ability to generate more
interactions with the target enzyme that could result in improved
affinity and selectivity of the conjugates, when compared with
single-site inhibitors. Examples of bisubstrate inhibitors include,
but are not limited to, nucleotide-peptide conjugates, adenosine
derivative-peptide conjugates, and conjugates of peptides with
potent ATP-competitive inhibitors.
Protein Transduction Domains (PTD)/Cell Permeable Proteins
(CPP)
[0060] The plasma membrane presents a formidable barrier to the
introduction of macromolecules into cells. For nearly all
therapeutics to exert their effects, at least one cellular membrane
must be traversed. Traditional small molecule pharmaceutical
development relies on the chance discovery of membrane permeable
molecules with the ability to modulate protein function. Although
small molecules remain the dominant therapeutic paradigm, many of
these molecules suffer from lack of specificity, side effects, and
toxicity. Information-rich macromolecules, which have protein
modulatory functions far superior to those of small molecules, can
be created using rational drug design based on molecular, cellular,
and structural data. However, the plasma membrane is impermeable to
most molecules of size greater than 500 Da. Therefore, the ability
of cell penetrating peptides, such as the basic domain of
Trans-Activator of Transcription (Tat), to cross the cell membrane
and deliver macromolecular cargo in vivo, can greatly facilitate
the rational design of therapeutic proteins, peptides, and nucleic
acids.
[0061] Protein transduction domains (PTDs) are a class of peptides
capable of penetrating the plasma membrane of mammalian cells and
of transporting compounds of many types and molecular weights
across the membrane. These compounds include effector molecules,
such as proteins, DNA, conjugated peptides, oligonucleotides, and
small particles such as liposomes. When PTDs are chemically linked
or fused to other proteins, the resulting fusion peptides still are
able to enter cells. Although the exact mechanism of transduction
is unknown, internalization of these proteins is not believed to be
receptor-mediated or transporter-mediated. PTDs are generally 10-16
amino acids in length and may be grouped according to their
composition, such as, for example, peptides rich in arginine and/or
lysine.
[0062] The use of PTDs capable of transporting effector molecules
into cells has become increasingly attractive in the design of
drugs as they promote the cellular uptake of cargo molecules. These
cell-penetrating peptides, generally categorized as amphipathic
(meaning having both a polar and a nonpolar end) or cationic
(meaning of or relating to containing net positively charged atoms)
depending on their sequence, provide a non-invasive delivery
technology for macromolecules. PTDs often are referred to as
"Trojan peptides", "membrane translocating sequences", or "cell
permeable proteins" (CPPs). PTDs also may be used to assist novel
HSPB1 kinase inhibitors to penetrate cell membranes. (see U.S.
application Ser. No. 11/972,459, entitled "Polypeptide Inhibitors
of HSPB1 Kinase and Uses Therefor," filed Jan. 10, 2008, and Ser.
No. 12/188,109, entitled "Kinase Inhibitors and Uses Thereof,"
filed Aug. 7, 2008, the contents of each application are
incorporated by reference in their entirety herein).
Viral PTD Containing Proteins
[0063] The first proteins to be described as having transduction
properties were of viral origin. These proteins still are the most
commonly accepted models for PTD action. The HIV-1 Transactivator
of Transcription (Tat) and HSV-1 VP 22 protein are the best
characterized viral PTD containing proteins.
[0064] Tat (HIV-1 trans-activator gene product) is an 86-amino acid
polypeptide, which acts as a powerful transcription factor of the
integrated HIV-1 genome. Tat acts on the viral genome, stimulating
viral replication in latently infected cells. The translocation
properties of the Tat protein enable it to activate quiescent
infected cells, and it may be involved in priming of uninfected
cells for subsequent infection by regulating many cellular genes,
including cytokines. The minimal PTD of Tat is the 9 amino acid
protein sequence RKKRRQRRR (TAT49-57; SEQ ID NO: 20). Studies
utilizing a longer fragment of Tat demonstrated successful
transduction of fusion proteins up to 120 kDa. The addition of
multiple Tat-PTDs as well as synthetic Tat derivatives has been
demonstrated to mediate membrane translocation. Tat PTD containing
fusion proteins have been used as therapeutic moieties in
experiments involving cancer, transporting a death-protein into
cells, and disease models of neurodegenerative disorders.
[0065] The mechanism used by transducing peptides to permeate cell
membranes has been the subject of considerable interest in recent
years, as researchers have sought to understand the biology behind
transduction. Early reports that Tat transduction occurred by a
nonendocytic mechanism have largely been dismissed as artifactual
though other cell-penetrating peptides might be taken up by way of
direct membrane disruption. The recent findings that transduction
of Tat and other PTDs occurs by way of macropinocytosis, a
specialized form of endocytosis, has created a new paradigm in the
study of these peptides. Enhanced knowledge of the mechanism of
transduction helped improve transduction efficiency with the
ultimate goal of clinical success (Snyder E. and Dowdy, S., Pharm
Res., 21(3):389-393, 2004).
[0066] The current model for Tat-mediated protein transduction is a
multistep process that involves binding of Tat to the cell surface,
stimulation of macropinocytosis, uptake of Tat and cargo into
macropinosomes, and endosomal escape into the cytoplasm. The first
step, binding to the cell surface, is thought to be through
ubiquitous glycan chains on the cell surface. Stimulation of
macropinocytosis by Tat occurs by an unknown mechanism that might
include binding to a cell surface protein or occur by way of
proteoglycans or glycolipids. Uptake by way of macropinocytosis, a
form of fluid phase endocytosis used by all cell types, is required
for Tat and polyarginine transduction. The final step in Tat
transduction is escape from macropinosomes into the cytoplasm; this
process is likely to be dependent on the pH drop in endosomes that,
along with other factors, facilitates a perturbation of the
membrane by Tat and release of Tat and its cargo (i.e. peptide,
protein or drug etc.) to the cytoplasm (Snyder E. and Dowdy, S.,
Pharm Res., 21(3):389-393, 2004).
[0067] VP22 is the HSV-1 tegument protein, a structural part of the
HSV virion. VP22 is capable of receptor independent translocation
and accumulates in the nucleus. This property of VP22 classifies
the protein as a PTD containing peptide. Fusion proteins comprising
full length VP22 have been translocated efficiently across the
plasma membrane.
Homeoproteins with Intercellular Translocation Properties
[0068] Homeoproteins are highly conserved, transactivating
transcription factors involved in morphological processes. They
bind to DNA through a specific sequence of 60 amino acids. The
DNA-binding homeodomain is the most highly conserved sequence of
the homeoprotein. Several homeoproteins have been described as
exhibiting PTD-like activity; they are capable of efficient
translocation across cell membranes in an energy-independent and
endocytosis-independent manner without cell type specificity.
[0069] The Antennapedia protein (Antp) is a trans-activating factor
capable of translocation across cell membranes; the minimal
sequence capable of translocation is a 16 amino acid peptide
corresponding to the third helix of the protein's homeodomain (HD).
The internalization of this helix occurs at 4.degree. C.,
suggesting that this process is not endocytosis dependent. Peptides
of up to 100 amino acids produced as fusion proteins with AntpHD
penetrate cell membranes.
[0070] Other homeodomains capable of translocation include Fushi
tarazu (Ftz) and Engrailed (En) homeodomain. Many homeodomains
share a highly conserved third helix.
Human PTDs
[0071] Human PTDs may circumvent potential immunogenicity issues
upon introduction into a human patient. Peptides with PTD sequences
include: Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX,
MOX-2, and FtzPTD. These proteins all share the sequence found in
AntpPTD. Other PTDs include Islet-1, Interleukin-1 (IL-1), Tumor
Necrosis Factor (TNF), and the hydrophobic sequence from
Kaposi-fibroblast growth factor or Fibroblast Growth Factor-4
(FGF-4) signal peptide, which is capable of energy-, receptor-, and
endocytosis-independent translocation. Unconfirmed PTDs include
members of the Fibroblast Growth Factor (FGF) family. FGFs are
polypeptide growth factors that regulate proliferation and
differentiation of a wide variety of cells. Several publications
have reported that basic fibroblast growth factor (FGF-2) exhibits
an unconventional internalization similar to that of VP-22, Tat,
and homeodomains. It has also been reported that acidic FGF (FGF-1)
translocated cell membranes at temperatures as low as 4.degree. C.
However, no conclusive evidence exists about the domain responsible
for internalization or the translocation properties of fusion
proteins (Beerens, A. et al., Curr Gene Ther., 3(5):486-494,
2003).
Synthetic PTDs
[0072] Several peptides have been synthesized in an attempt to
create more potent PTDs and to elucidate the mechanisms by which
PTDs transport proteins across cell membranes. Many of these
synthetic PTDs are based on existing and well documented peptides,
while others are selected for their basic residues and/or positive
charges, which are thought to be crucial for PTD function. A few of
these synthetic PTDs showed better translocation properties than
the existing ones (Beerens, A. et al., Curr Gene Ther.,
3(5):486-494, 2003). Exemplary Tat-derived synthetic PTDs include,
for example, but are not limited to, WLRRIKAWLRRIKA (SEQ ID NO:
12); WLRRIKA (SEQ ID NO: 13); YGRKKRRQRRR (SEQ ID NO: 14);
WLRRIKAWLRRI (SEQ ID NO: 15); FAKLAARLYR (SEQ ID NO: 16);
KAFAKLAARLYR (SEQ ID NO: 17); and HRRIKAWLKKI (SEQ ID NO: 18).
Compositions Comprising PTDs Fused to MK2 Inhibitor Peptide
Therapeutic Domains (TD)
[0073] Several MK2 inhibitor peptides (TD) have been synthesized,
fused to synthetic PTDs and the use of compositions comprising
these fused polypeptides has been studied. These polypeptides
include, but are not limited to, YARAAARQARAKALARQLGVAA (SEQ ID NO:
1; MMI-0100), YARAAARQARAKALNRQLGVA (SEQ ID NO: 19; MMI-0200),
FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3; MMI-0300),
KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4; MMI-0400),
HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7; MMI-0500),
YARAAARDARAKALNRQLAVAA (SEQ ID NO: 23; MMI-0600), and
YARAAARQARAKALNRQLAVA (SEQ ID NO: 24; MMI-0600-2). Both in vitro
and in vivo studies have shown that these polypeptides can be
useful in the treatment of various diseases, disorders and
conditions. These include, without limitation, hyperplasia and
neoplasm (U.S. Pat. Nos. 8,536,303 and 8,741,849) inflammatory
disorders (U.S. application Ser. No. 12/634,476 and U.S.
application Ser. No. 13/934,933), adhesions (U.S. application Ser.
No. 12/582,516), failure of a vascular graft due to neospasm (U.S.
application Ser. No. 13/114,872), improving neurite outgrowth (U.S.
application Ser. No. 12/844,815), a cutaneous scar (U.S.
application Ser. No. 13/829,876), failure of a coronary artery
bypass vascular graft (U.S. application Ser. No. 13/700,087) and
interstitial lung disease and pulmonary fibrosis (U.S. application
Ser. No. 13/445,759).
[0074] Peptide compositions present a number of particular
challenges to formulation scientists (R. W. Payne and M. C.
Manning, "Peptide formulation: challenges and strategies,"
Innovations in Pharmaceutical Technology, 64-68 (2009)). First,
since peptides do not have a globular structure that can sequester
reactive groups, the side chains of nearly all residues in a
peptide are fully solvent exposed, and can exhibit chemical
degradation through hydrolytic reactions, for example, oxidation
and deamidation. Second, the conformation in aqueous solution may
have little similarity to the structure found when bound to a
receptor. Third, many peptides tend to be monomeric at very low
concentration, but may self-assemble as the concentration is
increased and behave as if in a highly associated state, but these
structures are too transient or fluxional to provide any increase
in long-term stability. Fourth, the propensity of peptides to
self-associate is connected with their physical instablity, meaning
their likelihood of forming aggregates. Moreover, excipients
present in a peptide formulation can chemically degrade, interact
with various surfaces during manufacturing, interact with the
container or closure, or interact with the peptide itself, thereby
negatively affecting critical properties of the preparation (Lars
Hovgaard, and Sven Frokjaer, "Pharmaceutical Formulation
Development of Peptides and Proteins, 2.sup.nd Ed., CRC Press
(2012) pp. 212-213).
[0075] The described invention provides effective formulations
comprising a cell-penetrating peptide fused to a peptide-based
inhibitor of MK2.
SUMMARY OF THE INVENTION
[0076] According to one aspect, the described invention provides a
pharmaceutical formulation comprising a therapeutic amount of a
polypeptide of amino acid sequence YARAAARQARAKALARQLGVAA; SEQ ID
NO: 1 or a functional equivalent thereof, wherein the formulation
is characterized by preservation of stability and bioavailability
of the polypeptide.
[0077] According to one embodiment, the pharmaceutical formulation
is a particulate pharmaceutical formulation. According to another
embodiment, the pharmaceutical formulation is an aerosolized
pharmaceutical formulation. According to another embodiment, the
formulation is prepared by a process of spray drying. According to
another embodiment, the pharmaceutical formulation comprises 1% w/w
solids. According to another embodiment, the pharmaceutical
formulation comprises 5% w/w solids. According to another
embodiment, the pharmaceutical formulation further comprises
trehalose. According to another embodiment, the polypeptide of
amino acid sequence YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or the
functional equivalent thereof and the trehalose are in a ratio of
80/20 respectively. According to another embodiment, the MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or the functional equivalent
thereof and the trehalose are in a ratio of 92.5/7.5 respectively.
According to another embodiment, the pharmaceutical formulation is
delivered to a subject via a dry powder inhalation device
(DPI).
[0078] According to one embodiment, the pharmaceutical formulation
further comprises saline. According to another embodiment, the the
saline is NaCl. According to another embodiment, the polypeptide of
amino acid sequence YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or the
functional equivalent thereof is at a concentration of 0.7 mg/mL.
According to another embodiment, the polypeptide of amino acid
sequence YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or the functional
equivalent thereof is at a concentration of 7.0 mg/mL. According to
another embodiment, the pharmaceutical formulation is delivered to
a subject via a nebulizer.
[0079] According to one embodiment, the pharmaceutical formulation
comprises an ionic complex of a polypeptide of amino acid sequence
YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or a functional equivalent
thereof and a nano-polyplex polymer, the ionic complex being
characterized by dissociation of the ionic complex in intracellular
compartments selected by intracellular pH conditions such that
bioactivity and stability of the peptide is preserved.
[0080] According to another aspect, the described invention
provides a method for treating a vascular graft-induced intimal
hyperplasia in a subject in need of such treatment, the method
comprising administering the pharmaceutical formulation comprising
an ionic complex of a polypeptide of amino acid sequence
YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or a functional equivalent
thereof and a nano-polyplex polymer, the ionic complex being
characterized by dissociation of the ionic complex in intracellular
compartments selected by intracellular pH conditions such that
bioactivity and stability of the peptide is preserved, comprising a
therapeutic amount of a polypeptide of amino sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent
thereof, and a nano-polyplex polymer, wherein the therapeutic
amount is effective to inhibit MK2; and to treat a vascular
graft-induced intimal hyperplasia.
[0081] According to one embodiment, the nano-polyplex polymer is
anionic and endosomolytic. According to another embodiment, the
nano-polyplex polymer is poly(propylacrylic acid) (PPAA). According
to another embodiment, the nano-polyplex polymer is poly(acrylic
acid) (PAA). According to another embodiment, the pharmaceutical
formulation comprises a charge ratio (CR) of the polypeptide of
amino acid sequence YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or a
functional equivalent thereof to PPAA selected from the group
consisting of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1,
1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10. According
to another embodiment, the the charge ratio (CR) is 1:3. According
to another embodiment, the pharmaceutical formulation is delivered
to a subject via an implantation device. According to another
embodiment, the pharmaceutical formulation is delivered to a
subject topically. According to another embodiment, the
pharmaceutical formulation is delivered to a subject
parenterally.
[0082] According to one embodiment, the functional equivalent is
made from a fusion between a first polypeptide that is a protein
transduction domain (PTD) and a second polypeptide that is a
therapeutic domain (TD). According to another embodiment, the
protein transduction domain (PTD) is selected from the group
consisting of a polypeptide of amino acid sequence YARAAARQARA (SEQ
ID NO: 11), FAKLAARLYR (SEQ ID NO: 16), and KAFAKLAARLYR (SEQ ID
NO: 17), and a second polypeptide that is a therapeutic domain (TD)
of amino acid sequence KALARQLGVAA (SEQ ID NO: 2).
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0084] FIG. 1 shows the technical characteristics of the blister
lidding-push through.
[0085] FIG. 2 shows the technical characteristics of the
Formpack.RTM.-4PLY.
[0086] FIG. 3 shows a dynamic vapor sorption isotherm for a
MMI-0100 5% solids formulation.
[0087] FIG. 4 shows a chromatogram of an MMI-0100 working
standard.
[0088] FIG. 5 shows an EPIC inhaler device. On the left is an
assembled device (base unit with attached flow channel). The
inhaler is tethered to an external drive box (pictured on the
right) which contains the electronics.
[0089] FIG. 6 shows a particle size distribution plot of initial
aerosol performance results for a MMI-0100 5% formulation at 1 mg
and 2 mg.
[0090] FIG. 7 shows a particle size distribution plot of fill
weights up to 10 mg for MMI-0100 1% solids formulation (after
optimization).
[0091] FIG. 8 shows a linearity plot of fine particle dose (FPD)
from 5 to 10 mg of MMI-0100 1% solids formulation.
[0092] FIG. 9 shows a particle size distribution plot of
MMI-0100/Trehalose variant formulations.
[0093] FIG. 10 shows a particle size distribution plot of MMI-0100
1% solids formulation after 4 weeks storage in blisters at
40.degree. C./75% RH.
[0094] FIG. 11 shows a particle size distribution plot of recovered
drug at 40.degree. C./75% relative humidity (RH) for the MMI-0100
1% solids formulation.
[0095] FIG. 12 shows a particle size distribution plot of recovered
drug at 25.degree. C./60% RH for the MMI-0100 1% solids
formulation.
[0096] FIG. 13 shows a particle size distribution plot of recovered
drug at 4 weeks for the MMI-0100 1% solids formulation.
[0097] FIG. 14 shows a particle size distribution plot of recovered
drug at 40.degree. C./75% relative humidity (RH) for the MMI-0100
5% solids formulation.
[0098] FIG. 15 shows a particle size distribution plot of recovered
drug at 25.degree. C./60% RH for the MMI-0100 5% solids
formulation.
[0099] FIG. 16 shows a particle size distribution plot of recovered
drug at 4 weeks for the MMI-0100 5% solids formulation.
[0100] FIG. 17 shows a particle size distribution plot of recovered
drug at 40.degree. C./75% relative humidity (RH) for the MMI-0100
1% solids, 7.5% Trehalose formulation.
[0101] FIG. 18 shows a particle size distribution plot of recovered
drug at 25.degree. C./60% RH for the MMI-0100 1% solids, 7.5%
Trehalose formulation.
[0102] FIG. 19 shows a particle size distribution plot of recovered
drug at 4 weeks for the MMI-0100 1% solids, 7.5% Trehalose
formulation.
[0103] FIG. 20 shows a particle size distribution plot of recovered
drug at 40.degree. C./75% relative humidity (RH) for the MMI-0100
1% solids, 20% Trehalose formulation.
[0104] FIG. 21 shows a particle size distribution plot of recovered
drug at 25.degree. C./60% RH for the MMI-0100 1% solids, 20%
Trehalose formulation.
[0105] FIG. 22 shows a particle size distribution plot of recovered
drug at 4 weeks for the MMI-0100 1% solids, 20% Trehalose
formulation.
[0106] FIG. 23 shows a chromatogram of the sample solvent.
[0107] FIG. 24 shows a chromatogram of the limit of quantitation
(LOQ).
[0108] FIG. 25 shows a chromatogram of the 11 .mu.g/mL working
standard (full scale).
[0109] FIG. 26 shows a chromatogram of the 11 .mu.g/mL working
standard (expanded scale).
[0110] FIG. 27 shows a schematic of a laser diffraction device.
[0111] FIG. 28 shows a bar graph representing percent recovery of
MMI-0100 after extraction times of 0.5, 1, 2, 3 and 4 hours.
[0112] FIG. 29 shows the linear correlation between the filled drug
amount and the delivered dose (DD) (respirable dose <5 .mu.m)
nebulized using Nebulizer Type 1.
[0113] FIG. 30 shows the linear correlation between the filled drug
amount and the delivered dose (DD) (respirable dose <5 .mu.m)
nebulized using Nebulizer Type 2.
[0114] FIG. 31 shows a bar graph representing nebulization time of
different fill volumes and concentrations nebulized using Nebulizer
Type 1 and Nebulizer Type 2.
[0115] FIG. 32 shows a bar graph representing delivered dose of
different fill volumes and concentrations nebulized using Nebulizer
Type 1 and Nebulizer Type 2.
[0116] FIG. 33 shows a bar graph representing respirable dose <5
.mu.m of different fill volumes and concentrations nebulized using
Nebulizer Type 1 and Nebulizer Type 2.
[0117] FIG. 34 shows a schematic of the p38-MK2 pathway.
[0118] FIG. 35 shows MMI-0100 (MK2i)-NP synthesis and
characterization. a) MK2i-NP synthesis scheme. b) MK2i-NPs were
designed and optimized to mediate endosome escape and release
peptide therapeutics intracellularly. c) Treatment comparison
summary: MK2i-NPs were formulated with an endosomolytic PPAA
polymer whereas the NE-MK2i-NPs were formulated with a PAA polymer
that is structurally similar to PPAA but is not endosomolytic due
to its lower pKa. Both the MK2i-NPs and NE-MK2i-NPs are made with
the MK2i peptide with the sequence shown (red=modified TAT mimetic
cell penetrating peptide sequence, green=MK2 inhibitory sequence).
d) Zeta potential of polyplexes prepared at different charge ratios
([NH3+]/[COO-]). For imaging and uptake studies, Alexa NPs were
formulated from MK2i peptide labeled with an Alexa-488 fluorophore.
NE-NPs are formulated with a non-endosomolytic (NE) PAA polymer.
Values shown are an average of at least 3 independent measurements.
e) MK2i-NPs undergo pH-triggered disassembly in the endosomal pH
range as demonstrated by DLS analysis.
[0119] FIG. 36 shows MMI-0100 (MK2i)-NP formulations increase
cellular uptake, extend intracellular retention, and reduce
endo-lysosomal colocalization of MK2i. a) Flow cytometric
quantification of cellular uptake and retention of fluorescently
labeled MMI-0100 (MK2i), MK2i-NPs, and NE-MK2i-NPs. n=3. b)
Representative flow histograms demonstrate increased cellular
uptake and longer intracellular retention of fluorescently labeled
MK2i peptide delivered via MK2i-NPs. c) Red blood cell hemolysis
assay shows that MK2i-NPs have similar pH-dependent membrane
disruptive activity to the PPAA polymer while membrane disruption
of NE-MK2i-NPs and the MK2i peptide is negligible in the range
tested. d) Representative confocal microscopy images of Alexa-488
labeled MK2i colocalization with Lysotracker red 24 hours after 2
hours of treatment demonstrate that MK2i-NPs have reduced
endo-lysosomal colocalization. Scale bars=20 .mu.m. e)
Quantification of MK2i peptide colocalization with the
endolysosomal dye Lysotracker red 0, 12, and 24 hours after
treatment, n.gtoreq.3 independent images.
[0120] FIG. 37 shows ex vivo treatment with MK2-NPs reduces reduces
neointima formation and alters phosphorylation of molecules
downstream of MK2 in human saphenous vein. a) MK2i-NP formulation
increased delivery of Alexa 568-MK2i to HSV tissue ex vivo, scale
bars=200 .mu.m. b) Representative microscopy images of Verhoeff
Van-Gieson (VVG) stained human saphenous vein sections that were
treated for 2 hours and maintained in organ culture for 14 days.
MK2i-NPs potently blocked neointima formation. Red bars demarcate
intimal thickness. Scale bars=100 .mu.m. c) Quantification of
intimal thickness from VVG stained histological sections;
measurements are average of 6-12 radially parallel measurements
from at least 3 vein rings from separate donors. d) Representative
western blots showing the phosphorylation of MK2 substrates hnRNP
A0, CREB, and HSP27. e-g) Quantification of western blot analysis
from n.gtoreq.3 separate donors demonstrating that MK2i-NPs
enhanced MK2i mediated inhibition of several factors activated
downstream of MK2 that are implicated in migration and
inflammation.
[0121] FIG. 38 shows MMI-0100 (MK2i)-NP formulation enhances
MMI-0100 (MK2i) bioactivity in HCAVSMCs. a) MK2i-NP treatment
blocked TNF.alpha. production in HCAVSMCs stimulated with ANG II.
All data is normalized to cell number (data shown in supplementary
FIG. 11). NT=no treatment, n=4. b) MK2i-NP treatment blocked
migration in human coronary artery vascular smooth muscle cells
(HCAVSMCs) stimulated with the chemoattractant PDGF-BB (50 ng/mL)
24 hours after formation of a scratch wound, n=3. c) MK2i-NPs
inhibited cell migration towards the chemoattractant PDGF-BB in a
Boyden Chamber assay 8 hours after seeding onto the membrane, n=7.
d) Representative microscopy images of stained transwell insert
membranes for each treatment group.
[0122] FIG. 39 shows intraoperative treatment with MMI-0100
(MK2i)-NPs reduces neointima formation and macrophage persistence
in vivo in transplanted vein grafts. a) MK2i-NP treatment reduced
neointima formation as shown in representative images of Verhoeff
Van Gieson stained histological sections of vein grafts. b)
Quantification of intimal thickness in perfusion fixed jugular vein
interposition grafts 28 days post-op. n.gtoreq.7 grafts per
treatment group. c) MK2i-NP treatment also reduced persistence of
macrophages in the neointima as shown using RAM-11
immunohistochemsitry on vein grafts. Arrows demarcate positively
stained cells. Left column scale bar=100 .mu.m, right column zoomed
view scale bar=50 .mu.m. d) Quantification of RAM-11 positive
macrophage staining in jugular vein graft sections, n=16
histological images from 4 vein segments per treatment group.
[0123] FIG. 40 shows electrospray-ionization mass spectrometry
(ESI-MS) mass spectrum for the HPLC-purified CPP-MMI-0100 (MK2i)
fusion peptide (YARAAARQARAKALARQLGVAA (SEQ ID NO: 1), MW=2283.67
g/mol). The mass spectrum shows three major peaks each
corresponding to the fragmentation of the full peptide
sequence.
[0124] FIG. 41 shows .sup.1H NMR spectrum of A) poly(acrylic acid)
(PAA) and B) poly(propylacrylic acid) (PPAA) homopolymer in
D.sub.6MSO. Molecular weight was determined by comparing the area
of peaks associated with the chain transfer agent (i.e. peaks c,d
for PAA and peak b for PPAA) to peaks associated acrylic
acid/propylacrylic acid (i.e. peak a for PAA and peak c for PPAA):
PAA degree of polymerization=106, PPAA degree of
polymerization=190.
[0125] FIG. 42 shows gel permeation chromatography (GPC)
chromatograms of A) poly(acrylic acid) (PAA): degree of
polymerization=150, PDI=1.27, d.eta./dC=0.09 (mL/g) and B)
poly(propylacrylic acid) (PPAA): degree of polymerization=193,
PDI=1.471, d.eta./dC=0.087 (mL/g) polymers in DMF. The trace shows
UV absorbance at the characteristic absorption peak of the
trithiocarbonate moiety (310 nm) present in the
4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylvpentanoic acid (ECT)
chain transfer agent utilized in the polymerization.
[0126] FIG. 43 shows A) Dynamic light scattering analysis and B)
representative TEM images of uranyl acetate counterstained MMI-0100
(MK2i)-NPs. Scale bar=100 nm.
[0127] FIG. 44 shows a bar graph representing a full data set for
pH-dependent red blood cell membrane disruption. Red blood cell
hemolysis assay shows that MMI-0100 (MK2i)-NPs have similar
pH-dependent and dose-dependent membrane disruptive activity to the
PPAA polymer but NE-MK2i-NPs and the MK2i peptide alone do not.
[0128] FIG. 45 shows a bar graph representing average size of
intracellular compartments containing MMI-0100 (MK2i) 24 hours
after treatment with different peptide formulations. Compartment
area was quantified with ImageJ software. *p<0.001 vs. MK2 and
NE-MK2i-NPs, n=50 vesicles from at least 3 different images.
[0129] FIG. 46 shows a bar graph representing a full dose response
data set of intimal thickness measurements of human saphenous vein
(HSV) explants treated for 2 hours and then maintained in organ
culture for 14 days, n.gtoreq.3 from at least 3 different donors.
*p.ltoreq.0.01 compared to no treatment control (NT),
**p.ltoreq.0.001 compared to NT, p.ltoreq.0.05.
[0130] FIG. 47 shows a bar graph representing tissue viability in
HSV rings treated for 2 hours and maintained in organ culture for 1
or 14 days as assessed through an MTT assay. n.gtoreq.3 vein rings
from at least 3 separate donors.
[0131] FIG. 48 shows a bar graph representing TNF.alpha. production
in HCAVSMCs stimulated with ANG II for 6 hours, treated for two
hours with MMI-0100 (MK2i)-NPs, NE-MK2i-NPs, or the MMI-0100 (MK2i)
peptide alone and cultured for 24 hours in fresh media. All data is
normalized to cell number. NT=no treatment. *p<0.05 compared to
NT+TNF.alpha. group, p<0.05 compared to MK2i at the same
concentration, #p<0.05 compared to NE-MK2i-NPs at the same
concentration, n=4.
[0132] FIG. 49 shows a bar graph representing MMI-0100 (MK2i)-NPs
partially block TNF.alpha.-induced increase in IL-6 production in
HCAVSMCs. Cells were stimulated with TNF.alpha. for 6 hours,
treated for two hours with MK2i-NPs or MMI-0100 (MK2i) peptide
alone, and cultured for 24 hours in fresh media. All data is
normalized to cell number. NT=no treatment. *p<0.05 compared to
NT+TNF.alpha. group, p<0.05 compared to MK2i at the same
concentration, n=4.
[0133] FIG. 50 shows a bar graph representing cell viability in
HCAVSMCs stimulated with 10 .mu.M ANG II for 6 hours, treated for
two hours with MMI-0100 (MK2i)-NPs, NE-MK2i-NPs, or the MMI-0100
(MK2i) peptide alone and cultured for 24 hours in fresh media.
NT=no treatment, n=4.
[0134] FIG. 51 shows a bar graph representing cell viability in
HCAVSMCs stimulated with TNF.alpha. for 6 hours, treated for two
hours with MMI-0100 (MK2i)-NPs or MMI-0100 (MK2i) peptide alone,
and cultured for 24 hours in fresh media. n=4.
[0135] FIG. 52 shows a bar graph representing cell proliferation in
HCAVSMCs stimulated treated for 30 minutes with MMI-0100 (MK2i)
peptide alone, MK2i-NPs, or NE-MK2i-NPs and cultured for 24 hours
in fresh media with (+) or without (-) 50 ng/mL PDGF-BB. NT=no
treatment, n=4.
[0136] FIG. 53 shows representative RAM-11 staining images of
rabbit jugular vein graft explants for each treatment group. Arrows
demarcate positively stained cells. Left column scale bar=100
.mu.m, right column zoomed view scale bar=50 .mu.m.
[0137] FIG. 54 shows (A) Flow cytometric quantification of HCAVSMC
uptake and retention of fluorescently labeled MK2i, MK2i-NPs, and
NE-MK2i-NPs. Data are means.+-.SEM (n=3). P values determined by
single factor ANOVA. (B) Quantification of intracellular MK2i
half-life (t1/2) by exponential decay nonlinear regression analysis
of intracellular peptide fluorescence 0 and 5 days following
treatment removal. (C and D) Longitudinal quantification (C) and
representative flow histograms and subsets (D) used to calculate
the percentage of HCAVSMCs positive for MK2i internalization
following removal of treatment with free MK2i, MK2i-NPs, or
NE-MK2i-NPs. Data are means.+-.SEM (n=3). * P<0.01, **
P<0.001 vs. MK2i; P<0.01, P<0.001 vs. NE-MK2i-NPs; single
factor ANOVA.
[0138] FIG. 55 shows (A and B) Flow cytometric quantification (A)
and representative flow histograms (B) of endothelial cell uptake
of fluorescently labeled MK2i, MK2i-NPs, and NE-MK2i-NPs. Data are
means.+-.SEM (n=3). P values determined by single factor ANOVA. (C)
Quantification and representative images of endothelial cell
migration immediately after treatment removal determined by Boyden
transwell migration assay. (D) Quantification of MK2i-treated VSMC
migration in the presence of the chemoattractant PDGF-BB. Migration
was determined by calculating percent wound closure 24 hours after
scratch wound application in vitro. (C and D) Data are means.+-.SEM
(n=3). P values determined by single factor ANOVA.
[0139] FIG. 56 shows bar graphs representing MK2i-NP and MK2i
treatment effects on vascular smooth muscle and endothelial
monocyte chemoattractant protein-1 (MCP-1) production over time.
Quantification of MCP-1 production over time relative to untreated
controls in both (A) vascular smooth muscle cells (VSMCs) and (B)
endothelial cells (ECs). Cells were treated for 2 hours and then
cultured in fresh medium after MK2i treatment removal. After 3 or 5
days cells were stimulated with 20 ng/ml TNF.alpha. for 24 hours
and supernatants were collected for cytokine analysis. All
treatments used a 10 .mu.M dose of MK2i. Data are means.+-.SEM
(n=4). P values determined by single factor ANOVA.
[0140] FIG. 57 shows a bar graph representing MK2i-NP
internalization. MK2i-NP internalization is not affected by
membrane bound NPs as shown by minimal differences in MK2i-NP
uptake in vascular smooth muscle cells (VSMCs) that either had
extracellular fluorescence quenched by trypan blue and/or were
extensively washed with cell scrub buffer to remove any
extracellular NPs following treatment removal.
[0141] FIG. 58 shows (A) Quantification of MK2i peptide
colocalization with the endolysosomal dye Lysotracker red 0, 12,
and 24 hours after treatment, n.gtoreq.3 independent images; (B)
average size of intracellular compartments containing MK2i 24 hours
after treatment with different peptide formulations. Compartment
area was quantified with ImageJ software. n=50 vesicles from at
least 3 different images.
[0142] FIG. 59 shows (a) immunofluorescence microscopy images of
human saphenous vein cross sections treated with Alexa-568 labeled
MK2i, MK2i-NPs, or NE-MK2i-NPs (red) and stained for the vascular
smooth muscle marker .alpha.-smooth muscle actin (green) showing
MK2i-NP colocalization with .alpha.-smooth muscle actin; (b) zoomed
insets from images in (a); (c) zoomed immunofluorescence microscopy
images of human saphenous vein treated with Alexa-568 labeled MK2i,
MK2i-NPs, or NE-MK2i-NPs (red) and stained for the endothelial
marker CD31 (green) demonstrating MK2i colocalization with
endothelial cells; (d) zoomed insets showing MK2i penetration into
the vessel wall for all treatment groups; (e) pixel intensity
distribution of the images shown in (a) demonstrating increased
MK2i uptake (red channel) in vessels treated with MK2i-NPs.
[0143] FIG. 60 shows (a-b) immunofluorescence microscopy images of
human saphenous vein treated with Alexa-568 labeled MK2i, MK2i-NPs,
or NE-MK2i-NPs (red) and stained for the vascular smooth muscle
cell marker .alpha.-smooth muscle actin (green) showing MK2i-NP
colocalization with .alpha.-smooth muscle actin; (c)
immunofluorescence microscopy of demonstrating increased uptake and
penetration of MK2i-NPs into the vessel wall relative to the MK2i
and NE-MK2i-NP treated vessels.
[0144] FIG. 61 shows (a) MK2i-NPs inhibited vascular smooth muscle
cell migration towards the chemoattractant PDGF-BB in a Boyden
Chamber assay 8 hours after seeding onto the membrane. NT=no
treatment; (b) MK2i-NPs inhibited endothelial cell migration
towards the chemoattractant VEGF in a Boyden Chamber assay 8 hours
after seeding onto the membrane; (c) MK2i-NP treatment blocked
TNF.alpha. production in HCAVSMCs stimulated with ANG II (all data
is normalized to cell number); (d) MK2i-NP treatment showed
sustained inhibition of TNF.alpha. stimulated production of MCP-1
in both vascular smooth muscle and endothelial cells whereas
treatment with free MK2i or NE-MK2i-NPs did not; (e) MK2i-NPs
showed sustained inhibition of vascular smooth muscle cell
migration towards the chemoattractant PDGF-BB 5 days after
treatment removal.
[0145] FIG. 62 shows (a) MK2i-NP treatment reduced neointima
formation as shown in representative images of Verhoeff Van Gieson
stained histological sections of vein grafts; (b) quantification of
intimal thickness in perfusion fixed jugular vein interposition
grafts 28 days post-op. n.gtoreq.7 grafts per treatment group; (c)
MK2i-NP treatment reduced proliferation of intimal cells as shown
using ki67 immunohistochemistry on vein grafts; (d) quantification
of ki67 positive nuclear staining in jugular vein graft sections
normalized to intimal nuclei number; (e) MK2i-NP treatment
maintained higher intimal expression of the contractile marker
.alpha.-smooth muscle actin; (f) quantification of intimal
.alpha.-smooth muscle actin positive staining in jugular vein graft
sections normalized to intimal nuclei number; (g) MK2i-NP treatment
reduced intimal expression of the synthetic vascular smooth muscle
phenotypic marker vimentin; (h) quantification of intimal vimentin
positive staining in jugular vein graft sections normalized to
intimal nuclei number.
[0146] FIG. 63 shows (a) representative RAM-11 staining images of
rabbit jugular vein graft explants for each treatment group. Arrows
demarcate positively stained cells. Left column scale bar=100
.mu.m, right column zoomed view scale bar=50 .mu.m; (b) example
images from the color deconvolution method utilized to quantify
positive RAM-11 staining in the intima of rabbit jugular vein
explants; (c) quantification of intimal RAM-11 positive macrophage
staining in jugular vein graft sections, n=16 histological images
from 4 vein segments per treatment group.
[0147] FIG. 64 shows electrospray-ionization mass spectrometry
(ESI-MS) mass spectra for the HPLC-purified (A) MK2i peptide
(sequence: YARAAARQARA-KALARQLGVAA, MW=2283.7 g/mol) and (B)
p-HSP20 peptide (sequence: YARAAARQARA-WLRRAsAPLPGLK, MW=2731
g/mol). The mass spectra show three major peaks each corresponding
to the fragmentation of the full peptide sequence.
[0148] FIG. 65 shows (A) Z-average diameter (bars) and zeta
potential (circles) of MK2i-NPs prepared at a different charge
ratios (CR=[NH.sub.3.sup.+].sub.MK2i: [COO.sup.-].sub.PPAA).
Asterisks (*) denote a unimodal size distribution and the white bar
represents the MK2i-NP formulation that yielded a unimodal size
distribution with minimal size and polydispersity; (B)
representative DLS trace of lead MK2i-NP formulation (CR=1:3); (C)
representative TEM image of uranyl acetate stained MK2i-NPs, scale
bar=200 nm; (D) synthesis and characterization summary for lead
MK2i-NP formulation. CR=charge ratio, D.sub.h=hydrodynamic
diameter, .zeta.=zeta potential.
[0149] FIG. 66 shows A) Z-average diameter (bars) and zeta
potential (circles) of p-HSP20-NPs prepared at a different charge
ratios (CR=[NH.sub.3.sup.+].sub.p-HSP20: [COO.sup.-].sub.PPAA).
Asterisks (*) denote a unimodal size distribution, and the white
bar represents the p-HSP20-NP formulation that yielded a unimodal
size distribution with minimal size and polydispersity; (B)
Representative DLS trace of lead p-HSP20-NP formulation (CR=3:1);
(C) Representative TEM image of uranyl acetate stained p-HSP20-NPs,
scale bar=200 nm (D) Synthesis and characterization summary for
lead p-HSP20-NP formulation. CR=charge ratio, D.sub.h=hydrodynamic
diameter, .zeta.=zeta potential.
[0150] FIG. 67 shows a bar graph representing NP cytocompatibility.
The cytotoxicity of MK2i-NPs and p-HSP20-NPs was compared to the
corresponding dose of free peptide in HCAVSMCs. Cells were treated
for 2 hours and then allowed to incubate in fresh medium for 24
hours prior to running the cytotoxicity assay. *p<0.05 vs. NT,
n=4 mean.+-.SEM.
[0151] FIG. 68 shows NP uptake and retention. Flow cytometric
quantification of peptide uptake and retention of (A) MK2i-NPs vs.
MK2i and (B) p-HSP20-NPs vs. HSP20 at a 10 .mu.M dose of peptide
after 30 minutes of treatment. MK2i-NPs achieved .about.70 fold
increase in peptide uptake at the same concentration whereas
p-HSP20-NPs achieved a .about.35 fold increase in uptake; (C,D)
representative flow histograms of HCAVSMCs immediately after
treatment and (E,F) representative flow histograms demonstrating
that formulation into NPs increased peptide cellular retention
after 3 days of culture in fresh medium post-treatment. The
percentages overwritten on A-B represent the % retention at 3 days
relative to 0 days post-treatment.
[0152] FIG. 69 shows NP Endosomal Escape and Cytosolic Peptide
Delivery. (A) Experimental design for separation of vascular smooth
muscle cell cytosol and intracellular organelles using digitonin
semi-permeabilization. Conditions for semi-permeabilization were
optimized as shown in FIG. 70; (B) Western blot validation of the
optimized digitonin semi-permeabilization procedure confirmed
separation of the cytosolic proteins mitogen-activated protein
kinase kinase 1/2 (MEK1/2) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) from the endo-lysosomal markers early
endosomal antigen 1 (EEA1) and lysosomal-associated protein 1
(LAMP1); (C and D) comparison of the intracellular distribution of
(C) MK2i and (D) p-HSP20 peptides when delivered alone or
formulated into nano-polyplexes demonstrating increased cytosolic
delivery of the NP formulations. Significant inhibition of NP
mediated cytosolic peptide delivery when the endosomal
acidification inhibitor bafilomycin was added verified the
pH-dependent endosomal escape mechanism of the NPs.
[0153] FIG. 70 shows a bar graph representing Digitonin
semi-permeabilization optimization. The conditions for the
digitonin semi-permeabilization procedure to separate cytosolic
components from intracellular organelles (i.e., endo-lysosomal
compartments) were optimized based upon LDH release following 10
minutes of incubation with various concentrations of digitonin at
0.degree. C. on rotary shaker operating at 100 RPM. 25 ug/mL
digitonin was chosen as the optimal condition as no significant
increase in release of cytosolic LDH was seen at higher
concentrations.
[0154] FIG. 71 shows inhibition of F-actin stress fiber formation
in vascular smooth muscle cells. (A) F-actin stress fiber
quantification in HCAVSMCs pre-treated with p-HSP20-NPs, free
p-HSP20 peptide, MK2i-NPs or free MK2i for 1 hour and then
stimulated with ANG II for 2 hours. The number of stress fibers per
cell was calculated from three intensity profiles taken from the
axis transverse to the cellular polarity from n.gtoreq.36 ROIs from
n.gtoreq.12 different cells for each treatment group, *p<0.05,
**p<0.01, ***p<0.001 vs. NT+ANG II; p<0.1, p<0.01,
p<0.001 vs. the free peptide at the same concentration; (B)
representative fluorescence microscopy images of F-actin stress
fiber formation in ANG II-stimulated HCAVSMCs and the corresponding
intensity profile derived from the line shown in the image. The
asterisk denotes the left side of the intensity profile shown. Gain
settings were kept constant for all images obtained.
[0155] FIG. 72 shows inhibition of F-actin stress fiber formation
by MK2i-NPs. (A) F-actin stress fiber quantification in HCAVSMCs
pre-treated with MK2i-NPs or free MK2i for 1 hour and then
stimulated with ANG II for 2 hours. Data represents n.gtoreq.12
cells from 2 separate experiments: *p<0.05 vs. NT+ANG II
**p<0.001 vs. NT+ANG II, p<0.05 vs. MK2i at same
concentration; (B) representative fluorescence microscopy images of
F-actin stress fiber formation in ANG II-stimulated HCAVSMCs after
1 hour treatment with free MK2i or MK2i-NPs.
[0156] FIG. 73 shows inhibition of F-actin stress fiber formation
by p-HSP20-NPs. (A) F-actin stress fiber quantification in HCAVSMCs
pre-treated with p-HSP20-NPs or free p-HSP20 for 1 hour and then
stimulated with ANG II for 2 hours. Data represents n.gtoreq.12
cells from 2 separate experiments: *p<0.05, **p<0.01,
***p<0.001 vs. NT+ANG II, p<0.05, p<0.001 vs p-HSP20 at
same concentration; (B) representative fluorescence microscopy
images of F-actin stress fiber formation in ANG II-stimulated
HCAVSMCs after 1 hour treatment with free p-HSP20 or
p-HSP-20-NPs.
[0157] FIG. 74 shows MK2i-NP & p-HSP20-NP treatment inhibits
vasoconstriction and enhances vasorelaxation. (A) Experimental
design for inhibition of contraction studies: HSV rings are
initially contracted with PE and then relaxed. After 2 hours of
treatment with NPs, free peptide, or control, post treatment
contraction is measured; (B) quantification of MK2i and MK2i-NP
mediated inhibition of contraction. PPAA polymer equivalent to the
highest dose of MK2i-NPs was included as a vehicle control; (C)
quantification of p-HSP20 and p-HSP20-NP mediated inhibition of
contraction; (D) experimental design for vasorelaxation studies:
HSV rings are initially contracted with PE and subsequently relaxed
with SNP. HSV rings are then treated for two hours with NPs, free
peptide, or control and then contracted and relaxed under the same
conditions to compare post-treatment to pre-treatment relaxation;
(E) quantification of MK2i and MK2i-NP enhanced vasorelaxation.
PPAA polymer equivalent to the highest dose of MK2i-NPs was
included as a vehicle control; (F) quantification of p-HSP20 and
p-HSP20-NP enhanced vasorelaxation. For B,C,E,F: p<0.05;
*p<0.05, **p<0.01, ***p<0.01 vs. NT, n.gtoreq.3 separate
donors; (G) F-actin visualization in Alexa-488 phalloidin stained
cryosections of human saphenous vein explants obtained from a
single donor (n=1) pretreated with 100 .mu.M MK2i or MK2i-NPs, 500
.mu.M p-HSP20 or p-HSP20-NPs and subsequently stimulated with
ANG-II enabling visualization of decreased F-actin in samples
treated with the NP formulations.
[0158] FIG. 75 shows a schematic of the Mechanisms of action of
MAPKAP Kinase 2 (MK2) and Heat Shock Protein 20 (HSP20) in actin
mediated vasconstriction and vasorelaxation. MK2 is activated by
cellular stress (e.g. mechanical trauma, cytokines, oxidative
stress, etc.) through p38 MAPK. Phosphorylated MK2 activates a
number of downstream effectors: 1) phosphorylation of heat shock
protein 27 (HSP27) results in capping of filamentous actin thereby
inhibiting actin depolymerization and vasorelaxation. 2)
phosphorylation of Lim Kinase (LIMK) results in phosphorylation and
deactivation of cofilin which prevents actin degradation and
inhibits vasorelaxation. The MK2 inhibitory peptide (MK2i) binds to
MK2 preventing the activation of these downstream effectors and
promoting vasorelaxation. HSP20 is phosphorylated by cyclic
nucleotide-dependent protein kinases (PKA and PKG) resulting in
binding to and displacement of phosphorylated coflin from the
14-3-3 protein. This displacement allows for cofilin to be
dephosphorylated by phosphatases such as slingshot, resulting in
the activation of cofilin and concomitant cofilin-mediated
depolymerization of filamentous actin. The phospho-HSP20 peptide
mimetic (p-HSP20) recapitulates the activity of phosphorylated
HSP20, ultimately leading to vasorelaxation.
[0159] FIG. 76 shows a schematic of the mechanism of endosomolytic
nano-polyplex cytosolic peptide delivery.
[0160] FIG. 77 shows HPLC chromatograms of diluent (A) and MMI-0100
standard at 1 mg/mL (B).
[0161] FIG. 78 shows a linearity plot of MMI-0100 concentration
(mg/mL) versus Peak Area.
[0162] FIG. 79 is summary of MMI-0100 assay recovery and impurity
growth at 25.degree. C. (A) pH versus percent (%) recovery at 7
days and 14 days; (B) pH versus rate (percent (%) impurity growth
per day) at 7 days and 14 days.
[0163] FIG. 80 is summary of MMI-0100 assay recovery and impurity
growth at 40.degree. C. (A) pH versus percent (%) recovery at 1
day, 2 days, 7 days and 14 days; (B) pH versus rate (percent (%)
impurity growth per day) at 1 day, 2 days, 7 days and 14 days.
[0164] FIG. 81 is summary of MMI-0100 assay recovery and impurity
growth at 60.degree. C. [0165] (A) pH versus percent (%) recovery
at 1 day, 2 days and 7 days; (B) pH versus rate (percent (%)
impurity growth per day) at 1 day, 2 days and 7 days.
DETAILED DESCRIPTION OF THE INVENTION
Glossary
[0166] The term "active" as used herein refers to the ingredient,
component or constituent of the compositions of the present
invention responsible for the intended therapeutic effect. The term
"active ingredient" ("AI", "active pharmaceutical ingredient",
"API", or "bulk active") is the substance in a drug that is
pharmaceutically active. As used herein, the phrase "additional
active ingredient" refers to an agent, other than a compound of the
described composition, that exerts a pharmacological, or any other
beneficial activity.
[0167] The term "Actual Label Claim (ALC)" as used herein refers to
the actual amount of drug substance present, based on the potency
of the formulation and the target fill weight; equal to [(potency,
in %)/100%].times.(target fill weight, in mg).times.(1,000
.mu.g/mg).
[0168] The term "actuation" as used herein refers to the act of
propelling; to put in motion or action.
[0169] The term "admixture" or "blend" as used herein generally
refers to a physical combination of two or more different
components.
[0170] The term "administer" or "administering" as used herein
means to give or to apply, and includes in vivo administration, as
well as administration directly to tissue ex vivo. Generally,
administration may be systemic, e.g., orally, buccally,
parenterally, topically, by inhalation or insufflation (i.e.,
through the mouth or through the nose), rectally in dosage unit
formulations containing conventional nontoxic pharmaceutically
acceptable carriers, adjuvants, and vehicles as desired, or locally
by means such as, but not limited to, injection, implantation,
grafting, topical application, or parenterally.
[0171] The term "agent" as used herein refers generally to
compounds that are contained in or on the long-acting formulation.
Agent may include an antibody or nucleic acid or an excipient or,
more generally, any additive in the long-acting formulation.
"Agent" includes a single such compound and is also intended to
include a plurality of such compounds.
[0172] The term "agonist" as used herein refers to a chemical
substance capable of activating a receptor to induce a
pharmacological response. Receptors can be activated or inactivated
by either endogenous or exogenous agonists and antagonists,
resulting in stimulating or inhibiting a biological response. A
physiological agonist is a substance that creates the same bodily
responses, but does not bind to the same receptor. An endogenous
agonist for a particular receptor is a compound naturally produced
by the body which binds to and activates that receptor. A
superagonist is a compound that is capable of producing a greater
maximal response than the endogenous agonist for the target
receptor, and thus has an efficiency greater than 100%. This does
not necessarily mean that it is more potent than the endogenous
agonist, but is rather a comparison of the maximum possible
response that can be produced inside a cell following receptor
binding. Full agonists bind and activate a receptor, displaying
full efficacy at that receptor. Partial agonists also bind and
activate a given receptor, but have only partial efficacy at the
receptor relative to a full agonist. An inverse agonist is an agent
which binds to the same receptor binding-site as an agonist for
that receptor and reverses constitutive activity of receptors.
Inverse agonists exert the opposite pharmacological effect of a
receptor agonist. An irreversible agonist is a type of agonist that
binds permanently to a receptor in such a manner that the receptor
is permanently activated. It is distinct from a mere agonist in
that the association of an agonist to a receptor is reversible,
whereas the binding of an irreversible agonist to a receptor is
believed to be irreversible. This causes the compound to produce a
brief burst of agonist activity, followed by desensitization and
internalization of the receptor, which with long-term treatment
produces an effect more like an antagonist. A selective agonist is
specific for one certain type of receptor.
[0173] The term "Andersen Cascade Impactor" (ACI) as used herein
refers to an impactor used for the testing of inhaled products.
Cascade impactors operate on the principle of inertial impaction.
Each stage of the impactor comprises a series of nozzles or jets
through which the sample laden air is drawn, directing any airborne
sample towards the surface of the collection plate for that
particular stage. Whether a particular particle impacts on that
stage is dependent on its aerodynamic diameter. Particles having
sufficient inertia will impact on that particular stage collection
plate, while smaller particles will remain entrained in the air
stream and pass to the next stage where the process is repeated.
The stages are normally assembled in a stack or row in order of
decreasing particle size. As the jets get smaller, the air velocity
increases such that smaller particles are collected. At the end of
the test, the particle mass relating to each stage is recovered
using a suitable solvent and then analysed usually using HPLC to
determine the amount of drug actually present.
[0174] The term "antagonist" as used herein refers to a substance
that interferes with the effects of another substance. Functional
or physiological antagonism occurs when two substances produce
opposite effects on the same physiological function. Chemical
antagonism or inactivation is a reaction between two substances to
neutralize their effects. Dispositional antagonism is the
alteration of the disposition of a substance (its absorption,
biotransformation, distribution, or excretion) so that less of the
agent reaches the target or its persistence there is reduced.
Antagonism at the receptor for a substance entails the blockade of
the effect of an antagonist with an appropriate antagonist that
competes for the same site.
[0175] The term "bioactive agent" as used herein refers to a
compound of interest contained in or on a pharmaceutical
formulation or dosage form that is used for pharmaceutical or
medicinal purposes to provide some form of therapeutic effect or
elicit some type of biologic response or activity. "Bioactive
agent" includes a single such agent and is also intended to include
a plurality of bioactive agents including, for example,
combinations of two or more bioactive agents.
[0176] The term "bioavailable" as used herein refers to the rate
and extent to which an active ingredient is absorbed from a drug
product and becomes available at the site of action.
[0177] The term "biocompatible" as used herein refers to a material
that is generally non-toxic to the recipient and does not possess
any significant untoward effects to the subject and, further, that
any metabolites or degradation products of the material are
non-toxic to the subject. Typically a substance that is
"biocompatible" causes no clinically relevant tissue irritation,
injury, toxic reaction, or immunological reaction to living
tissue.
[0178] The term "biodegradable" as used herein refers to a material
that will erode to soluble species or that will degrade under
physiologic conditions to smaller units or chemical species that
are, themselves, non-toxic (biocompatible) to the subject and
capable of being metabolized, eliminated, or excreted by the
subject.
[0179] The term "biomimetic" as used herein refers to materials,
substances, devices, processes, or systems that imitate or "mimic"
natural materials made by living organisms.
[0180] The term "blister" or "blister pack" as used herein refers
to a unit dose package commonly constructed from a formed cavity
containing one or more individual doses.
[0181] The term "% blister clearance" as used herein refers to the
percentage of powder emitted from the blister during actuation, in
%, equal to the [(Initial weight-Final Weight)/Fill
Weight]*100%.
[0182] The term "carrier" as used herein refers to a material that
does not cause significant irritation to an organism and does not
abrogate the biological activity and properties of the peptide of
the composition of the described invention. Carriers must be of
sufficiently high purity and of sufficiently low toxicity to render
them suitable for administration to the mammal being treated. The
carrier can be inert, or it can possess pharmaceutical benefits.
The terms "excipient", "carrier", or "vehicle" are used
interchangeably to refer to carrier materials suitable for
formulation and administration of pharmaceutically acceptable
compositions described herein. Carriers and vehicles useful herein
include any such materials know in the art which are nontoxic and
do not interact with other components.
[0183] The term "component" as used herein refers to a constituent
part, element or ingredient.
[0184] The term "composition" as used herein refers to a product of
the described invention that comprises all active and inert
ingredients.
[0185] The term "condition", as used herein, refers to a variety of
health states and is meant to include disorders or diseases caused
by any underlying mechanism or disorder, injury, and the promotion
of healthy tissues and organs.
[0186] The term "contact" and all its grammatical forms as used
herein refers to a state or condition of touching or of immediate
or local proximity.
[0187] The term "controlled release" as used herein refers to refer
to any drug-containing formulation in which the manner and profile
of drug release from the formulation are regulated. This refers to
immediate as well as non-immediate release formulations, with
non-immediate release formulations including, but not limited to,
sustained release and delayed release formulations.
[0188] The term "delayed release" as used herein in its
conventional sense refers to a formulation in which there is a time
delay between administration of the formulation and the release of
the therapeutic agent therefrom. "Delayed release" may or may not
involve gradual release of the therapeutic agent over an extended
period of time, and thus may or may not be "sustained release."
[0189] The term "Delivered Dose (DD)" as used herein refers to the
amount of drug substance recovered from, for example, the
extraction of the dose sampling apparatus (DSA), dose uniformity
sampling apparatus (DUSA), Andersen Cascade Impactor (ACI), or Next
Generation Pharmaceutical Impactor (NGI), in mg or .mu.g. It is
equivalent to the amount of drug substance ex-device (i.e., it does
not include the amount of drug substance retained in a blister
and/or flow channel).
[0190] The term"derived delivered dose (DDD)" as used herein refers
to the amount of drug ex-device obtained from impactor testing, as
opposed to the amount of drug ex-device obtained from Delivered
Dose Uniformity (DDU) testing.
[0191] The term "% Delivered Dose" as used herein refers to a
percentage of Actual Label Claim (ALC); equal to
(DD/ALC).times.100%.
[0192] The term "disease" or "disorder", as used herein, refers to
an impairment of health or a condition of abnormal functioning.
[0193] The term "disposed", as used herein, refers to being placed,
arranged or distributed in a particular fashion.
[0194] The term "drug" as used herein refers to a therapeutic agent
or any substance, other than food, used in the prevention,
diagnosis, alleviation, treatment, or cure of disease.
[0195] The term "dry powder inhaler" or "DPI" as used herein refers
to a device similar to a metered-dose inhaler, but where the drug
is in powder form. The patient exhales out a full breath, places
the lips around the mouthpiece, and then quickly breathes in the
powder. Dry powder inhalers do not require the timing and
coordination that are necessary with MDIs.
[0196] The term "effective amount" refers to the amount necessary
or sufficient to realize a desired biologic effect.
[0197] The term "excipient" is used herein to include any other
agent or compound that may be contained in a long-acting
formulation that is not the bioactive agent. As such, an excipient
should be pharmaceutically or biologically acceptable or relevant
(for example, an excipient should generally be non-toxic to the
subject). "Excipient" includes a single such compound and is also
intended to include a plurality of such compounds.
[0198] The term "fill weight" as used herein refers to the actual
amount of powder (e.g., in mg or .mu.g) weighed into each blister
before actuation.
[0199] The term "final weight" as used herein refers to the weight
of the sealed blister and powder after actuation.
[0200] The term "fine particle dose (FPD)" as used herein refers to
the amount of drug substance (e.g., in mg or ug) recovered below a
specified cut-off diameter of an impactor (e.g., ACI or NGI);
equivalent to respirable dose.
[0201] The term "fine particle fraction (actual) as used herein
refers to the FPD normalized to the theoretical amount of drug
present in the blister(s) closed; equal to (FPD/[(fill
weight).times.(potency)].times.100%.
[0202] The term "fine particle fraction (Nominal Label Claim) as
used herein refers to the FPD normalized to the NLC; equal to
[(FPD)/(NLC).times.100%].
[0203] The term "fine particle fraction (Delivered Dose) as used
herein refers to the FPD normalized to the DD; equal to
[(FPD)/(DD).times.100%].
[0204] The terms "formulation" as used herein refers to a mixture
prepared according to a specific procedure, formula or rule.
[0205] The terms "functional equivalent" or "functionally
equivalent" are used interchangeably herein to refer to substances,
molecules, polynucleotides, proteins, peptides, or polypeptides
having similar or identical effects or use. A polypeptide
functionally equivalent to polypeptide YARAAARQARAKALARQLGVAA (SEQ
ID NO: 1), for example, may have a biologic activity, e.g., an
inhibitory activity, kinetic parameters, salt inhibition, a
cofactor-dependent activity, and/or a functional unit size that is
substantially similar or identical to the expressed polypeptide of
SEQ ID NO: 1.
[0206] Examples of polypeptides functionally equivalent to
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited
to, a polypeptide of amino acid sequence FAKLAARLYRKALARQLGVAA (SEQ
ID NO: 3), a polypeptide of amino acid sequence
KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4), a polypeptide of amino acid
sequence YARAAARQARAKALARQLAVA (SEQ ID NO: 5), a polypeptide of
amino acid sequence YARAAARQARAKALARQLGVA (SEQ ID NO: 6), a
polypeptide of amino acid sequence HRRIKAWLKKIKALARQLGVAA (SEQ ID
NO: 7), a polypeptide of amino acid sequence YARAAARQARAKALNRQLGVA
(SEQ ID NO: 19), a polypeptide of amino acid sequence
YARAAARDARAKALNRQLAVAA (SEQ ID NO: 23) and a polypeptide of amino
acid sequence YARAAARQARAKALNRQLAVA (SEQ ID NO: 24).
[0207] The MMI-0100 peptide of amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) described in the present
invention comprises a fusion protein in which a protein
transduction domain (PTD; YARAAARQARA; SEQ ID NO: 11) is
operatively linked to a therapeutic domain (KALARQLGVAA; SEQ ID NO:
2) in order to enhance therapeutic efficacy.
[0208] Examples of polypeptides functionally equivalent to the
therapeutic domain (TD; KALARQLGVAA; SEQ ID NO: 2) of the
polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are
not limited to, a polypeptide of amino acid sequence KALARQLAVA
(SEQ ID NO: 8), a polypeptide of amino acid sequence KALARQLGVA
(SEQ ID NO: 9), a polypeptide of amino acid sequence KALARQLGVAA
(SEQ ID NO: 10), a polypeptide of amino acid sequence KALNRQLAVAA
(SEQ ID NO: 25) and a polypeptide of amino acid sequence KALNRQLAVA
(SEQ ID NO: 26).
[0209] Examples of polypeptides functionally equivalent to the
protein transduction domain (PTD; YARAAARQARA; SEQ ID NO: 11) of
the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but
are not limited to, a polypeptide of amino acid sequence
WLRRIKAWLRRIKA (SEQ ID NO: 12), a polypeptide of amino acid
sequence WLRRIKA (SEQ ID NO: 13), a polypeptide of amino acid
sequence YGRKKRRQRRR (SEQ ID NO: 14), a polypeptide of amino acid
sequence WLRRIKAWLRRI (SEQ ID NO: 15), a polypeptide of amino acid
sequence FAKLAARLYR (SEQ ID NO: 16), a polypeptide of amino acid
sequence KAFAKLAARLYR (SEQ ID NO: 17), and a polypeptide of amino
acid sequence HRRIKAWLKKI (SEQ ID NO: 18).
[0210] The term "gene delivery vehicle" as used herein refers to a
component that facilitates delivery to a cell of a coding sequence
for expression of a polypeptide in the cell. The gene delivery
vehicle can be any component or vehicle capable of accomplishing
the delivery of a gene or cDNA to a cell, for example, a liposome,
a virus particle, or an expression vector.
[0211] The term "Geometric Standard Deviation (GSD)" as used herein
refers to a dimensionless number equal to the ratio between the
mass median aerodynamic diameter (MMAD) and either 84% or 16% of
the diameter size distribution (e.g., MMAD=2 .mu.m; 84%=4 .mu.m;
GSD=4/2=2.0.) The MMAD, together with the GSD, describe the
particle size distribution.
[0212] The term "granulation" as used herein refers to a process
whereby small red, grain-like prominences form on a raw surface in
the process of healing.
[0213] The term "hydrophilic" as used herein refers to a material
or substance having an affinity for polar substances, such as
water. The term "lipophilic" as used herein refers to a material or
substance preferring or possessing an affinity for a non-polar
environment compared to a polar or aqueous environment.
[0214] The term "inhalation" as used herein refers to the act of
drawing in a medicated vapor with the breath.
[0215] The term "inhalation delivery device" as used herein refers
to any device that produces small droplets or an aerosol from a
liquid or dry powder aerosol formulation and is used for
administration through the mouth in order to achieve pulmonary
administration of a drug, e.g., in solution, powder, and the like.
Examples of an inhalation delivery device include, but are not
limited to, a nebulizer, a metered-dose inhaler, and a dry powder
inhaler (DPI).
[0216] The term "insufflation" as used herein refers to the act of
delivering air, a gas, or a powder under pressure to a cavity or
chamber of the body. For example, nasal insufflation relates to the
act of delivering air, a gas, or a powder under pressure through
the nose.
[0217] The terms "inhibiting", "inhibit" or "inhibition" are used
herein to refer to reducing the amount or rate of a process, to
stopping the process entirely, or to decreasing, limiting, or
blocking the action or function thereof. Inhibition may include a
reduction or decrease of the amount, rate, action function, or
process of a substance by at least 5%, at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, or at least 99%.
[0218] The term "inhibitor" as used herein refers to a second
molecule that binds to a first molecule thereby decreasing the
first molecule's activity. Enzyme inhibitors are molecules that
bind to enzymes thereby decreasing enzyme activity. The binding of
an inhibitor may stop substrate from entering the active site of
the enzyme and/or hinder the enzyme from catalyzing its reaction.
Inhibitor binding is either reversible or irreversible.
Irreversible inhibitors usually react with the enzyme and change it
chemically, for example, by modifying key amino acid residues
needed for enzymatic activity. In contrast, reversible inhibitors
bind non-covalently and produce different types of inhibition
depending on whether these inhibitors bind the enzyme, the
enzyme-substrate complex, or both. Enzyme inhibitors often are
evaluated by their specificity and potency.
[0219] The term "initial weight" as used herein refers to the
weight of the scaled blister and powder before actuation (e.g., in
mg).
[0220] The term "injury," as used herein, refers to damage or harm
to a structure or function of the body caused by an outside agent
or force, which may be physical or chemical.
[0221] The term "isolated" is used herein to refer to material,
such as, but not limited to, a nucleic acid, peptide, polypeptide,
or protein, which is: (1) substantially or essentially free from
components that normally accompany or interact with it as found in
its naturally occurring environment. The terms "substantially free"
or "essentially free" are used herein to refer to considerably or
significantly free of, or more than about 95% free of, or more than
about 99% free of. The isolated material optionally comprises
material not found with the material in its natural environment; or
(2) if the material is in its natural environment, the material has
been synthetically (non-naturally) altered by deliberate human
intervention to a composition and/or placed at a location in the
cell (e.g., genome or subcellular organelle) not native to a
material found in that environment. The alteration to yield the
synthetic material may be performed on the material within, or
removed, from its natural state.
[0222] The term "LPM" or "L/min" as used herein refers to liters
per minute.
[0223] The term "mass balance" as used herein refers to the total
amount of drug substance recovered from each component of an
extraction, including the amount left in, for example, the inhaler.
The mass balance can be expressed as a percentage of Actual Fill
Weight equal to [(Metered Dose)/(Actual Fill
Weight.times.Potency)].times.100%.
[0224] The term "Mass Median Aerodynamic Diameter (MMAD)" as used
herein refers to particle size distribution statistically, based on
the weight and size of the particle. For example, 50% of particles
by weight will be smaller than the median diameter (and 50% of
particles will be larger than the median diameter).
[0225] The term "metered dose" as used herein refers to the
delivery of a specific amount of a drug to a target. For example,
delivery of an aerosolized drug to the lungs.
[0226] The term "metered-dose inhaler", "MDI", or "puffer" as used
herein refers to a pressurized, hand-held device that uses
propellants to deliver a specific amount of medicine ("metered
dose") to the lungs of a patient. The term "propellant" as used
herein refers to a material that is used to expel a substance
usually by gas pressure through a convergent, divergent nozzle. The
pressure may be from a compressed gas, or a gas produced by a
chemical reaction. The exhaust material may be a gas, liquid,
plasma, or, before the chemical reaction, a solid, liquid or gel.
Propellants used in pressurized metered dose inhalers are liquified
gases, traditionally chlorofluorocarbons (CFCs) and increasingly
hydrofluoroalkanes (HFAs). Suitable propellants include, for
example, a chlorofluorocarbon (CFC), such as trichlorofluoromethane
(also referred to as propellant 11), dichlorodifluoromethane (also
referred to as propellant 12), and
1,2-dichloro-1,1,2,2-tetrafluoroethane (also referred to as
propellant 114), a hydrochlorofluorocarbon, a hydrofluorocarbon
(HFC), such as 1,1,1,2-tetrafluoroethane (also referred to as
propellant 134a, HFC-134a, or HFA-134a) and
1,1,1,2,3,3,3-heptafluoropropane (also referred to as propellant
227, HFC-227, or HFA-227), carbon dioxide, dimethyl ether, butane,
propane, or mixtures thereof. In other embodiments, the propellant
includes a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or mixtures thereof. In other embodiments, a
hydrofluorocarbon is used as the propellant. In other embodiments,
HFC-227 and/or HFC-134a are used as the propellant.
[0227] The term "MK2 kinase" or "MK2" as used herein refers to
mitogen-activated protein kinase-activated protein kinase 2 (also
referred to as "MAPKAPK2", "MAPKAP-K2", "MK2"), which is a member
of the serine/threonine (Ser/Thr) protein kinase family.
[0228] The terms "MMI-0100", "MMI-0100 peptide", "MMI-0100
polypeptide", "MK2 inhibitor", "MK2i", "MK2i peptide", "MK2i
polypeptide" and the like, are used interchangeably herein to refer
to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).
[0229] The term "nebulizer" as used herein refers to a device used
to administer liquid medication in the form of a mist inhaled into
the lungs.
[0230] The term "Nominal Label Claim (NLC)" as used herein refers
to the intended amount of drug substance present per actuation
based upon target potency and target blister fill weight.
[0231] The term "nucleic acid" is used herein to refer to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0232] The term "nucleotide" is used herein to refer to a chemical
compound that consists of a heterocyclic base, a sugar, and one or
more phosphate groups. In the most common nucleotides, the base is
a derivative of purine or pyrimidine, and the sugar is the pentose
deoxyribose or ribose. Nucleotides are the monomers of nucleic
acids, with three or more bonding together in order to form a
nucleic acid. Nucleotides are the structural units of RNA, DNA, and
several cofactors, including, but not limited to, CoA, FAD, DMN,
NAD, and NADP. Purines include adenine (A), and guanine (G);
pyrimidines include cytosine (C), thymine (T), and uracil (U).
[0233] The following terms are used herein to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity."
[0234] (a) The term "reference sequence" refers to a sequence used
as a basis for sequence comparison. A reference sequence may be a
subset or the entirety of a specified sequence; for example, as a
segment of a full-length cDNA or gene sequence, or the complete
cDNA or gene sequence.
[0235] (b) The term "comparison window" refers to a contiguous and
specified segment of a polynucleotide sequence, wherein the
polynucleotide sequence may be compared to a reference sequence and
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length, and optionally can be at least 30 contiguous
nucleotides in length, at least 40 contiguous nucleotides in
length, at least 50 contiguous nucleotides in length, at least 100
contiguous nucleotides in length, or longer. Those of skill in the
art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence, a
gap penalty typically is introduced and is subtracted from the
number of matches.
[0236] Methods of alignment of sequences for comparison are
well-known in the art. Optimal alignment of sequences for
comparison may be conducted by the local homology algorithm of
Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology
alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443
(1970); by the search for similarity method of Pearson and Lipman,
Proc. Natl. Acad. Sci. 85:2444 (1988); by computerized
implementations of these algorithms, including, but not limited to:
CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group (GCG), 575
Science Dr., Madison, Wis., USA; the CLUSTAL program is well
described by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and
Sharp, CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids
Research 16:10881-90 (1988); Huang, et al., Computer Applications
in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in
Molecular Biology 24:307-331 (1994). The BLAST family of programs,
which can be used for database similarity searches, includes:
BLASTN for nucleotide query sequences against nucleotide database
sequences; BLASTX for nucleotide query sequences against protein
database sequences; BLASTP for protein query sequences against
protein database sequences; TBLASTN for protein query sequences
against nucleotide database sequences; and TBLASTX for nucleotide
query sequences against nucleotide database sequences. See, Current
Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0237] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using the BLAST 2.0
suite of programs using default parameters. Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997). Software for performing
BLAST analyses is publicly available, e.g., through the National
Center for Biotechnology-Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits then
are extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always>0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a word length (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a word length (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915).
[0238] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. BLAST searches assume that proteins may be
modeled as random sequences. However, many real proteins comprise
regions of nonrandom sequences which may be homopolymeric tracts,
short-period repeats, or regions enriched in one or more amino
acids. Such low-complexity regions may be aligned between unrelated
proteins even though other regions of the protein are entirely
dissimilar A number of low-complexity filter programs may be
employed to reduce such low-complexity alignments. For example, the
SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU
(Claverie and States, Comput. Chem., 17:191-201 (1993))
low-complexity filters may be employed alone or in combination.
[0239] (c) The term "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences is used herein
to refer to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions that are not
identical often differ by conservative amino acid substitutions,
i.e., where amino acid residues are substituted for other amino
acid residues with similar chemical properties (e.g. charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well-known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988)
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0240] (d) The term "percentage of sequence identity" is used
herein mean the value determined by comparing two optimally aligned
sequences over a comparison window, wherein the portion of the
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison, and multiplying the result by 100 to yield
the percentage of sequence identity.
[0241] (e) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, at least 80% sequence identity, at
least 90% sequence identity and at least 95% sequence identity,
compared to a reference sequence using one of the alignment
programs described using standard parameters. One of skill will
recognize that these values may be adjusted appropriately to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, or at least 70%,
at least 80%, at least 90%, or at least 95%. Another indication
that nucleotide sequences are substantially identical is if two
molecules hybridize to each other under stringent conditions.
However, nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is that the polypeptide that the first nucleic acid
encodes is immunologically cross reactive with the polypeptide
encoded by the second nucleic acid.
[0242] The term "operatively linked" as used herein refers to a
linkage in which two or more protein domains or peptides are
ligated or combined via recombinant DNA technology or chemical
reaction such that each protein domain or polypeptide of the
resulting fusion peptide retains its original function. For
example, SEQ ID NO: 1 is constructed by operatively linking a
protein transduction domain (SEQ ID NO: 26) with a therapeutic
domain (SEQ ID NO: 2), thereby creating a fusion peptide that
possesses both the cell penetrating function of SEQ ID NO: 26 and
the MK2 kinase inhibitor function of SEQ ID NO: 2.
[0243] The term "particle" as used herein refers to an extremely
small constituent, e.g., a nanoparticle or microparticle) that may
contain in whole or in part at least one therapeutic agent as
described herein. The term "microparticle" is used herein to refer
generally to a variety of substantially structures having sizes
from about 10 nm to 2000 microns (2 millimeters) and includes a
microcapsule, microsphere, nanoparticle, nanocapsule, nanosphere as
well as particles, in general, that are less than about 2000
microns (2 millimeters). The particles may contain therapeutic
agent(s) in a core surrounded by a coating. Therapeutic agent(s)
also may be dispersed throughout the particles. Therapeutic
agent(s) also may be adsorbed into the particles. The particles may
be of any order release kinetics, including zero order release,
first order release, second order release, delayed release,
sustained release, immediate release, etc., and any combination
thereof. The particle may include, in addition to therapeutic
agent(s), any of those materials routinely used in the art of
pharmacy and medicine, including, but not limited to, erodible,
nonerodible, biodegradable, or nonbiodegradable material or
combinations thereof. The particles may be microcapsules that
contain the active agent in a solution or in a semi-solid state.
The particles may be of virtually any shape.
[0244] The term "pharmaceutically acceptable salt" means those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response and
the like and are commensurate with a reasonable benefit/risk
ratio.
[0245] The terms "pharmaceutical formulation" or "pharmaceutical
composition" as used herein refer to a formulation or a composition
that is employed to prevent, reduce in intensity, cure or otherwise
treat a target condition or disease.
[0246] The term "prevent" as used herein refers to the keeping,
hindering or averting of an event, act or action from happening,
occurring, or arising.
[0247] The term "prodrug" as used herein means a peptide or
derivative which is in an inactive form and which is converted to
an active form by biological conversion following administration to
a subject.
[0248] The term "recombinant" as used herein refers to a substance
produced by genetic engineering.
[0249] The term "reduce", "reduced", "to reduce" or "reducing" as
used herein refer to a diminution, a decrease, an attenuation or
abatement of the degree, intensity, extent, size, amount, density
or number.
[0250] The term "similar" is used interchangeably with the terms
analogous, comparable, or resembling, meaning having traits or
characteristics in common.
[0251] The term "stability" of a pharmaceutical product as used
herein refers to the capability of a particular formulation to
remain within its physical, chemical, microbiological, therapeutic
and toxicological specifications.
[0252] The term "susceptible" as used herein refers to a member of
a population at risk.
[0253] The terms "subject" or "individual" or "patient" are used
interchangeably to refer to a member of an animal species of
mammalian origin, including but not limited to, a mouse, a rat, a
cat, a goat, a sheep, a horse, a hamster, a ferret, a platypus, a
pig, a dog, a guinea pig, a rabbit and a primate, such as, for
example, a monkey, an ape, or a human.
[0254] The phrase "subject in need thereof" as used herein refers
to a patient that (i) will be administered a formulation containing
at least one therapeutic peptide agent, (ii) is receiving a
formulation containing at least one therapeutic peptide agent; or
(iii) has received a formulation containing at least one
therapeutic agent, unless the context and usage of the phrase
indicates otherwise.
[0255] The term "sustained release" (also referred to as "extended
release") is used herein in its conventional sense to refer to a
drug formulation that provides for gradual release of a therapeutic
agent over an extended period of time, and that preferably,
although not necessarily, results in substantially constant levels
of the agent over an extended time period.
[0256] The term "symptom" as used herein refers to a phenomenon
that arises from and accompanies a particular disease or disorder
and serves as an indication of it.
[0257] The term "syndrome," as used herein, refers to a pattern of
symptoms indicative of some disease or condition.
[0258] The term "therapeutic agent" as used herein refers to a
drug, molecule, nucleic acid, protein, composition or other
substance that provides a therapeutic effect. The terms
"therapeutic agent" and "active agent" are used
interchangeably.
[0259] The term "therapeutic component" as used herein refers to a
therapeutically effective dosage (i.e., dose and frequency of
administration) that eliminates, reduces, or prevents the
progression of a particular disease manifestation in a percentage
of a population. An example of a commonly used therapeutic
component is the ED.sub.50 which describes the dose in a particular
dosage that is therapeutically effective for a particular disease
manifestation in 50% of a population.
[0260] The term "therapeutic effect" as used herein refers to a
consequence of treatment, the results of which are judged to be
desirable and beneficial. A therapeutic effect may include,
directly or indirectly, the arrest, reduction, or elimination of a
disease manifestation. A therapeutic effect may also include,
directly or indirectly, the arrest reduction or elimination of the
progression of a disease manifestation.
[0261] The term "therapeutically effective amount" or an "amount
effective" of one or more of the active agents is an amount that is
sufficient to provide the intended benefit of treatment. An
effective amount of the active agents that can be employed ranges
from generally 0.1 mg/kg body weight and about 50 mg/kg body
weight. However, dosage levels are based on a variety of factors,
including the type of injury, the age, weight, sex, medical
condition of the patient, the severity of the condition, the route
of administration, and the particular active agent employed. Thus
the dosage regimen may vary widely, but can be determined routinely
by a surgeon using standard methods.
[0262] The term "treat" or "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
disease, condition, disorder or injury, substantially ameliorating
clinical or esthetical symptoms of a disease, condition, disorder
or injury, substantially preventing the appearance of clinical or
esthetical symptoms of a disease, condition, disorder or injury,
and protecting from harmful or annoying symptoms. The term "treat"
or "treating" as used herein further refers to accomplishing one or
more of the following: (a) reducing the severity of the disease,
condition, disorder or injury; (b) limiting development of symptoms
characteristic of the disease, condition, disorder or injury being
treated; (c) limiting worsening of symptoms characteristic of the
disease, condition, disorder or injury being treated; (d) limiting
recurrence of the disease, condition, disorder or injury in
patients that have previously had the disease, condition, disorder
or injury; and (e) limiting recurrence of symptoms in patients that
were previously symptomatic for the disease, condition, disorder or
injury.
[0263] The terms "variants", "mutants", and "derivatives" are used
herein to refer to nucleotide or polypeptide sequences with
substantial identity to a reference nucleotide or polypeptide
sequence. The differences in the sequences may be the result of
changes, either naturally or by design, in sequence or structure.
Natural changes may arise during the course of normal replication
or duplication in nature of the particular nucleic acid sequence.
Designed changes may be specifically designed and introduced into
the sequence for specific purposes. Such specific changes may be
made in vitro using a variety of mutagenesis techniques. Such
sequence variants generated specifically may be referred to as
"mutants" or "derivatives" of the original sequence.
[0264] A skilled artisan likewise can produce polypeptide variants
of polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) having single
or multiple amino acid substitutions, deletions, additions or
replacements, but functionally equivalent to SEQ ID NO: 1. These
variants may include inter alia: (a) variants in which one or more
amino acid residues are substituted with conservative or
non-conservative amino acids; (b) variants in which one or more
amino acids are added; (c) variants in which at least one amino
acid includes a substituent group; (d) variants in which amino acid
residues from one species are substituted for the corresponding
residue in another species, either at conserved or non-conserved
positions; and (d) variants in which a target protein is fused with
another peptide or polypeptide such as a fusion partner, a protein
tag or other chemical moiety, that may confer useful properties to
the target protein, for example, an epitope for an antibody. The
techniques for obtaining such variants, including, but not limited
to, genetic (suppressions, deletions, mutations, etc.), chemical,
and enzymatic techniques, are known to the skilled artisan. As used
herein, the term "mutation" refers to a change of the DNA sequence
within a gene or chromosome of an organism resulting in the
creation of a new character or trait not found in the parental
type, or the process by which such a change occurs in a chromosome,
either through an alteration in the nucleotide sequence of the DNA
coding for a gene or through a change in the physical arrangement
of a chromosome. Three mechanisms of mutation include substitution
(exchange of one base pair for another), addition (the insertion of
one or more bases into a sequence), and deletion (loss of one or
more base pairs).
[0265] The term "vehicle" as used herein refers to a substance that
facilitates the use of a drug or other material that is mixed with
it.
[0266] According to one embodiment, the described invention
provides a pharmaceutical formulation comprising an inhibitor of
MK2 kinase. According to another embodiment, the MK2 inhibitor is a
polypeptide. According to another embodiment, the polypeptide
includes, but is not limited to, MMI-0100 (YARAAARQARAKALARQLGVAA
(SEQ ID NO: 1)) or its functional equivalents.
[0267] According to one embodiment, the pharmaceutical formulation
comprises a neat spray dried dispersion comprising MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a functional equivalent
thereof, 5% w/w solids. According to another embodiment, the
pharmaceutical formulation comprises a neat spray dried dispersion
comprising MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a
functional equivalent thereof, 1% w/w solids. According to another
embodiment, the pharmaceutical formulation comprises a spray dried
dispersion comprising 80/20 MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ
ID NO: 1) or a functional equivalent thereof/trehalose. According
to another embodiment, the pharmaceutical formulation comprises a
spray dried dispersion comprising 92.5/7.5 MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a functional equivalent
thereof/trehalose.
[0268] A spray-dried dispersion (SDD) is a single-phase, amorphous
molecular dispersion of a drug in a polymer matrix. It is a solid
solution with a compound (e.g., drug) moleculary "dissolved" in a
solid matrix. SDDs are obtained by dissolving drug and polymer in
an organic solvent to obtain a solution and then spray-drying the
solution. The use of spray drying for pharmaceutical applications
results in amorphous dispersions with increased solubility of
Biopharmaceutics Classification System (BCS) class II (high
permeability, low solubility) and class IV (low permeability, low
solubility) drugs. Formulation and process conditions are selected
so that the solvent quickly evaporates from the droplets, thus
allowing insufficient time for phase separation or crystallization.
SDDs have demonstrated long-term stability and manufacturability.
For example, shelf lives of more than 2 years have been
consistently demonstrated with SDDs. Advantages of SDDs include,
but are not limited to, enhanced oral bioavailabilty of poorly
water-soluble compounds, delivery using traditional solid dosage
forms (e.g., tablets and capsules), a reproducible, controllable
and scalable manufacturing process and broad applicability to
structurally diverse insoluble compounds with a wide range of
physical properties.
[0269] According to one embodiment, the pharmaceutical formulation
comprises MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a
functional equivalent thereof and 0.9% NaCl (saline). According to
another embodiment, the pharmaceutical formulation comprises 7
mg/mL, 6 mg/mL, 5 mg/mL, 4 mg/mL, 3 mg/mL, 2 mg/mL, or 1 mg/mL
MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a functional
equivalent thereof. According to anther embodiment, the
pharmaceutical formulation comprises 0.9 mg/mL, 0.8 mg/mL, 0.7
mg/mL, 0.6 mg/mL, 0.5 mg/mL, 0.4 mg/mL, 0.3 mg/mL, 0.2 mg/mL, or
0.1 mg/mL MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a
functional equivalent thereof. According to another embodiment, the
formulation comparing MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO:
1) or a functional equivalent thereof is a liquid formulation.
According to another embodiment, the liquid formulation is
aerosolized.
[0270] According to one embodiment, the pharmaceutical formulation
comprises MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a
functional equivalent thereof and glycerin.
[0271] According to one embodiment, the pharmaceutical formulation
comprises MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a
functional equivalent thereof and a nano-polyplex polymer.
According to another embodiment, the nano-polyplex polymer is
poly(acrylic acid) (PAA). According to another embodiment, the
nano-polyplex polymer is poly(propylacrylic acid) (PPAA). According
to another embodiment, the pharmaceutical formulation comprises a
charge ratio (CR) of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
or a functional equivalent thereof to PPAA
([NH.sub.3.sup.+].sub.MK2i:[COO.sup.-].sub.PPAA) selected from the
group consisting of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1,
1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10.
According to another embodiment, the pharmaceutical formulation
comprises a charge ratio of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID
NO: 1) or a functional equivalent thereof to PPAA
([NH.sub.3.sup.+].sub.MK2i:[COO.sup.-].sub.PPAA) of 1:3.
[0272] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1)
has a substantial sequence identity to amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).
[0273] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1)
has at least 80 percent sequence identity to amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to another
embodiment, the functional equivalent of the polypeptide
YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1) has at least 90
percent sequence identity to amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to another
embodiment, the functional equivalent of the polypeptide
YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1) has at least 95
percent sequence identity to amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).
[0274] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1)
is a polypeptide of amino acid sequence YARAAARQARAKALNRQLGVA
(MMI-0200; SEQ ID NO: 19)
[0275] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1)
is a polypeptide of amino acid sequence FAKLAARLYRKALARQLGVAA
(MMI-0300; SEQ ID NO: 3).
[0276] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
polypeptide of amino acid sequence KAFAKLAARLYRKALARQLGVAA
(MMI-0400; SEQ ID NO: 4).
[0277] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
polypeptide of amino acid sequence YARAAARQARAKALARQLAVA (SEQ ID
NO: 5).
[0278] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
polypeptide of amino acid sequence YARAAARQARAKALARQLGVA (SEQ ID
NO: 6).
[0279] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
polypeptide of amino acid sequence HRRIKAWLKKIKALARQLGVAA
(MMI-0500; SEQ ID NO: 7).
[0280] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
polypeptide of amino acid sequence YARAAARQARAKALNRQLAVAA (MMI0600,
SEQ ID NO: 23)
[0281] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
polypeptide of amino acid sequence YARAAARQARAKALNRQLAVA
(MMI0600-2, SEQ ID NO: 24).
[0282] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
fusion peptide comprising a first polypeptide operatively linked to
a second polypeptide, wherein the first polypeptide is of amino
acid sequence YARAAARQARA (SEQ ID NO: 11), and the second
polypeptide comprises a therapeutic domain whose sequence has a
substantial identity to amino acid sequence KALARQLGVAA (SEQ ID NO:
2).
[0283] According to another embodiment, the second polypeptide has
at least 70 percent sequence identity to amino acid sequence
KALARQLGVAA (SEQ ID NO: 2), and the pharmaceutical formulation
inhibits the kinase activity of Mitogen-Activated Protein
Kinase-Activated Protein Kinase 2 (MK2). According to another
embodiment, the second polypeptide has at least 80 percent sequence
identity to amino acid sequence KALARQLGVAA (SEQ ID NO: 2), and the
pharmaceutical formulation inhibits the kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2).
According to another embodiment, the second polypeptide has at
least 90 percent sequence identity to amino acid sequence
KALARQLGVAA (SEQ ID NO: 2), and the pharmaceutical formulation
inhibits the kinase activity of Mitogen-Activated Protein
Kinase-Activated Protein Kinase 2 (MK2). According to another
embodiment, the second polypeptide has at least 95 percent sequence
identity to amino acid sequence KALARQLGVAA (SEQ ID NO: 2), and the
pharmaceutical formulation inhibits the kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2
(MK2).
[0284] According to another embodiment, the second polypeptide is a
polypeptide of amino acid sequence KALARQLAVA (SEQ ID NO: 8).
[0285] According to another embodiment, the second polypeptide is a
polypeptide of amino acid sequence KALARQLGVA (SEQ ID NO: 9).
[0286] According to another embodiment, the second polypeptide is a
polypeptide of amino acid sequence KALNRQLAVAA (SEQ ID NO: 25)
[0287] According to another embodiment, the second polypeptide is a
polypeptide of amino acid sequence KALNRQLAVA (SEQ ID NO: 26).
[0288] According to another embodiment, the second polypeptide is a
polypeptide of amino acid sequence KALARQLGVAA (SEQ ID NO: 10);
see, e.g., U.S. Published Application No. 2009-0196927, U.S.
Published Application No. 2009-0149389, and U.S. Published
Application No 2010-0158968, each of which is incorporated herein
by reference in its entirety.
[0289] According to another embodiment, the functional equivalent
of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a
fusion peptide comprising a first polypeptide operatively linked to
a second polypeptide, wherein the first polypeptide comprises a
protein transduction domain functionally equivalent to YARAAARQARA
(SEQ ID NO: 11), and the second polypeptide is of amino acid
sequence KALARQLGVAA (SEQ ID NO: 2).
[0290] According to another embodiment, the first polypeptide is a
polypeptide of amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO:
12).
[0291] According to another embodiment, first polypeptide is a
polypeptide of amino acid sequence WLRRIKA (SEQ ID NO: 13).
[0292] According to another embodiment, the first polypeptide is a
polypeptide of amino acid sequence YGRKKRRQRRR (SEQ ID NO: 14).
[0293] According to another embodiment, the first polypeptide is a
polypeptide of amino acid sequence WLRRIKAWLRRI (SEQ ID NO:
15).
[0294] According to another embodiment, the first polypeptide is a
polypeptide of amino acid sequence FAKLAARLYR (SEQ ID NO: 16).
[0295] According to another embodiment, the first polypeptide is a
polypeptide of amino acid sequence KAFAKLAARLYR (SEQ ID NO:
17).
[0296] According to another embodiment, the first polypeptide is a
polypeptide of amino acid sequence HRRIKAWLKKI (SEQ ID NO: 18).
[0297] According to some embodiments, in order to enhance drug
efficacy and to prevent accumulation of the polypeptide
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or its functional equivalent
in non-target tissues, the polypeptide of the present invention of
amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or its
functional equivalent can be linked or associated with a targeting
moiety, which directs the polypeptide to a specific cell type or
tissue. Examples of the targeting moiety include, but are not
limited to, (i) a ligand for a known or unknown receptor or (ii) a
compound, a peptide, or a monoclonal antibody that binds to a
specific molecular target, e.g., a peptide or carbohydrate,
expressed on the surface of a specific cell type.
[0298] According to some embodiments, the polypeptide of the
described invention is chemically synthesized. Such a synthetic
polypeptide, prepared using the well known techniques of solid
phase, liquid phase, or peptide condensation techniques, or any
combination thereof, may include natural and unnatural amino acids.
Amino acids used for peptide synthesis may be standard Boc
(N-.alpha.-amino protected N-.alpha.-t-butyloxycarbonyl) amino acid
resin with the standard deprotecting, neutralization, coupling and
wash protocols of the original solid phase procedure of Merrifield
(1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile
N-.alpha.-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino
acids first described by Carpino and Han (1972, J. Org. Chem.
37:3403-3409). Both Fmoc and Boc N-.alpha.-amino protected amino
acids can be obtained from Sigma, Cambridge Research Biochemical,
or other chemical companies familiar to those skilled in the art.
In addition, the polypeptide may be synthesized with other
N-.alpha.-protecting groups that are familiar to those skilled in
this art. Solid phase peptide synthesis may be accomplished by
techniques familiar to those in the art and provided, for example,
in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition,
Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int.
J. Pept. Protein Res. 35:161-214, or using automated synthesizers,
each incorporated by reference herein in its entirety.
[0299] According to some embodiments, the polypeptide of the
invention comprises D-amino acids (which are resistant to L-amino
acid-specific proteases in vivo), a combination of D- and L-amino
acids, and various "designer" amino acids (e.g., .beta.-methyl
amino acids, C-.alpha.-methyl amino acids, and N-.alpha.-methyl
amino acids, etc.) to convey special properties. Examples of
synthetic amino acid substitutions include ornithine for lysine,
and norleucine for leucine or isoleucine.
[0300] According to some embodiments, the polypeptide may be linked
to other compounds to promote an increased half-life in vivo, such
as polyethylene glycol or dextran. Such linkage can be covalent or
non-covalent as is understood by those of skill in the art.
According to some other embodiments, the polypeptide may be
encapsulated in a micelle, such as a micelle made of
poly(ethyleneglycol)-block-poly(polypropylenglycol) or
poly(ethyleneglycol)-block-polylactide. According to some other
embodiments, the polypeptide may be encapsulated in degradable
nano- or micro-particles composed of degradable polyesters
including, but not limited to, polylactic acid, polyglycolide, and
polycaprolactone.
[0301] According to one embodiment, the pharmaceutical formulation
of the described invention may be administered by an inhalation
device. Examples of the inhalation device that can be used for
administering the pharmaceutical formulation includes, but is not
limited to, a nebulizer, a metered-dose inhaler, a dry powder
inhaler and an aqueous droplet inhaler.
[0302] Nebulizers, which actively aerosolize a liquid formulation
and operate continuously once loaded, require either compressed air
or an electrical supply. Exemplary nebulizers include, a vibrating
mesh nebulizer, a jet nebulizer (also known as an atomizer) and an
ultrasonic wave nebulizer. Exemplary vibrating mesh nebulizers
include, but are not limited to, Respironics i-Neb, Omron MicroAir,
Beurer Nebulizer IH50 and Aerogen Aeroneb. Acorn-I, Acorn-II,
AquaTower, AVA-NEB, Cirrhus, Dart, DeVilbiss 646, Downdraft, Fan
Jet, MB-5, Misty Neb, Salter Labs 8900, Sidestream, Updraft-II, and
Whisper Jet are examples of a jet nebulizer. Exemplary ultrasonic
nebulizers include, but are not limited to, an Omron NE-U17
nebulizer and a Beurer Nebulizer IH30.
[0303] Metered-dose inhalers (MDI) use a propellant to deliver a
fixed volume of liquid solution or suspension to a patient in the
form of a spray.
[0304] Dry powder inhalers (DPI) contain an active drug mixed with
an excipient containing much larger particles (e.g., lactose) to
which the drug attaches. During aerosolization, the active drug is
stripped from the carrier and inhaled while the the carrier
particles impact on the mouth and throat and are ingested. DPIs
synchronize drug delivery with inhalation.
[0305] According to one embodiment, the polypeptide of the
described invention may be in the form of a dispersible dry powder
for delivery by inhalation or insufflation (either through the
mouth or through the nose, respectively). Dry powder compositions
may be prepared by processes known in the art, such as
lyophilization and jet milling, as disclosed in International
Patent Publication No. WO 91/16038 and as disclosed in U.S. Pat.
No. 6,921,527, the disclosures of which are incorporated by
reference. The composition of the described invention is placed
within a suitable dosage receptacle in an amount sufficient to
provide a subject with a unit dosage treatment. The dosage
receptacle is one that fits within a suitable inhalation device to
allow for the aerosolization of the dry powder composition by
dispersion into a gas stream to form an aerosol and then capturing
the aerosol so produced in a chamber having a mouthpiece attached
for subsequent inhalation by a subject in need of treatment. Such a
dosage receptacle includes any container enclosing the composition,
such as gelatin or plastic capsules, with a removable portion that
allows a stream of gas (e.g., air) to be directed into the
container to disperse the dry powder composition. Such containers
are exemplified by those shown in U.S. Pat. No. 4,227,522; U.S.
Pat. No. 4,192,309; and U.S. Pat. No. 4,105,027. Suitable
containers also include those used in conjunction with Glaxo's
Ventolin.RTM. Rotohaler brand powder inhaler or Fison's
Spinhaler.RTM. brand powder inhaler. Another suitable unit-dose
container which provides a superior moisture barrier is formed from
an aluminum foil plastic laminate. The pharmaceutical-based powder
is filled by weight or by volume into the depression in the
formable foil and hermetically sealed with a covering foil-plastic
laminate. Such a container for use with a powder inhalation device
is described in U.S. Pat. No. 4,778,054 and is used with Glaxo's
Diskhaler.RTM. (U.S. Pat. Nos. 4,627,432; 4,811,731; and
5,035,237). All of these references are incorporated herein by
reference in their entireties.
[0306] Aqueous droplet inhalers (ADI) deliver a pre-metered dose of
liquid formulation without using a propellant. ADIs actively
aerosolize liquid producing a soft mist of fine particles. Berodual
Respimat.RTM. (Boehringer Ingelheim Pharma Gmbh & Co.) is an
exemplary aqueous droplet inhaler.
[0307] According to one embodiment, the polypeptide of the
described invention may be in the form of a nebulization solution.
According to another embodiment, the nebulization formulation does
not contain mannitol. According to one embodiment, the nebulization
solution is delivered by a nebulizer.
[0308] According to another embodiment, the polypeptide may be
prepared in a solid form (including granules, powders or
suppositories) or in a liquid form (e.g., solutions, suspensions,
or emulsions).
[0309] According to another embodiment, the polypeptide of the
described invention may be in the form of a nano-polyplex.
According to one embodiment, the nan-polyplex polymer is anionic.
According to another embodiment, the nano-polyplex polymer is an
endosomolytic polymer. Exemplary nano-polyplex polymers include,
but are not limited to, chitosan, polyethyleneimine (PEI),
polyethylene oxide (PEO), poly(organophos-phazene), poly(acrylic
acid) (PAA) and poly(propylacrylic acid) (PPAA).
[0310] According to one embodiment, the formulation of the
described invention may be delivered by implanting a biomedical
device. The biomedical device includes, but is not limited to, a
graft. According to another embodiment, the formulation may be
disposed on or in the graft. According to another embodiment, the
graft includes, but is not limited to, a vascular graft. According
to another embodiment, the formulation may be delivered
parenterally. According to another embodiment, the formulation may
be delivered topically.
[0311] According to another embodiment, the formulation of the
described invention comprises a carrier. The carrier can include,
but is not limited to, a release agent, such as a sustained release
or delayed release carrier. According to such embodiments, the
carrier can be any material capable of sustained or delayed release
of the polypeptide to provide a more efficient administration,
e.g., resulting in less frequent and/or decreased dosage of the
polypeptide, improving ease of handling, and extending or delaying
effects on diseases, disorders, conditions, syndromes, and the
like. Non-limiting examples of such carriers include liposomes,
microsponges, microspheres, or microcapsules of natural and
synthetic polymers and the like. Liposomes may be formed from a
variety of phospholipids, including, but not limited to,
cholesterol, stearylamines or phosphatidylcholines.
[0312] According to another embodiment, the polypeptide of the
invention may be applied in a variety of solutions. A suitable
formulation is sterile, dissolves sufficient amounts of the
therapeutic polypeptide, preserves stability of the therapeutic
polypeptide, and is not harmful for the proposed application. For
example, the compositions of the described invention may be
formulated as aqueous suspensions wherein the active ingredient(s)
is (are) in admixture with excipients suitable for the manufacture
of aqueous suspensions.
[0313] Such excipients include, without limitation, suspending
agents (e.g., sodium carboxymethylcellulose, methylcellulose,
hydroxy-propylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, and gum acacia), dispersing
or wetting agents including, a naturally-occurring phosphatide
(e.g., lecithin), or condensation products of an alkylene oxide
with fatty acids (e.g., polyoxyethylene stearate), or condensation
products of ethylene oxide with long chain aliphatic alcohols
(e.g., heptadecaethyl-eneoxycetanol), or condensation products of
ethylene oxide with partial esters derived from fatty acids and a
hexitol (e.g., polyoxyethylene sorbitol monooleate), or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides (e.g., polyethylene
sorbitan monooleate).
[0314] Compositions of the described invention also may be
formulated as oily suspensions by suspending the active ingredient
in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or
coconut oil) or in a mineral oil (e.g., liquid paraffin). The oily
suspensions may contain a thickening agent (e.g., beeswax, hard
paraffin or cetyl alcohol).
[0315] Compositions of the described invention also may be
formulated in the form of dispersible powders and granules suitable
for preparation of an aqueous suspension by the addition of water.
The active ingredient in such powders and granules is provided in
admixture with a dispersing or wetting agent, suspending agent, and
one or more preservatives. Suitable dispersing or wetting agents
and suspending agents are exemplified by those already mentioned
above. Additional excipients also may be present.
[0316] Compositions of the described invention also may be in the
form of an emulsion. An emulsion is a two-phase system prepared by
combining two immiscible liquid carriers, one of which is disbursed
uniformly throughout the other and consists of globules that have
diameters equal to or greater than those of the largest colloidal
particles. The globule size is critical and must be such that the
system achieves maximum stability. Usually, separation of the two
phases will not occur unless a third substance, an emulsifying
agent, is incorporated. Thus, a basic emulsion contains at least
three components, the two immiscible liquid carriers and the
emulsifying agent, as well as the active ingredient. Most emulsions
incorporate an aqueous phase into a non-aqueous phase (or vice
versa). However, it is possible to prepare emulsions that are
basically non-aqueous, for example, anionic and cationic
surfactants of the non-aqueous immiscible system glycerin and olive
oil. Thus, the compositions of the invention may be in the form of
an oil-in-water emulsion. The oily phase may be a vegetable oil,
for example, olive oil or arachis oil, or a mineral oil, for
example a liquid paraffin, or a mixture thereof. Suitable
emulsifying agents may be naturally-occurring gums, for example,
gum acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the partial esters with
ethylene oxide, for example, polyoxyethylene sorbitan
monooleate.
[0317] According to some embodiments, pharmaceutical formulations
of the described invention are capable of inhibiting a kinase
activity of Mitogen-Activated Protein Kinase-Activated Protein
Kinase 2 (MK2). According to some embodiments, pharmaceutical
formulations of the described invention inhibit at least 50% of the
kinase activity of MK2 kinase. According to some embodiments,
pharmaceutical formulations of the described invention inhibit at
least 55% of the kinase activity of MK2 kinase. According to some
embodiments, pharmaceutical formulations of the described invention
inhibit at least 60% of the kinase activity of MK2 kinase.
According to some embodiments, pharmaceutical formulations or the
described invention inhibit at least 65% of the kinase activity of
MK2 kinase. According to some embodiments, pharmaceutical
formulations of the described invention inhibit at least 70% of the
kinase activity of MK2 kinase. According to some embodiments,
pharmaceutical formulations of the described invention inhibit at
least 75% of the kinase activity of MK2 kinase. According to some
embodiments, pharmaceutical formulations of the described invention
inhibit at least 80% of the kinase activity of MK2 kinase.
According to some embodiments, pharmaceutical formulations of the
described invention inhibit at least 85% of the kinase activity of
MK2 kinase. According to some embodiments, pharmaceutical
formulations of the described invention inhibit at least 90% of the
kinase activity of MK2 kinase. According to some embodiments,
pharmaceutical formulations of the described invention inhibit at
least 95% of the kinase activity of MK2 kinase.
[0318] According to another embodiment, the pharmaceutical
formulation is effective to inhibit a kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 3 (MK3).
According to some such embodiments, the pharmaceutical formulation
inhibits at least 50% of the kinase activity of MK3 kinase.
According to some such embodiments, the pharmaceutical formulation
inhibits at least 55% of the kinase activity of MK3 kinase.
According to some such embodiments, the pharmaceutical formulation
inhibits at least 60% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 65% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 70% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 75% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 80% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 85% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 90% of the kinase activity of MK3 kinase.
According to another embodiment, the pharmaceutical formulation
inhibits at least 95% of the kinase activity of MK3 kinase.
[0319] According to another embodiment, the pharmaceutical
formulation is effective to inhibit a kinase activity of
calcium/calmodulin-dependent protein kinase I (CaMKI). According to
some such embodiments, the pharmaceutical formulation further
inhibits at least 50% of the kinase activity of
Ca2+/calmodulin-dependent protein kinase I (CaMKI). According to
some such embodiments, the pharmaceutical formulation further
inhibits at least 55% of the kinase activity of
Ca2+/calmodulin-dependent protein kinase I (CaMKI). According to
some such embodiments, the pharmaceutical formulation further
inhibits at least 60% of the kinase activity of
Ca2+/calmodulin-dependent protein kinase I (CaMKI). According to
another embodiment, the pharmaceutical formulation further inhibits
at least 65% of the kinase activity of Ca2+/calmodulin-dependent
protein kinase I (CaMKI). According to another embodiment, the
pharmaceutical formulation further inhibits at least 70% of the
kinase activity of Ca2+/calmodulin-dependent protein kinase I
(CaMKI). According to another embodiment, the pharmaceutical
formulation further inhibits at least 75% of the kinase activity of
Ca2+/calmodulin-dependent protein kinase I (CaMKI). According to
another embodiment, the pharmaceutical formulation further inhibits
at least 80% of the kinase activity of Ca2+/calmodulin-dependent
protein kinase I (CaMKI). According to another embodiment, the
pharmaceutical formulation further inhibits at least 85% of the
kinase activity of Ca2+/calmodulin-dependent protein kinase I
(CaMKI). According to another embodiment, the pharmaceutical
formulation further inhibits at least 90% of the kinase activity of
Ca2+/calmodulin-dependent protein kinase I (CaMKI). According to
another embodiment, the pharmaceutical formulation further inhibits
at least 95% of the kinase activity of Ca2+/calmodulin-dependent
protein kinase I (CaMKI).
[0320] According to another embodiment, the pharmaceutical
formulation is capable of inhibiting a kinase activity of BDNF/NT-3
growth factors receptor (TrkB). According to some such embodiments,
the pharmaceutical further inhibits at least 50% of the kinase
activity of BDNF/NT-3 growth factors receptor (TrkB). According to
some such embodiments, the pharmaceutical further inhibits at least
55% of the kinase activity of BDNF/NT-3 growth factors receptor
(TrkB). According to some such embodiments, the pharmaceutical
further inhibits at least 60% of the kinase activity of BDNF/NT-3
growth factors receptor (TrkB). According to another embodiment,
the pharmaceutical further inhibits at least 65% of the kinase
activity of BDNF/NT-3 growth factors receptor (TrkB). According to
another embodiment, the pharmaceutical further inhibits at least
70% of the kinase activity of BDNF/NT-3 growth factors receptor
(TrkB). According to another embodiment, the pharmaceutical further
inhibits at least 75% of the kinase activity of BDNF/NT-3 growth
factors receptor (TrkB). According to another embodiment, the
pharmaceutical formulation inhibits at least 80% of the kinase
activity of BDNF/NT-3 growth factors receptor (TrkB). According to
another embodiment, the pharmaceutical formulation inhibits at
least 85% of the kinase activity of BDNF/NT-3 growth factors
receptor (TrkB). According to another embodiment, the
pharmaceutical formulation inhibits at least 90% of the kinase
activity of BDNF/NT-3 growth factors receptor (TrkB). According to
another embodiment, the pharmaceutical formulation inhibits at
least 95% of the kinase activity of BDNF/NT-3 growth factors
receptor (TrkB).
[0321] According to another embodiment, the pharmaceutical
formulation is effective to inhibit a kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2)
and a kinase activity of calcium/calmodulin-dependent protein
kinase I (CaMKI).
[0322] According to another embodiment, the pharmaceutical
formulation is effective to inhibit a kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2)
and a kinase activity of BDNF/NT-3 growth factors receptor
(TrkB).
[0323] According to another embodiment, the pharmaceutical
formulation is effective to inhibit a kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2),
a kinase activity of calcium/calmodulin-dependent protein kinase I
(CaMKI), and a kinase activity of BDNF/NT-3 growth factors receptor
(TrkB).
[0324] According to another embodiment, the pharmaceutical
formulation inhibits at least 65% of the kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2)
and at least 65% of the kinase activity of
calcium/calmodulin-dependent protein kinase I (CaMKI).
[0325] According to another embodiment, the pharmaceutical
formulation inhibits at least 65% of the kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2)
and at least 65% of the kinase activity of BDNF/NT-3 growth factors
receptor (TrkB).
[0326] According to another embodiment, the pharmaceutical
formulation inhibits at least 65% of the kinase activity of
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2),
at least 65% of the kinase activity of calcium/calmodulin-dependent
protein kinase I (CaMKI), and at least 65% of the kinase activity
of BDNF/NT-3 growth factors receptor (TrkB).
[0327] According to another embodiment, the pharmaceutical
formulation inhibits the kinase activity of at least one kinase
selected from the group of MK2, MK3, CaMKI, TrkB, without
substantially inhibiting the activity of one or more other selected
kinases from the remaining group listed in Table 1 herein.
TABLE-US-00001 TABLE 1 Kinase Profiling Assay MMI-0100 MMI-0200
MMI-0300 MMI-0400 MMI-0500 (SEQ ID NO: 1) (SEQ ID NO: 19) (SEQ ID
NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 7) (100 .mu.M) (100 .mu.M) (100
.mu.M) (100 .mu.M) (100 .mu.M) Abl(h) 136 107 69 84 16
Abl(H396P)(h) 130 121 101 105 51 Abl(M351T)(h) 128 119 90 121 61
Abl(Q252H)(h) 105 107 82 98 40 Abl(T315I)(h) 98 108 97 105 16
Abl(Y253F)(h) 104 102 86 78 29 ACK1(h) 106 97 104 95 64 ALK(h) 118
95 19 16 12 ALK4(h) 124 152 140 130 81 Arg(h) 89 82 72 84 22
AMPK.alpha.1(h) 107 108 71 87 35 AMPK.alpha.2(h) 121 88 54 58 9
ARKS(h) 108 93 78 69 20 ASK1(h) 100 101 80 69 -4 Aurora-A(h) 120
107 92 119 110 Aurora-B(h) 94 166 128 150 5 Axl(h) 81 99 52 41 12
Bmx(h) 62 76 N/D 26 45 BRK(h) 70 127 35 18 41 BrSK1(h) 100 93 67 76
72 BrSK2(h) 129 102 83 86 84 BTK(h) 112 100 102 94 18 BTK(R28H)(h)
91 104 74 24 10 CaMKI(h) 13 21 1 0 -1 CaMKII.beta.(h) 58 53 2 11 3
CaMKII.gamma.(h) 106 94 5 3 3 CaMKI.delta.(h) 59 47 10 17 0
CaMKII.delta.(h) 89 2 1 2 1 CaMKIV(h) 87 71 17 18 -1
CDK1/cyclinB(h) 96 115 73 74 57 CDK2/cyclinA(h) 97 114 86 92 87
CDK2/cyclinE(h) 106 112 94 83 19 CDK3/cyclinE(h) 106 104 94 92 8
CDK5/p25(h) 114 97 89 92 66 CDK5/p35(h) 94 92 79 76 59
CDK6/cyclinD3(h) 103 100 86 85 23 CDK7/cyclinH/MAT1(h) 89 67 65 47
15 CDK9/cyclin T1(h) 228 103 91 235 6 CHK1(h) 97 115 91 87 65
CHK2(h) 104 105 66 54 13 CHK2(I157T)(h) 97 85 43 41 3
CHK2(R145W)(h) 97 81 33 31 3 CK1.gamma.1(h) 110 98 111 116 109
CK1.gamma.2(h) 119 104 123 114 119 CK1.gamma.3(h) 105 96 125 115
114 CK1.delta.(h) 115 92 92 93 78 CK2(h) 90 83 90 101 93
CK2.alpha.2(h) 104 88 105 96 103 CLK2(h) 88 97 103 116 116 CLK3(h)
108 76 61 84 76 cKit(h) 95 110 53 43 45 cKit(D816V)(h) 117 118 60
35 30 cKit(D816H)(h) 79 106 126 143 194 cKit(V560G)(h) 94 115 102
124 198 cKit(V654A)(h) 69 113 134 150 223 CSK(h) 70 33 49 16 2
c-RAF(h) 97 115 107 102 19 cSRC(h) 70 32 26 14 30 DAPK1(h) 97 113
46 36 0 DAPK2(h) 41 92 32 16 3 DCAMKL2(h) 146 131 81 70 56 DDR2(h)
105 104 94 95 79 DMPK(h) 60 66 59 54 12 DRAK1(h) 47 34 14 14 8
DYRK2(h) 99 142 155 195 127 eEF-2K(h) 113 136 91 43 43 EGFR(h) 95
83 21 16 -1 EGFR(L858R)(h) 76 120 N/D 52 26 EGFR(L861Q)(h) 53 74 25
22 15 EGFR(T790M)(h) 106 113 100 106 70 EGFR(T790M,L858R)(h) 93 108
85 78 53 EphA1(h) 114 136 73 61 40 EphA2(h) 58 95 31 17 N/D
EphA3(h) 107 117 6 12 33 EphA4(h) 110 127 88 65 48 EphA5(h) 110 123
18 24 42 EphA7(h) 193 220 159 222 189 EphA8(h) 181 133 93 146 337
EphB2(h) 68 128 18 22 70 EphB1(h) 99 95 44 58 37 EphB3(h) 109 128
62 47 79 EphB4(h) 62 131 44 28 38 ErbB4(h) 73 82 40 0 2 FAK(h) 98
110 111 96 94 Fer(h) 117 101 130 108 196 Fes(h) 44 74 20 16 23
FGFR1(h) 120 97 55 59 18 FGFR1(V561M)(h) 108 72 74 74 113 FGFR2(h)
49 73 14 18 12 FGFR2(N549H)(h) 95 104 116 112 105 FGFR3(h) 73 208
102 0 10 FGFR4(h) 67 75 28 19 3 Fgr(h) 54 71 60 47 109 Flt1(h) 109
96 69 48 27 F1t3(D835Y)(h) 120 115 80 71 65 F1t3(h) 104 99 84 18 17
F1t4(h) 135 105 83 89 73 Fms(h) 89 92 45 37 14 Fms(Y969C)(h) 126 88
72 91 N/D Fyn(h) 71 75 74 54 83 GCK(h) 98 99 70 66 30 GRK5(h) 117
135 136 131 116 GRK6(h) 131 132 147 141 174 GRK7(h) 111 124 122 100
93 GSK3.alpha.(h) 183 119 157 164 175 GSK3.beta.(h) 113 132 205 202
238 Haspin(h) 127 71 48 36 25 Hck(h) 354 107 72 72 78 Hck(h)
activated 58 100 82 81 67 HIPK1(h) 94 115 74 91 47 HIPK2(h) 98 102
73 90 38 HIPK3(h) 105 105 93 105 85 IGF-1R(h) 102 49 119 90 117
IGF-1R(h), activated 126 94 80 77 45 IKK.alpha.(h) 108 104 93 87 50
IKK.beta.(h) 105 109 84 84 71 IR(h) 112 90 96 85 95 IR(h),
activated 127 105 79 59 90 IRR(h) 85 69 8 8 10 IRAK1(h) 97 101 95
93 5 IRAK4(h) 100 110 59 59 3
Itk(h) 99 98 77 63 7 JAK2(h) 89 131 133 119 49 JAK3(h) 150 117 121
122 95 JNK1.alpha.1(h) 91 106 97 98 109 JNK2.alpha.2(h) 114 109 98
96 81 JNK3(h) 104 90 89 70 171 KDR(h) 100 110 101 94 15 Lck(h) 346
113 -2 228 359 Lck(h) activated 106 90 243 216 76 LIMK1(h) 103 109
88 92 87 LKB1(h) 111 99 101 89 51 LOK(h) 37 67 37 18 7 Lyn(h) 113
98 69 3 31 MAPK1(h) 108 97 107 100 102 MAPK2(h) 98 105 98 93 60
MAPKAP-K2(h) 19 35 5 5 9 MAPKAP-K3(h) 27 39 3 7 9 MEK1(h) 86 116 77
77 21 MARK1(h) 109 102 132 120 110 MELK(h) 74 59 16 17 0 Mer(h) 47
90 52 50 17 Met(h) 104 71 65 62 27 Met(D1246H)(h) 99 139 125 68 150
Met(D1246N)(h) 114 149 82 31 90 Met(M1268T)(h) 114 143 255 265 239
Met(Y1248C)(h) 77 141 84 36 73 Met(Y1248D)(h) 87 118 102 31 218
Met(Y1248H)(h) 88 153 117 63 126 MINK(h) 96 103 48 52 5 MKK6(h) 74
98 48 44 18 MKK7.beta.(h) 137 117 100 94 102 MLCK(h) 85 103 2 1 0
MLK1(h) 77 84 40 33 43 Mnk2(h) 94 106 89 86 6 MRCK.alpha.(h) 98 103
104 97 5 MRCK.beta.(h) 103 102 83 71 -10 MSK1(h) 52 50 32 28 8
MSK2(h) 105 88 56 52 14 MSSK1(h) 82 100 77 75 22 MST1(h) 85 72 14 6
3 MST2(h) 98 104 19 11 2 MST3(h) 104 95 45 36 4 mTOR(h) 102 110 91
93 135 mTOR/FKBP12(h) 117 118 145 125 140 MuSK(h) 85 106 93 93 27
NEK2(h) 102 97 78 61 0 NEK3(h) 100 100 92 85 20 NEK6(h) 109 98 82
85 49 NEK7(h) 97 96 84 87 89 NEK11(h) 102 95 53 33 2 NLK(h) 100 106
87 90 19 p70S6K(h) 89 84 35 33 3 PAK2(h) 71 69 65 59 44 PAK4(h) 92
98 94 89 86 PAK3(h) N/D 50 140 121 102 PAK5(h) 97 100 110 117 125
PAK6(h) 121 105 104 100 107 PAR-1B.alpha.(h) 62 110 113 109 97
PASK(h) 81 60 29 28 9 PDGFR.alpha.(h) 104 108 65 40 40
PDGFR.alpha.(D842V)(h) 103 107 114 118 170 PDGFR.alpha.(V561D)(h)
58 106 82 100 146 PDGFR.beta.(h) 116 137 81 53 40 PDK1(h) 144 143
135 159 178 PhK.gamma.2(h) 62 86 46 38 16 Pim-1(h) 44 18 8 7 0
Pim-2(h) 117 74 76 92 46 Pim-3(h) 98 94 80 80 37 PKA(h) 138 110 119
119 118 PKB.alpha.(h) 140 110 57 67 30 PKB.beta.(h) 284 250 84 98
21 PKB.gamma.(h) 105 103 20 41 20 PKC.alpha.(h) 94 100 89 86 3
PKC.beta.I(h) 88 98 78 78 1 PKC.beta.II(h) 102 100 82 75 3
PKC.gamma.(h) 94 101 89 79 6 PKC.delta.(h) 100 101 101 90 61 PKC
(h) 102 98 79 59 23 PKC.eta.(h) 105 101 103 98 45 PKC(h) 110 97 68
46 7 PKC.mu.(h) 79 73 22 14 10 PKC.theta.(h) 102 101 88 76 62
PKC.zeta.(h) 82 98 81 75 7 PKD2(h) 84 78 33 25 10 PKG1.alpha.(h) 82
70 64 58 25 PKG1.beta.(h) 71 57 50 53 24 Plk1(h) 109 128 115 119
104 Plk3(h) 107 107 127 129 122 PRAK(h) 159 115 128 118 95 PRK2(h)
72 74 33 27 7 PrKX(h) 84 112 61 76 57 PTK5(h) 135 108 132 129 96
Pyk2(h) 113 127 47 34 46 Ret(h) 108 96 140 145 174 Ret(V804L)(h)
113 100 79 73 20 Ret(V804M)(h) 92 105 95 87 36 RIPK2(h) 92 98 97 98
30 ROCK-I(h) 99 117 79 73 17 ROCK-II(h) 102 85 74 77 2 Ron (h) 117
120 93 79 46 Ros(h) 107 86 95 99 150 Rse(h) 109 88 88 89 63 Rsk1(h)
86 102 46 54 34 Rsk2(h) 65 101 51 38 14 Rsk3(h) 76 109 76 71 23
Rsk4(h) 99 125 90 91 29 SAPK2a(h) 110 107 90 85 52 SAPK2a(T106M)(h)
101 100 97 99 32 SAPK2b(h) 99 95 81 82 42 SAPK3(h) 106 97 84 79 24
SAPK4(h) 98 106 96 91 48 SGK(h) 128 115 48 54 2 SGK2(h) 103 119 56
98 -1 SGK3(h) 95 58 10 8 -3
[0328] According to some embodiments, inhibitory profiles of
MMI-0100 (SEQ ID NO: 1) and its functional equivalents in vivo
depend on dosages, routes of administration, and cell types
responding to the inhibitors.
[0329] According to some embodiments, the pharmaceutical
formulation inhibits less than 65% of the kinase activity of the
other selected kinase(s). According to some embodiments, the
pharmaceutical formulation inhibits less than 60% of the kinase
activity of the other selected kinase(s). According to some
embodiments, the pharmaceutical formulation inhibits less than 55%
of the kinase activity of the other selected kinase(s). According
to another embodiment, the pharmaceutical formulation inhibits less
than 50% of the kinase activity of the other selected kinase(s).
According to some embodiments, the pharmaceutical formulation
inhibits less than 45% of the kinase activity of the other selected
kinase(s). According to another embodiment, the pharmaceutical
formulation inhibits less than 40% of the kinase activity of the
other selected kinase(s). According to some embodiments, the
pharmaceutical formulation inhibits less than 35% of the kinase
activity of the other selected kinase(s). According to some
embodiments, the pharmaceutical formulation inhibits less than 30%
of the kinase activity of the other selected kinase(s). According
to some embodiments, the pharmaceutical formulation inhibits less
than 25% of the kinase activity of the other selected kinase(s).
According to another embodiment, the pharmaceutical formulation
inhibits less than 20% of the kinase activity of the other selected
kinase(s). According to another embodiment, the pharmaceutical
formulation inhibits less than 15% of the kinase activity of the
other selected kinase(s). According to another embodiment, the
pharmaceutical formulation inhibits less than 10% of the kinase
activity of the other selected kinase(s). According to another
embodiment, the pharmaceutical formulation inhibits less than 5% of
the kinase activity of the other selected kinase(s). According to
another embodiment, the pharmaceutical formulation increases the
kinase activity of the other selected kinases.
[0330] According to the embodiments of the immediately preceding
paragraph, the one or more other selected kinase that is not
substantially inhibited is selected from the group of
Ca2+/calmodulin-dependent protein kinase II (CaMKII, including its
subunit CaMKII.delta.), Proto-oncogene serine/threonine-protein
kinase (PIM-1), cellular-Sarcoma (c-SRC), Spleen Tyrosine Kinase
(SYK), c-Src Tyrosine Kinase (CSK), and Insulin-like Growth Factor
1 Receptor (IGF-1R).
[0331] According to some embodiments, kinases that are
substantially inhibited (i.e., kinases whose kinase activity is
inhibited by at least 65%) by at least one MMI inhibitor (i.e., at
least one of MMI-0100 (SEQ ID NO: 1), MMI-0200 (SEQ ID NO: 19),
MMI-0300 (SEQ ID NO: 3), MMI-0400 (SEQ ID NO: 4), and MMI-0500 (SEQ
ID NO: 7)) of the present invention is selected from the group
consisting of: Abelson murine leukemia viral oncogene homolog 1
(Abl), Abelson murine leukemia viral oncogene homolog 1 (T3151)
(Abl (T3151)), Abelson murine leukemia viral oncogene homolog 1
(Y253F) (Abl (Y253F)), Anaplastic lymphoma kinase (ALK),
Abelson-related gene (Arg), 5'-AMP-activated protein kinase
catalytic subunit alpha-1 (AMPK.alpha.1), 5'-AMP-activated protein
kinase catalytic subunit alpha-2 (AMPK.alpha.2), AMPK-related
protein kinase 5 (ARKS), Apoptosis signal regulating kinase 1
(ASK1), Aurora kinase B (Aurora-B), AXL receptor tyrosine kinase
(Axl), Bone marrow tyrosine kinase gene in chromosome X protein
(Bmx), Breast tumor kinase (BRK), Bruton's tyrosine kinase (BTK),
Bruton's tyrosine kinase (R28H) (BTK (R28H)),
Ca2.sup.+/calmodulin-dependent protein kinase I (CaMKI),
Ca2.sup.+/calmodulin-dependent protein kinase 11.beta.
(CaMII.beta.), Ca2.sup.+/calmodulin-dependent protein kinase
II.gamma. (CaMKII.gamma.), Ca2.sup.+/calmodulin-dependent protein
kinase 6 (CaMKI.delta.), Ca2.sup.+/calmodulin-dependent protein
kinase II.delta. (CaMKII.delta.), Ca2.sup.+/calmodulin-dependent
protein kinase IV (CaMKIV), Cell devision kinase 2 (CDK2/cyclinE),
Cell devision kinase 3 (CDK3/cyclinE), Cell devision kinase 6
(CDK6/cyclinD3), Cell devision kinase 7 (CDK7/cyclinH/MAT1), Cell
devision kinase 9 (CDK9/cyclin T1), Checkpoint kinase 2 (CHK2),
Checkpoint kinase 2 (1157T) (CHK2 (1157T)), Checkpoint kinase 2
(R145W) (CHK2 (R145W)), Proto-oncogene tyrosine-protein kinase cKit
(D816V) (cKit (D816V)), C-src tyrosine kinase (CSK), Raf
proto-oncogene serine/threonine protein kinase (c-RAF),
Proto-oncogene tyrosine-protein kinase (cSRC), Death-associated
protein kinase 1 (DAPK1), Death-associated protein kinase 2
(DAPK2), Dystrophia myotonica-protein kinase (DMPK), DAP
kinase-related apoptosis-inducing protein kinase 1 (DRAK1),
Epidermal growth factor receptor (EGFR), Epidermal growth factor
receptor (EGFR L858R), Epidermal growth factor receptor L861Q (EGFR
(L861Q)), Eph receptor A2 (EphA2) (EphA2), Eph receptor A3 (EphA3),
Eph receptor A5 (EphA5), Eph receptor B2 (EphB2), Eph receptor B4
(EphB4), Erythroblastic leukemia viral oncogene homolog 4 (ErbB4),
c-Fes protein tyrosine kinase (Fes), Fibroblast growth factor
receptor 2 (FGFR2), Fibroblast growth factor receptor 3 (FGFR3),
Fibroblast growth factor receptor 4 (FGFR4), Fms-like tyrosine
kinase receptor-3 (Flt3), FMS proto-oncogene (Fms), Haploid germ
cell-specific nuclear protein kinase (Haspin), Insulin
receptor-related receptor (IRR), Interleukin-1 receptor-associated
kinase 1 (IRAK1), Interleukin-1 receptor-associated kinase 4
(IRAK4), IL2-inducible T-cell kinase (Itk), Kinase insert domain
receptor (KDR), Lymphocyte cell-specific protein-tyrosine kinase
(Lck), Lymphocyte-oriented kinase (LOK), Lyn tyrosine protein
kinase (Lyn), MAP kinase-activated protein kinase 2 (MK2), MAP
kinase-activated protein kinase 3 (MK3), MEK1, Maternal embryonic
leucine zipper kinase (MELK), c-Mer proto-oncogene tyrosine kinase
(Mer), c-Met proto-oncogene tyrosine kinase (Met), c-Met
proto-oncogene tyrosine kinase D1246N (Met (D1246N)), c-Met
proto-oncogene tyrosine kinase Y1248D (Met Y1248D),
Misshapen/NIK-related kinase (MINK), MAP kinase kinase 6 (MKK6),
Myosin light-chain kinase (MLCK), Mixed lineage kinase 1 (MLK1),
MAP kinase signal-integrating kinase 2 (MnK2), Myotonic dystrophy
kinase-related CDC42-binding kinase alpha (MRCK.alpha.), Myotonic
dystrophy kinase-related CDC42-binding kinase beta (MRCK.beta.),
Mitogen- and stress-activated protein kinase 1 (MSK1), Mitogen- and
stress-activated protein kinase 2 (MSK2), Muscle-specific serine
kinase 1 (MSSK1), Mammalian STE20-like protein kinase 1 (MST1),
Mammalian STE20-like protein kinase 2 (MST2), Mammalian STE20-like
protein kinase 3 (MST3), Muscle, skeletal receptor tyrosine-protein
kinase (MuSK), Never in mitosis A-related kinase 2 (NEK2), Never in
mitosis A-related kinase 3 (NEK3), Never in mitosis A-related
kinase 11 (NEK11), 70 kDa ribosomal protein S6 kinase 1 (p70S6K),
PAS domain containing serine/threonine kinase (PASK), Phosphorylase
kinase subunit gamma-2 (PhK.gamma.2), Pim-1 kinase (Pim-1), Protein
kinase B alpha (PKB.alpha.), Protein kinase B beta (PKB.beta.),
Protein kinase B gamma (PKB.gamma.), Protein kinase C, alpha
(PKC.alpha.), Protein kinase C, beta1 (PKC.beta.1), Protein kinase
C, beta II (PKC.beta.II), Protein kinase C, gamma (PKC.gamma.),
Protein kinase C, epsilon (PKC.epsilon.), Protein kinase C, iota
(PCK), Protein kinase C, mu (PKC.mu.), Protein kinase C, zeta
(PKC.zeta.), protein kinase D2 (PKD2), cGMP-dependent protein
kinase 1 alpha (PKG1.alpha.), cGMP-dependent protein kinase 1 beta
(PKG1.beta.), Protein-kinase C-related kinase 2 (PRK2),
Proline-rich tyrosine kinase 2 (Pyk2), Proto-oncogene
tyrosine-protein kinase receptor Ret V804L (Ret (V804L)),
Receptor-interacting serine-threonine kinase 2 (RIPK2),
Rho-associated protein kinase I (ROCK-I), Rho-associated protein
kinase II (ROCK-II), Ribosomal protein S6 kinase 1 (Rsk1),
Ribosomal protein S6 kinase 2 (Rsk2), Ribosomal protein S6 kinase 3
(Rsk3), Ribosomal protein S6 kinase 4 (Rsk4), Stress-activated
protein kinase 2A T106M (SAPK2a, T106M), Stress-activated protein
kinase 3 (SAPK3), Serum/glucocorticoid regulated kinase (SGK),
Serum/glucocorticoid regulated kinase 2 (SGK2),
Serum/glucocorticoid-regulated kinase 3 (SGK3), Proto-oncogene
tyrosine-protein kinase Src 1-530 (Src, 1-530),
Serine/threonine-protein kinase 33 (STK33), Spleen tyrosine kinase
(Syk), Thousand and one amino acid protein 1 (TAO1), Thousand and
one amino acid protein 2 (TAO2), Thousand and one amino acid
protein 3 (TAO3), TANK-binding kinase 1 (TBK1), Tec protein
tyrosine kinase (Tec), Tunica interna endothelial cell kinase 2
(Tie2), Tyrosine kinase receptor A (TrkA), BDNF/NT-3 growth factors
receptor (TrkB), TXK tyrosine kinase (Txk), WNK lysine deficient
protein kinase 2 (WNK2), WNK lysine deficient protein kinase 3
(WNK3), Yamaguchi sarcoma viral oncogene homolog 1 (Yes),
Zeta-chain (TCR) Associated Protein kinase 70 kDa (ZAP-70), and ZIP
kinase (ZIPK).
[0332] According to some other embodiments, kinases that are
substantially inhibited (i.e., kinases whose kinase activity is
inhibited by at least 65%) by at least two MMI inhibitors (i.e., at
least two of MMI-0100 (SEQ ID NO: 1), MMI-0200 (SEQ ID NO: 19),
MMI-0300 (SEQ ID NO: 3), MMI-0400 (SEQ ID NO: 4), and MMI-0500 (SEQ
ID NO: 7)) of the present invention is selected from the group
consisting of: Anaplastic lymphoma kinase (ALK), Breast tumor
kinase (BRK), Bruton's tyrosine kinase (BTK),
Ca.sup.2+/calmodulin-dependent protein kinase I (including
CaMKI.delta.), Ca.sup.2+/calmodulin-dependent protein kinase II
(CaMKII, including CaMKII.beta., CaMKII.delta. and CaMKII.gamma.),
Ca.sup.2+/calmodulin-dependent protein kinase IV (CaMKIV),
Checkpoint kinase 2 (CHK2 (R145W)), Proto-oncogene tyrosine-protein
kinase cKit (D816V) (cKit (D816V)), C-src tyrosine kinase (CSK),
Proto-oncogene tyrosine-protein kinase (cSRC), Death-associated
protein kinase 1 (DAPK1), Death-associated protein kinase 2
(DAPK2), DAP kinase-related apoptosis-inducing protein kinase 1
(DRAK1), Epidermal growth factor receptor (EGFR), Epidermal growth
factor receptor L861Q (EGFR (L861Q)), Eph receptor A2 (EphA2), Eph
receptor A3 (EphA3), Eph receptor A5 (EphA5), Eph receptor B2
(EphB2), Erythroblastic leukemia viral oncogene homolog 4 (ErbB4),
c-Fes protein tyrosine kinase (Fes), Fibroblast growth factor
receptor 2 (FGFR2), Fibroblast growth factor receptor 3 (FGFR3),
and Fibroblast growth factor receptor 4 (FGFR4), Fms-like tyrosine
kinase receptor-3 (Flt3), Insulin receptor-related receptor (IRR),
Lymphocyte-oriented kinase (LOK), Lyn tyrosine protein kinase
(Lyn), MAP kinase-activated protein kinase 2 (MK2), MAP
kinase-activated protein kinase 3 (MK3), Maternal embryonic leucine
zipper kinase (MELK), Myosin light-chain kinase (MLCK), Mitogen-
and stress-activated protein kinase (MSK1), Mammalian STE20-like
protein kinase 1 (MST1), Mammalian STE20-like protein kinase 2
(MST2), Never in mitosis A-related kinase 11(NEK11), 70 kDa
ribosomal protein S6 kinase 1 (p70S6K), PAS domain containing
serine/threonine kinase (PASK), Pim-1 kinase (Pim-1), Protein
kinase B, gamma (PKB.gamma.), Protein kinase C, mu (PKC.mu.),
protein kinase D2 (PKD2), Protein-kinase C-related kinase 2 (PRK2),
Serum/glucocorticoid-regulated kinase 3 (SGK3), Proto-oncogene
tyrosine-protein kinase Src (Src), Spleen tyrosine kinase (Syk),
Tec protein tyrosine kinase (Tec), Tunica interna endothelial cell
kinase 2 (Tie2), Tyrosine kinase receptor A (TrkA), BDNF/NT-3
growth factors receptor (TrkB), Zeta-chain (TCR) Associated Protein
kinase 70 kDa (ZAP-70), and ZIP kinase (ZIPK).
[0333] According to some embodiments, the pharmaceutical
formulation comprises a small-molecule inhibitor of MK2, including,
but not limited to:
##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010##
or a combination thereof.
[0334] According to some embodiments, the polypeptide of amino acid
sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) and its functional
equivalents are effective to reduce a level of TGF-.beta.
expression, infiltration of immunomodulatory cells, or both.
[0335] According to another embodiment, pharmaceutical formulations
of the described invention are effective to reduce infiltration of
one or more types of inflammatory or stem cells, including, without
limitation, monocytes, fibrocytes, macrophages, lymphocytes, and
mast or dendritic cells, into the wound.
[0336] According to another embodiment, the cell type is
characterized by expression of cell surface marker(s) including,
without limitation, CD4 and/or CD8.
[0337] According to some embodiments, the therapeutic amount of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
of an amount from about 0.000001 mg/kg body weight to about 100
mg/kg body weight. According to another embodiment, the therapeutic
amount of the therapeutic inhibitory peptide of the pharmaceutical
formulation is of an amount from about 0.00001 mg/kg body weight to
about 100 mg/kg body weight. According to another embodiment, the
therapeutic amount of the therapeutic inhibitory peptide of the
pharmaceutical formulation is of an amount from about 0.0001 mg/kg
body weight to about 100 mg/kg body weight. According to another
embodiment, the therapeutic amount of the therapeutic inhibitory
peptide of the pharmaceutical formulation is of an amount from
about 0.001 mg/kg body weight to about 10 mg/kg body weight.
According to another embodiment, the therapeutic amount of the
therapeutic inhibitory peptide of the pharmaceutical formulation is
of an amount from about 0.01 mg/kg body weight to about 10 mg/kg
body weight. According to another embodiment, the therapeutic
amount of the therapeutic inhibitory peptide of the pharmaceutical
formulation is of an amount from about 0.1 mg/kg (or 100 mg/kg)
body weight to about 10 mg/kg body weight. According to another
embodiment, the therapeutic amount of the therapeutic inhibitory
peptide of the pharmaceutical formulation is of an amount from
about 1 mg/kg body weight to about 10 mg/kg body weight. According
to another embodiment, the therapeutic amount of the therapeutic
inhibitory peptide of the pharmaceutical formulation is of an
amount from about 10 mg/kg body weight to about 100 mg/kg body
weight. According to another embodiment, the therapeutic amount of
the therapeutic inhibitory peptide of the pharmaceutical
formulation is of an amount from about 2 mg/kg body weight to about
10 mg/kg body weight. According to another embodiment, the
therapeutic amount of the therapeutic inhibitory peptide of the
pharmaceutical formulation is of an amount from about 3 mg/kg body
weight to about 10 mg/kg body weight. According to another
embodiment, the therapeutic amount of the therapeutic inhibitory
peptide of the pharmaceutical formulation is of an amount from
about 4 mg/kg body weight to about 10 mg/kg body weight. According
to another embodiment, the therapeutic amount of the therapeutic
inhibitory peptide of the pharmaceutical formulation is of an
amount from about 5 mg/kg body weight to about 10 mg/kg body
weight. According to another embodiment, the therapeutic amount of
the therapeutic inhibitory peptide of the pharmaceutical
formulation is of an amount from about 60 mg/kg body weight to
about 100 mg/kg body weight. According to another embodiment, the
therapeutic amount of the therapeutic inhibitory peptide of the
pharmaceutical formulation is of an amount from about 70 mg/kg body
weight to about 100 mg/kg body weight. According to another
embodiment, the therapeutic amount of the therapeutic inhibitory
peptide of the pharmaceutical formulation is of an amount from
about 80 mg/kg body weight to about 100 mg/kg body weight.
According to another embodiment, the therapeutic amount of the
therapeutic inhibitory peptide of the pharmaceutical formulation is
of an amount from about 90 mg/kg body weight to about 100 mg/kg
body weight. According to another embodiment, the therapeutic
amount of the therapeutic inhibitor peptide of the pharmaceutical
formulation is of an amount from about 0.000001 mg/kg body weight
to about 90 mg/kg body weight. According to another embodiment, the
therapeutic amount of the therapeutic inhibitor peptide of the
pharmaceutical formulation is of an amount from about 0.000001
mg/kg body weight to about 80 mg/kg body weight. According to
another embodiment, the therapeutic amount of the therapeutic
inhibitor peptide of the pharmaceutical formulation is of an amount
from about 0.000001 mg/kg body weight to about 70 mg/kg body
weight. According to another embodiment, the therapeutic amount of
the therapeutic inhibitor peptide of the pharmaceutical formulation
is of an amount from about 0.000001 mg/kg body weight to about 60
mg/kg body weight. According to another embodiment, the therapeutic
amount of the therapeutic inhibitor peptide of the pharmaceutical
formulation is of an amount from about 0.000001 mg/kg body weight
to about 50 mg/kg body weight. According to another embodiment, the
therapeutic amount of the therapeutic inhibitor peptide of the
pharmaceutical formulation is of an amount from about 0.000001
mg/kg body weight to about 40 mg/kg body weight. According to
another embodiment, the therapeutic amount of the therapeutic
inhibitor peptide is of an amount from about 0.000001 mg/kg body
weight to about 30 mg/kg body weight. According to another
embodiment, the therapeutic amount of the therapeutic inhibitor
peptide of the pharmaceutical formulation is of an amount from
about 0.000001 mg/kg body weight to about 20 mg/kg body weight.
According to another embodiment, the therapeutic amount of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
of an amount from about 0.000001 mg/kg body weight to about 10
mg/kg body weight. According to another embodiment, the therapeutic
amount of the therapeutic inhibitor peptide of the pharmaceutical
formulation is of an amount from about 0.000001 mg/kg body weight
to about 1 mg/kg body weight. According to another embodiment, the
therapeutic amount of the therapeutic inhibitor peptide of the
pharmaceutical formulation is of an amount from about 0.000001
mg/kg body weight to about 0.1 mg/kg body weight. According to
another embodiment, the therapeutic amount of the therapeutic
inhibitor peptide of the pharmaceutical formulation is of an amount
from about 0.000001 mg/kg body weight to about 0.1 mg/kg body
weight. According to another embodiment, the therapeutic amount of
the therapeutic inhibitor peptide of the pharmaceutical formulation
is of an amount from about 0.000001 mg/kg body weight to about 0.01
mg/kg body weight. According to another embodiment, the therapeutic
amount of the therapeutic inhibitor peptide of the pharmaceutical
formulation is of an amount from about 0.000001 mg/kg body weight
to about 0.001 mg/kg body weight. According to another embodiment,
the therapeutic amount of the therapeutic inhibitor peptide of the
pharmaceutical formulation is of an amount from about 0.000001
mg/kg body weight to about 0.0001 mg/kg body weight. According to
another embodiment, the therapeutic amount of the therapeutic
inhibitor peptide of the pharmaceutical formulation is of an amount
from about 0.000001 mg/kg body weight to about 0.00001 mg/kg body
weight.
[0338] According to some other embodiments, the therapeutic dose of
the therapeutic inhibitor peptide of the pharmaceutical formulation
ranges from 1 .mu.g/kg/day to 25 .mu.g/kg/day. According to some
other embodiments, the therapeutic dose of the therapeutic
inhibitor peptide of the pharmaceutical formulation ranges from 1
.mu.g/kg/day to 2 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 2
.mu.g/kg/day to 3 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 3
.mu.g/kg/day to 4 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical ranges from 4 .mu.g/kg/day to 5
.mu.g/kg/day. According to some other embodiments, the therapeutic
dose of the therapeutic inhibitor peptide of the pharmaceutical
formulation ranges from 5 .mu.g/kg/day to 6 .mu.g/kg/day. According
to some other embodiments, the therapeutic dose of the therapeutic
inhibitor peptide of the pharmaceutical formulation ranges from 6
.mu.g/kg/day to 7 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 7
.mu.g/kg/day to 8 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 8
.mu.g/kg/day to 9 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 9
.mu.g/kg/day to 10 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 1
.mu.g/kg/day to 5 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 5
.mu.g/kg/day to 10 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 10
.mu.g/kg/day to 15 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 15
.mu.g/kg/day to 20 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 25
.mu.g/kg/day to 30 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 30
.mu.g/kg/day to 35 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 35
.mu.g/kg/day to 40 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 40
.mu.g/kg/day to 45 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 45
.mu.g/kg/day to 50 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 50
.mu.g/kg/day to 55 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 55
.mu.g/kg/day to 60 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 60
.mu.g/kg/day to 65 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 65
.mu.g/kg/day to 70 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 70
.mu.g/kg/day to 75 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 80
.mu.g/kg/day to 85 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 85
.mu.g/kg/day to 90 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 90
.mu.g/kg/day to 95 .mu.g/kg/day. According to some other
embodiments, the therapeutic dose of the therapeutic inhibitor
peptide of the pharmaceutical formulation ranges from 95
.mu.g/kg/day to 100 .mu.g/kg/day.
[0339] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
1 .mu.g/kg/day.
[0340] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
2 .mu.g/kg/day.
[0341] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
3 .mu.g/kg/day.
[0342] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
4 .mu.g/kg/day.
[0343] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
5 .mu.g/kg/day.
[0344] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
6 .mu.g/kg/day.
[0345] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
7 .mu.g/kg/day.
[0346] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
8 .mu.g/kg/day.
[0347] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
9 .mu.g/kg/day.
[0348] According to another embodiment, the therapeutic dose of the
therapeutic inhibitor peptide of the pharmaceutical formulation is
10 .mu.g/kg/day.
[0349] The polypeptide of amino acid sequence
YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent
thereof may be administered in the form of a pharmaceutically
acceptable salt. When used in medicine the salts should be
pharmaceutically acceptable, but non-pharmaceutically acceptable
salts may conveniently be used to prepare pharmaceutically
acceptable salts thereof. Such salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic,
salicylic, p-toluene sulphonic, tartaric, citric, methane
sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and
benzene sulphonic. Also, such salts may be prepared as alkaline
metal or alkaline earth salts, such as sodium, potassium or calcium
salts of the carboxylic acid group. Pharmaceutically acceptable
salts are well-known. For example, P. H. Stahl, et al. describe
pharmaceutically acceptable salts in detail in "Handbook of
Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH,
Zurich, Switzerland: 2002). The salts may be prepared in situ
during the final isolation and purification of the compounds
described within the described invention or may be prepared by
separately reacting a free base function with a suitable organic
acid. Representative acid addition salts include, but are not
limited to, acetate, adipate, alginate, citrate, aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,
camphorsufonate, digluconate, glycerophosphate, hemisulfate,
heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethansulfonate(isethionate), lactate,
maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate,
oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate,
picrate, pivalate, propionate, succinate, tartrate, thiocyanate,
phosphate, glutamate, bicarbonate, p-toluenesulfonate and
undecanoate. Also, the basic nitrogen-containing groups may be
quaternized with such agents as lower alkyl halides such as methyl,
ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl
sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long
chain halides such as decyl, lauryl, myristyl and stearyl
chlorides, bromides and iodides; arylalkyl halides like benzyl and
phenethyl bromides and others. Water or oil-soluble or dispersible
products are thereby obtained. Examples of acids which may be
employed to form pharmaceutically acceptable acid addition salts
include such inorganic acids as hydrochloric acid, hydrobromic
acid, sulphuric acid and phosphoric acid and such organic acids as
oxalic acid, maleic acid, succinic acid and citric acid. Basic
addition salts may be prepared in situ during the final isolation
and purification of compounds described within the invention by
reacting a carboxylic acid-containing moiety with a suitable base
such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation or with ammonia or an
organic primary, secondary or tertiary amine. Pharmaceutically
acceptable salts include, but are not limited to, cations based on
alkali metals or alkaline earth metals such as lithium, sodium,
potassium, calcium, magnesium and aluminum salts and the like and
nontoxic quaternary ammonia and amine cations including ammonium,
tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, diethylamine,
ethylamine and the like. Other representative organic amines useful
for the formation of base addition salts include ethylenediamine,
ethanolamine, diethanolamine, piperidine, piperazine and the like.
Pharmaceutically acceptable salts also may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium or magnesium) salts of carboxylic acids may also be
made.
[0350] The formulations may be presented conveniently in unit
dosage form and may be prepared by methods known in the art of
pharmacy. Such methods include the step of bringing into
association a therapeutic agent(s), or a pharmaceutically
acceptable salt or solvate thereof ("active compound") with the
carrier which constitutes one or more accessory agents. In general,
the formulations are prepared by uniformly and intimately bringing
into association the active agent with liquid carriers or finely
divided solid carriers or both and then, if necessary, shaping the
product into the desired formulation.
[0351] According to some embodiments, the carrier is a controlled
release carrier. The term "controlled release" is intended to refer
to any drug-containing formulation in which the manner and profile
of drug release from the formulation are controlled. This includes
immediate as well as non-immediate release formulations, with
non-immediate release formulations including, but not limited to,
sustained release and delayed release formulations. According to
some embodiments, the controlled release of the pharmaceutical
formulation is mediated by changes in temperature. According to
some other embodiments, the controlled release of the
pharmaceutical formulation is mediated by changes in pH.
[0352] Injectable depot forms may be made by forming
microencapsulated matrices of a therapeutic agent/drug in
biodegradable polymers such as, but not limited to, polyesters
(polyglycolide, polylactic acid and combinations thereof),
polyester polyethylene glycol copolymers, polyamino-derived
biopolymers, polyanhydrides, polyorthoesters, polyphosphazenes,
sucrose acetate isobutyrate (SAIB), photopolymerizable biopolymers,
naturally-occurring biopolymers, protein polymers, collagen, and
polysaccharides. Depending upon the ratio of drug to polymer and
the nature of the particular polymer employed, the rate of drug
release may be controlled. Such long acting formulations may be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in acceptable oil) or ion exchange resins,
or as sparingly soluble derivatives, for example, as a sparingly
soluble salt. Depot injectable formulations also are prepared by
entrapping the drug in liposomes or microemulsions which are
compatible with body tissues.
[0353] According to some embodiments, the carrier is a delayed
release carrier. According to another embodiment, the delayed
release carrier comprises a biodegradable polymer. According to
another embodiment, the biodegradable polymer is a synthetic
polymer. According to another embodiment, the biodegradable polymer
is a naturally occurring polymer.
[0354] According to some embodiments, the carrier is a sustained
release carrier. According to another embodiment, the
sustained-release carrier comprises a biodegradable polymer.
According to another embodiment, the biodegradable polymer is a
synthetic polymer. According to another embodiment, the
biodegradable polymer is a naturally occurring polymer.
[0355] According to some embodiments, the carrier is a short-term
release carrier. The term "short-term" release, as used herein,
means that an implant is constructed and arranged to deliver
therapeutic levels of the active ingredient for about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
or 23 hours. According to some other embodiments, the short term
release carrier delivers therapeutic levels of the active
ingredient for about 1, 2, 3, or 4 days.
[0356] According to some embodiments, the carrier is a long-term
release carrier. The term "long-term" release, as used herein,
means that an implant is constructed and arranged to deliver
therapeutic levels of the active ingredient for at least about 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 29, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 48, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, or 60 days. According to another embodiment, the
long-term-release carrier comprises a biodegradable polymer.
According to another embodiment, the biodegradable polymer is a
synthetic polymer.
[0357] According to some embodiments, the carrier comprises
particles. According to some embodiments, formulations as described
herein are contained in the particle. According to some
embodiments, formulations as described herein are contained on the
particle. According to some embodiments, formulations as described
herein are contained both in and on the particle.
[0358] The formulations also may contain appropriate adjuvants,
including, without limitation, preservative agents, wetting agents,
emulsifying agents, and dispersing agents. Prevention of the action
of microorganisms may be ensured by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, and the like. It also may be desirable to include
isotonic agents, for example, sugars, sodium chloride and the like.
Prolonged absorption of the injectable pharmaceutical form may be
brought about by the use of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0359] According to some embodiments, the polypeptides of the
present invention can be covalently attached to polyethylene glycol
(PEG) polymer chains. According to some other embodiments, the
polypeptides of the present invention are stapled with hydrocarbons
to generate hydrocarbon-stapled peptides that are capable of
forming stable alpha-helical structure (Schafmeister, C. et al., J.
Am. Chem. Soc., 2000, 122, 5891-5892, incorporated herein by
reference in its entirety).
[0360] According to some other embodiments, the polypeptides of the
present invention are encapsulated or entrapped into microspheres,
nanocapsules, liposomes, or microemulsions, or comprises d-amino
acids in order to increase stability, to lengthen delivery, or to
alter activity of the peptides. These techniques can lengthen the
stability and release simultaneously by hours to days, or delay the
uptake of the drug by nearby cells.
[0361] The formulations of therapeutic agent(s) may be administered
in pharmaceutically acceptable solutions, which may routinely
contain pharmaceutically acceptable concentrations of salt,
buffering agents, preservatives, compatible carriers, adjuvants,
and optionally other therapeutic ingredients.
[0362] According to some embodiments, the pharmaceutical
formulation further comprises at least one additional therapeutic
agent.
[0363] According to some such embodiments, the additional
therapeutic agent comprises EXC001 (an anti-sense RNA against
connective tissue growth factor (CTGF)), AZX100 (a phosphopeptide
analog of Heat Shock Protein 20 (HSP20)), PRM-151 (recombinant
human serum amyloid P/Pentaxin 2), PXL01 (a synthetic peptide
derived from human lactoferrin), DSC127 (an angiotensin analog),
RXI-109 (a self-delivering RNAi compound that targets connective
tissue growth factor (CTGF)), TCA (trichloroacetic acid), Botulium
toxin type A, or a combination thereof.
[0364] According to another embodiment, the additional therapeutic
agent is an anti-inflammatory agent.
[0365] According to some embodiments, the anti-inflammatory agent
is a steroidal anti-inflammatory agent. The term "steroidal
anti-inflammatory agent", as used herein, refer to any one of
numerous compounds containing a 17-carbon 4-ring system and
includes the sterols, various hormones (as anabolic steroids), and
glycosides. Representative examples of steroidal anti-inflammatory
drugs include, without limitation, corticosteroids such as
hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone,
dexamethasone-phosphate, beclomethasone dipropionates, clobetasol
valerate, desonide, desoxymethasone, desoxycorticosterone acetate,
dexamethasone, dichlorisone, diflucortolone valerate,
fluadrenolone, fluclorolone acetonide, flumethasone pivalate,
fluosinolone acetonide, fluocinonide, flucortine butylesters,
fluocortolone, fluprednidene (fluprednylidene) acetate,
flurandrenolone, halcinonide, hydrocortisone acetate,
hydrocortisone butyrate, methylprednisolone, triamcinolone
acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone,
difluorosone diacetate, fluradrenolone, fludrocortisone,
diflorosone diacetate, fluradrenolone acetonide, medrysone,
amcinafel, amcinafide, betamethasone and the balance of its esters,
chloroprednisone, chlorprednisone acetate, clocortelone,
clescinolone, dichlorisone, diflurprednate, flucloronide,
flunisolide, fluoromethalone, fluperolone, fluprednisolone,
hydrocortisone valerate, hydrocortisone cyclopentylpropionate,
hydrocortamate, meprednisone, paramethasone, prednisolone,
prednisone, beclomethasone dipropionate, triamcinolone, and
mixtures thereof.
[0366] According to another embodiment, the anti-inflammatory agent
is a nonsteroidal anti-inflammatory agent. The term "non-steroidal
anti-inflammatory agent" as used herein refers to a large group of
agents that are aspirin-like in their action, including, but not
limited to, ibuprofen (Advil.RTM.), naproxen sodium (Aleve.RTM.),
and acetaminophen (Tylenol.RTM.). Additional examples of
non-steroidal anti-inflammatory agents that are usable in the
context of the described invention include, without limitation,
oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam, and
CP-14,304; disalcid, benorylate, trilisate, safapryn, solprin,
diflunisal, and fendosal; acetic acid derivatives, such as
diclofenac, fenclofenac, indomethacin, sulindac, tolmetin,
isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac,
zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates,
such as mefenamic, meclofenamic, flufenamic, niflumic, and
tolfenamic acids; propionic acid derivatives, such as benoxaprofen,
flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen,
pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,
tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles,
such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone,
and trimethazone. Mixtures of these non-steroidal anti-inflammatory
agents also may be employed, as well as the dermatologically
acceptable salts and esters of these agents. For example,
etofenamate, a flufenamic acid derivative, is particularly useful
for topical application.
[0367] According to another embodiment, the anti-inflammatory agent
includes, without limitation, Transforming Growth Factor-beta3
(TGF-.beta.3), an anti-Tumor Necrosis Factor-alpha (TNF-.alpha.)
agent, or a combination thereof.
[0368] According to some embodiments, the additional agent is an
analgesic agent. According to some embodiments, the analgesic agent
relieves pain by elevating the pain threshold without disturbing
consciousness or altering other sensory modalities. According to
some such embodiments, the analgesic agent is a non-opioid
analgesic. "Non-opioid analgesics" are natural or synthetic
substances that reduce pain but are not opioid analgesics. Examples
of non-opioid analgesics include, but are not limited to, etodolac,
indomethacin, sulindac, tolmetin, nabumetone, piroxicam,
acetaminophen, fenoprofen, flurbiprofen, ibuprofen, ketoprofen,
naproxen, naproxen sodium, oxaprozin, aspirin, choline magnesium
trisalicylate, diflunisal, meclofenamic acid, mefenamic acid, and
phenylbutazone. According to some other embodiments, the analgesic
is an opioid analgesic. "Opioid analgesics", "opioid", or "narcotic
analgesics" are natural or synthetic substances that bind to opioid
receptors in the central nervous system, producing an agonist
action. Examples of opioid analgesics include, but are not limited
to, codeine, fentanyl, hydromorphone, levorphanol, meperidine,
methadone, morphine, oxycodone, oxymorphone, propoxyphene,
buprenorphine, butorphanol, dezocine, nalbuphine, and
pentazocine.
[0369] According to another embodiment, the additional agent is an
anti-infective agent. According to another embodiment, the
anti-infective agent is an antibiotic agent. The term "antibiotic
agent" as used herein means any of a group of chemical substances
having the capacity to inhibit the growth of, or to destroy
bacteria, and other microorganisms, used chiefly in the treatment
of infectious diseases. Examples of antibiotic agents include, but
are not limited to, Penicillin G; Methicillin; Nafcillin;
Oxacillin; Cloxacillin; Dicloxacillin; Ampicillin; Amoxicillin;
Ticarcillin; Carbenicillin; Mezlocillin; Azlocillin; Piperacillin;
Imipenem; Aztreonam; Cephalothin; Cefaclor; Cefoxitin; Cefuroxime;
Cefonicid; Cefmetazole; Cefotetan; Cefprozil; Loracarbef;
Cefetamet; Cefoperazone; Cefotaxime; Ceftizoxime; Ceftriaxone;
Ceftazidime; Cefepime; Cefixime; Cefpodoxime; Cefsulodin;
Fleroxacin; Nalidixic acid; Norfloxacin; Ciprofloxacin; Ofloxacin;
Enoxacin; Lomefloxacin; Cinoxacin; Doxycycline; Minocycline;
Tetracycline; Amikacin; Gentamicin; Kanamycin; Netilmicin;
Tobramycin; Streptomycin; Azithromycin; Clarithromycin;
Erythromycin; Erythromycin estolate; Erythromycin ethyl succinate;
Erythromycin glucoheptonate; Erythromycin lactobionate;
Erythromycin stearate; Vancomycin; Teicoplanin; Chloramphenicol;
Clindamycin; Trimethoprim; Sulfamethoxazole; Nitrofurantoin;
Rifampin; Mupirocin; Metronidazole; Cephalexin; Roxithromycin;
Co-amoxiclavuanate; combinations of Piperacillin and Tazobactam;
and their various salts, acids, bases, and other derivatives.
Anti-bacterial antibiotic agents include, but are not limited to,
penicillins, cephalosporins, carbacephems, cephamycins,
carbapenems, monobactams, aminoglycosides, glycopeptides,
quinolones, tetracyclines, macrolides, and fluoroquinolones.
[0370] Other examples of at least one additional therapeutic agent
include, but are not limited to, rose hip oil, vitamin E,
5-fluorouracil, bleomycin, onion extract, pentoxifylline,
prolyl-4-hydroxylase, verapamil, tacrolimus, tamoxifen, tretinoin,
colchicine, a calcium antagonist, tranilst, zinc, an antibiotic,
and a combination thereof.
[0371] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein also can be used in the practice or testing of the described
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0372] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges which may
independently be included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
[0373] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
references unless the context clearly dictates otherwise. All
technical and scientific terms used herein have the same
meaning.
[0374] The publications discussed herein, the contents of which are
incorporated herein by reference, are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the
described invention is not entitled to antedate such publication by
virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0375] The described invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof and, accordingly, reference should be made to the appended
claims, rather than to the foregoing specification, as indicating
the scope of the invention.
Examples
[0376] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Materials and Methods
[0377] A. Dry Powder Formulations of MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
[0378] MMI-0100 Formulations:
[0379] MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), Lyophilized
(American Peptide, Inc., Sunnyvale Calif.) Lot number 100429, Date
of Manufacture 29 Jun. 2010, 500 mg.
[0380] Neat Spray Dried MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID
NO: 1), 5% w/w solids (Bend Research, Bend Oreg.) Lot Number BREC
00708-003A, Date of Manufacture 27 Jul. 2012, 1 g.
[0381] Neat Spray Dried MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID
NO: 1), 1% w/w solids (Bend Research, Bend Oreg.) Lot Number BREC
00708-003B, Date of Manufacture 27 Jul. 2012, 1 g.
[0382] Spray Dried 80/20 MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID
NO: 1)/Trehalose (Santa Cruz Biotechnology, Inc. Dallas Tex.), 1%
w/w solids (Bend Research, Bend Oreg.) Lot Number BREC 00708-011C,
Date of Manufacture w/c 10 Sep. 2012, 500 mg.
[0383] Spray Dried 92.5/7.5 MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ
ID NO: 1)/Trehalose (Santa Cruz Biotechnology, Inc. Dallas Tex.),
1% w/w solids (Bend Research, Bend Oreg.) Lot Number BREC
00708-011F, Date of Manufacture w/c 10 Sep. 2012, 500 mg.
[0384] Rapid HPLC and NGI Sample Extraction Method
[0385] Materials and Equipment
[0386] Water, Millipore or equivalent
[0387] Acetonitrile, HPLC grade
[0388] Methanol, HPLC grade
[0389] Trifluoroacetic Acid
[0390] Tween 20
[0391] MMI-0100 Neat lyophilized drug substance
[0392] Microbalance (Mettler-Toledo, Columbus Ohio)
[0393] Next Generation Impactor (NGI) (MSP Corp, Shoreview
Minn.)
[0394] Dose Unit Sampling Apparatus (Copley, Nottingham UK)
[0395] TPK Controller (Copley, Nottingham UK)
[0396] HPLC System
[0397] HPLC instrument (Waters Alliance 2695, Milford Mass.) with
thermostatted column compartment or column oven and sample
compartments
[0398] Column: Supelco, Ascentis Express.RTM. Peptide ES-C18,
50.times.4.6 mm (Sigma-Aldrich, St Louis Mo.)
[0399] Flow rate: 1.5 mL/min
[0400] Injection volume: 40 ILL
[0401] Column Temperature: 40.degree. C.
[0402] Sample Temperature: 5.degree. C.
[0403] Detector Wavelength: 215 nm
[0404] Mobile Phase A: 0.1% IT A in Water (72%)
[0405] Mobile Phase B: 0.1% TFA in 1:1 Methanol:Acetonitrile
(28%)
[0406] Run time: 3 minutes. Retention time of MMI-0100 is about
2.35 minutes.
[0407] Solution Preparation
[0408] Mobile Phase A: 0.1% TFA in Water
[0409] Pipet 2.0 mL of TFA into 1000 mL of water in a 2 L
volumetric flask and dilute to volume with water. Mix and degas.
Alternate volumes may be prepared provided that proportions are
kept equal.
[0410] Mobile Phase B: 0.1% TFA in 1:1 Methanol:Acetonitrile
[0411] Pipet 1.0 mL of TFA into 500 mL of methanol in a 1 L
volumetric flask and dilute to volume with methanol. Mix and degas.
Alternate volumes may be prepared provided that proportions are
kept equal. Pipet 1.0 mL of TFA into 500 mL of acetonitrile in a 1
L volumetric flask and dilute to volume with acetonitrile. Mix and
degas. Alternate volumes may be prepared provided that proportions
are kept equal. Mix the above prepared solutions for 2,000 mL of
mobile phase.
[0412] Sample Solvent: 0.02% Tween 20 in Water
[0413] Using a graduated wide-mouth TC pipette transfer 0.8 mL of
Tween 20 into a 4,000 mL volumetric flask containing approximately
3,000 mL of water. Tween 20 is viscous. Be sure to rinse the
pipette with the water into the flask several times to flush the
Tween 20 out of the pipette. Dilute to volume with water. Mix
well.
[0414] Coating Solution: 5% Tween 20 in Methanol
[0415] Using a graduated wide-mouth TC pipette transfer 5 mL of
Tween 20 into a 100.0 mL volumetric flask containing approximately
75 mL of methanol. Tween 20 is viscous. Be sure to rinse the
pipette with methanol into the flask several times to flush the
Tween 20 out of the pipette. Dilute to volume with methanol. Mix
well.
[0416] NOTE: MMI-0100 is hygroscopic. All handling of the neat drug
substance should be performed in a glove box maintained at 5%
relative humidity.
[0417] NOTE: Lyophilized MMI-0100 is stored between -10.degree. C.
and -20.degree. C. Prior to use, the lyophilized MMI-0100 should be
thawed in a desiccator or a glove box maintained at 5% relative
humidity for at least 2 hours.
[0418] Standard Stock Solution--1.1 mg/mL
[0419] Weigh an amount of MMI-0100 equivalent to 11 mg of pure
MMI-0100, into an appropriate weighing vessel. The actual weight
needed can be calculated by dividing 11 mg by the purity factor
reported on the Certificate of Analysis. The amount of MMI-0100
actually weighed out should be within .+-.0.250 mg of this
calculated weight. Record the weight of MMI-0100 (as is) plus the
weighing vessel as W.sub.i. Transfer the MMI-0100 to a 10.0 mL
volumetric flask. Place the empty weighing vessel onto the balance
and record the weight (W.sub.f). The standard amount is equal to
W.sub.i-W.sub.f. Add approximately 6 mL of sample solvent. Swirl
the volumetric flask to dissolve and dilute to volume with sample
solvent. Mix well and immediately transfer the solution to a
polypropylene centrifuge tube. Prepare a second solution for check
standard stock solution.
[0420] Working Standard Solution--110 .mu.g/mL
[0421] Pipette 5.0 mL of standard stock solution into a 50-mL
volumetric flask. Dilute to volume with sample solvent and
immediately transfer the solution to a polypropylene centrifuge
tube. Final concentration: 110 .mu.g/mL.
[0422] Working Standard Solution--11 .mu.g/mL
[0423] Pipette 5.0 mL of standard stock solution into a 50-mL
volumetric flask. Dilute to volume with sample solvent. Final
concentration: 11 .mu.g/mL.
[0424] Limit of Quantification (LOQ) Solution
[0425] Pipette 1.0 mL of 110 .mu.g/mL working standard solution
into a 50-mL volumetric flask. Dilute to volume with sample
solvent. Final concentration: 2.2 .mu.g/mL.
[0426] Procedure
[0427] Equilibrate the HPLC with mobile phase until a stable signal
is achieved. Perform system suitability and sample injections using
one of the following sequences as appropriate.
[0428] NOTE: The HPLC autosampler temperature is maintained at
5.degree. C. MMI-0100 sample solutions should be transferred to the
HPLC immediately after preparation and allowed to thermally
equilibrate for at least 10-15 minutes prior to injecting.
[0429] NOTE: Glass will absorb the MMI-0100 peptide from solution.
Only polypropylene HPLC vials should be used for analysis.
[0430] NGI Samples
[0431] Sample solvent (1.times.)
[0432] LOQ solution (6.times.)
[0433] 11 .mu.g/mL Working Standard (5.times.)
[0434] 11 .mu.g/mL Check Standard (1.times.)
[0435] NGI Samples--1 replicate, Blister through MOC
(1.times.each)
[0436] 11 .mu.g/mL Working Standard (1.times.)
[0437] Additional replicates of NGI samples
[0438] 11 .mu.g/mL Working Standard (1.times.after each NGI
replicate)
[0439] System suitability is achieved if the following target
requirements are met.
[0440] Sample solvent peaks: none detected at retention time of
MMI-0100
[0441] LOQ solution: % RSD (relative standard deviation) for n=6
injections should be .ltoreq.10%
[0442] First n=5 injections of Working standard:
[0443] % RSD should be .ltoreq.1.5%
[0444] Tailing factor should be .ltoreq.2.0
[0445] k' should be >2.0. Use the first peak in the solvent
front as the to void time.
[0446] Theoretical plates should be recorded for information
only.
[0447] Check standard: 98.0-102.0%
[0448] Working Standard injections through run: % RSD of all
working standard injections should be .ltoreq.2.0%
[0449] NGI Sample Preparation
[0450] Blisters for NGI analysis should be dosed according to the
normal use instructions for the inhaler used in the study.
[0451] Blisters, Flow Channel, Throat, and NGI Impaction Cups
should be extracted with sample solvent using normal lab practices
for the stages with extraction volumes listed in Table 2.
TABLE-US-00002 TABLE 2 Summary of Test Solutions Test Solution
Volume (mL) Blister 20.0 Flow Channel 5.0 Throat 20.0 Preseparator
Insert 10.0 Preseparator Base 10.0 Impaction Cups 1 through 3 10.0
Impaction Cups 4 through 6 20.0 Impaction Cup 7 10.0 Microorifice
Collector 5.0 (MOC)
[0452] NGI Impaction Cups do not need to be covered when mixing.
Mixing time should be 3 minutes.
[0453] Preseparator Extraction
[0454] The Preseparator is not extracted into volumetric
glassware.
[0455] Preseparator Top: The Preseparator Top is not extracted.
[0456] Preseparator Insert: The Preseparator Insert will have 10.0
mL of sample solvent added to the central cup during dosing. This
solution will be mixed briefly by pipette in the central cup prior
to transfer to HPLC vial with no additional dilution.
[0457] Preseparator Base: Close the Preseparator Base tightly with
a stopper. Add 10.0 mL sample solvent to the flat portion of the
base. Rinse the entire surface area of the flat portion several
times by pipette. Using this same sample solution, rinse the inner
wall of the stem of the base. Mix the sample solution by
pipette.
[0458] Calculations
[0459] Calculate check standard accuracy using the following
equation:
(A.sub.check
standard)(C.sub.standard)(100%)/(A.sub.standard)(C.sub.check
standard)
[0460] Where:
[0461] A.sub.check standard=Peak area of the MMI-0100 peak in the
check standard solution
[0462] C.sub.standard=Concentration of MMI-0100 in the working
standard solution
[0463] A.sub.standard=Mean peak area of the MMI-0100 peak in the
first five (5) injections of the working standard solution
[0464] C.sub.check standard=Concentration of MMI-0100 in the check
standard solution
[0465] Calculate the amount of MMI-0100 in individual test
solutions in .mu.g using the following equation:
(A.sub.sample)(C.sub.standard)(V.sub.sample)(P)/(A.sub.standard)
[0466] Where:
[0467] A.sub.sample=Peak area of the MMI-0100 peak in the test
solution
[0468] C.sub.standard=Concentration of MMI-0100 in the working
standard solution
[0469] P=Potency factor of the reference substance (if
appilcable)
[0470] A.sub.standard=Mean peak area of the MMI-0100 peak in the
first five (5) injections of the working standard solution
[0471] The blister and device parameters listed in Table 3 were
used as a starting point for optimization of aerosol
performance.
TABLE-US-00003 TABLE 3 Blister, device and test conditions (Final
Conditions) Blister Information Blister design 4.5 mm flat-top
blisters (manufactured at MDTx (Monmouth Junction NJ) with Rohrer
750 equipment Filling information Blister filling inside a glove
box at ambient room temperature and <5% relative humidity Fill
weight Target fill weight .+-.5% (95%-105% target) Foils: Blister,
Lidding Blister Lidding Material and Blister Forming Material:
Alcan (Shelbyville KY) (FIG. 1 and FIG. 2) Sealing Parameters ST3
Sealer Sealing Temperature: 136.degree. C. Sealing Time: 0.5 sec
Pressure set at 100 psi Vacuum Cooling Time: 5 sec Sealing
Information ST3 Sealer was contained in a glove box with
microbalance; blisters were sealed immediately following filling
Blister Stamping Blisters stamped to 15 mm flange using Arbor Press
Blister Height Ames Pneumatic AG-698 (Ames IA) Measurement Air
Guage Range: 4.48-4.63 mm Device Information Platform #(s) EPIC
S0361F-24 Flow channel S0619 Electronics Function generators Drive
Scheme F1 = 39.8 kHz, F2 = 54.0 kHz, 100 Hz Modulation. 90/10 Duty
Cycle Drive Voltage 240 V Transducer on- 2 .times. 2 sec time
Piercing Tool # A0101A-5 (4 .times. 0.011'' OD pins in square
pattern Flow rate 25 L/min
[0472] B. Nebulizer Formulations of MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
[0473] MMI-0100 Formulations:
[0474] Formulation A: 7 mg/mL; 1.8 g of lyophilized peptide weighed
into a volumetric flask containing 200 mL of 0.9% saline.
[0475] Formulation B: 0.7 mg/mL; 0.18 g of lyophilized peptide
weighed into a volumetric flask containing 200 mL of 0.9%
saline.
[0476] Instruments:
[0477] Malvern MasterSizer X V2.15: Malvern Instruments GmbH,
Munchen II
[0478] HPLC Alliance 2695 with column oven, 2487 dual absorbance
detector and
[0479] chromatographic data system (Empower 3); Waters
[0480] Mass flow controller 0-30 l/min, e.g. PR4000; MKS
[0481] Measuring system for relative humidity and temperature, e.g.
testo 645
[0482] Digital manometer, e.g. testo 525
[0483] Precision balance, e.g. Excellence X56035 DR, Mettler
Toledo
[0484] Conditioning system
[0485] Bubble Flow Meter, e.g. Gilibrator2, Gillian
[0486] Breath simulator Z
[0487] Filter pads (Polypropylene)
[0488] Filter casings
[0489] Laboratory shaker e.g. 3015, IKA Werke
[0490] Temperature-/Humidity sensor, e.g. Testo 645, Testo
[0491] Gas meter, G4, Elster Instromet
[0492] Pipette Research 1000, Eppendorf
[0493] Multipette stream, Eppendorf
[0494] Waterbath, e.g. F12; Julabo
[0495] Magnet stirrer, e.g. IKA RCT basic
[0496] Rheometer, e.g. Rheostressl, Haake
[0497] Tensiometer, e.g. science line t60, Sita Messtechnik
[0498] Osmomat, e.g. Gonotec auto
[0499] C. Nano-polyplex Formulations of MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
[0500] Synthesis of Cell Penetrant MK2 Inhibitory Peptide
[0501] MK2 inhibitory peptide (MK2i) MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) was synthesized on a PS3
peptide synthesizer (Protein Technologies, Inc. Tucson, Ariz.)
utilizing standard Fmoc Chemistry. N-methylpyrrolidone (NMP,
Fischer Scientific) was utilized as a solvent in all peptide
syntheses. HCTU (1H-Benzotriazolium
1-[bis(dimethylamino)methylene]-5chloro-,hexafluorophosphate
(1-),3-oxide) was used as an activator (Chempep, Wellington, Fla.)
in the presence of N-methylmorpholine. All amino acids were double
coupled in order to maximize yield and purity. Peptides were
cleaved/deprotected in trifluoroacetic acid
(TFA)/Phenol/H.sub.2O/triisopropylsilane (88/5/5/2). The peptide
was then further purified by reverse phase HPLC on a Waters 1525
binary HPLC pump outfitted with an extended flow kit, a Waters 2489
UV/Visible detector, and a phenomenex Luna C18(2) AXIA packed
column (100A, 250.times.21.2 mm, 5 micron). A) HPLC grade water
with 0.05% formic acid and B) HPLC grade acetonitrile were used as
the mobile phase, and the peptide was purified utilizing a 90% A to
90% B gradient over 25 mins (16 mL/min). Acetonitrile was removed
from purified fractions with a rotary evaporator, and the purified
fractions were then lyophilized. Peptide purity was verified
through electrospray ionization mass spectrometry (ESI-MS) on a
Waters Synapt ESI-MS.
[0502] Monomer and Polymer Synthesis
[0503] All reagents were purchased from Sigma and were of
analytical grade unless otherwise stated. 2-propylacrylic acid was
synthesized according to the procedure outlined by Ferrito et al.
(Macromolecular Syntheses 11, 59-62 (1992)) utilizing diethyl
propylmalonate (Alfa Aesar) as a precursor. The
4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylvpentanoic acid (ECT)
chain transfer agent (CTA) was synthesized as describe by
Convertine et al. (J. Control Release 133,221-229 (2009)).
Reversible addition-fragmentation chain transfer (RAFT)
polymerization of the poly(propylacrylic acid) (PPAA) homopolymer
was carried out in bulk under a nitrogen atmosphere at 70.degree.
C. for 48 hours using 2,2'-azo-bis-isobutyrylnitrile (AIBN) as the
free radical initiator. The reaction mix was put through three
freeze-vacuum-thaw cycles and purged with nitrogen for thirty
minutes prior to polymerization. The molar ratio of chain transfer
agent (CTA) to AIBN was 1 to 1, and the monomer to CTA ratio was
set so that a degree of polymerization (DP) of 190 would be
achieved at 100% conversion. Following polymerization, the
resultant polymer was dissolved in dimethylformamide (DMF) and
precipitated into ether 5 times before drying overnight in vacuo.
RAFT polymerization of the poly(acrylic acid) (PAA) homopolymer was
carried out in distilled dioxane under a nitrogen atmosphere at
70.degree. C. for 18 hours using AIBN as the free radical
initiator. The reaction mix was purged with nitrogen for thirty
minutes prior to polymerization. The molar ratio of CTA to AIBN was
5 to 1 and the monomer to CTA ratio was set so that a degree of
polymerization of 150 would be achieved at 100% conversion.
Following polymerization, the resulting polymer was dissolved in
dioxane and precipitated into ether 5 times before drying overnight
in vacuo. Gel permeation chromatography (GPC, Agilent) was used to
determine molecular weight and polydispersity (M.sub.w/M.sub.n,
PDI) of the PPAA and PAA homopolymers using HPLC-grade DMF
containing 0.1% LiBr at 60.degree. C. as the mobile phase.
Molecular weight calculations were performed with ASTRA V software
(Wyatt Technology) and were based on experimentally-determined
dn/dc values determined through offline injections of the polymer
through a refractive index detector (calculated PPAA dn/dc=0.087
mL/g, DP=193 (GPC), PDI=1.47 (GPC); calculated PAA dn/dc=0.09 mL/g,
DP=150 (GPC), PDI=1.27 (GPC)). Polymer purity and molecular weight
were verified through NMR spectroscopy utilizing D.sub.6MSO as a
solvent (PPAA DP=190 (H.sup.1 NMR); PAA DP=106 (H.sup.1 NMR)).
[0504] MMI-0100 Nano-Polyplex (MK2i-NP) and Phosphor-HSP20
Nano-Plex (HSP20-NP) Synthesis and Characterization
[0505] PPAA was dissolved in 1 M NaOH and diluted into a phosphate
buffer (pH 8) to obtain a stock solution. Purified MMI-0100 peptide
was dissolved in phosphate buffer (pH 8). The MMI-0100 peptide and
PPAA polymer were mixed at a range of charge ratios (CRs) from
[NH.sub.3.sup.+]:[COO.sup.-]=10:1 to 1:10 to form MK2i-NPs. The
resulting polyplexes were syringe filtered through 0.45 .mu.m
polytetrafluoroethylene (PTFE) filter, and the hydrodynamic
diameter and .zeta.-potential were characterized on a Malvern
Zetasizer Nano-ZS with a reusable dip cell kit (Malvern Instruments
Ltd., Worcestershire, U.K.).
[0506] A CR of 1:3 was then chosen as the optimal MK2i-NP
formulation, whereas a charge ratio of 3:1 was chosen as the lead
p-HSP20-NP formulation. These formulations were used in subsequent
in vitro, ex vivo, and in vivo studies. Nano-polyplexes formulated
at the same CR with the non-endosomolytic polymer PAA (i.e.,
NE-MK2i-NPs) were analyzed by dynamic light scattering (DLS) and
used as a vehicle control in all subsequent studies. In order to
verify the sizes indicated by DLS analysis, MK2i-NPs and HSP20-NPs
were visualized through transmission electron microscopy (TEM)
imaging. TEM samples were prepared by inverting carbon film-backed
copper grids (Ted Pella) onto a 20 .mu.L droplet of aqueous
polyplex suspensions (1 mg/mL) and blotted dry. All samples were
then inverted onto a 20 .mu.L droplet of 3% Uranyl Acetate and
stained for 2 min. After blotting the sample dry, samples were
desiccated in vacuo for 2 hr prior to imaging on a Philips CM20
system operating at 200 kV. Images were collected using a
charge-coupled device (CCD) camera with AMT Image capture Engine
software (Advanced Microscopy Techniques, Danvers, Mass.). The
pH-dependent size changes of polypexes at a CR of 1:3 were then
quantified by DLS analysis at various pH values in PBS -/- (i.e. pH
7.4, 6.8, 6.2, and 5.6).
[0507] pH-Dependent Membrane Disruption Hemolysis Assay
[0508] To assess the endosomal disruptive potential of MK2i-NPs, a
red blood cell hemolysis assay was utilized as previously described
by Henry et al. (Biomacromolecules 7,2407-2414 (2006)) to measure
MK2i-NP pH-dependent disruption of lipid bilayers. Whole human
blood was drawn from an anonymous donor, and plasma was removed
through centrifugation and saline washes. The remaining
erythrocytes were washed three times with 150 mM NaCl and
resuspended into phosphate buffers corresponding to physiologic (pH
7.4), early endosome (pH 6.8), early/late endosome (pH 6.2), and
late endosome/lysosomal (pH 5.8) environments. MK2i-NPs,
NE-MK2i-NPs, MMI-0100 (MK2i) peptide alone (1-40 .mu.g/mL), PBS
(negative control), or 1% Triton X-100 (positive control) were
added to the erythrocyte suspensions and incubated at 37.degree. C.
for 1 hour. Intact erythrocytes were pelleted via centrifugation,
and supernatant was transferred to a new 96-well plate. The
hemoglobin content within the supernatant was then measured via
absorbance at 541 nm. Percent hemolysis was determined relative to
Triton X-100 and PBS controls.
[0509] Cell Culture
[0510] Primary human coronary artery vascular smooth muscle cells
(HCAVSMCs) were obtained from Lonza. HCAVSMCs were cultured in
complete growth medium [vascular cell basal medium (ATCC)
supplemented with 5% FBS, human basic fibroblast growth factor
(bFGF, 5 ng/mL), human insulin (5 .mu.g/mL), ascorbic acid (50
.mu.g/mL), L-glutamine (10 mM), human epidermal growth factor (EGF,
5 ng/mL), and 1% penicillin-streptomycin].
[0511] All cultures were maintained in 75 cm.sup.2 polystyrene
tissue culture flasks in a 37.degree. C. and 5% CO.sub.2
environment with cell culture media refreshed every other day.
Cells were grown to 80-90% confluence prior to being harvested and
passaged. All cells were seeded at a density of 20,000-30,000
cells/cm.sup.2, as required for each specific experiment. Only
cells from early passages (numbers 3-8) were used in
experiments.
[0512] Inflammatory Cytokine Analysis
[0513] 200 .mu.L of cell suspension (at 10,000 cells/well) was
seeded onto 96-well plates to yield an approximate 70% confluence
per well. Cells were allowed to adhere to the plate overnight.
[0514] Tumor Necrosis Factor-.alpha. ELISA
[0515] HCAVSMCs were treated in low serum media (DMEM, 1% FBS, and
1% P/S, to achieve cellular quiescence) with 10 .mu.M ANG-II for 4
hours followed by treatment with MK2i-NPs, MK2i, or NE-MK2i-NPs for
2 hours. Following treatment, each well was aspirated and
supplemented with fresh medium. After 24 hours, 100 .mu.L of
supernatant was collected and frozen at -80.degree. C. until
cytokine analysis was performed. A Human TNF-.alpha. (cat #900-K25)
ELISA development kit (Peprotech; Rocky Hill, N.J.) was used to
measure cytokine levels in supernatant collected from treated cells
according to the manufacturer's protocol. Briefly, microtiter
plates (Nunc MaxiSorp, cat. #439454) were prepared by diluting
polyclonal capture antibody with phosphate-buffered saline (PBS;
Gibco BRL, cat. #14200-075) (1.times., pH 7.20) to a concentration
of 1 .mu.g/mL and adding 100 .mu.L of the diluted capture antibody
to each well of the microtiter plate. The plate was sealed and
incubated overnight at room temperature. After incubation, the
wells were aspirated and washed 4 times with 300 .mu.L of wash
buffer (0.05% Tween-20 (Sigma, cat. # P7949) in PBS) per well.
Next, 300 .mu.L of blocking buffer (1% bovine serum albumin (BSA;
Sigma, cat. # A-7030) in PBS) was added to each well and the
microtiter plate was incubated for 1 hour at room temperature.
After incubation, the wells were aspirated and washed 4 times with
300 .mu.L of wash buffer per well. Next, TNF-.alpha. standard was
serially diluted from 0.01 .mu.g/mL to 0 .mu.g/mL in diluent (0.05%
Tween-20, 0.1% BSA in PBS). Diluted standard and samples were added
(100 .mu.L/well) to the microtiter plate in triplicate and the
plate was incubated for 2 hours at room temperature. Wells were
aspirated and the plate was washed 4 times with wash buffer. After
washing, 100 .mu.L of biotinylated detection antibody (at a
concentration of 0.5 .mu.g/mL; 500 ng/mL in diluent) was added to
each well and the microtiter plate was incubated for 2 hours at
room temperature. Following incubation, wells were aspirated and
washed 4 times with wash buffer. Avidin-HRP conjugate (Sigma, cat.
# A-7419) was diluted 1:2000 in diluent and added to each well of
the plate (100 .mu.L/well). The plate was incubated for 30 minutes
at room temperature. After incubation, the wells were aspirated and
the plate was washed 4 times with wash buffer. Next, 100 .mu.L of
ABTS liquid substrate solution (Sigma, cat. # A3219) was added to
each well and the plate was incubated at room temperature for color
development. Plates were read with a plate reader (Molecular
Devices) at 405 nm (650 nm wavelength correction). All data were
then normalized to cell viability determined by a CytoTox-ONE.TM.
Homogenous Membrane Integrity assay (Promega) according to the
manufacturer's protocol. Briefly, 200 .mu.L of a HCAVSMC cell
suspension was seeded (at 10,000 cells/well) onto a 96-well plate
to yield an approximate 70% confluence per well. Cells were allowed
to adhere to the plate overnight. Next, the plate was equilibrated
to 22.degree. C. for approximately 30 minutes. Following
equilibration, 200 .mu.L of YtoTox-ONE.TM. reagent was added to
each well, the plate was shaken for 30 seconds and then incubated
for 10 minutes at 22.degree. C. After incubation, 100 .mu.L of Stop
Solution was added to each well, the plate was shaken for 10
seconds and fluorescence was recorded at an excitation wavelength
of 560 nm and an emission wavelength of 590 nm using a plate reader
(Molecular Devices).
[0516] Interleukin-6 ELISA
[0517] HCAVSMCs were treated in low serum media with 20 ng/mL
TNF-.alpha. for 4 hours followed by treatment with MK2i-NPs,
MMI-0100 (MK2i), or NE-MK2i-NPs for 2 hours. Following treatment,
each well was aspirated and supplemented with fresh medium. After
24 hours, 100 .mu.L of supernatant was collected and frozen at
-80.degree. C. until cytokine analysis could be performed. A human
IL-6 (cat #900-K16) ELISA development kit (Peprotech; Rocky Hill,
N.J.) was used to measure cytokine levels in supernatant collected
from treated cells according to the manufacturer's protocol.
Briefly, microtiter plates (Nunc MaxiSorp, cat. #439454) were
prepared by diluting polyclonal capture antibody with
phosphate-buffered saline (PBS; Gibco BRL, cat. #14200-075)
(1.times., pH 7.20) to a concentration of 1 .mu.g/mL and adding 100
.mu.L of the diluted capture antibody to each well of the
microtiter plate. The plate was sealed and incubated overnight at
room temperature. After incubation, the wells were aspirated and
washed 4 times with 300 .mu.L of wash buffer (0.05% Tween-20
(Sigma, cat. # P7949) in PBS) per well. Next, 300 .mu.L of blocking
buffer (1% bovine serum albumin (BSA; Sigma, cat. # A-7030) in PBS)
was added to each well and the microtiter plate was incubated for 1
hour at room temperature. After incubation, the wells were
aspirated and washed 4 times with 300 .mu.L of wash buffer per
well. Next, IL-6 standard was serially diluted from 0.01 .mu.g/mL
to 0 .mu.g/mL in diluent (0.05% Tween-20, 0.1% BSA in PBS). Diluted
standard and samples were added (100 .mu.L/well) to the microtiter
plate in triplicate and the plate was incubated for 2 hours at room
temperature. Wells were aspirated and the plate was washed 4 times
with wash buffer. After washing, 100 .mu.L of biotinylated
detection antibody (at a concentration of 0.5 .mu.g/mL; 500 ng/mL
in diluent) was added to each well and the microtiter plate was
incubated for 2 hours at room temperature. Following incubation,
wells were aspirated and washed 4 times with wash buffer.
Avidin-HRP conjugate (Sigma, cat. # A-7419) was diluted 1:2000 in
diluent and added to each well of the plate (100 .mu.L/well). The
plate was incubated for 30 minutes at room temperature. After
incubation, the wells were aspirated and the plate was washed 4
times with wash buffer. Next, 100 .mu.L of ABTS liquid substrate
solution (Sigma, cat. # A3219) was added to each well and the plate
was incubated at room temperature for color development. Plates
were read with a plate reader (Molecular Devices) at 405 nm (650 nm
wavelength correction). All data were then normalized to cell
viability determined by a CytoTox-ONE Homogenous Membrane Integrity
assay (Promega) according to the manufacturer's protocol.
Monocyte Chemoattractant Protein-1 (MCP-1) ELISA
[0518] HCAVSMCs were treated in low serum media with MK2i-NPs,
MK2i, or NE-MK2i-NPs for 2 hours. Following treatment, each well
was aspirated and supplemented with fresh medium. After 3 or 5
days, cells were stimulated with TNF-.alpha. (20 ng/ml) for 24
hours. Following stimulation, 100 .mu.l of supernatant was
collected and frozen at -80.degree. C. until cytokine analysis
could be performed. A human monocyte chemoattractant protein-1 (cat
#EH2MCP1) ELISA development kit (ThermoFisher Scientific/Pierce
Biotechnology; Rockford, Ill.) was used to measure cytokine levels
in supernatant collected from treated cells according to the
manufacturer's protocol. Briefly, 50 .mu.L of standard diluent was
added to each well of the anti-human MCP-1 precoated 96-well strip
plate. Next, 50 .mu.L of standards or samples were added to the
strip plate in duplicate, the strip plate was covered with an
adhesive plate sealer and incubated at room temperature for 1 hour.
Following incubation, the strip plate was washed three times with
Wash Buffer. After washing, 100 .mu.L of Biotinylated Antibody
Reagent was added to each well of the strip plate, the plate was
covered with an adhesive plate sealer and incubated at room
temperature for 1 hour. Following incubation, the strip plate was
washed three times with Wash Buffer. Next, 100 .mu.L of
Streptavidin-HRP Solution was added to each well of the strip
plate, the strip plate was covered with an adhesive plate sealer
and incubated at room temperature for 30 minutes. Following
incubation, the strip plate was washed three times with Wash
Buffer. After washing, 100 .mu.L of TMB Substrate Solution was
added to each well of the strip plate and the strip plate was
developed at room temperature for 20 minutes. Next, 100 .mu.L of
Stop Solution was added to each well of the strip plate. Absorbance
was measured on a plate reader (Molecular Devices) at 450 nm (550
nm wavelength correction) and results were calculated using
curve-fitting statistical software.
[0519] Migration Assays
[0520] Scratch Wound Chemokinesis Assay
[0521] HCAVSMCs were seeded in Lab-TEK II 8-well chambered
coverglass at a density of 20,000 cells/well in 250 .mu.l low serum
growth media and allowed to adhere overnight to achieve a nearly
confluent (90-95%) monolayer. Cells were treated with MK2i-NPs,
NE-MK2i-NPs, MMI-0100 (MK2i) peptide or PBS -/- for 30 minutes.
Following treatment, scratch wounds were made with a 10 uL pipette
tip through the middle of each cell monolayer. The media was then
replaced with low serum growth media containing a CellTracker.TM.
Green BODIPY.RTM. dye (Invitrogen) according to the manufacturer's
protocol for thirty minutes to enable visualization of migrating
cells. Following treatment with the dye, media was replaced with
low serum growth media containing 50 ng/ml platelet-derived growth
factor-BB (PDGF-BB) (or with PBS -/- for the negative control).
Scratch wound areas were then imaged at 0, 3, 6, 12, and 24 hours
using a Nikon Eclipse Ti inverted fluorescence microscope (Nikon
Instruments Inc, Melville, N.Y.) with NIS Elements imaging
software. Wound closure was calculated with imageJ software by
quantifying the scratch wound area around the periphery of
migrating cells normalized to the original scratch wound area.
Scratch wound assays for each treatment group were performed in 3
independent experiments.
[0522] Boyden Chamber Chemotaxis Assay
[0523] HCAVSMCs were seeded in a 24 well plate at a density of
30,000 cells/well in low serum media (DMEM, 1% FBS, and 1% P/S) and
allowed to adhere overnight. Cells were treated for 30 mins with
MK2i-NPs, NE-MK2i-NPs, MMI-0100 (MK2i) peptide, or PBS. Following
treatment, each well was washed 2.times. with PBS -/-, trypsinized,
resuspended in 100 .mu.l low serum growth media, and plated onto
6.5 mm, 8 .mu.m pore polycarbonate inserts (Corning) in a 24 well
plate with 600 .mu.l low serum growth media containing 50 ng/ml
PDGF-BB (or PBS -/- for the negative control) in the lower chamber.
Cells were allowed to migrate for 8 hours, and then cells on the
upper side of each insert were gently removed with a cotton swab.
Cells on the lower side of each insert were then fixed and stained
using a Modified Giemsa Differential Quik Stain Kit (Polysciences).
Inserts were fixed in solution A for at least 10 seconds, dipped 5
times in solution B, and then dipped 5 times in solution C. After
staining, 4 images were taken from the four quadrants of each
insert, and the number of cells/high power field were quantified in
ImageJ by thresholding each image and manually counting the cells.
Each treatment was performed in triplicate, and average cell
#/field was calculated.
[0524] Cell Proliferation Assay
[0525] HCAVSMCs were seeded in a 96 well plate at 10,000 cells/well
in low serum media (DMEM, 1% FBS, and 1% P/S) and allowed to adhere
overnight. Cells were treated for 30 minutes with MK2i-NPs,
NE-MK2i-NPs, MMI-0100 (MK2i) peptide or PBS -/- (for positive and
negative controls). Each treatment was then aspirated and replaced
with 100 .mu.l low serum growth media .+-.50 ng/mL PDGF-BB. After
24 hours of incubation, a CellTiter 96.RTM. Aqueous Non-Radioactive
Cell Proliferation Assay (Promega) was performed according to the
manufacturer's protocol. Briefly, 100 .mu.l phenazine methosulfate
(PMS) solution was added to 2.0 ml MTS solution and mixed. 20 .mu.l
of PMS/MTS solution was then added to each well of the 96 well
plate containing 100 .mu.l medium, and the plate was incubated for
4 hours at 37.degree. C. in a humidified, 5% CO.sub.2 atmosphere.
Following incubation, the absorbance of each well was recorded at
490 nm with a TECAN Infinite M1000 Pro plate reader to determine
the relative proliferation rate of all treatment groups.
[0526] Microscopic Analysis of Cellular Uptake and Intracellular
Trafficking
[0527] An amine-reactive Alexa-488 succinimidyl ester was dissolved
in DMSO and mixed at a 1 to 3 molar ratio with the MMI-0100 (MK2i)
peptide in 100 mM sodium bicarbonate buffer (pH=8.3). Unreacted
fluorophore and organic solvent were removed using a PD-10 miditrap
G-10 desalting column, and the fluorescently labeled peptide was
lyophilized. PPAA and PAA polymers were mixed with fluorescently
labeled MMI-0100 (MK2i) peptide at a CR of
[NH.sub.3.sup.+]/[COO.sup.-]=1:3 and syringe filtered through a
0.45 .mu.m PTFE filter to form fluorescent MK2i-NPs and control
NE-MK2i-NPs, respectively. Fluorescent MK2i-NP and NE-MK2i-NP
hydrodynamic diameter and surface charge were measured by DLS and
Zeta potential analysis, respectively. Fluorescent MK2i-NPs,
NE-MK2i-NPs, or MMI-0100 (MK2i) peptide alone were applied to
HCAVSMCs grown on Lab-Tek II 8-well chambered coverglass (Thermo
Scientific Nunc) at a concentration of 10 .mu.M MMI-0100 (MK2i)
peptide in DMEM media supplemented with 1% FBS and 1% P/S. Cells
were treated for 2 hours, washed 2.times. with PBS -/-, and media
was replaced. Cells were then incubated for an additional 0, 2, 4,
10, or 22 hours in fresh media. For the final two hours of
incubation, 50 nM Lysotracker Red DND-99 (Invitrogen) was added to
each well in order to visualize acidic endo/lysosomal vesicles
within cells. After incubation, cells were washed with 0.1% trypan
blue for 1 minute to quench extracellular fluorescence followed by
2 additional washes with PBS -/-. Cells were then imaged using a
LSM 710 META fluorescence microscope with ZEN imaging software
(Carl Zeiss Thornwood, N.Y.). Gain settings were kept constant for
all images acquired.
[0528] All images were processed using ImageJ and colocalization
was analyzed using Just Another Colocalization Plugin (JACoP)(62).
Mander's overlap coefficients (the fraction of pixels with positive
pixel values in both fluorescent channels) were then calculated for
n.gtoreq.3 separate images for each treatment group to quantify
colocalization. To determine treatment effects on the size of the
compartments where the peptide was found, the free hand selection
tool in ImageJ was used to outline n.gtoreq.50 individual
intracellular compartments for each treatment group, and the area
of each was quantified and averaged.
[0529] Flow Cytometric Quantification of Intracellular Uptake and
Retention
[0530] HCAVSMCs were grown to 80-90% confluence, harvested, and
seeded at 20,000 cells/well in a 24 well plate and allowed to
adhere overnight in low serum media (DMEM, 1% FBS, and 1% P/S).
Fluorescent MMI-0100 (MK2i) peptide, MK2i-NPs, and NE-MK2i-NPs were
synthesized as noted above for microscopy analysis, and HCAVSMCs
were treated at a concentration of 10 .mu.M MMI-0100 (MK2i) for 2
hours. Following treatment, cells were washed with PBS -/-, washed
with CellScrub buffer (Genlantis) for 10 minutes at room
temperature to remove extracellular polyplexes and/or peptide,
washed 2.times. in PBS -/-, and given fresh complete growth media.
Cells were then incubated for an additional 0, 12, 24, 72, or 120
hours. Cells were then washed with PBS -/-, trypsinized, and
resuspended in 0.1% Trypan blue in PBS (-/-) for analysis on a
FACSCalibur flow cytometer (Becton Dickinson) with BD CellQuest.TM.
Pro software (V 5.2). Data was exported and analyzed with FlowJo
software (V 7.6.4). All samples were run in triplicate.
[0531] For MK2i-NP and HSP20-NP studies, An amine-reactive
Alexa-488 succinimidyl ester (Life Technologies) was dissolved in
DMSO and mixed at a 1 to 3 molar ratio with the MK2i or p-HSP20
peptide in 100 mM sodium bicarbonate buffer (pH=8.3) and allowed to
react for 3 hours. Unreacted fluorophore and organic solvent were
removed using a PD-10 miditrap G-10 desalting column, and the
fluorescently labeled MK2i and p-HSP20 peptides were lyophilized.
PPAA polymer was mixed with fluorescently labeled MK2i peptide at a
CR of [NH.sub.3.sup.+]/[COO.sup.-]=1:3 and syringe filtered through
a 0.45 .mu.m PTFE filter to form fluorescent MK2i-NPs. Similarly,
PPAA was mixed with fluorescently labeled p-HSP20 at a CR of
[NH.sub.3.sup.+]/[COO.sup.-]=1:3 and syringe filtered through a
0.45 .mu.m PTFE filter to form fluorescent HSP20-NPs. HCAVSMCs were
grown to 80-90% confluence, harvested, and seeded at 20,000
cells/well in a 24 well plate and allowed to adhere overnight.
HCAVSMCs were treated with fluorescent MK2i peptide, MK2i-NPs,
p-HSP20 peptide, p-HSP20-NPs, or PBS as a control at a
concentration of 10 .mu.M peptide in Opti-MEM medium supplemented
with 1% penicillin-streptomycin for 30 minutes. Following
treatment, cells were washed 2.times. in PBS, and either
immediately harvested or incubated in complete growth media for an
additional 72 hours. Cells were harvested with 0.05% trypsin-EDTA,
centrifuged, and suspended in 0.1% Trypan blue in PBS (-/-) for
analysis on a FACSCalibur flow cytometer (Becton Dickinson) with BD
CellQuest.TM. Pro software (V 5.2). Data was exported and analyzed
with FlowJo software (V 7.6.4). All samples were run in
triplicate.
[0532] The intracellular MK2i half-life (t.sub.1/2) was calculated
by exponential decay nonlinear regression analysis of intracellular
peptide fluorescence at 0 and 5 days following treatment removal
using the exponential decay function [where N=intracellular
fluorescence and .lamda.=the decay rate]:
N(t)=N.sub.oe.sup.-.lamda.t (eq. 51)
And calculating the t.sub.1/2 from the decay constant of each
exponential decay function as follows:
t.sub.1/2=ln(2)/.lamda. (eq. 52)
[0533] Human Saphenous Vein (HSV)
[0534] De-identified, discarded segments of HSV were collected from
consented patients undergoing coronary or peripheral vascular
bypass surgeries. Following surgical resection, HSV segments were
stored in saline solution until the end of the surgical procedure,
at which time they were placed in cold transplant harvest buffer
(100 mM potassium lactobionate, 25 mM KH.sub.2PO.sub.4, 5 mM
MgSO.sub.4, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM
allopurinol, 50 g/L hydroxyethyl starch, pH 7.4). All HSV segments
were used within 24 hours of harvest. Utilizing sterile technique
in a sterile culture hood, HSV segments were transferred to a 60 mm
Petri dish. The end of each segment (0.5 mm) was removed with a
blade, and excess adventitial and adipose tissue was removed with
minimal manipulation. HSV segments were cut into consecutive rings
with an approximate width of 1.0 mm to be utilized in organ culture
experiments. Two rings from each segment were immediately fixed in
10% formalin at 37.degree. C. for 30 min to obtain pre-culture
intimal thickness measurements.
[0535] Prior to experiments, HSV viability was confirmed. HSV rings
were weighed and their lengths recorded. HSV rings were then
suspended in a muscle bath containing a bicarbonate buffer (120 mM
NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM glucose, 1.5
mM CaCl2, and 25 mM Na2HCO3, pH 7.4) equilibrated with 95% 02 and
5% CO2 at 37.degree. C. The rings were stretched and the length
progressively adjusted until maximal tension was obtained49.
Normalized reactivity was obtained by determining the passive
length-tension relationship for each vessel segment. Rings were
maintained at a resting tension of 1 g, which produces maximal
responses to contractile agonists, as previously determined, and
equilibrated for 2 h in buffer. Force measurements were obtained
using a Radnoti Glass Technology (Monrovia, Calif.) force
transducer (159901A) interfaced with a Powerlab data acquisition
system and LabChart software (AD Instruments, Colorado Springs,
Colo.).
[0536] HSV rings were initially isometrically contracted with 110
mM KCl (with equimolar replacement of NaCl in bicarbonate buffer),
and the generated force was measured. 110 mM KCl causes membrane
depolarization, leading to contraction of vessels containing
functionally viable smooth muscle. After vessel viability was
verified with multiple KCl challenges, additional rings were cut to
be utilized in smooth muscle physiology experiments and for F-actin
staining.
[0537] HSV Smooth Muscle Physiology Studies
[0538] Inhibition of HSV Contraction
[0539] Viable HSV rings were washed, allowed to equilibrate in
bicarbonate solution for 30 min, and then contracted with
phenylephrine (PE, 1 .mu.M). All rings were washed and equilibrated
in fresh buffer and allowed to relax until baseline contraction was
achieved. Rings were then incubated with either MK2i peptide,
MK2i-NPs, p-HSP20 peptide, p-HSP20-NPs, or buffer alone for 2 h.
Treated HSV rings were then contracted with the same doses of PE,
and the forces generated were again recorded. Measured force was
normalized for ring weight and length and percent inhibition of
contraction was calculated by dividing the post-treatment
contractile force with the pre-treatment contractile force;
pre-treatment force generated with 1 .mu.M PE was set as 100%
contraction. Data was obtained in HSV from n.gtoreq.3 separate
patients.
[0540] Enhanced HSV Vasorelaxation
[0541] Viable HSV rings were washed and allowed to equilibrate in
bicarbonate solution for 30 min, and then contracted with
phenylephrine (PE, 1 .mu.M). Rings were relaxed with a cumulative
log dose of sodium nitroprusside (SNP, 0.1-10 .mu.M), a nitric
oxide donor, and the resulting decrease in contractile force was
recorded over time. All rings were again washed and equilibrated in
buffer for 15 min. Rings were then incubated with either MK2i
peptide, MK2i-NPs, p-HSP20, p-HSP20-NPs, or buffer alone for 2 h,
followed by treatment with the same doses of PE and SNP. The forces
generated were again recorded, and measured force was normalized
for ring weight and length and percent relaxation was calculated;
force generated with 100 .mu.M PE was set as 0% relaxation. Data
was obtained in HSV from n.gtoreq.3 separate patients.
[0542] Actin Staining of Angiotensin II Stimulated HSV
[0543] Viable HSV rings were placed in a 24 well plate in RPMI
medium supplemented with 10% FBS and 1% penicillin-streptomycin and
allowed to equilibrate in an incubator at 37.degree. C. and 5%
CO.sub.2 for several hours. HSV rings were then treated with 100
.mu.M MK2i peptide, 100 .mu.M MK2i-NPs, 500 .mu.M p-HSP20, or 500
.mu.M p-HSP20-NPs or PBS -/- as a negative control for 30 minutes
in Opti-MEM medium supplemented with 1% penicillin-streptomycin and
then washed 2.times. in PBS -/-. Subsequently, treated HSV rings
were stimulated with 10 .mu.M angiotensin II for 2 hours and then
washed 2.times. in PBS -/-. HSV rings were then immediately fixed
in 4% paraformaldehyde for 4 hours at 37.degree. C. HSV rings were
then incubated overnight in 30% sucrose in 1.times.PBS -/-. HSV
rings were washed 2.times. in PBS -/-, embedded in OCT and frozen.
10 micron cryosections were cut from the midportion of each HSV
rings and placed onto SuperFrost Plus microscope slides (Fisher
Scientific). The slides were then stained and imaged according to
the procedure stated in the F-actin stress fiber assay section
above. Full HSV sections were compiled through the image stitching
capability in the NIS Elements software.
[0544] HSV Organ Culture and Assay for Ex Vivo Intimal Hyperplasia
(IH)
[0545] Prior to organ culture experiments, HSV viability was
confirmed. HSV rings were weighed and their lengths recorded. HSV
rings were then suspended in a muscle bath containing a bicarbonate
buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO.sub.4, 1.0 mM
NaH.sub.2PO.sub.4, 10 mM glucose, 1.5 mM CaCl.sub.2, and 25 mM
Na.sub.2HCO.sub.3, pH 7.4) equilibrated with 95% O.sub.2 and 5%
CO.sub.2 at 37.degree. C. The rings were stretched and the length
progressively adjusted until maximal tension was obtained.
Normalized reactivity was obtained by determining the passive
length-tension relationship for each vessel segment. Rings were
maintained at a resting tension of 1 g, which produces maximal
responses to contractile agonists, as previously determined, and
equilibrated for 2 hr in buffer. Force measurements were obtained
using a Radnoti Glass Technology (Monrovia, Calif.) force
transducer (159901A) interfaced with a Powerlab data acquisition
system and Chart software (AD Instruments, Colorado Springs,
Colo.).
[0546] HSV rings were initially contracted with 110 mM KCl (with
equimolar replacement of NaCl in bicarbonate buffer) and the force
generated was measured. 110 mM KCl causes membrane depolarization,
leading to contraction of vessels containing functionally viable
smooth muscle. After vessel viability was verified with multiple
KCl challenges, additional rings were cut and placed in a 24 well
plate and maintained in RPMI 1640 medium supplemented with 30% FBS,
1% L-glutamine and 1% penicillin/streptomycin for 14 days at
37.degree. C. in an atmosphere of 5% CO2 in air. The rings were
untreated, treated with MK2i-NPs, NE-MK2i-NPs, MMI-0100 (MK2i)
peptide, or buffer alone for 2 hours, washed, and given fresh
media. The culture medium without treatments was replaced every 2
days for 14 days.
[0547] HSV Viability
[0548] To ensure that the treatments did not impact tissue
viability, an MTT assay (Life Technologies) for assessing cell
viability was performed on HSV rings at 1 and 14 days after
treatment. HSV rings were prepared and treated as noted above, and
following 1 or 14 days of organ culture, HSV rings were weighed and
then placed in 250 .mu.L of 0.01% methyl tetrazolium dissolved in
DPBS. The rings were placed in a 37.degree. C. incubator for 1
hour. The reaction was stopped by placing the rings into distilled
water. The rings were then placed into 1 mL of CelloSolve and
incubated at 37.degree. C. overnight. Following incubation, rings
were mixed in solution, and the CelloSolve was extracted and placed
into a cuvette where the optical density at 570 nm was determined.
Relative viability calculations were based on the optical density
normalized to the wet weight of the ring.
[0549] Vessel Morphometry
[0550] After 14 days of organ culture, vein segments were fixed in
0.5 ml of 10% formalin at 37.degree. C. for 30 min and embedded in
paraffin for sectioning. Beginning at the midportion of each ring,
5 transverse sections, spaced 5 .mu.m apart, were cut from each
specimen. Sections were then stained with Verhoeff-van Gieson
stain. Histology sections were imaged using a Nikon Eclipse Ti
inverted fluorescence microscope (Nikon Instruments Inc, Melville,
N.Y.), and 6 radially parallel measurements of intimal and medial
thickness were randomly taken from each section using NIS Elements
imaging software (total of 6-12 measurements per ring, n.gtoreq.3
rings per treatment group from separate donors). Intima was defined
as tissue on the luminal side of the internal elastic lamina or the
chaotic organization of the cells contained within it, whereas the
medial layer was contained between the intimal layer and the
external elastic lamina. Intimal and medial thickening was measured
for each section at 10.times. magnification with the microscope's
computerized image analysis software.
[0551] Microscopic Analysis of MK2i Delivery to HSV
[0552] After verifying viability, HSV rings were treated with
Alexa-568 labeled MMI-0100 (MK2i) peptide, MK2i-NPs, or NE-MK2i-NPs
for 30 minutes, washed 2.times. in PBS -/-, and immediately
embedded in optimal cutting temperature (OCT) compound (Fisher
Scientific) and frozen over dry ice. 5 .mu.m cryosections were cut
from the middle of each treated vessel and mounted on microscope
slides for analysis of peptide delivery into the vessel wall.
Immunofluorescence staining was then carried out with CD31 and
.alpha.-SMA primary antibodies and a FAM labeled secondary
antibody. Microscopy images were obtained using a Nikon Eclipse Ti
inverted fluorescence microscope or a LSM 710 META fluorescence
microscope with ZEN imaging software (Carl Zeiss Thornwood, N.Y.).
Gain settings were kept constant for all images acquired for every
treatment group, and images were stitched together in Adobe
Photoshop to provide a macroscopic image of the entire section of
the HSV ring.
[0553] Western Blot Analysis
[0554] Following 2 hours of treatment with MMI-0100 (MK2i) peptide,
a portion of the treated HSV rings was snap-frozen with liquid
nitrogen, pulverized, and homogenized using urea-DTT-CHAPS buffer.
For analysis of heterogeneous nuclear ribonucleoprotein A0 (hnRNP
A0) phosphorylation, treated HSV rings were maintained in organ
culture in fresh media for 24 hours prior to homogenization. For
analysis of CREB and HSP27 phosphorylation, HSV rings were frozen
after the 2 hour treatment. Lysates were centrifuged (6000 g, 20
minutes), and the supernatant was collected for evaluation of hnRNP
A0, cAMP response element-binding (CREB) protein, and heat shock
protein 27 (HSP27) phosphorylation. Equal amounts of protein (20
.mu.g per lane) were loaded on 15, 10, or 4-20% SDS-PAGE gels;
proteins were electrophoretically separated, and then transferred
to Immobilon membranes (Millipore, Billerica, Mass.). For hnRNP A0
phosphorylation, membranes were probed overnight at 4.degree. C.
with primary antibodies for phospho-hnRNP A0 (Millipore) and
unphosphorylated hnRNP A0 (Santa Cruz). For CREB phosphorylation,
membranes were probed overnight at 4.degree. C. with primary
antibodies for phospho-CREB (abcam) and unphosphorylated CREB
(abcam). For HSP27 phsophorylation membranes were probed overnight
at 4.degree. C. with primary antibodies for phospho-HSP27
(Epitomics) and unphosphorylated HSP27 (Santa Cruz). After washing,
the membranes were incubated with appropriate secondary antibodies
(Li-Cor) for 1 hour at room temperature. The secondary antibody was
imaged using the Odyssey direct infrared fluorescence imaging
system (Li-Cor) and densitometrically quantified with LiCor Odyssey
software v2.1 at 800 and 680 nm wavelengths. For each biological
replicate, all treated samples were normalized to untreated control
tissue.
[0555] For MK2i-NP and HSP20-NP studies, western blot analysis of
the cytosolic and organelle fractions from the digitonin
semi-permeabilization procedure was performed. Briefly, cytosolic
and organelle fractions were concentrated on a centrifuge using
Vivacon 500 DNA concentrators (2000 MWCO). Equal amounts of protein
(20 .mu.g per lane) were loaded on 4-20% SDS-PAGE gels; proteins
were electrophoretically separated and then transferred to
Immobilon membranes. The membranes were then probed overnight at
4.degree. C. with primary antibodies for the cytosolic proteins
mitogen-activated protein kinase kinase 1/2 (MEK1/2) and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the
endo-lysosomal markers early endosomal antigen 1 (EEA1) and
lysosomal-associated protein 1 (LAMP1). All antibodies were
obtained from Cell Signaling Technologies. After washing, the
membranes were incubated with appropriate secondary antibodies
(Li-Cor) for 1 hour at room temperature. The secondary antibody was
imaged using the Odyssey direct infrared fluorescence imaging
system and densitometrically quantified with LiCor Odyssey software
v2.1 at 800 and 680 nm wavelengths.
[0556] Rabbit Bilateral Jugular Vein Graft Interposition Model
[0557] Male New Zealand White rabbits (3.0-3.5 kg; n=24) were
anesthetized b an intramuscular injection with ketamine
hydrochloride (1.4 mg/kg) and xylazine (0.2 mg/kg). Anesthesia was
maintained with endotracheal intubation and inhaled isoflurane
(2.0-5.0%). A high-dose IV heparin bolus (250 U/kg) was
administered immediately prior to carotid cross clamp. The
operative procedure was performed with aseptic technique under
optical magnification (magnification .times.2.5).
[0558] Vein bypass grafts were constructed with an anastomotic cuff
technique as described by Jiang et al. (Am. J. Physiol. Heart Circ.
Phyisol. 286, H240-245 (21004). Briefly, polymer cuffs consisting
of a 2.0-mm body loop were fashioned from a 4-Fr introducer sheath
(Terumo Medical, Elkton, Md.). Following ligation of smaller
tributary vessels, the external jugular veins were harvested
(3.0-4.0 cm in length) for creation of an interposition graft into
the common carotid artery. Jugular vein ends were passed through a
cuff, everted, and fixed with 6-0 silk. Vein grafts were
subsequently treated for 30 minutes in 2 mL of Heparin Plasma-Lyte
solution containing either 30 .mu.M MK2i-NP, 30 .mu.M MMI-0100
(MK2i) peptide, or PBS (no treatment). Following treatment, the
carotid artery lumen was exposed with a 2.0-cm arteriotomy, and the
cuffed, reversed vein ends were inserted. A 3-0 silk was used to
secure the artery around the cuff. Finally, 1.0 cm of carotid
artery back wall was cut away between the cuffs to permit vein
graft extension.
[0559] Rabbits were euthanized at 28 days post-operatively, and
vein grafts were perfusion fixed in situ with 10% neutral buffered
formalin under .about.50 mm Hg pressure with a roller pump. Vein
grafts were subsequently excised and sectioned into four segments
avoiding the tissue overlying the cuff in order to allow for
evaluation of morphological variation along the length of the
graft. Histological sections were prepared, and intimal and medial
thicknesses were quantified by taking 3 measurements from each
quadrant of each vessel section (12 measurements/segment=48
measurements/graft). Separate sections were stained with the rabbit
macrophage antibody RAM-11 (Dako) to evaluate treatment effect on
the infiltration of immune cells into the intima of each graft.
Macrophage positive staining in the intima was quantified by
manually counting the number of positively stained cells in the
intima of stained graft sections. 16 histological images from 4
different graft sections were analyzed for each treatment
group.
[0560] Cytotoxicity Assay
[0561] 200 .mu.L of cell suspension (at 10,000 cells/well) were
seeded onto 96-well plates to yield an approximate 70% confluence
per well. Cells were allowed to adhere to the plate overnight.
Cells were then treated with 10, 50, 100, and 500 .mu.M doses of
MK2i-NPs, p-HSP20-NPs, MK2i peptide, p-HSP20 peptide, or PBS as a
control treatment for 2 hours in Opti-MEM medium supplemented with
1% penicillin-streptomycin. Treatments were subsequently removed
and the cells were cultured in fresh complete growth medium for 24
hours. Cells were then washed 2.times. with PBS +/+ and cell
viability was then determined by a CytoTox-ONE Homogenous Membrane
Integrity assay (Promega) according to the manufacturer's protocol.
Briefly, 100 .mu.L of Ambion KDalert Lysis Buffer was added to each
well, and then 100 .mu.L of freshly prepared CytoTox-ONE reagent
was added to each well. After 10 minutes of incubation, 50 .mu.L of
stop solution was added, and the fluorescence of each well
(.lamda..sub.ex=560 nm, .lamda..sub.em=590 nm) was determined with
a TECAN Infinite M1000 Pro plate reader.
[0562] F-Actin Stress Fiber Assay
[0563] HCAVSMCs were seeded in Lab-Tek II 8-well chambered
coverglass (Thermo Scientific Nunc) at 15,000 cells/well and
allowed to adhere overnight. Cells were then treated in low serum
media (Optimem, 1% FBS, and 1% P/S) with MK2i-NPs, p-HSP20-NPs,
MK2i peptide, p-HSP20 peptide, or at concentrations of 10, 25, and
50 .mu.M (PBS -/- as a control) for 1 hour. Following treatment,
cells were washed 2.times. with PBS -/- and subsequently treated
with 1 .mu.M Angiotensin II (Sigma Aldrich) or PBS -/- (negative
control) for 2 hours. After ANG-II stimulation cells were washed
2.times. with PBS, fixed in 4% paraformaldehyde for 5 minutes,
permeabilized with 0.4% Triton-X 100 for 10 minutes, and blocked
with 1% BSA in PBS -/- for 15 minutes. Cells were then stained with
Hoechst solution (1/5000 dilution in PBS -/-) for 10 minutes
followed by staining with Alexa-488-Phallodin (Life Technologies)
for 30 minutes according to the manufacturer's instructions.
Stained coverslips were then inverted onto glass cover slides with
ProLong Gold Antifade mounting medium (Invitrogen). Slides were
allowed to dry for 24 hours prior to sealing and imaging. Treated
cells were imaged using a Nikon Eclipse Ti inverted fluorescence
microscope (Nikon Instruments Inc, Melville, N.Y.) with NIS
Elements imaging software. Gain settings and exposure times were
kept constant for all images taken. The number of stress fibers per
cell was quantified as previously described.sup.48. Briefly, in the
NIS elements software, 3 separate intensity profiles were generated
across the axis of stained cells perpendicular to the cell's
polarity. Prior to image analysis, the background noise from each
image was removed using a rolling ball background subtraction
filter with a radius of 70 pixels. A fluorescence level of 2000 RFU
was set as the threshold for positive F-actin fiber staining as the
background fluorescence outside of the stained cells was never
greater than this value. The stress fibers per cell were then
quantified from the average of 3 intensity profiles from n.gtoreq.6
cells from 2 separate experiments for each treatment group (total
n.gtoreq.36 ROIs for each treatment group). Relative quantification
of cellular F-actin content was further quantified using imageJ
software to free hand select individual cells and to calculate the
relative fluorescence intensity of n.gtoreq.12 cells from 2
separate experiments for each treatment group.
[0564] Quantification of Cytosolic Vs. Organelle Bound Peptide
Through Semi-Permeabilization
[0565] In order to quantify the cytosolic bioavailability of the
MK2i and HSP20 peptides a method to separate cytosolic and
organelle bound (i.e. endosomal, lysosomal, golgi, etc.) peptide
was adapted from the methods developed by Liu et al40. The
procedure was optimized for this experiment based upon LDH release
from HCAVSMCs treated with varying concentrations of digitonin
(Calbiochem) in buffer (150 mM NaCl, 0.2 mM EDTA, 20 mM HEPES-NaOH
(pH 7.4), 2 mM DTT and 2 mM MgCL2) on ice for 10 mins on a rotary
shaker operating at 100 RPM (supplementary FIG. 3). A concentration
of 25 .mu.g/mL was then chosen as the optimal digitonin
concentration for selective semi-permeabilization of the HCAVSMC
membrane and subsequently used for the analysis of intracellular
peptide distribution.
[0566] To quantify intracellular distribution of the MK2i and
p-HSP20 peptides, HCAVSMCs were seeded into a 96 well plate at a
density of 20,000 cells/cm2 and allowed to adhere overnight in
complete growth medium. A portion of the cells were pretreated with
500 nM Bafilomycin A1 (Sigma) for 30 minutes, and the Bafilomycin
was included in subsequent peptide/NP treatment and in the
post-treatment incubation phase to inhibit endosomal acidification.
Cells were then treated with Alexa-488 labeled MK2i peptide,
MK2i-NPs, p-HSP20 peptide, p-HSP20-NPs at a concentration of 10
.mu.M peptide (or PBS -/- as a control) in Opti-MEM medium
supplemented with 1% penicillin-streptomycin with or without 500 nM
Bafilomycin A1 for 30 minutes. Treatments were removed and cells
were incubated in fresh medium with or without 500 nM Bafilomycin
A1 for 6 hours. Each well was then washed 1.times. with ice cold
PBS +/+ and then subsequently incubated with 20 uL of 25 .mu.g/mL
digitonin solution at 0.degree. C. (on ice) on a rotary shaker
operating at 100 RPM for 10 minutes. The supernatant from each well
was then transferred to a new 96 well plate, and each well was
washed with 80 .mu.L ice cold PBS +/+ which was then transferred to
the 96 well plate containing the digitonin (cytosolic) fractions.
100 uL of 1% triton X-100 in PBS -/- was then added to each well to
obtain a 96 well plate containing all non-cytosolic (i.e. organelle
bound) cellular components, and the fluorescence of each well
(.lamda.ex=495 nm, .lamda.em=519 nm) was determined with a TECAN
Infinite M1000 Pro plate reader. Readings were normalized to cell
number and cytosolic content as determined by a CytoTox-ONE
Homogenous Membrane Integrity assay (Promega) according to the
manufacturer's protocol (section 4.5).
[0567] Statistics
[0568] Statistical analysis was performed with one-way ANOVA
followed by Tukey's post-hoc test to compare experimental groups.
Analyses were done with OriginPro 8 software (Originlab,
Northampton, Mass.) or Minitab 16 software (State College, Pa.).
Statistical significance was accepted within a 95% confidence
limit. Results are presented as arithmetic mean.+-.SEM graphically
and p-values are included within figures or in the figure
legends.
Example 1. Dry Powder Formulations of MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
[0569] Gravimetric clearance testing of blisters filled with 1 and
2 mg of neat spray dried MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID
NO: 1) 5% solids formulation was used to optimize and determine
aerosol performance. Blisters were filled inside a glove box at
4-5% relative humidity. Sealing of blisters occurred inside the
glove box using a bench-top heat sealer. An EPIC style inhaler was
coupled with function generators to perform all aerosol tests.
Table 3 contains information on final (optimal) blister, device and
test conditions.
[0570] Uptake of water by the spray dried formulation was
investigated. Dynamic Vapor Sorption (DVS) Isotherm confirmed the
rapid uptake of water in the spray dried formulation as the %
relative humidity (RH) was increased over time (FIG. 3). In order
to maintain less than 3% water in the formulation, the DVS
indicated that the material should be handled at less than 20% RH
(FIG. 3). In addition, a controlled charge dissipating unit was
installed inside a glove box to neutralize the positively charged
filling station (i.e., due to the glove box's polycarbonate
construction) Negative ions were released by a pulsed DC controller
around the vicinity of filling to neutralize positive charges.
[0571] A rapid HPLC method was developed to determine, for example,
formulation impurities and the concentration of MMI-0100 contained
in the formulations. Briefly, a Supelco Ascentis Express.RTM.
Peptide ES-C18 column was used. The flow rate, column temperature,
and mobile phase were adjusted to give a run time of 3 minutes.
Evaluation of the linearity of the rapid HPLC method indicated
acceptable linearity between 6.5 to 32 mg/mL based on % response
factor of the mean response factor for the 10.8 .mu.g/mL working
standard (97.0 to 101.4% of the mean response factor). A decrease
in linearity was observed at 2.2 .mu.g/mL, however, this decrease
was at an acceptable level for limit of quantitation (LOQ) to
quantitate low deposition next generation impactor (NGI) stages
(such as microorifice collector (MOC)). An example chromatogram of
the working standard is shown in FIG. 4. A summary of the final
HPLC method parameters are listed below:
Column: Supelco Ascentis Express.RTM. Peptide ES-C18, 50.times.4.6
mm, 2.7 Jim
[0572] Flow rate: 1.5 mL/min Injection volume: 40 .mu.L
Column Temperature: 40.degree. C.
Sample Temperature: 5.degree. C.
Detector Wavelength: 215 nm
Mobile Phase A: 0.1% TFA in Water (72%)
Mobile Phase B: 0.1% TFA in 1:1 Methanol:Acetonitrile (28%).
[0573] A next generation impactor (NGI) method was developed. NGI
cups were coated with 5% Tween 20 in methanol. Blisters filled with
formulation neat spray dried MMI-0100 5% w/w solids were dosed at
15 L/min using an EPIC device as outlined in Table 3. Although the
use of a preseparator is not typically required for non-lactose
based formulations, it was included to collect possible large
aggregates. All NGI components were initially extracted with 10 mL
of 0.02% Tween 20 in water as the sample solvent. Adjustments to
the extraction volumes varied throughout the project based on the
fill weight and amount of MMI-0100 dosed into the impactor.
Recovery was assessed and method alterations were developed to
maintain recoveries of greater than 85%.
[0574] Aerosol performance was evaluated and optimized using an
EPIC inhaler similar to the device shown in FIG. 5. A function
generator set-up was used affording greater flexibility in drive
scheme development. Blisters filled with 1 and 2 mg of the 5%
solids and 2 mg of the 1% solids formulation were evaluated for
gravimetric clearance to assess powder clearance from the blister
and device. A drive scheme consisting of a single pulse of 2.0
second duration was used with the EPIC inhaler with a standard EPIC
flow channel. Since the baseline clearance results were acceptable
(>80% mass cleared from the blister), NGI tests were performed
to evaluate the aerodynamic particle size distribution (PSD). Table
4 contains the aerosol results summary. FIG. 6 shows the particle
size distribution plots.
TABLE-US-00004 TABLE 4 Initial Aerosol Performance Results 1.0 mg
Fill Weight 2.0 mg Fill Weight 5% Solids - Spray Dried MMI-0100 5%
w/w solids Gravimetric Clearance = 88.3% Gravimetric Clearance =
Loaded Dose = 775 .mu.g 83.0% Delivered Dose = 500 .mu.g Loaded
Dose = 1563 .mu.g Fine Particle Dose (FPD) .ltoreq.5.0 .mu.m = 379
.mu.g Delivered Dose = 1105 .mu.g FPD .ltoreq.3.0 .mu.m = 206 .mu.g
FPD .ltoreq.5.0 .mu.m = 650 .mu.g Mass Median Aerodynamic Diameter
FPD .ltoreq.3.0 .mu.m = 305 .mu.g (MMAD) = 3.2 .mu.m MMAD = 3.8
.mu.m 1% Solids - Spray Dried MMI-0100 1% w/w Solids Not tested
(higher fill weights were Gravimetric Clearance = preferred to
maximize the FPD) 88.3% Loaded Dose = 1580 .mu.g Delivered Dose =
1183 .mu.g FPD .ltoreq.5.0 .mu.m = 508 .mu.g FPD .ltoreq.3.0 .mu.m
= 352 .mu.g MMAD = 3.6 .mu.m
[0575] The results of initial aerosol performance testing indicated
that the 5% and 1% solids formulations can be disbursed from the
inhaler with good efficiency and offered a good starting point for
optimization to reduce the MMAD values closer to the target of 2
.mu.m. The results at 1.0 mg fill weight were closest to the target
MMAD at 3.2 .mu.m. At the 2 mg dose level, the 1% solids
formulation provided a finer distribution as shown by the higher
Fine Particle Dose (FPD)<3.0 .mu.m and particle size
distribution centered around stage 5 of the NGI (FIG. 6). With
additional optimization, the 1% solids formulation was more likely
to meet the aerosol performance targets as defined at the onset of
the project and therefore was selected as the lead formulation
moving forward.
[0576] In an effort to increase the fill weight above 2 mg,
modifications were made to the existing flow channel of the inhaler
to increase the air velocity over the pierced holes of the blister.
Without being bound by theory, an increase in the air velocity is
thought to increase the rate of clearance of particles from the
blister. The gravimetric clearance of blisters filled with up to 10
mg of the 1% solids formulation was found to be acceptable
(>90%) at a flow rate of 25 L/min. Three NGI tests were
performed at fill weights of 5 and 8 mg, and a single NGI was
performed to assess the feasibility of dosing 10 mg. These results
are summarized in Table 5 and FIG. 7. Error bars are included for
the 5 and 8 mg fill weights. The 5 mg aerosol performance tests
were highly reproducible.
TABLE-US-00005 TABLE 5 Aerosol Performance Results at Fill Weights
up to 10 mg After Optimization Fill FPD .ltoreq. FPF .ltoreq. 5.0
FPD .ltoreq. FPF .ltoreq. 3.0 Weight % Delivered 5.0 .mu.m .mu.m (%
of 3.0 .mu.m .mu.m (% of MMAD (mg) Clearance Dose (.mu.g) (.mu.g)
Delivered) (.mu.g) Delivered) (.mu.m) 5 92.1 3135 2791 89.0 2362
75.3 2.1 8 94.9 5249 4615 87.9 3675 70.0 2.2 10 95.3 6575 5839 88.8
4658 70.8 2.2 *FPF = Fine Particle Fraction
[0577] Device optimization permitted efficient formulation
dispersion as noted by increased Fine Particle Dose (FPD),
decreased MMAD, and decreased throat and pre-separator retention.
The resulting MMADs of 2.1 to 2.2 .mu.m met the project target and
the successful delivery of 10 mg of formulation results in a Fine
Particle Dose <3.0 .mu.m of 4.7 mg. The results from 5 to 10 mg
also indicate dose linearity which will allow for adjustment of
both the fill weight and number of blisters to achieve the required
clinical doses (See FIG. 8 for linearity plot).
[0578] Using identical device conditions, the formulations co-spray
dried with 7.5 and 20% Trehalose (Santa Cruz Biotechnology, Inc.
Dallas Tex.) were screened for aerosol performance by performing a
single NGI for each at a fill weight of 5 mg. The results are
summarized and compared to the neat formulation at 5 mg in Table 6
and FIG. 9.
TABLE-US-00006 TABLE 6 Aerosol Performance Results with Trehalose
Variants at a Fill Weight of 5 mg FPD .ltoreq. FPF .ltoreq. 5.0
.mu.m FPD .ltoreq. FPF .ltoreq. 3.0 .mu.m % % Delivered 5.0 .mu.m
(% of 3.0 .mu.m (% of MMAD Trehalose Clearance Dose (.mu.g) (.mu.g)
Delivered) (.mu.g) Delivered) (.mu.m) 0 (Neat) 92.1 3135 2791 89.0
2362 75.3 2.1 7.5 90.4 2812 2370 84.3 2087 74.2 2.0 20 91.6 2423
2248 92.8 1967 81.2 2.0
[0579] The trehalose variants at percentages of 7.5 and 20% showed
very similar aerosol distribution compared to the neat formulation
at the same fill weight. This demonstrated that MMI-0100 can be
successfully co-spray dried with trehalose and efficiently
dispersed from the inhaler with little or no change in performance
over the neat formulation.
[0580] Two stability studies were conducted to assess the effect of
various conditions on the aerosol performance and impurities of the
MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) formulations.
Blisters were filled with 5 mg of each of the four formulations
(Neat Spray MMI-0100 5% w/w solids; Neat Spray MMI-0100 1% w/w
solids; Spray Dried 80/20 MMI-0100/Tehalose 1% w/w solids; Spray
Dried 92/5/7.5 MMI-0100/Trehalose 1% w/w solids). Blisters were
placed in a 1.times.5 blister holder and sealed into an aluminum
pouch. The pouch blisters were stored, pulled and tested for
aerosol performance (n=3 NGI tests per pull condition) according to
Table 7.
TABLE-US-00007 TABLE 7 Blister Stability Storage and Pull Schedule
Storage Condition 0 (Initial) 2 weeks 4 weeks Ambient X N/A N/A
40.degree. C.75% Relative humidity N/A X X 25.degree. C./60%
Relative humidity N/A X X 2-8.degree. C. N/A N/A X
[0581] Chemical stability in blisters was tested using 5% solids
neat MMI-0100 formulation. Blisters were filled with 10 mg, placed
in a 1.times.5 blister holder and sealed into an aluminum pouch.
The pouched blisters were stored at 40.degree. C./75% relative
humidity, pulled at 2 and 4 weeks, and tested for assay and
impurities.
[0582] Bulk stability was tested using approximately 50 mg of 1%
and 5% solid MMI-0100 formulations. Formulations were transferred
to amber glass vials, caps were wrapped with parafilm and the
entire vial was placed into an aluminum overwrap pouch and sealed.
For trehalose variants, the original glass bottle was treated in a
similar manner. Each vial was placed into a stability chamber at
40.degree. C./75% relative humidity and pulled after 4 weeks for
assay and impurities testing.
[0583] The stability results with respect to aerosol performance
(n=3 NGI) for all four formulations stored in single dose blisters
with overwrap pouch at 5 mg fill weight are presented in Table 8,
Table 9, and Table 10.
TABLE-US-00008 TABLE 8 Stability Results for Formulations after 4
Weeks Storage in Blisters at 2-8.degree. C. MMI- MMI- 92.5/7.5
80/20 0100 0100 MMI-0100/ MMI-0100/ 1% 5% Trehalose Trehalose w/w
w/w 1% w/w 1% w/w solids solids solids solids % solids in water on
spray drying 1 5 1 1 % Trehalose 0 0 7.5 20 Mean Theoretical Drug
Load (.mu.g) 3948 3913 3647 3162 % Gravimetric Clearance 95.8 95.8
95.4 94.8 Derived Delivered Dose (DDD) (.mu.g) 3260 3292 2976 2599
% DDD of Initial 96.8 93.6 99.5 99.6 FPD .ltoreq. 5.0 .mu.m (.mu.g)
2886 2613 2634 2344 FPD .ltoreq. 5.0 .mu.m (.mu.g) (% of Initial)
97.5 93.4 101.0 99.7 FPD .ltoreq. 3.0 .mu.m (.mu.g) 2437 1769 2230
2032 FPD .ltoreq. 3.0 .mu.m (.mu.g) (% of Initial) 98.4 95.2 100.8
100.7 MMAD (.mu.m) 2.1 2.7 2.1 2.0 Geometric Standard Deviation
(GSD) 1.6 1.6 1.6 1.5
TABLE-US-00009 TABLE 9 Stability Results for Formulations after 4
Weeks Storage in Blisters at 25.degree. C./60% RH MMI- MMI-
92.5/7.5 80/20 0100 0100 MMI-0100/ MMI-0100/ 1% 5% Trehalose
Trehalose w/w w/w 1% w/w 1% w/w solids solids solids solids %
solids in water on spray drying 1 5 1 1 % Trehalose 0 0 7.5 20 Mean
Theoretical Drug Load (.mu.g) 3982 3925 3578 3077 % Gravimetric
Clearance 95.2 95.4 95.8 95.7 Derived Delivered Dose (DDD) (.mu.g)
3256 3402 2941 2525 % DDD of Initial 96.7 96.8 98.4 96.7 FPD
.ltoreq. 5.0 .mu.m (.mu.g) 2840 2720 2522 2291 FPD .ltoreq. 5.0
.mu.m (.mu.g) (% of Initial) 95.9 97.2 96.7 97.4 FPD .ltoreq. 3.0
.mu.m (.mu.g) 2375 1783 2134 1978 FPD .ltoreq. 3.0 .mu.m (.mu.g) (%
of Initial) 95.9 95.9 96.4 98.0 MMAD (.mu.m) 2.1 2.7 2.1 2.0
Geometric Standard Deviation (GSD) 1.6 1.5 1.7 1.5
TABLE-US-00010 TABLE 10 Stability Results for Formulations after 4
Weeks Storage in Blisters at 40.degree. C./75% RH MMI- MMI-
92.5/7.5 80/20 0100 0100 MMI-0100/ MMI-0100/ 1% 5% Trehalose
Trehalose w/w w/w 1% w/w 1% w/w solids solids solids solids %
solids in water on spray drying 1 5 1 1 % Trehalose 0 0 7.5 20 Mean
Theoretical Drug Load (.mu.g) 3925 3914 3606 3145 % Gravimetric
Clearance 95.2 94.4 94.2 94.3 Derived Delivered Dose (DDD) (.mu.g)
3223 3333 2939 2532 % DDD of Initial 95.7 94.8 98.3 97.0 FPD
.ltoreq. 5.0 .mu.m (.mu.g) 2742 2499 2472 2222 FPD .ltoreq. 5.0
.mu.m (.mu.g) (% of Initial) 92.6 89.3 94.8 94.5 FPD .ltoreq. 3.0
.mu.m (.mu.g) 2309 1695 2096 1890 FPD .ltoreq. 3.0 .mu.m (.mu.g) (%
of Initial) 93.3 91.1 94.7 93.6 MMAD (.mu.m) 2.1 2.7 2.1 2.1
Geometric Standard Deviation (GSD) 1.7 1.7 1.8 1.6
[0584] The results indicate a less than 10% change in aerosol
performance from the initial time point for all formulation
variants except for the 5% solids formulation (10.7% change). The
three MMI-0100 formulations containing 1% solids are stable for up
to 4 weeks at 40.degree. C./75% RH giving them an effective shelf
life of 3-4 months at ambient conditions when placed in an overwrap
pouch. There was essentially no difference in performance from the
addition of trehalose to the formulation with either the 7.5% or
the 20% variants, in terms of aerosol performance. A representative
particle size distribution plot from the 1% solids/0% trehalose
formulation after 4 weeks storage at 40.degree. C./75% RH is shown
in FIG. 10. The particle size distribution for each of the
formulations at each stability condition at 0, 2, and 4 weeks as
well as a complete listing of the aerosol results can be found in
FIGS. 11-22. The impurities and MMI-0100 content for the 5% solids
formulation were also assessed after storage in single dose
blisters within a foil overwrap pouch after 2 and 4 weeks at
40.degree. C./75% RH. The 5% formulation was used for this study
based upon available remaining supply of material. The results are
summarized in Table 11.
TABLE-US-00011 TABLE 11 Impurities and Content Summary for Single
Dose Blisters- 5% Solids Formulation Impurities Assay Total
Impurity % Sample Total Peaks Content (% Area) Content Initial
Initial 6 0.9 100.6 2 Weeks 40.degree. C./75% RH 6 0.9 100.0
25.degree. C./60% RH 6 0.9 100.9 2-8.degree. C. N/A N/A N/A 4 Weeks
40.degree. C./75% RH 7 1.2 99.4 25.degree. C./60% RH 7 0.9 100.2
2-8.degree. C. 6 0.8 100.4
[0585] There was a slight decrease in assay content at 40.degree.
C./75% RH after 4 weeks (from 100.6 to 99.4%) with one extra
unidentified peak detected in the impurity profile. The impurity
profile and % content were stable at all other time-points and
conditions. This data also supports an effective shelf life of 3-4
months at ambient conditions for the 5% solids formulation.
[0586] The assay and impurity profile of the formulations stored in
bulk in glass jars after 4 weeks storage at 40.degree. C./75% RH is
summarized in Table 12. There was not enough available formulation
to determine aerosol performance of samples stored in bulk (by
filling and dosing blisters after the time point). Again, due to
limited stock of formulation, the trehalose containing formulations
were not assessed at the initial time point. Initial results for
neat formulations were determined during method transfer of the
assay/impurity method. Samples were handled/prepared in the same
manner.
TABLE-US-00012 TABLE 12 Impurities and Content Summary for
Formulations Stored in Glass for 4 Weeks at 40.degree. C./75% RH
Impurities Assay Total Impurity % Sample Total Peaks Content (%
Area) Content Initial 92.5/7.5 MMI- N/A N/A N/A 0100/Trehalose 1%
w/w solids 80/20 MMI- N/A N/A N/A 0100/Trehalose 1% w/w solids
MMI-0100 6 0.9 100.5 1% w/w solids MMI-0100 6 0.9 100.6 5% w/w
solids 4 Weeks 92.5/7.5 MMI- 6 1.0 96.2 0100/Trehalose 1% w/w
solids 80/20 MMI- 6 1.0 97.2 0100/Trehalose 1% w/w solids MMI-0100
6 1.1 100.4 1% w/w solids MMI-0100 7 1.4 98.7 5% w/w solids
[0587] The stability results for the 1% solids formulation stored
in glass bottles exhibited little change from the initial results.
The 5% solids formulation showed some increase in impurity content
from 0.9 to 1.4% with a corresponding decrease in assay content
from 100.6 to 98.7%, and an increase in the number of peaks
observed (from 6 to 7). The trehalose containing formulations were
not tested at initial, but the results after 4 weeks are in the
range of the results obtained for the 1% neat formulation, in terms
of total impurities and number of peaks. Without being bound by
theory, based on the improved stability of the 1% neat formulations
when stored in bulk in glass, it is possible that the 1%
formulations would also be stable in blisters, in terms of chemical
stability (based on the data for the 5% neat formulation in
blisters).
Example 2. Nebulizer Formulations of MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
[0588] In this study, aerosolization of MMI-0100 inhalation
formulations at two concentrations was characterized using an
electronic nebulizer containing a vibrating mesh with pore sizes of
3 .mu.m and 4 .mu.m (Type 1 and Type 2, respectively). Laser
defraction measurements were used to determine droplet size
distribution. Breath simulation experiments were performed to
determine delivered dose and nebulization time. In addition,
physicochemical parameters (e.g., viscosity, surface tension,
osmolality and density) were determined. The study design is
outlined in Table 13.
TABLE-US-00013 TABLE 13 Nebulizer Formulation Study Design Task
Description of Task 1 Lyophilized MMI-0100 peptide Two
concentrations of MMI-0100 solutions were prepared by dissolving
the lyophilized MMI-0100 peptide in 0.9% NaCl (saline): Formulation
A: 7 mg/mL; Formulation B: 0.7 mg/mL in order to cover the range of
theoretical delivered dose of 5-200 .mu.g/kg 2 Physicochemical
characterization was performed on both formulations with respect
to: Viscosity Surface tension Osmolality Density 3 Laser
diffraction measurements of MMI-0100 formulations upon nebulization
with Nebulizer Type 1 and Nebulizer Type 2. For each nebulizer
type, three were analyzed in duplicate (=12 measurements per
concentration for 24 measurements) Target fill volume was 2 mL of
Formulation A and B each Information obtained from these
experiments included: Mass Median Diameter (MMD (.mu.g)) Respirable
fraction (RF (droplets <5 .mu.m (%))) Geometric Standard
Deviation (GSD) Total output rate (TOR (mg/min)) 4 Breath
simulation upon nebulization of two (2) fill volumes (1 mL and 4
mL) of MMI-0100 of each formulation (Formulation A and Formulation
B) using Nebulizer Type 1 and Nebulizer Type 2). For each nebulizer
type, three were analyzed in duplicate (=48 measurements). Adult
breathing pattern was applied: Tidal volume = 500 mL; Frequency =
15 breath/min; Inhalation/Exhalation ratio = 1 Results obtained
were: Nebulization time (min) -Respirable Dose (RD (.mu.g in
droplets <5 .mu.m) = dose which is expected to reach lungs)
calculated from laser diffraction measurement and breath simulation
Samples from breath simulation experiments were analyzed using HPLC
Prior to breath simulation experiments, filter recovery tests for
method qualification were conducted (n = 3)
[0589] Assessment of geometric droplet size distribution was
performed by laser diffraction (Malvern MasterSizerX). FIG. 27
shows a schematic of the laser diffraction test set-up. Fill volume
was 2 mL for each test solution. Before testing of the
formulations, the nebulizer was tested with 0.9% NaCl (saline)
solution. Results of the laser diffraction measurements are
displayed in Table 14.
TABLE-US-00014 TABLE 14 Comparison of Values of Laser Diffraction
Measurements for the Tested MMI-0100 Formulations and 0.9% Saline
Solution Results RF < MMD 5 .mu.m RF < 3.3 .mu.m TOR
Formulation (.mu.m) GSD (%) (%) (mg/mL) Nebulizer Type 1 B Mean
3.30 1.51 83.79 50.09 353.17 (0.7 mg/mL) SD 0.07 0.00 1.28 1.91
17.79 RSD 2.15 0.19 1.53 3.82 5.04 Nebulizer Type 2 Mean 4.39 1.63
61.90 28.34 900.50 SD 0.23 0.10 6.17 1.26 193.39 RSD 5.27 6.18 9.96
4.46 21.48 Nebulizer Type 1 A Mean 3.03 1.53 86.40 57.12 352.33
(7.0 mg/mL) SD 0.07 0.01 1.26 2.00 44.16 RSD 2.37 0.68 1.46 3.50
12.53 Nebulizer Type 2 Mean 4.03 1.65 67.58 35.15 797.17 SD 0.10
0.02 1.61 1.57 35.92 RSD 2.55 0.93 2.38 4.46 4.51 Nebulizer Type 1
0.9% Saline Mean 3.26 1.56 82.33 50.99 370 SD 0.04 0.01 0.78 0.95
54 RSD 1.2 0.5 0.9 1.9 14.7 Nebulizer Type 2 Mean 4.44 1.68 59.70
28.95 922 SD 0.21 0.06 4.12 1.72 79 RSD 4.7 3.8 6.9 6.0 8.5 TOR =
total output rate (mg/mL); mass of aerosol delivered per minute; SD
= standard deviation; RSD = relative standard deviation
[0590] Filter recovery was determined using 0.9% saline for sample
extraction from inhalation filters. Briefly, approximately 1,000 mg
of formulation A (7.0 mg/mL) was nebulized on an inhalation filter
(n=3) while a constant airflow was applied to the filter by a pump.
After the application of Formulation A, the filter pads were placed
in a 50 mL conical tube containing 30 mL of 0.9% saline and shaken
at 250 rpm for up to 4 hours. Samples (approximately 800 .mu.L)
were collected after 0.5, 1, 2, 3 and 4 hours. Results of the
filter recovery experiment are displayed in Table 15 and and
graphically represented in FIG. 28.
TABLE-US-00015 TABLE 15 MMI-0100 Recovery (%) from Filter
Extraction After Increasing Extraction Times with 0.9% Saline
Recovery (%) of MMI-0100 after different extraction times Filter
No. 0.5 hr 1 hr 2 hr 3 hr 4 hr #1 97.1 97.0 99.2 97.2 96.0 #2 94.3
93.6 95.1 94.9 92.9 #3 96.5 96.2 97.5 97.0 96.0 Mean 96.0 95.6 97.2
96.4 95.0 SD 1.2 1.5 1.7 1.1 1.5 SD = standard deviation
[0591] A maximum of roughly 96% recovery was reached after 0.5 hr
extraction time. Longer extraction times (1, 2, 3 and 4 hr) did not
improve recovery.
[0592] Breath simulations were conducted using an adult breathing
pattern (Tidal Volume: 500 mL, Breath per minute: 15;
Inhalation/Exhalation ratio: 50:50). Table 16 contains fill volumes
chosen to meet desired respirable doses of 5-200 .mu.g/kg (assuming
an average weight of 70 kg).
TABLE-US-00016 TABLE 16 Fill Volumes for Formulation A and
Formulation B Formuation A B Concentration 7 mg/mL 0.7 mg/mL Fill
Volume 1 mL 4 mL 1 mL 4 mL
[0593] Fill volumes were loaded into a medication cup of a
nebulizer connected to a sinus pump. Inspiratory filters were
installed between the nebulizer, including the mouth piece and the
pump, and fixed with rubber connectors. The nebulizer filled with
the formulation was driven until the automatic shut off stopped the
device. The MMI-0100-containing aerosol was collected on inhalation
polypropylene inhalation filters. After nebulization, the
inhalation filters were removed from the filter casings with
forceps and were put in glass vials with plastic screw caps. The
filter casings were rinsed with 0.9% saline and the saline was
collected in 50 mL conical tubes. Corresponding filters were
transferred to the conical tubes containing 0.9% saline and shaken
at 250 rpm for 0.5 hr. After 0.5 hr., HPLC analysis was used to
determine extracted MMI-0100 from the filters. The nebulizer was
rinsed several times with 0.9% saline and the saline was collected
in a glass beaker.
[0594] Peptide content of the saline samples was determined by
gradient HPLC with linear standard calibration. The HPLC instrument
and settings were as follows:
[0595] HPLC with column oven, UV detector and chromatographic data
system;
[0596] Zorbax 300SB, 3.5 .mu.m, 150.times.3.0 mm (L.times.ID)
column (or equivalent);
[0597] Column temperature: 25.degree. C.;
[0598] Sample temperature: 4.degree. C.;
[0599] Flow: 0.5 mL/min;
[0600] Mobile Phase A: 0.1% trifluoroacetic acid (TFA) in
water;
[0601] Mobile Phase B: 0.1% TFA in acetonitrile/methanol
(50:50);
[0602] Injection volume: 20 .mu.L;
[0603] Run time: 15 minutes; and
[0604] Detector wavelength: 215 nm.
[0605] The HPLC gradient used is shown in Table 17.
TABLE-US-00017 TABLE 17 Gradient Table Time (mm) Flow (mL/min) %
Phase A % Phase B Curve 0.00 0.50 68.0 32.0 6 5.00 0.50 63.0 37.0 6
6.00 0.50 10.0 90.0 6 9.00 0.50 10.0 90.0 6 10.00 0.50 68.0 32.0 6
15.00 0.50 68.0 32.0 6
[0606] Accuracy by recovery and method precision experiments were
performed. MMI-0100 was weighed and dissolved in 0.9% saline and
determined by the HPLC method described. Samples 2 and 4 from the
accuracy by recovery experiment were divided into six (6) vials
each and used in the method precision experiment. The results of
these experiments are shown in Tables 18 and 19. This HPLC method
was able to determine an MMI-0100 peptide content in the range of
12-600 .mu.g/mL.
TABLE-US-00018 TABLE 18 Accuracy by Recovery Concentration
Concentration (target) (actual) Recovery Sample (.mu.g/mL)
(.mu.g/mL) (%) 1 600.43 589.53 98.18 2 300.22 286.53 95.44 3 120.09
123.78 103.08 4 48.03 49.60 103.25 5 24.02 24.69 102.80 Mean 100.55
SD 3.55 RSD (%) 3.5 SD = standard deviation RSD = relative standard
deviation
TABLE-US-00019 TABLE 19 Method Precision Vial Sample 2
Concentration Sample 4 Concentration No. (.mu.g/mL) (.mu.g/mL) Vial
1 284.62 49.62 Vial 2 285.72 49.76 Vial 3 285.86 49.6 Vial 4 287.09
49.63 Vial 5 288.24 49.77 Vial 6 287.67 49.19 Mean 286.53 49.60 SD
1.36 0.21 RSD 0.48 0.43
[0607] Results of the breath simulation experiments are summarized
in Tables 20 and 21 and FIGS. 29-33.
TABLE-US-00020 TABLE 20 Breath Simulation Data Summarized for
Nebulizer Type 1 Formulation B: MMI-0100 (0.7 mg/mL) A: MMI-0100
(7.0 mg/mL) Label claim 700 .mu.g/mL 7000 .mu.g/mL Fill volume mL 1
mL 4 mL 1 mL 4 mL Number of n = 6 n = 6 n = 6 n = 6 replicates
Filled drug mg 0.72 2.84 6.93 27.68 amount (based on determined
values of the formulations) Deposition of Nebulized Formulation DD
mg 0.43 1.81 5.13 20.07 SD 0.03 0.05 0.24 0.57 DD % 59.8 63.6 74.0
72.5 SD 3.5 1.6 3.4 2.0 Residue % 0.0 6.3 5.0 7.3 SD 0.0 0.8 0.7
0.8 Nebulized Time Time min 3.34 11.39 3.40 15.81 SD 0.33 1.28 0.57
1.96 Caluculated Values RD < 5 .mu.m mg 0.36 1.51 4.43 17.34 SD
0.02 0.06 0.24 0.69 RD < 5 .mu.m % 50.07 53.28 63.96 62.66 SD
2.94 2.19 3.55 2.49 RD < 3.3 .mu.m mg 0.22 0.91 2.93 11.47 SD
0.02 0.07 0.19 0.63 RD < 3.3 .mu.m % 29.92 31.87 42.29 41.43 SD
2.19 2.32 2.80 2.28 DD = delivered dose SD = standard deviation RD
= respirable dose
TABLE-US-00021 TABLE 21 Breath Simulation Data Summarized for
Nebulizer Type 2 Formulation B: MMI-0100 (0.7 mg/mL) A: MMI-0100
(7.0 mg/mL) Label claim 700 .mu.g/mL 7000 .mu.g/mL Fill volume mL 1
mL 4 mL 1 mL 4 mL Number of n = 6 n = 6 n = 6 n = 6 replicates
Filled drug mg 0.71 2.84 6.97 27.59 amount (based on determined
values of the formulations) Deposition of Nebulized Formulation DD
mg 0.34 1.50 4.71 17.62 SD 0.01 0.07 0.38 0.57 DD % 48.1 52.7 67.6
63.9 SD 1.6 2.3 5.6 2.3 Residue % 2.6 17.4 15.1 17.1 SD 6.4 2.8 5.1
3.0 Nebulized Time Time min 1.16 3.99 1.28 4.37 SD 0.09 0.23 0.14
0.36 Caluculated Values RD < 5 .mu.m mg 0.21 0.93 3.19 11.91 SD
0.03 0.11 0.40 1.16 RD < 5 .mu.m % 29.81 32.69 45.74 43.19 SD
3.61 3.93 5.79 4.42 RD < 3.3 .mu.m mg 0.10 0.42 1.66 6.20 SD
0.01 0.03 0.21 0.60 RD < 3.3 .mu.m % 13.63 14.95 23.82 22.47 SD
0.94 1.07 3.12 2.28 DD = delivered dose SD = standard deviation RD
= respirable dose
[0608] The Delivered Dose (DD [mg] or [%]) represents the amount of
MMI-0100 delivered to the patient assuming a specified breathing
pattern. The respirable doses <x pm (RD <x .mu.m [mg] or [%])
gives the amount of MMI-0100 contained in the part of the droplets
<x pm. The droplet size defines where the particles in the
aerosol cloud are likely to deposit. Without being bound by theory,
it is assumed that, to be therapeutically effective, particles
should be in the range of 1-5 .mu.m in order to deposit in the
lungs. In contrast, particles with >5 .mu.m will generally
impact in the oropharynx and be swallowed, whereas particles below
<1 .mu.m will remain entrained in the air stream and be exhaled.
Respirable dose is calculated by multiplying the DD [mg] with the
percentage of the Respirable Fraction (RF [%]]) determined by laser
diffraction measurement.
[0609] FIGS. 29 and 30 show that there is a linear correlation
between the filled drug amount and the amount of drug delivered (DD
[mg]) as well as the amount respired into the lungs given as the
respirable dose <5 .mu.m (RD<5 .mu.m). The linearity is given
for both nebulizer devices (Nebulizer Type 1 and Nebulizer Type 2).
Based on the results, nebulization performance appears to be
independent of formulation concentration.
[0610] Physicochemical characterization was performed on both
MMI-0100 formulations with respect to osmolality, viscosity,
surface tension and density. The results of each experiment are
shown in Table 22.
TABLE-US-00022 TABLE 22 Physicochemical Characterization of
Formulation A and Formulation B Formulation A B Concentration mg/mL
7.0 0.7 Osmolality Osmol/kg 0.297 (SD 0.001) 0.286 (SD 0.001)
Dynamic Viscosity mPa s 1.04 (SD 0.01) 0.99 (SD 0.01) (20.degree.
C.) Surface Tension mN/m 65.0 (SD 0.2) 67.5 (SD 0.1) Density
(23.8.degree. C.) g/cm.sup.3 1.0047 1.0031 SD = standard
deviation
[0611] The results of these experiments indicate that the mass
median diameter (MMD) for Formulation A (Nebulizer Type 1=3.0
.mu.m; Nebulizer Type 2=4.0 .mu.m) was slightly less than that of
Formulation B (Nebulizer Type 1=3.3 .mu.m; Nebulizer Type 2=4.4
.mu.m). These values were comparable to the data determined for
pure 0.9% saline. Likewise, geometrical standard deviation (GSD),
respirable fraction (RF) and total output rate (TOR) values were
also slightly less for Formulation A as compared to Formulation B.
A linear correlation was found to exist between the delivered dose
(respirable dose <5 .mu.m) and the filled MMI-0100 amount.
Without being bound by theory, this correlation can be used to
calculate the amount of MMI-0100 administered to a patient via a
nebulizer device.
Example 3. Nano-Polyplex (NP) Formulations of MMI-0100
(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)
[0612] Synthesis and Physicochemical Characterization of MMI-0100
(MK2i)-NPs
[0613] Formulation of the positively charged, CPP-based MMI-0100
(MK2i) peptide with the anionic, endosomolytic polymer
poly(propylacrylic acid) (PPAA) was conceptualized as a method to
enhance peptide endolysosomal escape and therapeutic potency. This
approach was inspired by the convention for nonviral delivery of
nucleic acids, which is based on electrostatic formation of
polyplexes between anionic nucleic acids and positively charged CPP
sequences, lipids, or polymeric transfection agents to enhance
uptake and endosome escape (K. A. Mislick et al., Proc Natl Acad
Sci USA 93, 12349-12354 (1996); J. P. Richard et al., J Biol Chem
280, 15300-15306 (2005); C. E. Nelson et al., ACS Nano 7, 8870-8880
(2013)).
[0614] MMI-0100 (MK2i) peptide (YARAAARQARAKALARQLGVAA; SEQ ID NO:
1) was synthesized via solid phase synthesis and purity was
verified through electrospray-ionization mass spectrometry (FIG.
40). Reversible addition fragmentation chain transfer (RAFT)
polymerization was utilized to synthesize poly(acrylic acid) (PAA)
[DP=150 (GPC), DP=106 (H.sup.1 NMR), PDI=1.27 (GPC) FIGS. 41A and
42A] and poly(propylacrylic acid) (PPAA) [DP=193 (GPC), DP=190
(H.sup.1 NMR), PDI=1.47 (GPC) FIGS. 41B and 42B]. NPs were formed
by simple mixing of the PAA or PPAA homopolymers with the MMI-0100
(MK2i) peptide in PBS at pH 8.0, which is between the pKa values of
the primary amines present on the MMI-0100 (MK2i) peptide and the
carboxylic acid moieties in the PPAA polymer; this ensures optimal
solubility and net charge on both molecules (FIG. 35A). PPAA was
utilized because of its well-defined pH-dependent membrane
disruptive activity (R. A. Jones et al., Biochem J 372, 65-75
(2003); C. A. Lackey et al., Bioconjugate Chemistry 13, 996-1001
(2002); N. Murthy et al., J Control Release 61, 137-143 (1999); S.
Foster et al., Bioconjug Chem 21, 22015-2212 (2010)) and previous
safe use in animal model (S. Foster et al., Bioconjug Chem 21,
2205-2212 (2010); E. Crownover et al., J Control Release 155,
167-174 (2011) (FIG. 35B). PAA was utilized as a vector control as
it is an anionic polymer with structural similarity to PPAA but
lacks pH-response in a physiologically relevant range due to its
lower pKa (pKa-4.3) (FIG. 35C).
[0615] To determine optimal nanoparticle formulation conditions, a
library of MK2i-NPs was prepared at a range of charge ratios [i.e.
CR=([NH.sub.3.sup.+].sub.MK2i:[COO.sup.-].sub.PPAA)], and the size
distribution and particle surface charge were characterized through
dynamic light scattering (DLS) and .zeta.-potential analysis,
respectively. Table 23 contains a size summary of MMI-0100
(MK2i)-NPs prepared at different charge ratios as determined by DLS
analysis. As expected, MK2i-NP .zeta.-potential was directly
proportional to the CR, with an apparent isoelectric point at CR
.about.2:1 (FIG. 35D). The CR also significantly affected MMI-0100
(MK2i)-NP size, with only a narrow range of CRs yielding a unimodal
size distribution (i.e. CR=1:2 and 1:3, supplementary table 1). A
CR of 1:3 was chosen as the optimal formulation as this ratio
consistently yielded a unimodal size distribution with minimal
particle size and polydispersity (d.sub.h=119.+-.28 nm,
.zeta.=-11.9.+-.3.2 mV). Non-endosomolytic MK2i nano-polyplexes
(NE-MK2i-NPs) were formulated with PAA as a vehicle control for
biological studies. NE-MK2i-NPs prepared at CR=1:3 with PAA had
size and .zeta.-potential statistically equivalent to the
endosomolytic MK2i-NPs (d.sub.h=114.+-.38 nm, .zeta.=-16.4.+-.5.1
mV). Fluorescent MMI-0100 (MK2i)-NPs and NE-MK2i-NPs were prepared
with an Alexa-488 conjugated MMI-0100 (MK2i) peptide at a CR of 1:3
in order to enable intracellular tracking and yielded similar size
and charge to the unlabeled NPs. NPs prepared at a CR=1:3 were also
characterized through TEM imaging (FIG. 43), which was in agreement
with DLS results. The PPAA-MK2i formulations yielded net negatively
charged NPs.
TABLE-US-00023 TABLE 23 Size Summary of MMI-0100 (MK2i)-NPs
Prepared at Different Charge Ratios NH2:COOH Z-ave Diameter (nm)
10:1 10.32 .+-. 2.63* 2:1 52.1 .+-. 46.86* 1:1 970.6 .+-. 662.4
1:1.5 465.1 .+-. 138.4* 1:2 474.2 .+-. 32.59 1:3 118.8 .+-. 26.76
1:4 607.4 .+-. 285.2* 1:5 213.0 .+-. 67.95* 1:10 21.57 .+-. 9.89*
1:3 (Alexa) 168.5 .+-. 24.63 1:3 (NE) 113.7 .+-. 14.47 1:3 (NE
Alexa) 197.4 .+-. 12.85 *indicate multimodal size distributions
(multiple peaks).
1:3 (Alexa) polyplexes were formulated with an Alexa488-conjugated
MMI-0100 (MK2i) peptide to use in cellular uptake studies. 1:3 (NE)
polyplexes were formulated with a non-endosomolytic (NE)
poly(acrylic acid) polymer that does not exhibit pH-dependent
membrane disruptive activity in the endosomal pH range as a vehicle
control.
[0616] MMI-0100 (MK2i)-NP unpackaging under endolysosomal
conditions was assessed using DLS at a range of pHs and revealed
that the MK2i-NPs dissociated as the pH was lowered from
extracellular pH toward the pKa of the carboxylic acids
(pH.about.6.7) on PPAA, which also correlates to early endosomal
conditions (A. Sorkin et al., Nat Rev Mol Cell Biol 3, 600-614
(2002)) (FIG. 35E). Without being bound by theory, it is
hypothesized that at the lower pH, the PPAA polymer becomes
protonated/deionized, and the net positive charge on the peptide
causes electrostatic repulsion and disassembly of the MK2i-NPs. NP
disassembly under early endosome-like conditions reduces the
possibility that peptide bioactivity and/or PPAA endosomal membrane
disruptive function is sterically hindered by polymer-peptide
interactions.
[0617] MMI-0100 (MK2i)-NP Cell Internalization, Endosome Escape,
and Intracellular Retention
[0618] Quantity of MMI-0100 (MK2i)-NP uptake and intracellular
retention over time were assessed through flow cytometric analysis
of human coronary artery vascular smooth muscle cells (HCAVSMCs)
treated for 2 hours, washed, and maintained in fresh medium for 5
days. More than an order of magnitude increase in peptide uptake
was measured in MK2i-NP treated cells compared to NE-MK2i-NPs and
MMI-0100 (MK2i) (FIG. 36A and FIG. 54A). Because NE-MK2i-NP uptake
was equivalent to the free peptide, these data indicate that
differences in cell internalization are due to NP formulation and
independent of particle morphology and charge. Enhanced peptide
delivery via the MK2i-NP formulation was also detected in analogous
studies on endothelial cells suggesting that this is not a cell
type-specific observation (FIG. 56). Half-life calculations (FIG.
54B) showed that MK2i-NPs increased the intracellular half-life of
the MK2i peptide by over an order of magnitude from 4 days to 58
days. Additionally, HCAVSMCs treated with MMI-0100 (MK2i)-NPs
demonstrated longer peptide intracellular retention compared to
NE-MK2i-NP and MK2i treated cells, likely due to a higher rate of
peptide degradation in the endolysosomal pathway and/or exocytotic
recycling out of the cell (I. R. Ruttekolk et al., Mol Pharmaceut
9, 1077-1086 (2012)) (FIG. 36B). Interestingly, MK2i-NPs showed an
increase in fluorescence over the first 72 hours of incubation
following treatment/washing. It was verified that this effect was
not due to delayed internalization of MK2i-NPs bound to the outer
membrane of the cells but that this increase in fluorescence is due
to an Alexa-488 self-quenching mechanism (W. H. t. Humphries et
al., Anal Biochem 424, 178-183 (2012)); increased fluorescence over
time may be due to diminished quenching as the MMI-0100 (MK2i) is
unpackaged from the NPs intracellularly (FIG. 57).
[0619] To gain clarity into the mechanism of improved intracellular
retention of peptide delivered via MMI-0100 (MK2i)-NPs, a red blood
cell hemolysis assay (B. C. Evans et al., J Vis Exp, e50166 (2013))
and microscopy/colocalization studies were used to assess
pH-dependent membrane disruptive activity and endosomal escape of
MK2i-NPs. PPAA disrupts erythrocyte membranes at pHs at or below
its pKa (.about.6.7) (FIG. 36C). At extracellular (7.4) and early
endosomal (6.8) pH, MK2i-NPs showed little membrane disruptive
activity. However, at pH representative of late endosomes (6.2) and
lysosomes (5.6), a significant increase in hemolysis was observed.
The hemolytic behavior of the MK2i-NPs at late endosome/lysosomal
pH was directly proportional to polymer concentration (FIG. 44),
with >90% erythrocyte lysis occurring at 40 .mu.g/mL MK2i-NPs at
pH 5.6. MK2i-NPs retain the inherent membrane disruptive activity
of the PPAA polymer, although formulation into NPs partially masked
the membrane disruptive activity relative to free PPAA at pH 6.8.
As expected, neither the MK2i peptide alone nor the
non-endosomolytic NE-MK2i-NP formulation displayed any membrane
disruptive activity in the endolysosomal pH range.
[0620] MK2i-NP endosomal escape was imaged and quantified in vitro
in HCAVSMCs (FIG. 36D). Approximately 90% of the MK2i delivered as
free peptide or via NE-MK2i-NPs colocalized with the Lysotracker
dye, while MK2i-NP formulation significantly reduced MK2i
endolysosomal colocalization. Longitudinal quantification of
MK2i/Lysotracker colocalization following a 2-hr treatment and wash
revealed significantly reduced MK2i/Lysotracker colocalization for
the MK2i-NP formulations at all time points (FIG. 36E).
Interestingly, quantification of compartment size revealed that
NE-MK2i-NP or MK2i treated cells showed MK2i localization within
smaller vesicles representative of endosomes, whereas MK2i
delivered via MK2i-NPs was found within larger compartments,
potentially representative of the cytosol or disrupted vesicles
(FIGS. 45 and 58B).
[0621] The NP formulation significantly increased peptide uptake by
vascular smooth muscle cells (VSMCs) relative to the free,
CPP-based MMI-0100 (MK2i) peptide (FIG. 36A). Without being bound
by theory, the in vitro comparisons of MMI-0100 (MK2i)-NPs and
NE-MK2i-NPs shown in FIG. 35 suggest that the high levels of
MK2i-NP cell internalization was dependent on the specific
formulation of PPAA, rather than purely dictated by NP morphology
and surface charge. The .alpha.-alkyl substitution of the propyl
moiety makes PPAA more lipophilic/hydrophobic relative to acrylic
acid, suggesting that the observed differences in uptake may be the
result of increased hydrophobic interactions of MMI-0100 (MK2i)-NPs
with the cell membrane. Hydrophobic interactions may
nonspecifically trigger MK2i-NP cell internalization, or MK2i-NP
internalization may be mediated by VSMC scavenger receptors that
are upregulated in settings of vascular stress and that internalize
negatively charged/hydrophobic particles (e.g., LDL).
[0622] In addition to efficient cell internalization, avoiding
endolysosomal degradation and extracellular recycling is vital to
optimizing therapeutic potency and longevity of action of
cytosolically-active peptides (C. L. Duvall et al., Mol Pharm 7,
468-476 (2010)). This sustained therapeutic effect is of particular
importance for a peptide-based vein graft therapeutic where a
single, intraoperative treatment should achieve prolonged
bioactivity throughout the post-transplant inflammatory and healing
phases. To this end, the MK2i-NP formulation significantly improved
intracellular retention of the MMI-0100 (MK2i) peptide (FIGS. 36A
and B). This enhanced retention is achieved through the
pH-dependent membrane disruptive activity of PPAA, which is ideally
tuned for directing endolysosomal escape (FIG. 36C-E). Cell imaging
studies supported the endosomolytic function of PPAA and showed
that peptide delivered via MK2i-NPs had significantly decreased
colocalization with an endolysosomal dye (FIG. 36D,E). Avoiding
endosomal entrapment was associated with increased longevity of
intracellular peptide retention. Estimation of the intracellular
half-life (T.sub.1/2) of MMI-0100 (MK2i) based upon exponential
decay nonlinear regression analysis of intracellular peptide
fluorescence at 0 and 5 days following treatment removal revealed
that intracellular T.sub.1/2 was increased 14-fold by incorporation
into MK2i-NPs (MK2i-NP T.sub.1/2=57.8 days vs. MK2i T.sub.1/2=4.1
days) (data not shown).
[0623] MMI-0100 (MK2i)-NPs improved peptide potency based on
shifting the dose response curve (i.e., increased potency
.about.10-fold in most assays, FIG. 38). However, the longer
intracellular half-life of MK2i peptide via the NP formulation may
also enable superior longevity of action and improve, for example,
long-term graft patency. Without being bound by theory, the
intracellular half-life of MMI-0100 (MKi) delivered via NPs is
expected to be therapeutically relevant, as TGF-.beta.-mediated
transdifferentiation and cell migration mediated by the p38 MAPK
pathway has been found to contribute to pathological vein graft
remodeling out to 35 days post-transplant (A. V. Bakin et al., J
Cell Sci 115, 3193-3206 (2002)). Other studies on the kinetics of
intimal hyperplasia (IH) pathogenesis in rabbit and canine models
detected an initial burst in cellular proliferation during the
first week, followed by continued graft adaptation that reaches
steady state by week 12 (M. Kalra et al., J Vasc Res 37, 576-584
(2000); R. M. Zwolak et al., J Vasc Surg 5, 126-136 (1987)). The
extended half-life achieved with MK2i-NPs is expected to yield
significantly improved long-term performance following a single
treatment prior to, for example, implantation, by inhibiting
underlying signaling pathways and accelerating resolution of
inflammation and the time required to reach steady-state
conditions.
[0624] MK2i-NP delivery of peptide into intact human saphenous vein
(HSV) was also assessed. The results of this experiment suggested
that uptake occurs in both endothelial and smooth muscle cells. As
expected, MK2i-NPs and controls showed more concentrated uptake at
the luminal and adventitial surfaces that act as diffusion barriers
(FIG. 59). MK2i penetration into the intimal and medial layers was
verified by colocalization with the smooth muscle marker
.alpha.-SMA (FIG. 60 a-b). Furthermore, in accordance with in vitro
results, MK2i-NPs increased the overall peptide uptake within the
vessel wall (FIG. 60c; FIG. 59e).
[0625] Inhibition of Intimal Hyperplasia (IH in Human Saphenous
Vein (HSV)
[0626] To confirm efficient delivery and MMI-0100 (MK2i)-NP
bioactivity in three dimensional human vascular graft tissue, an ex
vivo organ culture model of vein IH was completed using human
saphenous vein (HSV). HSV rings were cut from HSV samples that were
confirmed to be viable based on contractile response to KCl
challenges in a muscle bath. Rings were treated for 2 hours,
washed, and maintained in high serum conditions that accelerate
neointima formation. An Alexa-568 conjugated MK2i peptide was used
to visualize peptide delivery to the vessel wall immediately
following treatment, and, similar to the in vitro results, MMI-0100
(MK2i)-NPs consistently increased peptide delivery relative to free
MMI-0100 (MK2i) (FIG. 37A). After 14 days in culture, Verhoeff-Van
Gieson (VVG) staining of the elastic laminae was performed on
tissue sections (FIG. 37B). Quantification of intimal thickness of
samples from multiple human donors revealed that MK2i-NPs
significantly inhibited IH in a dose-dependent fashion and at an
order of magnitude lower peptide dose than free MMI-0100 (MK2i)
(FIG. 37C and FIG. 46). Furthermore, MK2i-NP therapy at 100 .mu.M
MK2i was the only treatment that fully abrogated IH, yielding
intimal thickness statistically equivalent to control tissues
prepared for histology immediately after harvest (p=0.49). MTT
assays were performed 1 and 14 days post-treatment and verified
that organ culture results were not affected by treatment effects
on tissue viability (FIG. 47). Treatment of human saphenous vein
with 100 .mu.M MK2i-NPs completely abrogated neointimal growth over
2 weeks in the ex-vivo organ culture model of IH.
[0627] Mechanistic Elucidation of MMI-0100 (MK2i)-NP
Bioactivity
[0628] To elucidate the mechanism by which MK2i-NPs reduced IH in
human vein, phosphorylation of hnRNP A0 and CREB was first assessed
using Western blot analysis. Downstream of MK2, hnRNP A0 stabilizes
the mRNA and increases translation of inflammatory cytokines (S.
Rousseau et al., EMBO J 21, 6505-6514 (2002); N. Ronkina et al.,
Biochem Pharmacol 80, 1915-1920 (2010); E. Hitti et al., Mol Cell
Biol 26, 2399-2407 (2006)), and CREB binds to cAMP-responsive
elements to promote expression of genes that induce smooth muscle
cell migration (S. Jalvy et al., Circulation Research 100,
1292-1299 (2007); H. Ono et al., Arterioscl Throm Vas 24, 1634-1639
(2004)), proliferation (P. Molnar et al., J Cell Commun Signal 8,
29-37 (2014); K. Nakanishi et al., Journal of Vascular Surgery 57,
182-U254 (2013)), and production of the inflammatory cytokines such
as IL-6 (G. L. Lee et al., Arterioscl Throm Vas 32, 2751-+(2012)).
MMI-0100 (MK2i)-NPs significantly reduced both hnRNP A0 and CREB
phosphorylation in HSV (FIG. 37D,E). In further support of this
mechanism, MK2i-NPs also significantly inhibited secretion of the
primary hnRNP A0 target TNF.alpha. (S. Rousseau et al., EMBO J 21,
6505-6514 (2002)) in vitro in angiotensin-II stimulated HCAVSMCs
(FIG. 38A, FIG. 48). In this study, MK2i-NPs achieved TNF.alpha.
inhibition equivalent to NE-MK2i-NP and MK2i at an order of
magnitude lower dose (i.e. 10 .mu.M MMI-0100 (MK2i) produced an
effect equivalent to 100 .mu.M MMI-0100 (MK2i)), and 100 .mu.M
MK2i-NPs fully abrogated Angiotensin II-stimulated TNF.alpha.
production. It was also confirmed that MK2i-NPs significantly
reduced production of IL-6, a CREB target gene (G. L. Lee et al.,
Arterioscl Throm Vas 32, 2751-+(2012)), in TNF.alpha.-stimulated
HCAVSMCs. This study also showed that MK2i-NPs were significantly
more bioactive than free MK2i (FIG. 49). None of the in vitro
treatments resulted in significant toxicity as assessed by tissue
viability at 1 and 14 days post-treatment compared to untreated
controls (FIGS. 50 and 51).
[0629] It was also confirmed that MK2i-NPs significantly decreased
phosphorylation of HSP-27 (FIG. 37D,F), which along with CREB, is
believed to promote pathological vascular smooth muscle cell
migration characteristic of IH (T. Zarubin et al., Cell Res 15,
11-18 (2005); H. F. Chen et al., Mol Cell Biochem 327, 1-6 (2009);
L. B. Lopes et al., J Vasc Surg 52, 1596-1607 (2010)).
[0630] The effects of MK2i-NPs on HCAVSMC migration in the presence
of the chemokine PDGF-BB were also investigated in vitro using both
scratch wound chemokinetic and Boyden chamber chemotactic migration
assays (FIG. 38B,-D). MK2i-NPs significantly inhibited cell
migration and did so at an order of magnitude lower dose than free
MMI-0100 (MK2i) peptide. MK2i-NPs did not significantly affect
HCAVSMC proliferation, confirming that these results were not
attributable to treatment effects on cell growth (FIG. 52).
Additionally, MK2i-NPs potently inhibited both vascular smooth
muscle (VSMC) and endothelial cell (EC) migration (FIG. 61a-d), and
MK2i-NPs were significantly more potent at inhibiting VSMC
migration compared to the free MK2i peptide (FIG. 61a). These
results correlated with the MK2i-NP inhibition of CREB and HSP27
phosphorylation detected in human vascular tissue.
[0631] An ex vivo organ culture model of IH in HSV also revealed
that MK2i-NPs significantly inhibited neointima formation in a
dose-dependent fashion and at an order of magnitude lower peptide
dose than free MK2i (FIGS. 37b and c; FIGS. 48-51).
[0632] These studies also validated the broad anti-inflammatory and
anti-migratory mechanism of action of MMI-0100 (MK2i)-NPs (FIG. 38)
and confirmed the utility of targeting the p38-MK2 pathway to
inhibit multiple factors underlying IH pathogenesis. MK2i-NPs were
shown to modulate pro-inflammatory mediators activated downstream
of MK2 such as hnRNP A0 and CREB. MMI-0100 (MK2i)-NP decreased
hnRNP A0 phosphorylation in human tissue, which correlated to a
decrease in angiotensin-II stimulated production of the
pro-inflammatory cytokines TNF-.alpha. and IL-6 in vitro. MK2i-NPs
were also shown to modulate migration-related pathways in human
tissue, as demonstrated by reduced phosphorylation of HSP27, which
triggers VSMC transition to a migratory and fibrotic myofibroblast
phenotype and causes vein graft vasoconstriction. The effects of
HSP27 are mediated through regulation of cytoskeleton dynamics,
which impacts migration towards pathologically relevant stimuli
such as angiotensin II and PDGF. Additionally, MK2i-NPs decreased
phosphorylation of the CREB transcription factor, which is also
known to contribute to VSMC migration and lead to the pathological
VSMC phenotype characteristic of IH (See, e.g., H. F. Chen et al.,
Mole Cell Biochem 327, 1-6 (2009); K. Nakanishi et al., Journal of
Vascular Surgery 57, 182-U254 (2013); G. L. Lee et al., Arterioscl
Throm Vas 32, 2751-+(2012); L. C. Fuchs et al., Am J Physiol-Reg I
279, R492-R498 (2000)). Inhibition of activation of HSP27 and CREB
correlated to reduced VSMC migration towards PDGF in vitro.
[0633] Because the intracellular half-life of MK2i was
significantly higher when delivered via MK2i-NPs, in vitro
bioactivity assays were also carried out at 3 and 5 days
post-treatment to assess the impact of the NP formulation on
longevity of peptide therapeutic action. In accord with our
intracellular half-life calculations, the ability of the free MK2i
peptide to inhibit the production of monocyte chemoattractant
protein-1 (MCP-1, which is upregulated both through hnRNP A0 and by
TNF.alpha. (Rousseau S, Morrice N, Peggie M, Campbell D G, Gaestel
M, Cohen P. Inhibition of sapk2a/p38 prevents hnrnp a0
phosphorylation by mapkap-k2 and its interaction with cytokine
mrnas. EMBO J. 2002; 21:6505-6514; Mueller L, von Seggern L,
Schumacher J, Goumas F, Wilms C, Braun F, Broering D C. Tnf-alpha
similarly induces it-6 and mcp-1 in fibroblasts from colorectal
liver metastases and normal liver fibroblasts. Biochem Biophys Res
Commun. 2010; 397:586-591) and implicated in vein graft intinmal
hyperplasia (IH) (Stark V K, Hoch J R, Warner T F, Hullett D A.
Monocyte chemotactic protein-1 expression is associated with the
development of vein graft intimal hyperplasia. Arterioscl Throm
Vas. 1997; 17:1614-1621), was significantly decreased at 3 and 5
days post-treatment in both vascular smooth muscle cells (VSMC) and
endothelial cells (EC) (FIG. 61f-g). In contrast, MK2i-NPs
demonstrated sustained inhibitory bioactivity at 5 days
post-treatment in both cell types. Moreover, MK2i-NPs demonstrated
significant inhibition of VSMC migration 5 days post-treatment
whereas free MK2i or NE-MK2i-NPs showed minimal effect (FIG.
61h-i). The decrease in anti-inflammatory and anti-migratory
activity between days 3 and 5 corresponded with the calculated
intracellular half-life of the free MK2i peptide.
[0634] These results establish the relationship between MK2 and the
downstream pro-inflammatory and pro-migratory factors hnRNP A0,
CREB, and HSP27 in intact, human vascular tissue. The collective
anti-inflammatory and anti-migratory actions of MK2i-NPs emphasize
the utility of this therapy against a multifactorial process, for
example, like IH, which involves a complex interplay of cell
proliferation, migration, inflammation, and matrix synthesis.
Because this translationally-relevant MK2i-NP formulation (formed
by simple mixing; no complex syntheses, conjugations, or
purifications required) comprehensively targets multiple factors
involved in IH, it has potential to overcome the shortfalls of
prior therapeutic candidates with more narrow mechanisms of
action.
[0635] In Vivo Bioactivity in a Rabbit Vein Graft Interposition
Model
[0636] The therapeutic benefit of MMI-0100 (MK2i)-NPs in vivo was
assessed in a rabbit bilateral jugular vein graft interpositional
transplant model that employs a polymeric cuff method to induce
turbulent blood flow and accelerate graft IH. In this model,
jugular vein grafts were treated or given vehicle control for 30
minutes ex vivo, which is representative of the amount of time that
grafts are explanted during human revascularization procedures.
Grafts were harvested 28 days post-operatively, and VVG stained
histological sections were used for intimal thickness
quantification (FIG. 39A and FIG. 62a). Treatment with 30 .mu.M
MMI-0100 (MK2i)-NPs significantly inhibited neointima formation
compared to both untreated controls and the free MMI-0100 (MK2i)
peptide, which did not produce any significant change in neointima
formation relative to vehicle controls at the 30 .mu.M dose tested
(FIG. 39B and FIG. 62b).
[0637] To assess in vivo cell-based mechanisms underlying MK2i-NP
mediated inhibition of neointimal thickening, proliferating cell
nuclear antigen (PCNA), .alpha.-smooth muscle actin (.alpha.-SMA),
and vimentin stained histological sections were used to analyze
cellular proliferation and vascular smooth muscle cell phenotype.
Intimal PCNA staining was significantly decreased by .about.17-fold
in grafts treated with MK2i-NPs, whereas treatment with the free
MK2i were similar to untreated grafts (FIG. 62c-d). MK2i-NP treated
grafts also demonstrated increased staining intensity for
.alpha.-SMA, which is a marker for contractile SMC phenotype
(Rensen SSM, Doevendans PAFM, van Eys GJJM. Regulation and
characteristics of vascular smooth muscle cell phenotypic
diversity. Neth Heart J. 2007; 15:100-108), relative to untreated
grafts or grafts treated with free MK2i (FIG. 62f). Images of
.alpha.-SMA immunostained sections revealed that untreated and free
MK2i treatment groups showed sparse intimal staining (FIG. 62e),
indicating loss of the contractile VSMC phenotype and/or excess
production of extracellular matrix proteins, both of which are
implicated in vein graft IH. In agreement with increased
contractile marker expression, intimal expression of the synthetic
VSMC marker vimentin was also decreased in MK2i-NP treated grafts
but not in grafts treated with free MK2i peptide (FIG. 62g-h).
[0638] The number of residual inflammatory cells present in the
intima of the vein grafts 28-day post-transplant was assessed in
tissue sections using a rabbit macrophage specific antibody, RAM-11
(FIG. 39C, FIG. 53 and FIG. 63). Significantly less intimal
macrophages were detected in MK2i-NP treated grafts, suggesting
that MK2i-NPs blunted local macrophage recruitment and/or
persistence (FIG. 39D). This mechanism is potentially mediated
through decreased secretion of macrophage inflammatory protein 2
(MIP-2, also known as CXCL2) and/or monocyte chemoattractant
protein-1 (MCP-1) (A. Muto et al., Vascul Pharmacol 56, 47-55
(2012)), both of which attract inflammatory cells and are
upregulated either directly or indirectly through hnRNP A0 (S.
Rousseau et al., EMBO J 21, 6505-6514 (2002); L. Mueller et al.,
Biochem Biophys Res Commun 397, 586-591 (2010); R. N. Mitchell et
al., Circ Res 100, 967-978 (2007)). Our in vitro study results
showing that MK2i-NPs inhibited MCP-1 production in both smooth
muscle and endothelial cells support this mechanism. Though the
inflammatory response was predominately resolved in all samples at
28-days, macrophage persistence in untreated samples agrees with
previous observations that MCP-1 can be elevated even at 8 weeks
after vein grafting, resulting in local recruitment of monocytes
and pathogenesis of IH (V. K. Stark et al., Arterioscl Throm Vas
17, 1614-1621 (1997)). Treatment with 100 .mu.M MK2i-NPs completely
abrogated neointimal growth over 2 weeks in the rabbit transplant
model. Intraoperative treatment with 30 .mu.M MK2i-NPs
significantly reduced the number of macrophages and the degree of
IH in the grafts at 4 weeks post-transplant (FIG. 39).
Example 4. Synthesis, Characterization and Optimization of MK2i-NPs
and p-Hsp20-NPs
[0639] The MK2i peptide with the sequence YARAAARQARA-KALARQLGVAA
(SEQ ID NO: 1) and the p-HSP20 peptide with the sequence
YARAAARQARA-WLRRAsAPLPGLK (SEQ ID NO: 27) were synthesized via
solid phase synthesis, and purity was verified through
electrospray-ionization mass spectrometry (FIG. 64). Reversible
addition fragmentation chain transfer (RAFT) polymerization was
utilized to synthesize poly(propylacrylic acid) (PPAA) [DP=193
(GPC), DP=190 (H.sup.1 NMR), PDI=1.47 (GPC)]. NPs were formed by
simple mixing of the PPAA homopolymer with the MK2i or p-HSP20
peptides in PBS at pH 8.0, which is between the pKa values of the
primary amines present on the peptides (pKa-9-12 depending on the
amino acid residue) and the carboxylic acid moieties in the PPAA
polymer (pKa-6.7); this ensures optimal solubility and net charge
on both molecules to facilitate electrostatic complexation.
[0640] To assess the impact of nanoparticle formulation conditions,
a series of MK2i-NPs and p-HSP20-NPs were prepared at a range of
charge ratios [i.e.
CR=([NH.sub.3.sup.+].sub.MK2i/p-HSP20:[COO.sup.-].sub.PPAA)], and
the size distribution and particle surface charge were
characterized through dynamic light scattering (DLS) and
.zeta.-potential analysis, respectively. As expected, MK2i-NP and
p-HSP20-NP .zeta.-potential was directly proportional to the CR
(FIGS. 65A, 66A). The CR also significantly affected NP size, with
a narrow range of CRs yielding a unimodal size distribution (i.e.
CR=1:2 and 1:3 for MK2i-NPs (Table 24) and CR=3:1 for p-HSP20-NPs,
(Table 25). A CR of 1:3 was utilized in subsequent studies for the
MK2i-NP formulation, and a CR of 3:1 was utilized for the
p-HSP20-NP formulation; these charge ratios consistently yielded a
unimodal size distribution with minimal particle size and
polydispersity (MK2i-NP d.sub.h=119.+-.28 nm, .zeta.=-11.9.+-.3.2
mV, FIG. 65B; p-HSP20-NP d.sub.h=141.+-.6 nm, .zeta.=-7.5.+-.2.8
mV, FIG. 66B). This difference in the charge ratio that produced
unimodal particles between the two peptides may be attributable to
differences in peptide size, charge distribution, sequence
hydrophobicity, or secondary structures, and future analysis of a
broader library of peptides will be required to better understand
the structure-function relationships of these formulations.
Interestingly, both optimal NP formulations demonstrated a negative
.zeta.-potential, indicating that the cationic peptides are
sequestered in the core of the nanopolyplexes and the anionic PPAA
polymer is more preferentially localized to the particle surface.
The leading MK2i-NP and p-HSP20-NP formulations were also
characterized through TEM imaging (FIGS. 65C, 66C), which confirmed
the presence of nano-structures with size distributions in
accordance with DLS results. For subsequent in vitro and ex vivo
studies, these lead NP formulations (FIGS. 65D, 66D) were compared
to the corresponding free peptide.
TABLE-US-00024 TABLE 24 Size summary of MK2i-NPs prepared at
different charge ratios ([NH.sub.3.sup.+]/[COO.sup.-]) as
determined by DLS analysis NH.sub.3.sup.+:COO.sup.- Z-ave diameter
(nm) PDI 10:1 10.32 .+-. 2.63* 0.314 2:1 52.1 .+-. 46.86* 0.297 1:1
970.6 .+-. 662.4* 0.41 1:1.5 465.1 .+-. 138.4* 0.5465 1:2 474.2
.+-. 32.59 0.239 1:3 118.8 .+-. 26.76 0.271 1:4 607.4 .+-. 285.2*
0.662 1:5 213.0 .+-. 67.95* 0.407 1:10 21.57 .+-. 9.89* 0.355
Asterisks (*) indicate multimodal size distributions (multiple
peaks present). A CR of 1:3 was chosen as the lead MK2i-NP
formulation.
TABLE-US-00025 TABLE 25 Size summary of p-HSP20-NPs prepared at
different charge ratios ([NH.sub.3.sup.+]/[COO]) as determined by
DLS analysis NH.sub.3.sup.+:COO.sup.- Z-ave diameter (nm) PDI 10:1
659.4 .+-. 293.7* 0.594 5:1 238.3 .+-. 38.13* 0.574 4:1 169.1 .+-.
2.501* 0.591 3:1 141.0 .+-. 5.783 0.207 2:1 369.3 .+-. 69.83* 0.554
1:1 1018 .+-. 786.6* 0.903 1:2 1321 .+-. 1430* 0.662 1:3 1369 .+-.
255.9* 0.750 1:4 1772 .+-. 513* 0.470 1:5 1496 .+-. 602.9* 0.429
1:10 4246 .+-. 4428 0.741 Asterisks (*) indicate multimodal size
distributions (multiple peaks present). A CR of 3:1 was chosen as
the lead p-HSP20-NP formulation.
Example 5. NP In Vitro Biocompatibility, Uptake, Retention,
Trafficking and Bioactivity
[0641] The biocompatibility of the lead candidate MK2i-NP and
HSP20-NP formulations was compared to the corresponding free
peptide at a range of doses (10-500 .mu.M peptide) in human
coronary artery vascular smooth muscle cells (HCAVSMCs) in vitro.
HCAVSMCs were treated for 2 hours and then incubated in fresh
medium for 24 hours prior to running the cytotoxicity assay. No
significant cytotoxicity was evident for MK2i-NPs at all
concentrations tested, whereas the free MK2i peptide demonstrated
mild toxicity at the highest dose tested (76% cell viability at 500
.mu.M, FIG. 67). HSP20-NPs and the HSP20 peptide were found to be
biocompatible with the exception of mild cytotoxicity detected at
500 .mu.M (60% and 77% viability for p-HSP20-NPs and the free
p-HSP20 peptide, respectively).
[0642] Quantity of MK2i-NP and p-HSP20-NP uptake and intracellular
retention over time were assessed through flow cytometric analysis
of HCAVSMCs treated for 30 minutes, washed, and maintained in fresh
medium for 0 or 3 days. More than an order of magnitude increase in
uptake (.about.70-fold increase in MK2i uptake and .about.35-fold
increase in p-HSP20 uptake) was detected for both peptides when
incorporated into NPs (FIG. 68). Since the negative
.zeta.-potential of both NP formulations indicates that the PPAA
polymer is primarily exposed at the NP surface, this increase in
uptake is likely facilitated by the pH-responsive polymer. More
specifically, the .alpha.-alkyl substitution of the propyl moiety
imparts PPAA with lipophilic/hydrophobic character, suggesting that
the observed differences in uptake may be the result of increased
hydrophobic interactions of NPs with the cell membrane. In addition
to increased uptake, HCAVSMCs treated with MK2i-NPs or p-HSP20-NPs
demonstrated increased intracellular peptide retention 3 days after
treatment removal compared to the free MK2i or p-HSP20 peptide (82%
vs. 54% of initial uptake remaining for MK2i-NPs vs. free MK2i,
FIG. 4A,E; 70% vs. 35% retention of p-HSP20-NPs vs. free p-HSP20,
FIG. 68B,F). Intracellular retention of bioactive cargo can be
improved by reducing exocytosis of the intact peptide and/or
reducing degradation of the peptide in acidic endo-lysosomal
compartments18, 35. These optimized NP formulations are
intentionally designed to respond to the decreased pH encountered
in the endo-lysosomal trafficking pathway to facilitate cytosolic
peptide delivery, as the PPAA polymer has well-defined pH-dependent
endosomolytic activity36, 37, has previously demonstrated
biocompatibility in animal models38, and has been applied for
intracellular delivery of a pro-apoptotic anti-cancer peptide via a
multi-step bioconjugation of the PPAA polymer to the peptide
through a streptavidin linker39. Thus, a simplified electrostatic
complexation approach was utilized incorporating the PPAA polymer
to facilitate therapeutic endosome escape and retention in these
studies: PPAA undergoes a transition from an ionized, expanded
conformation at physiologic pH to a collapsed, hydrophobic globular
conformation in acidic/endosomal conditions. This transition
results in hydrophobic interactions with lipids in the endosomal
membrane and ultimately in endosomal escape and improved
intracellular retention and bioactivity of the therapeutic peptide
cargo.
[0643] To investigate the connection between increased peptide
intracellular retention and endosomal escape of peptides delivered
via the NP formulation, a digitonin-based, semi-permeabilization
technique40 was adapted and optimized for measuring the relative
quantity of cytosolic and vesicle-bound peptide for NP and free
peptide treated HCAVSMCs (FIG. 69A). Digitonin is a non-ionic
detergent that, under optimized conditions, results in the
selective semi-permeabilization of the cell membrane while leaving
intracellular organelles (e.g., endosomes and lysosomes) intact. An
optimized semi-permeabilization procedure was determined by
measuring the LDH (which is known to be localized to the cytosol)
quantity in the "cytosolic" and "organelle" fractions from HCAVSMCs
incubated with a range of concentrations of digitonin for 10
minutes on ice. (FIG. 70). Western blot analysis of the cytosolic
and organelle fractions collected using the optimized
semi-permeabilization protocol verified effective separation of the
cytosolic proteins mitogen-activated protein kinase kinase 1/2
(MEK1/2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from
the endo-lysosomal markers early endosomal antigen 1 (EEA1) and
lysosomal-associated protein 1 (LAMP1, FIG. 69B). Utilizing
fluorescently labeled MK2i and p-HSP20 peptides allowed for
quantification of the intracellular distribution of both peptides
following delivery in their free form versus via NP formulations.
This analysis verified that formulation into NPs not only increased
peptide uptake but also significantly increased the fraction of
internalized peptide in the cytosol; the net effect was an
approximately 8-fold increase in cytosolic MK2i delivery and
.about.29-fold increase in cytosolic p-HSP20 delivery (FIG. 69C,
D). In order to confirm that the increased cytosolic peptide
delivery is facilitated by the pH-dependent membrane disruptive
activity of PPAA in the NP formulations, cells were treated with
NPs in the presence of the vacuolar-type H+ ATPase inhibitor
Bafilomycin A1 to prevent endo-lysosomal acidification. Preventing
endosomal acidification markedly reduced the fraction of
internalized peptide in the cytosol for both NP formulations,
confirming that the mechanism of NP escape from edosomes is
pH-dependent (FIG. 69C, D). Bafilomycin treatment was found to have
negligible effects on the cytosolic fraction of internalized free
MK2i or p-HSP20 peptide (Data not shown: MK2i: 9.64%.+-.8.17%
cytosolic, p-HSP20: 7.36%.+-.8.28% cytosolic).
[0644] The efficacy of MK2i-NP and p-HSP20-NP mediated inhibition
of F-actin stress fiber formation was quantified in angiotensin-II
(ANG II) stimulated HCAVSMCs. Both NP formulations enhanced peptide
functional bioactivity as measured by a significant decrease in the
average number of stress fibers per cell (FIG. 71A). Qualitatively,
HCAVSMCs treated with the NP formulations and ANG II displayed cell
morphology and staining consistent with unstimulated control cells,
whereas HCAVSMCs treated with the free peptide demonstrated stress
fiber formation similar to ANG II-stimulated control cells (FIG.
71B). The total amount of F-actin per cell was also quantified
using Alexa-488 phalloidin, a stain that selectively binds to
filamentous but not globular, actin (FIGS. 72 and 73). This
analysis was consistent with the quantification of number of stress
fibers per cell and revealed that formulation into NPs
significantly enhanced stress fiber inhibitory activity of both
peptides.
Example 6. NP Effect on Smooth Muscle Physiology in Human Vascular
Tissue
[0645] The effect of the MK2i-NP and p-HSP20-NP formulations on
smooth muscle physiology in human vascular tissue was assessed in
order to evaluate these formulations as potential treatments for
vasospasm. For these studies, human saphenous vein (HSV) was
collected from consented patients undergoing bypass grafting
surgery and sectioned into rings. After verifying viability through
KCL challenge in a muscle bath, the ability of each NP formulation
to inhibit phenylephrine (PE) induced vasoconstriction was measured
in HSV rings using an organ bath system outfitted with a force
transducer. In an experimental design where vessels were
contracted, relaxed, treated, and then contracted again, untreated
control HSV rings displayed no changes in the second round of PE
induced contraction relative to the initial contraction. However,
intermediate treatment with the MK2i or p-HSP20 peptides
significantly inhibited the second PE-induced HSV contraction (FIG.
74A-C). Consistent with in vitro F-actin stress fiber results,
equivalent doses of peptide delivered via NP formulations
demonstrated significantly enhanced peptide-mediated inhibition of
contraction compared to the free peptide (FIG. 74C). Notably,
treatment with a dose of free PPAA polymer equivalent to the
highest NP dose administered showed negligible effects on
PE-induced HSV contraction (FIG. 74B) indicating that the enhanced
inhibitory activity is mediated through enhancement of peptide
bioactivity and is not a non-specific effect of the endosomolytic
polymer carrier. This ability of the peptide-NPs to potently
inhibit vasoconstriction demonstrates the translational potential
of these formulations as a prophylactic approach to prevent
vasospasm in applications such as coronary or peripheral bypass
grafting.
[0646] In addition to testing the efficacy of these NP formulations
as a prophylactic therapy, the ability of the MK2i- and p-HSP20-NPs
to enhance sodium nitroprusside (SNP) induced vasorelaxation was
evaluated as a potential salutary therapeutic intervention (e.g.,
to treat SAH induced vasospasm) in viable HSV explants (FIG. 74D).
Again, both NP formulations demonstrated an enhanced ability to
promote SNP-induced vasorelaxation at all concentrations tested
(FIG. 74E, F) whereas untreated HSV or HSV treated with the PPAA
polymer alone showed negligible differences in vasorelaxation (FIG.
74E). Because MK2i-NP and p-HSP20-NP formulations trigger
vasorelaxation through separate molecular mechanisms, combining
both peptides into a NP formulation represents a promising approach
for future studies because it may achieve a synergistic effect that
produces a therapeutic benefit at lower peptide doses.
[0647] In order to qualitatively assess the correlation of F-actin
stress fiber formation with the smooth muscle physiology results in
human tissue, HSV rings were pretreated with free peptide or the NP
formulations and then subsequently stimulated with ANG II prior to
F-actin staining with Alexa-488 phalloidin (FIG. 74G). In
concordance with the smooth muscle physiology results, HSV rings
treated with NP formulations showed diminished phalloidin staining
compared to HSV treated with the free peptide. Altogether, these
results indicate that MK2i- and p-HSP20-NPs significantly enhance
the ability of the MK2i and p-HSP20 peptide to inhibit
vasoconstriction and promote vasorelaxation by modulating actin
dynamics in human smooth muscle tissue.
[0648] The results of the experiments set forth above establish the
potential use of nanotechnology to enhance cell and tissue
delivery, bioactivity, and intracellular pharmacokinetics of
therapeutic peptides such as MMI-0100 (MK2i). In general, CPPs are
highly cationic, and thus, complexation with PPAA can potentially
serve as a generalized platform biotechnology to facilitate
intracellular delivery of therapeutic peptides.
Example 7. HPLC Method for Assay and Purity Determination of
MMI-0100 in Solution
[0649] The purpose of this study was to evaluate and optimize an
HPLC method for assay and purity determination of MMI-0100 in
solution by evaluating, among others, column wash steps, elution
gradient, precision (injection repeatability) and linearity.
[0650] The HPLC method conditions used are listed in Table 26.
TABLE-US-00026 TABLE 26 HPLC method conditions Column Grace, Vydec
C18, 5 .mu.m, 300 .ANG., 4.6 .times. 250 mm, polymeric, PN: 218TP54
with pre-column filter Mobile Phase (MP) % MP A: 0.1% TFA in DI
water MP B: 0.1% TFA in 1:1 methanol:acetonitrile (v/v) (MP A
filtered through 0.8 .mu.m membrane) Time (minutes) % MP A % MP B
Gradient 0 85 15 5.5 78 22 35 57 43 40 57 43 42 10 90 45 10 90 47
85 15 55 85 15 Flow Rate 1.0 mL/min Detection Wavelength
Ultraviolet (UV): 215 nm Column Temperature 25.degree. C. Sample
Temperature 5.degree. C. Injection Volume 20 .mu.L Run Time 64 min
HPLC Standard and Sample Tween 20, 0.02% (v/v) in water Diluent
("Diluent")
[0651] In order to maintain a clean column with a large number of
formulations, the column wash step (from 42-45 min) was extended by
7 min (from 42 to 52 min). Accordingly, the column equilibration
step was increased by 2 min (from 47-55 min to 54-64 min). The
elution condition under which the MMI-0100 elutes was not changed
(e.g. 0-40 min). The optimized gradient is listed Table 27.
TABLE-US-00027 TABLE 27 Optimized HPLC gradient Time (minutes) % MP
A % MP B Optimized 0 85 15 Gradient 5.5 78 22 35 57 43 40 57 43 42
10 90 52 10 90 54 85 15 64 85 15
[0652] Representative HPLC chromatograms of diluent and MMI-0100
standard at 1 mg/mL are shown in FIGS. 77 A and B respectively.
[0653] Precision
[0654] Precision (or injection repeatability) was evaluated by
injecting a solution containing 1.1 mg/mL MMI-0100 in 0.02% Tween
20 onto the HPLC for a total of six consecutive injections. The
retention time (RT), peak area, tailing factor and theoretical
plate for the MMI-0100 peak were recorded for each injection and
their respective relative standard deviations (RSDs) were
calculated. Precision test results are shown in Table 28. The RSD
for the RT and response factor from the six injections were less
than 2%, indicating that the method meets precision/injection
repeatability test criteria.
TABLE-US-00028 TABLE 28 Precision (injection repeatability) test
results Tailing Theor. Plates (1/2 Inj # RT PA RF (USP) Width
method) 1 25.21 12296 11032 2.588 21997 2 25.21 12283 11020 2.617
22756 3 25.19 12262 11002 2.584 21606 4 25.17 12228 10971 2.602
22301 5 25.16 12190 10937 2.625 22287 6 25.15 12191 10938 2.582
22272 AVG 25.18 12242 10983 2.600 22203 RSD (%) 0.1 0.4 0.4 0.7
1.7
[0655] Linearity
[0656] Linearity test solutions were prepared using a stock
solution of MMI-0100 with a series of dilutions. The stock solution
of MMI-0100 was prepared at 1.9 mg/mL (167% of the nominal
concentration of 1.1 mg/mL).
[0657] Actual Stock Preparation:
(24.0 mg of MMI-0100)*(Peptide Content from CoA 0.774)/(10 mL
Volumetric Flask).
[0658] Linearity solution preparation is detailed in Table 29.
Linearity test results are shown in Table 30 and FIG. 78. FIG. 78
shows a linearity plot of MMI-0100 concentration versus peak area.
The Y-intercept bias was calculated to be 0.5% using the formula
(y-int)/(Average Peak Area for 100% Nominal Concentration)*100
(62.339/12242*100=0.5%). For linearity, an acceptable correlation
coefficient (R.sup.2) value is >0.995 in a defined range and the
y-intercept bias must be .ltoreq.5% of the peak area obtained at
the nominal concentration. The linearity test results obtained meet
the R.sup.2 and y-intercept test criteria (Table 30 and FIG.
78).
TABLE-US-00029 TABLE 29 Linearity solution preparation Sol. Total
Solution Vol. Vol. Conc. % ID # Transferred (mL) (mL) (mg/mL)
Nominal STOCK 1.86 167 Linearity #1 Stock 4 5 1.49 133 Linearity #2
Stock 3 5 1.11 100 Linearity #3 Linearity #1 3 5 0.89 80 Linearity
#4 Linearity #3 3 5 0.53 48 Linearity #5 Linearity #4 1 2 0.27
24
TABLE-US-00030 TABLE 30 RF Diff from CONC Response Nominal Conc
(mg/mL) % Nominal RT PA Factor (%) 0.27 24 25.59 2822 10550 96 0.53
48 25.45 5795 10983 100 0.89 80 25.30 9837 11032 100 1.11 100 25.18
12242 10983 100 1.49 133 25.06 16167 10879 99 1.86 167 24.95 19958
10744 98
Example 8. Heat-Accelerated Stability Study of MMI-0100 in Various
Buffers with a pH Range from 4-8
[0659] The purpose of this study was to generate a pH-stability
profile MMI-0100, to determine solubility of MMI-0100 at 5.5. mg/mL
in select buffers, to determine the pH.sub.max (pH where MMI-0100
is most stable), to generate an impurity profile at pH.sub.max, to
determine the long-term prognosis at room temperature and
2-8.degree. C. or whether lyophilization is needed to maintain
stability, to compare DI water to buffered solutions in order to
determine which provides better solubility and stability for
MMI-0100 and to observe any apparent viscosity change or gelation
of MMI-0100 at 5.5 mg/mL.
[0660] MMI-0100 formulations prepared and tested are listed in
Table 31.
TABLE-US-00031 TABLE 31 MMI-0100 formulations Final Buffer MMI-
Buffer IIL Target 0100 (% Conc Limit* ID Buffer Stock Used pH w/w)
(% w/w) (% w/w) T-1 100 mM citric acid 4 0.55 0.11 0.44 T-2 100 mM
citric acid 5 0.55 0.11 0.44 T-3 100 mM citric acid 6 0.55 0.11
0.44 T-4 100 mM citric acid 6.5 0.55 0.11 0.44 T-5 DI water +
NaOH/HCl 7 0.55 NA NA for pH adjustment T-6 50 mM phosphoric acid +
7 0.55 0.05 NA NaOH for pH adjustment T-7 0.9% NaCl + NaOH/HCl 7
0.55 NA NA for pH adjustment T-8 L-lysine monohydrate 8 0.55 0.08
5.25
[0661] 0.4 mL of each MMI-0100 formulation was filled into a HPLC
vial (total 5 vials per each composition) and placed on stability
as described in Table 32.
TABLE-US-00032 TABLE 32 Stability conditions Condition Vial # Test
Schedule 2-8.degree. C. 1 Test as "Initial" 2-8.degree. C. 2 Store
at 2-8.degree. C. for 24 hours; Filter using (0.22 .mu.m filtered)
0.22 .mu.m SpinX; filtrate tested by HPLC 25.degree. C. 3 7 and 14
days 40.degree. C. 4 1, 2, 7 and 14 days 60.degree. C. 5 1, 2 and 7
days
[0662] Testing of stability samples included pH (initial only),
appearance, HPLC assay and impurities. Results are shown in Tables
33-49. CC=clear and colorless.
TABLE-US-00033 TABLE 33 Initial pH and appearance Appearance
Formulation ID Initial pH Initial 2-8.degree. C. (filtered), 24 hr
25.degree. C. .times. 7 d 25.degree. C. .times. 14 d T-1 4.1 CC
liquid No change No change No change T-2 5.0 CC liquid No change No
change No change T-3 6.0 CC liquid No change No change No change
T-4 6.6 CC liquid No change No change No change T-5 7.0 CC liquid
No change No change No change T-6 6.9 CC liquid No change No change
No change T-7 7.1 CC liquid No change Slightly Slightly cloudy
cloudy T-8 7.9 CC liquid No change No change No change
TABLE-US-00034 TABLE 34 Appearance Appearance Formulation
40.degree. C. .times. 40.degree. C. .times. 40.degree. C. .times.
40.degree. C. .times. ID Initial 1 d 2 d 7 d 14 d T-1 CC liquid No
change No change No change No change T-2 CC liquid No change No
change No change No change T-3 CC liquid No change No change No
change No change T-4 CC liquid No change No change Slightly
Slightly cloudy cloudy T-5 CC liquid No change No change No change
No change T-6 CC liquid No change No change Slightly Slightly brown
brown T-7 CC liquid No change No change Slightly Slightly cloudy
cloudy T-8 CC liquid No change No change No change No change
TABLE-US-00035 TABLE 35 Appearance Appearance Formulation ID
Initial 60.degree. C. .times. 1 d 60.degree. C. .times. 2 d
60.degree. C. .times. 7 d T-1 CC liquid No change No change No
change T-2 CC liquid No change No change No change T-3 CC liquid No
change No change No change T-4 CC liquid No change No change No
change T-5 CC liquid No change No change No change T-6 CC liquid No
change No change No change T-7 CC liquid No change No change
Slightly cloudy T-8 CC liquid No change No change No change
TABLE-US-00036 TABLE 36 Assay concentration Assay (Concentration
(mg/mL)) Formulation ID Initial 2-8.degree. C. Filtered 25.degree.
C. 7 d 25.degree. C. 14 d T-1 5.45 5.42 5.41 5.39 T-2 5.64 5.66
5.49 5.55 T-3 5.57 5.57 5.48 5.50 T-4 5.48 5.42 5.32 5.25 T-5 6.05
6.07 5.98 5.92 T-6 5.72 5.66 5.49 4.93 T-7 5.38 5.27 0.98 0 T-8
5.50 5.44 5.36 5.04
TABLE-US-00037 TABLE 37 Assay concentration Assay (Concentration
(mg/mL)) Formulation 40.degree. C. 40.degree. C. ID Initial 1 d
40.degree. C. 2 d 40.degree. C. 7 d 14 d T-1 5.45 5.48 5.45 5.23
5.14 T-2 5.64 5.76 5.55 5.48 5.48 T-3 5.57 5.56 5.58 5.33 5.35 T-4
5.48 5.49 5.38 4.49 3.64 T-5 6.05 6.04 6.08 6.12 6.00 T-6 5.72 5.70
5.72 0 0 T-7 5.38 5.21 5.21 5.04 5.00 T-8 5.50 5.26 5.46 5.13
5.03
TABLE-US-00038 TABLE 38 Assay concentration Assay (Concentration
(mg/mL)) Formulation ID Initial 60.degree. C. 1 d 60.degree. C. 2 d
60.degree. C. 7 d T-1 5.45 5.42 5.35 4.73 T-2 5.64 5.69 5.55 5.08
T-3 5.57 5.53 5.47 5.06 T-4 5.48 5.36 5.28 4.98 T-5 6.05 6.02 5.91
5.64 T-6 5.72 5.38 5.55 5.18 T-7 5.38 5.37 5.19 4.68 T-8 5.50 5.14
5.12 3.79
TABLE-US-00039 TABLE 39 Assay recovery Formulation Assay Recovery
(% conc. over initial conc.) ID Initial 2-8.degree. C. Filtered
25.degree. C. 7 d 25.degree. C. 14 d T-1 100 99 99 99 T-2 100 100
97 99 T-3 100 100 98 99 T-4 100 99 97 96 T-5 100 100 99 98 T-6 100
99 96 87 T-7 100 98 18 0 T-8 100 99 97 92
TABLE-US-00040 TABLE 40 Assay recovery Assay Recovery (% conc. over
initial conc.) Formulation 40.degree. C. ID Initial 40.degree. C. 1
d 40.degree. C. 2 d 40.degree. C. 7 d 14 d T-1 100 100 100 96 95
T-2 100 102 98 97 98 T-3 100 100 100 96 96 T-4 100 100 98 82 67 T-5
100 100 101 101 100 T-6 100 100 100 0 0 T-7 100 97 97 94 93 T-8 100
96 99 93 91
TABLE-US-00041 TABLE 41 Assay recovery Assay Recovery (% conc. over
initial conc.) Formulation ID Initial 60.degree. C. 1 d 60.degree.
C. 2 d 60.degree. C. 7 d T-1 100 100 98 87 T-2 100 101 98 90 T-3
100 99 98 91 T-4 100 98 96 91 T-5 100 99 98 93 T-6 100 97 97 91 T-7
100 100 96 87 T-8 100 94 93 69
TABLE-US-00042 TABLE 42 Impurity profile for pH 4 T = 1 d T = 2 d T
= 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree. C. 60.degree.
C. 40.degree. C. 60.degree. C. 25.degree. C. 40.degree. C.
60.degree. C. 25.degree. C. 40.degree. C. 25.14 1.00 99.07 99.06
98.66 97.47 98.17 96.40 98.63 96.44 91.31 98.00 94.84 18.36 0.69 ND
ND ND ND 0.31 ND 0.35 1.63 ND 0.77 2.20 21.61 0.83 ND ND 0.35 0.29
0.32 ND ND ND ND ND ND 22.78 0.90 ND ND ND ND ND ND ND ND 0.23 ND
ND 23.76 0.94 0.23 0.34 0.34 0.37 0.24 0.27 0.27 0.32 0.32 0.28
0.25 26.44 1.06 0.04 ND ND ND ND ND ND ND ND ND ND 26.74 1.07 0.16
0.16 ND 0.18 0.10 0.14 0.18 0.19 ND 0.16 0.10 27.34 1.10 0.17 0.21
0.16 0.19 0.17 0.23 0.13 0.15 0.18 0.16 0.18 28.21 1.14 0.14 0.12
0.23 0.23 0.14 0.45 0.23 0.15 0.77 0.17 0.38 28.52 1.15 0.20 0.11
ND ND 0.17 0.37 ND 0.15 0.37 ND 0.29 28.78 1.16 ND ND 0.26 0.26 ND
ND 0.21 ND ND 0.21 ND 29.99 1.21 ND ND ND 0.19 ND 0.53 ND ND ND ND
ND 30.23 1.22 ND ND ND 0.81 0.36 1.61 ND 0.97 6.82 0.25 1.76 Total
Imp 0.93 0.94 1.34 2.53 1.83 3.60 1.37 3.56 8.69 2.00 5.16
TABLE-US-00043 TABLE 43 Impurity profile for pH 5 T = 1 d T = 2 d T
= 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree. C. 60.degree.
C. 40.degree. C. 60.degree. C. 25.degree. C. 40.degree. C.
60.degree. C. 25.degree. C. 40.degree. C. 25.20 1.00 99.09 98.86
98.86 98.22 98.44 97.68 98.84 97.71 94.25 98.01 96.63 18.41 0.70 ND
ND ND ND 0.24 ND 0.20 0.37 ND 0.41 0.95 21.63 0.84 ND ND ND 0.39
0.20 ND ND ND ND ND ND 22.79 0.90 ND ND ND ND ND ND ND ND 0.35 ND
ND 23.83 0.95 0.29 0.32 0.34 0.38 0.28 0.31 0.28 0.29 0.37 0.28
0.30 26.80 1.06 0.14 0.20 0.19 ND 0.19 0.19 0.10 0.26 0.11 0.16
0.11 27.40 1.09 0.19 0.22 0.16 0.15 0.16 0.22 0.17 0.18 0.21 0.20
0.18 28.25 1.12 ND 0.18 0.24 ND ND ND ND 0.27 ND ND ND 28.59 1.13
0.12 0.22 0.21 0.13 0.23 0.28 0.16 0.29 0.31 0.27 036 28.69 1.16
0.16 ND ND 0.17 0.25 0.18 0.24 ND 0.15 0.33 0.22 29.89 1.21 ND ND
ND 0.12 ND ND ND ND ND ND ND 30.15 1.22 ND ND ND 0.44 ND 1.14 ND
0.63 4.24 0.35 1.25 Total Imp 0.94 1.14 1.14 1.78 1.56 2.32 1.16
2.29 5.75 1.99 3.37
TABLE-US-00044 TABLE 44 Impurity profile for pH 6 T = 1 d T = 2 d T
= 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree. C. 60.degree.
C. 40.degree. C. 60.degree. C. 25.degree. C. 40.degree. C.
60.degree. C. 25.degree. C. 40.degree. C. 25.26 1.00 99.05 98.88
98.67 98.44 98.46 97.99 98.81 97.90 96.72 98.94 96.73 18.28 0.69 ND
ND ND ND ND ND ND 0.57 ND ND 1.19 21.65 0.83 ND ND 0.34 0.35 0.46
0.30 ND ND ND ND 0.29 22.84 0.90 ND ND ND 0.27 ND 0.42 ND ND 0.69
ND 0.31 23.85 0.94 0.33 0.24 0.32 0.33 0.24 0.41 0.50 0.47 0.63
0.23 0.40 26.90 1.07 0.16 0.18 0.20 ND 0.20 0.18 0.15 0.16 0.08 ND
0.15 27.43 1.10 0.21 0.26 0.11 0.31 0.24 0.37 0.17 0.25 0.59 0.24
0.21 28.32 1.14 0.12 0.22 0.15 ND 0.22 0.20 0.20 0.39 0.32 0.26
0.38 28.63 1.15 0.13 0.22 0.21 0.31 0.17 0.13 0.17 0.25 0.24 ND
0.34 28.30 1.18 ND ND ND ND ND ND ND ND ND 0.33 ND 29.83 1.23 ND ND
ND ND ND ND ND ND 0.74 ND ND Total Imp 0.95 1.12 1.33 1.56 1.54
2.01 1.19 2.10 3.28 1.06 3.27
TABLE-US-00045 TABLE 45 Impurity profile for pH 6.5 T = 1 d T = 2 d
T = 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree. C.
60.degree. C. 40.degree. C. 60.degree. C. 25.degree. C. 40.degree.
C. 60.degree. C. 25.degree. C. 40.degree. C. 25.10 1.00 98.94 98.96
98.62 98.77 98.11 98.08 99.04 90.73 96.80 98.44 85.07 9.19 0.27 ND
ND ND ND ND ND ND 0.45 ND ND ND 9.97 0.31 ND ND ND ND ND ND ND 1.92
ND ND 4.79 14.93 0.54 ND ND ND ND ND ND ND ND ND ND 0.76 15.63 0.57
ND ND ND ND ND ND ND 0.63 ND ND 0.78 16.74 0.62 ND ND ND ND ND ND
ND 0.34 ND ND 0.64 17.36 0.65 ND ND ND ND ND ND ND 0.16 ND ND 0.77
18.42 0.69 ND ND ND ND ND ND ND 0.27 ND 0.53 ND 20.54 0.79 ND ND ND
ND 0.22 ND ND 0.45 ND ND ND 21.66 0.83 ND ND 0.34 ND 0.22 0.26 ND
ND ND ND ND 22.82 0.90 ND ND ND ND ND 0.32 ND 0.58 0.76 ND 1.21
23.80 0.94 0.29 0.32 0.32 0.34 0.63 0.40 0.41 2.83 0.73 0.29 3.72
26.14 1.06 ND ND ND ND ND ND ND ND 0.12 ND ND 26.70 1.07 0.11 0.17
0.13 0.14 0.17 0.17 0.15 0.26 0.06 0.12 ND 27.29 1.10 0.34 0.11
0.26 0.33 0.21 0.50 0.17 0.09 1.03 0.13 0.44 28.09 1.14 0.23 0.23
0.17 0.19 0.22 0.12 0.11 1.13 0.28 0.27 1.82 28.50 1.16 0.09 0.22
0.16 0.23 0.20 0.15 0.12 0.16 0.23 0.22 ND Total Imp 1.06 1.04 1.38
1.23 1.89 1.92 0.96 9.27 3.20 1.56 14.93
TABLE-US-00046 TABLE 46 Impurity profile for pH 7 in H.sub.2O T = 1
d T = 2 d T = 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree.
C. 60.degree. C. 40.degree. C. 60.degree. C. 25.degree. C.
40.degree. C. 60.degree. C. 25.degree. C. 40.degree. C. 25.04 1.00
98.79 98.40 98.80 98.93 98.91 98.96 99.02 98.90 97.88 98.91 98.23
18.48 0.70 ND ND ND ND ND ND ND 0.17 0.16 0.12 0.44 21.80 0.83 ND
0.53 0.18 ND ND ND ND ND ND ND ND 23.72 0.94 0.39 0.33 0.33 0.28
0.29 0.31 0.24 0.33 0.19 0.26 0.39 24.08 0.96 ND ND ND ND ND 0.08
ND ND ND ND ND 25.97 1.05 ND ND ND ND ND ND ND ND 0.07 ND ND 26.67
1.07 0.19 0.16 0.14 0.16 0.17 0.20 0.16 0.13 0.06 0.14 0.12 27.24
1.10 0.18 0.21 0.25 0.30 0.26 0.21 0.15 0.25 0.59 0.15 0.25 28.09
1.14 0.20 0.17 0.18 0.17 0.16 0.10 0.20 0.12 0.28 0.22 0.30 28.48
1.16 0.26 0.21 0.13 0.15 0.21 0.15 0.23 0.11 0.27 0.20 0.26 Total
Imp 1.21 1.60 1.20 1.07 1.09 1.04 0.98 1.10 2.12 1.09 1.77
TABLE-US-00047 TABLE 47 Impurity profile for pH 7 in
H.sub.3PO.sub.4 T = 1 d T = 2 d T = 7 d* T = 14 d RT (min) RRT T =
0 Filtrate 40.degree. C. 60.degree. C. 40.degree. C. 60.degree. C.
25.degree. C. 60.degree. C. 25.degree. C. 40.degree. C. 25.13 1.00
98.76 98.48 98.53 98.82 98.91 98.96 98.68 94.79 95.95 No 18.23 0.69
ND ND ND ND ND ND ND ND 0.44 Peak* 21.85 0.83 ND 0.40 0.39 ND ND ND
ND ND ND 22.61 0.90 ND ND ND ND ND ND ND 1.07 0.40 23.82 0.94 0.33
0.34 0.34 0.34 0.29 0.31 0.40 0.96 2.23 24.08 0.96 ND ND ND ND ND
0.08 ND ND ND 25.91 1.05 ND ND ND ND ND ND ND 0.44 ND 26.73 1.07
0.14 0.18 0.16 0.15 0.17 0.20 0.13 0.07 0.11 27.26 1.10 0.19 0.18
0.27 0.37 0.26 0.21 0.20 1.45 0.20 28.16 1.14 0.23 0.20 0.15 0.17
0.16 0.10 0.33 0.56 0.38 28.47 1.15 0.36 0.22 0.16 0.14 0.21 0.15
0.27 0.66 0.30 Total Imp 1.24 1.52 1.47 1.18 1.09 1.04 1.32 5.21
4.05 *No peak was observed for the pH 7 solutions in
H.sub.3PO.sub.4 at 40.degree. C. at 7 and 14 days
TABLE-US-00048 TABLE 48 Impurity profile for pH 7 in 0.9% NaCl T =
1 d T = 2 d T = 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree.
C. 60.degree. C. 40.degree. C. 60.degree. C. 25.degree. C.
40.degree. C. 60.degree. C. 25.degree. C. 40.degree. C. 25.10 1.00
98.78 98.39 98.52 98.76 98.79 98.51 38.60 97.96 96.44 No 97.37
18.36 0.69 ND ND ND ND ND ND ND 0.26 ND Peak 0.59 20.49 0.79 ND ND
ND ND ND ND ND 0.18 ND ND 21.73 0.83 ND 0.34 0.33 ND ND ND 57.88 ND
ND ND 22.75 0.88 ND ND ND ND ND ND 0.49 ND 0.54 ND 23.75 0.94 0.27
0.31 0.38 0.39 0.42 0.31 2.90 0.66 1.46 0.79 26.22 1.05 ND ND ND ND
ND ND 0.14 ND 0.72 ND 26.70 1.07 0.17 0.22 0.15 0.19 0.12 0.11 ND
0.07 0.05 0.10 27.27 1.10 0.29 0.18 0.20 0.27 0.24 0.51 ND 0.40
1.51 0.56 28.18 1.14 0.25 0.22 0.20 0.18 0.23 0.26 ND 0.25 0.39
0.23 28.41 1.15 0.24 0.34 0.22 0.21 0.20 0.30 ND 0.22 0.89 0.35
Total Imp 1.22 1.61 1.48 1.24 1.21 1.49 61.40 2.04 5.56 2.63 *No
peak was observed for the pH 7 solutions in 0.9% NaCl at 25.degree.
C. at 14 days
TABLE-US-00049 TABLE 49 Impurity profile for pH 8 T = 1 d T = 2 d T
= 7 d T = 14 d RT (min) RRT T = 0 Filtrate 40.degree. C. 60.degree.
C. 40.degree. C. 60.degree. C. 25.degree. C. 40.degree. C.
60.degree. C. 25.degree. C. 40.degree. C. 25.14 1.00 98.35 98.26
98.75 97.80 98.45 93.68 99.34 97.50 81.21 98.65 95.09 19.89 0.77 ND
ND ND ND ND 0.45 ND ND 1.16 ND 0.25 20.40 0.79 ND ND ND ND ND 0.26
ND ND 0.86 ND 0.25 20.84 0.81 ND ND ND ND ND 0.08 ND ND 0.31 ND ND
21.74 0.83 ND 0.53 ND ND ND ND ND ND ND ND ND 21.61 0.85 ND ND ND
ND ND 0.24 ND ND 0.68 ND ND 23.04 0.91 ND ND ND 0.24 ND 0.68 ND
0.50 2.43 ND 0.79 23.79 0.94 0.32 0.43 0.26 0.48 0.40 0.61 ND 0.61
1.49 0.57 1.05 23.91 0.95 ND ND ND 0.23 ND 0.69 ND ND 3.22 ND ND
24.62 0.99 ND ND ND ND ND 0.23 ND ND 0.77 ND ND 25.92 1.05 ND ND ND
ND ND 0.92 ND ND 2.57 ND 0.47 26.75 1.07 0.14 0.19 0.13 0.13 0.19
0.10 0.12 0.13 ND 0.13 ND 27.31 1.10 0.24 0.16 0.26 0.34 0.28 0.53
0.22 0.35 1.39 0.21 0.59 28.23 1.14 0.25 0.21 0.31 0.28 0.32 0.43
0.15 0.38 1.03 0.22 0.51 28.49 1.15 0.21 0.21 0.27 0.50 0.37 0.92
0.18 0.53 2.85 0.23 1.01 Total Imp 1.15 1.74 1.25 2.20 1.55 6.14
0.66 2.50 18.79 1.35 3.39
[0663] MMI-0100 formulation solutions pH 6.5 with citrate, pH 7
with phosphate, pH 7 with 0.9% NaCl and pH 8 with L-lysine showed
haziness, indicating the presence of precipitates.
[0664] FIGS. 79 A and B summarize the assay recovery and impurity
growth at 25.degree. C. FIGS. 80 A and B summarize the assay
recovery and impurity growth at 40.degree. C. FIGS. 81 A and B
summarize the assay recovery and impurity growth at 60.degree.
C.
[0665] The results of this study indicated that: [0666] i. MMI-0100
is most stable at pH 7; [0667] ii. phosphate and NaCl induced
precipitation of MMI-0100 at pH 7; [0668] iii. citrate induced
precipitation of MMI-0100 at pH 6.5; [0669] iv. in citrate,
MMI-0100 is most stable at pH 6; [0670] v. pH.sub.max for MMI-0100
is pH 7 and DI water (i.e. no buffer) was the best solution; [0671]
vi. in T-5, there were 5 impurities exceeding 0.2% detected at the
initial (T=0) testing; [0672] vii. in T-5 (pH 7 without a buffer),
the assay recovery was near 100% after 14 days at 40.degree. C. and
93% after 7 days at 60.degree. C., indicating the shelf life
(defined by T.sub.90) is likely to be 2 years at 25.degree. C. or 2
years at 5.degree. C.; [0673] viii. when T-5 reaches the T.sub.90
(e.g. 10% assay loss), 7 impurities may grow to exceed 0.1% (the
top three impurities were RRT=1.14, RRT=0.94 and RRT=0.70); [0674]
ix. after 7 days at 60.degree. C. in T-5, the assay loss was 7% and
total impurity was 2.12%, indicating that the impurities may have a
lower extinction coefficient at the detection wavelength of 215 nm;
and [0675] x. without being limited by theory, it is suspected that
the RRT=1.14, RRT=0.94 impurities are the deamination products
(Gin' and Gin') and RRT=0.70 impurity is a hydrolysis product.
Example 9. Evaluation of Stability of Several MMI-0100 Formulation
Solutions at pH 7 in DI Water Containing Various Osmotic Agents
and/or Lyoprotectants
[0676] The purpose of this study was to determine osmotic pressure
of non-buffered 0.7 mg/mL and 7 mg/mL solutions in water at pH 7,
select an osmotic agent(s) based on stability, calculate the
concentration of osmotic agent(s) needed to reach the iso-osmotic
pressure (e.g., Glycerin IIL limit for inhalation is 7.3%; Lactose
IIL limit for inhalation is 9%).
[0677] MMI-0100 formulation solutions were prepared as described in
Table 50.
TABLE-US-00050 TABLE 50 MMI-0100 formulation solutions Component
F-1 F-2 F-3 F-4 MMI 0.7 7 7 7 Glycerin 0 0 Amount needed 0 for
isotonicity Lactose 0 0 0 Amount needed for isotonicity DI-water,
qs qs qs qs qs Adjust pH to 7 7 .+-. 0.1 7 .+-. 0.1 7 .+-. 0.1 7
.+-. 0.1 with NaOH/HCl
[0678] 5 g of each MMI-0100 formulation solution was prepared. 0.7
mL of each formulation solution was added to an HPLC glass vial (5
vials each). One HPLC vial was used as T=0. The remaining 4 HPLC
vials were stored at 60.degree. C. and tested at 0, 1, 2 and 4
weeks. Results are shown in Tables 51-59.
TABLE-US-00051 TABLE 51 Appearance Formu- Appearance lation 1 week
2 weeks 4 weeks at ID Initial at 60.degree. C. at 60.degree. C.
60.degree. C. F-1 Clear, colorless liquid No change No change F-2
Clear, colorless liquid No change No change F-3 Clear, colorless
liquid No change No change F-4 Clear, colorless liquid No change No
change
TABLE-US-00052 TABLE 52 Osmotic pressure adjustment Osmotic Amount
of Final Osmotic Formulation Pressure Initial Osmotic Modifier
Added Pressure ID Modifier Pressure (mOsm) (% w/w) (mOsm) F-1 None
13 0 13 F-2 None 24 0 24 F-3 Glycerin 35 (before adding 2.2% 286
glycerin) F-4 Lactose 31 (before adding 7.6% 299 lactose)
TABLE-US-00053 TABLE 53 pH pH Formulation 1 week 2 weeks 4 weeks ID
Initial at 60.degree. C. at 60.degree. C. at 60.degree. C. F-1 7.0
8.3 8.1 F-2 7.1 6.9 7.1 F-3 7.1 7.0 7.1 F-4 7.0 6.6 6.3
TABLE-US-00054 TABLE 54 Concentration MMI-001 Concentration (mg/mL)
Formulation 1 week 2 weeks 4 weeks ID Initial at 60.degree. C. at
60.degree. C. at 60.degree. C. F-1 0.65 0.49 0.35 F-2 6.65 6.84
6.47 F-3 6.45 6.59 6.17 F-4 6.04 5.03 4.02
TABLE-US-00055 TABLE 55 Assay concentration (percent concentration
over initial concentration) Assay (% conc. over initial conc.) ID 1
week 2 weeks 4 weeks Formulation Initial at 60.degree. C. at
60.degree. C. at 60.degree. C. F-1 100 75 54 F-2 100 103 97 F-3 100
102 96 F-4 100 83 67
TABLE-US-00056 TABLE 56 Impurity profile for F-1 (peak area %) RT
(min) RRT T = 0 T = 1 wk at 60.degree. C. T = 2 wk at 60.degree. C.
24.364 1.00 97.21 76.65 53.59 6.791 0.28 2.44 2.82 2.75 19.706 0.79
ND 1.15 2.16 20.246 0.81 ND 0.96 2.04 20.644 0.83 ND 0.42 1.18
21.836 0.88 ND 0.84 4.56 22.432 0.90 ND 1.53 1.39 22.749 0.91 ND
2.47 5.09 22.955 0.94 0.34 1.49 3.62 23.633 0.95 ND 3.48 6.92
24.451 0.98 ND 0.77 2.18 25.784 1.04 ND 2.42 5.25 26.763 1.07 ND
1.26 2.66 27.767 1.12 ND 1.04 2.00 28.102 1.13 ND 2.70 4.61 Total
Imp 2.79 23.35 46.41 ND = Not Detected
TABLE-US-00057 TABLE 57 Ipurity profile for F-2 (peak area %) T = 1
wk T = 2 wk T = 4 wk RT (min) RRT T = 0 60.degree. C. 60.degree. C.
60.degree. C. 24.40 1.00 99.56 98.17 96.29 23.00 0.94 0.44 0.62
1.11 26.77 1.08 ND 1.21 2.60 Total Imp 0.44 1.83 3.71 ND = Not
Detected
TABLE-US-00058 TABLE 58 Impurity profile for F-3 (peak area %) T =
1 wk T = 2 wk T = 4 wk RT (min) RRT T = 0 60.degree. C. 60.degree.
C. 60.degree. C. 24.41 1.00 100 98.07 93.95 23.38 0.94 ND 0.50 1.12
26.05 1.04 ND ND 1.47 26.80 1.08 ND 1.43 3.05 28.25 1.13 ND ND 0.41
Total Imp 0 1.93 6.05 ND = Not Detected
TABLE-US-00059 TABLE 59 Impurity profile for F-4 (peak area %) T =
1 wk T = 2 wk T = 4 wk RT (min) RRT T = 0 60.degree. C. 60.degree.
C. 60.degree. C. 24.47 1.00 97.60 84.26 72.31 23.04 0.94 0.26 ND
0.85 23.94 0.95 ND ND 0.60 24.39 0.98 ND 11.11 19.45 25.42 1.04
2.14 3.81 4.88 26.78 1.07 ND 0.81 1.91 Total Imp 2.40 15.74 27.69
ND = Not Detected
[0679] The results of this study indicated that: [0680] i. MMI-0100
formulation solution at pH 7 without a buffer was capable of
maintaining its pH at 7 at the high concentration (7 mg/mL),
whereas the pH drifted up to about 8 at the lower concentration
(0.7 mg/mL), indicating that at 7 mg/mL strength, no pH buffer is
needed; [0681] ii. the addition of lactose resulted in pH drift
(down to about 6) and appeared to cause more degradation of
MMI-0100; [0682] iii. the addition of glycerin did not cause pH
drift in the high concentration formulation, thus, glycerin is
preferred over lactose; [0683] iv. the addition of glycerin to the
MMI-0100 formulation solution also caused slightly more degradation
of MMI-0100 (F-3) than the formulation solution without an osmotic
agent (F-2), thus, if an isosmotic formulation is not necessary,
the F-2 formulation solution would be preferred.
[0684] While the described invention has been described with
reference to the specific embodiments thereof it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adopt a particular situation,
material, composition of matter, process, process step or steps, to
the objective spirit and scope of the described invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
27122PRTUnknownMammalian 1Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg
Ala Lys Ala Leu Ala Arg 1 5 10 15 Gln Leu Gly Val Ala Ala 20
211PRTUnknownMammalian 2Lys Ala Leu Ala Arg Gln Leu Gly Val Ala Ala
1 5 10 321PRTUnknownMammalian 3Phe Ala Lys Leu Ala Ala Arg Leu Tyr
Arg Lys Ala Leu Ala Arg Gln 1 5 10 15 Leu Gly Val Ala Ala 20
423PRTUnknownMammalian 4Lys Ala Phe Ala Lys Leu Ala Ala Arg Leu Tyr
Arg Lys Ala Leu Ala 1 5 10 15 Arg Gln Leu Gly Val Ala Ala 20
521PRTUnknownMammalian 5Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala
Lys Ala Leu Ala Arg 1 5 10 15 Gln Leu Ala Val Ala 20
621PRTUnknownMammalian 6Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala
Lys Ala Leu Ala Arg 1 5 10 15 Gln Leu Gly Val Ala 20
722PRTUnknownMammalian 7His Arg Arg Ile Lys Ala Trp Leu Lys Lys Ile
Lys Ala Leu Ala Arg 1 5 10 15 Gln Leu Gly Val Ala Ala 20
810PRTUnknownMammalian 8Lys Ala Leu Ala Arg Gln Leu Ala Val Ala 1 5
10 910PRTUnknownMammalian 9Lys Ala Leu Ala Arg Gln Leu Gly Val Ala
1 5 10 1011PRTUnknownMammalian 10Lys Ala Leu Ala Arg Gln Leu Gly
Val Ala Ala 1 5 10 1111PRTUnknownMammalian 11Tyr Ala Arg Ala Ala
Ala Arg Gln Ala Arg Ala 1 5 10 1214PRTUnknownMammalian 12Trp Leu
Arg Arg Ile Lys Ala Trp Leu Arg Arg Ile Lys Ala 1 5 10
137PRTUnknownMammalian 13Trp Leu Arg Arg Ile Lys Ala 1 5
1411PRTUnknownMammalian 14Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg 1 5 10 1512PRTUnknownMammalian 15Trp Leu Arg Arg Ile Lys Ala
Trp Leu Arg Arg Ile 1 5 10 1610PRTUnknownMammalian 16Phe Ala Lys
Leu Ala Ala Arg Leu Tyr Arg 1 5 10 1712PRTUnknownMammalian 17Lys
Ala Phe Ala Lys Leu Ala Ala Arg Leu Tyr Arg 1 5 10
1811PRTUnknownMammalian 18His Arg Arg Ile Lys Ala Trp Leu Lys Lys
Ile 1 5 10 1921PRTUnknownMammalian 19Tyr Ala Arg Ala Ala Ala Arg
Gln Ala Arg Ala Lys Ala Leu Asn Arg 1 5 10 15 Gln Leu Gly Val Ala
20 209PRTUnknownMammalian 20Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5
2117PRTUnknownMammalianX(3)..(12)Any amino acid 21Lys Lys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Arg Arg Lys 1 5 10 15 Lys
227PRTUnknownMammalian 22Leu Leu Lys Arg Arg Lys Lys 1 5
2322PRTUnknownMammalian 23Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg
Ala Lys Ala Leu Asn Arg 1 5 10 15 Gln Leu Ala Val Ala Ala 20
2421PRTUnknownMammalian 24Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg
Ala Lys Ala Leu Asn Arg 1 5 10 15 Gln Leu Ala Val Ala 20
2511PRTUnknownMammalian 25Lys Ala Leu Asn Arg Gln Leu Ala Val Ala
Ala 1 5 10 2610PRTUnknownMammalian 26Lys Ala Leu Asn Arg Gln Leu
Ala Val Ala 1 5 10 2713PRTUnknownMammalian 27Trp Leu Arg Arg Ala
Ser Ala Pro Leu Pro Gly Leu Lys 1 5 10
* * * * *