US 2016O200782A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0200782 A1 Lander et al. (43) Pub. Date: Jul. 14, 2016

(54) FORMULATION OF MK2 INHIBITOR Publication Classification PEPTIDES (51) Int. Cl. (71) Applicant: MOERAE MATRIX, INC., C07K 4/47 (2006.01) Morristown, NJ (US) A647/32 (2006.01) A647/02 (2006.01) (72) Inventors: Cynthia Lander, Mendham, NJ (US); A647/26 (2006.01) Colleen Brophy, Nashville, TN (US); A619/00 (2006.01) Caryn Peterson, Encinitas, CA (US) (52) U.S. Cl. CPC ...... C07K 14/4703 (2013.01); A61K 47/26 (2013.01); A61 K9/0075 (2013.01); A61 K (21) Appl. No.: 14/991,531 47/02 (2013.01); A61K 9/0078 (2013.01); A61K 47/32 (2013.01); A61 K9/0019 (2013.01); A61 K38/00 (2013.01) (22) Filed: Jan. 8, 2016 (57) ABSTRACT The described invention provides pharmaceutical formula tions comprising a polypeptide of amino acid sequence Related U.S. Application Data YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a (60) Provisional application No. 62/101,190, filed on Jan. functional equivalent thereof with improved stability and bio 8, 2015. availability. Patent Application Publication Jul. 14, 2016 Sheet 1 of 85 US 2016/0200782 A1

Figarei.

Technical Data Sheet Bister Liciting - Push Through

: 8xxx 88.8 x 8x8. 8xxx xxx ::::::::88xx:8xxx 8888.

x 8xxx xxx 8x8.

8x8x8xxx xxxx 8x88: 8888.3888 8888 8xxx: 888 8xxx xxx xxx xxx 8:8xx$888 888. & &x & x8: 8:8;&x: 8&.

Patent Application Publication

& s

Patent Application Publication Jul. 14, 2016 Sheet 4 of 85 US 2016/0200782 A1

Figure 4

Patent Application Publication Jul. 14, 2016 Sheet 5 of 85 US 2016/0200782 A1

Figure 5 Patent Application Publication Jul. 14, 2016 Sheet 6 of 85 US 2016/0200782 A1

Figure 6

Patent Application Publication Jul. 14, 2016 Sheet 7 of 85 US 2016/0200782 A1

F.

-- is SOLIDS-5 g : - ; SOLDS-8 g -&- 8 SOLIDS-10 Rg 3.

2

Q Patent Application Publication Jul. 14, 2016 Sheet 8 of 85 US 2016/0200782 A1

Figure 3

«'s Sir * x 3 as

weig (g Patent Application Publication Jul. 14, 2016 Sheet 9 of 85 US 2016/0200782 A1

Figure 9

Patent Application Publication Jul. 14, 2016 Sheet 10 of 85 US 2016/0200782 A1

Figre 0

*&^is -8-3 weeks ''x'' 4 weeks

Patent Application Publication Jul. 14, 2016 Sheet 11 of 85 US 2016/0200782 A1

Figi'e 1

stice Size strities a Recovereirag Ci is ki x:is Xxxw8:8 x 8.8x8

Patent Application Publication Jul. 14, 2016 Sheet 12 of 85 US 2016/0200782 A1

Figure 12

1750 Xxinitial x2-weeks x weeks

1500 100013. Patent Application Publication Jul. 14, 2016 Sheet 13 of 85 US 2016/0200782 A1

Fig. re 13

xiiia x8°C; i3%8 x 23°Ci8%R x-8

Patent Application Publication Jul. 14, 2016 Sheet 14 of 85 US 2016/0200782 A1

Figure 4

x initial x2-weeks «i-weeks

Patent Application Publication Jul. 14, 2016 Sheet 15 of 85 US 2016/0200782 A1

Figure 15

x initis x-weeks x^4-weeks

1750 Patent Application Publication Jul. 14, 2016 Sheet 16 of 85 US 2016/0200782 A1

Figure: 35

xiii. xx; 9 is: 8 x 25 °C f : 8 x2-8

500 250 a00 3150 : ; : Patent Application Publication Jul. 14, 2016 Sheet 17 of 85 US 2016/0200782 A1

Figure i7

xiii. 8-yes xx weeks

Patent Application Publication Jul. 14, 2016 Sheet 18 of 85 US 2016/0200782 A1

Figure 33

xx initial x2-weeks «4-weeks Patent Application Publication Jul. 14, 2016 Sheet 19 of 85 US 2016/0200782 A1

Figare 3

xiiia xx; C F S: X&xis Ci 8%. R. x.8 °C Patent Application Publication Jul. 14, 2016 Sheet 20 of 85 US 2016/0200782 A1

Figure 20

xiritis xx2-weeks x4-weeks Patent Application Publication Jul. 14, 2016 Sheet 21 of 85 US 2016/0200782 A1

Figure 2

xiritis x3-weeks «4-weeks

Patent Application Publication Jul. 14, 2016 Sheet 22 of 85 US 2016/0200782 A1

Figure 22

itial xx4°C f is 9%. R. xx25°C 6% R x2-8°C

Patent Application Publication Jul. 14, 2016 Sheet 23 of 85 US 2016/0200782 A1

Figure 23

V- x-Mwww.--8ww.wawhm. www.------axw-Xww. 4. 3x-axas-X k w xxxx"S" " re-' "*g irrirrirrrrrrr," (, "", r r 888 Patent Application Publication Jul. 14, 2016 Sheet 24 of 85 US 2016/0200782 A1

Figure 24

Patent Application Publication Jul. 14, 2016 Sheet 25 of 85 US 2016/0200782 A1

Figure 25

Maxxx-xx-x-xx-aa-awaw------.--- Myx -

X -

\w- Mixx-M&^w;| - xxi-x-xx-xx-x**** rter?,x-rr re-r-s": ' ' ' ' ' ' ' ' ' ' ' ' '. w Patent Application Publication Jul. 14, 2016 Sheet 26 of 85 US 2016/0200782 A1

Figure 26

Patent Application Publication Jul. 14, 2016 Sheet 27 of 85 US 2016/0200782 A1

Figure 27

Scof (20 timin)

83 cities Afflew 20 ini i (in emperature: 23°C it 2°C Relative Humidity 50%. 5% 8::::::::::::::::::::::::::::::::::''...'...'...'...'...'''''''''''''''''''''''''''''''''''''''''www.www.

Netize Patent Application Publication Jul. 14, 2016 Sheet 28 of 85 US 2016/0200782 A1

Figure 23

3. 8 : xtractic 88 Patent Application Publication Jul. 14, 2016 Sheet 29 of 85 US 2016/0200782 A1

Figure 29

s, beivered Dose and Respirable Bose <5 in

.

.

8.8 s . is, s. 3. Filies - 38 g. & & Resan are linear bid or linear Riks Patent Application Publication Jul. 14, 2016 Sheet 30 of 85 US 2016/0200782 A1

Figure 30

evered ose and Respirate lose KS

y s 3,8839x - p s s ...'...' ------. -----' ' ' ' ' '.'.' ' ' ' '.' ' ' ------' ---->'------'X---'X'X'''''''v's

s 50 x e (, : s 3. File:-388 &ntag

& 8.383 & 8:3x: x 3.3883: :::::: i888: 83x38:8: Patent Application Publication Jul. 14, 2016 Sheet 31 of 85 US 2016/0200782 A1

Figure 31.

Nebulization time in

8. 888. 8 0.7 mg/ml. 0.7 mg/ml. : 70 mg/ml. 8 .g. 8::::::::::::: 8x8 8888:388x338

type 30 Type 40 Patent Application Publication Jul. 14, 2016 Sheet 32 of 85 US 2016/0200782 A1

Figure 32

evere exerg 2,

0.7 mg/ml. 7.0 mg/ml 7.0 mg/ml. wgie. 888 8:388x88

Type 30 Type 40 Patent Application Publication Jul. 14, 2016 Sheet 33 of 85 US 2016/0200782 A1

Figure 33

Respirable Dose <5 in

88: 0.7 mg/ml, 0.7 mg/ml. 7.0 mg/ml 8,888; i. 8:38:::::::::::::::::::::::::

Type 30 Type 40 Patent Application Publication Jul. 14, 2016 Sheet 34 of 85 US 2016/0200782 A1

Figure 34

Cytokines & with Factors Mechanical GO ... Environmental Tauma N ?/ Stress (i.e., Temp., ROS)

Migration & inflammatory WaSOConstriction Cytokine Production

Patent Application Publication Jul. 14, 2016 Sheet 36 of 85 US 2016/0200782 A1

Figurs 36

Cuantification of Different MK2 formutations uptake & Retention d 882: Colocalizati 8 MK2-NPs 8 AK2. Peptide & CNTRL-NFs w pro, KQX71. 88) : station procco.2 & toos: sists: w

iss s :

3. asses WW s 22 g 2 it catation title his *: post-treativek incusation time ------38's 238ts s s sf SF: ki w % Peptide Colocalization with lysatracker (pH 2. Red Blocal hemotiasy 8 pH 5.6 8 MK2-NP 8 RAK2 aptide & NE-MK2i-NR

px.ss arg sixtee 8% 88sixtegic Early endoso late endissotte, tysostine % 2.84 .. acces asses

g 88% 8.8%:. w 68% : 80% : & 40% : 40% S. 28% 20%

8% k-a----&-X X--- 3% PAA K-EP MK2 E-3- 2 24 inciation Eine his Patent Application Publication Jul. 14, 2016 Sheet 37 of 85 US 2016/0200782 A1

Figure 37

Stickess after is rex is of pac.004 six, 8. W. 895 M :ys

5

s it ex w to K2. MKPetics -N

hard P. A phosphorylation in Sw x: x:

hosphe-hir RNP AG 38.3x3

838...... hnRNP Aoi. ... is e : hospho-CRES

RE8

Phospho-HSP2

...-- tro Fiki- 2i reptics

HSP27 phosphorylation in SW 8 CREB piosphorylation in SW sty st : :. : :

0.

re

2- MK28&title Patent Application Publication Jul. 14, 2016 Sheet 38 of 85 US 2016/0200782 A1

Figure 38

Afts -stimulated NFc production Boyder answeisigration Assay s: s sy -o-it,30s...ac. isogo 200 - acces A. s

2 8 s

2- MK2, NE-MK2-teA - . f vK2.jpi. ifE. - G A: -S --- Stratic World Wigratic Assa s : y m S -- B

... pak: .039

3. ';

Patent Application Publication Jul. 14, 2016 Sheet 39 of 85 US 2016/0200782 A1

so ge ex xrs (Art SSN3OH. VINI Patent Application Publication Jul. 14, 2016 Sheet 40 of 85 US 2016/0200782 A1

:

xer w is career were rea s Ni3S A 830 8

S Patent Application Publication Jul. 14, 2016 Sheet 41 of 85 US 2016/0200782 A1

Figure 40

M+2-12 S83: $83.288 838& 83.38 88 $8:38: 3888 : $42,838

; 3388 $2.88: .." 33 Patent Application Publication Jul. 14, 2016 Sheet 42 of 85 US 2016/0200782 A1

Figure 4.

Patent Application Publication Jul. 14, 2016 Sheet 43 of 85 US 2016/0200782 A1

Figure 42

A AAGPC spectra

s 5 20 25 3. 3S 4) 45 inte (ins

3. PPAAGPC spectrum O.S :

S s 3. 8 63 s

3S 4) 45 Patent Application Publication Jul. 14, 2016 Sheet 44 of 85 US 2016/0200782 A1

Figure 43

&

. 8 Diarieter inst

NE-M82-9s diaster 2:4:33

88

g w k·

&r-rror-rrierrer----Giafeter Patent Application Publication Jul. 14, 2016 Sheet 45 of 85 US 2016/0200782 A1

Figure 44

Red Blood Cei Henrolysis Assay pH 7.4 8 ph, 6.8 8 pH 5.2 8 pH 5.6 Physiologic Early enclosene late endosorie lysosome

50%

se 40% 20%

------xx------:38-K-x.i.a. 3. 3.: gift 5 gami 4t isfy igfamil Sigfrn at tisfin agfmi Sigfrit &(gfmi i girl. Siginal 40 agfin M anananana --~~~~ A& MK2i-N MK2i Peptide NE-Wik2.N. Patent Application Publication Jul. 14, 2016 Sheet 46 of 85 US 2016/0200782 A1

Figure 45

--- traceluiar Compartet Size t

:

3

Viki-Ns K NE-K2-Ns

assssssssssssssstaaaaaa. ------Patent Application Publication Jul. 14, 2016 Sheet 47 of 85 US 2016/0200782 A1

Figure 46

SW intina hickness

a. precuit Nr. 50k Mitt as 8th Souvi Gosty lost 50g out 500. 19898M PFAA polymer (3. K2 E.-K.

Patent Application Publication Jul. 14, 2016 Sheet 48 of 85 US 2016/0200782 A1

2 *

8 4.s w

s

saw

M M M 10 M 100 IM 101 him MKi-NP V MK

Patent Application Publication Jul. 14, 2016 Sheet 49 of 85 US 2016/0200782 A1

Figure 48

N.F. ESA in ANG stiniated CAVSMCs N. : V & S M S S if 8 2,000 sk,

f i, s s s: 3,200 o e. s 8 s S 400 x :

m Mawawaw mease---ee-ee-a--M.- WK2. NE-Ki-N: kolkow - ANs - ANG

m-----axx-x-xx-x-xx-x-xx-xxxx Patent Application Publication Jul. 14, 2016 Sheet 50 of 85 US 2016/0200782 A1

Figure 49

intereukin-6 ESA in CAVSMCs 1400 Controls E 10 M S 25 M SOM

1200 7.

8

.

x: N Wiki- K to twox Kiror - Nes Patent Application Publication Jul. 14, 2016 Sheet 51 of 85 US 2016/0200782 A1

Figure 50

HCAVSMC Viability 28% N :: O M 25 M 850 V 800 M

%

8 %

6%

4%

%

% Patent Application Publication Jul. 14, 2016 Sheet 52 of 85 US 2016/0200782 A1

Figure S1

HCAVSMC Cell Viability Contro is 10 M 25 M S 50 M Patent Application Publication Jul. 14, 2016 Sheet 53 of 85 US 2016/0200782 A1

Figure S2

3.

l, 2. s 8s11Ya.

i. s

. s

s it i? 100pm ion copy of 100M ion loopy

Ki- K. E.-K. MK2-NP MK2, NE-MK2-NP Patent Application Publication Jul. 14, 2016 Sheet 54 of 85 US 2016/0200782 A1

e s & ae

i ar ---

---

is Saais e Patent Application Publication Jul. 14, 2016 Sheet 55 of 85 US 2016/0200782 A1

Figure 54.

st-seater A 82-Ns 8K2. Peptide &R's incaiation time

Stil s 2 Eirc.7 6 sciss g30014 w 5 assigge)01. pe,039cos s s e 3.

ar 333333-s-s-s-X-SS 3. 2 2 Post-treatment incubation time hrs 8 axa-Ps ex-R(2i Peptide -8. RE-K2-NPs x3x3 &:38: &:3.388 i 0000 3:33,888ys s:8.388:3 88.388: x:::::::::::::::::::::::::::::::::::::::::::::::------155 s --- ^x.

A.

m :------eeee-ee: 4. 60 S. 2 inciation time hrs) C -xxiii- &-Ki i8 EK2-p sits st s

2 5. 8 Post-treatentinctiation inners Patent Application Publication Jul. 14, 2016 Sheet 56 of 85 US 2016/0200782 A1

figure SS

axis, 382. gassiggs-r

s RK2 x-RAKai-N3. 3888-8 8. s roxexo-oo-eeee-eeeeeeeee, ------K&N Ree XEE-3K83-N3.

s: sec.02.assinaissaxxarwww.www.www.www.ww.www.www. sox{38.

six 3.838 w assess 3.338 88s... 888 33Cs, ... it...--ix. s-s-s sses:

R kx ox - Ss. fixes:

as

S. 3

a.

38 M. s 88: 338 siR test 883 as i. . sing 8x3 N-2-3 3. ... $288 4 stees Patent Application Publication Jul. 14, 2016 Sheet 57 of 85 US 2016/0200782 A1

Figure S6 A N (MK2i-NP x MK2 s:83.38 2% is p:::::::: 100% 80% s: :

%

8 -

3. says post-treat set s of N e Ki-N x Kei % past,3 pe,02 . so est.88 %, . x"; poor

3% .

is .

% : ------...-- Days post-treatent Patent Application Publication Jul. 14, 2016 Sheet 58 of 85 US 2016/0200782 A1

Figure 57 MK2-NP Membrane Binding vs. internalization 2,500

2, {{ 1,500 1,000

S

w Trypan blue/cell Scrub Patent Application Publication Jul. 14, 2016 Sheet 59 of 85 US 2016/0200782 A1

Figure 53 s % Peptide Colocalization with lysotracker (Ki-NP x. Kei eptice x NE-tyikai-NP gi. c.3. i es.c:8: s 83. gifts

8%

6.

cisatia tiers tracear Con partnet Size

g 3

8 s

E-Ki-Ms Patent Application Publication Jul. 14, 2016 Sheet 60 of 85 US 2016/0200782 A1

Patent Application Publication Jul. 14, 2016 Sheet 61 of 85 US 2016/0200782 A1

F. S. Ct.

- BE CHANNE as a RE CHANNE Patent Application Publication Jul. 14, 2016 Sheet 62 of 85 US 2016/0200782 A1

Figure 60

Patent Application Publication Jul. 14, 2016 Sheet 63 of 85 US 2016/0200782 A1

3. WSC Boyders rangwe Migration Assay f rites S.P. Presis s s . . . . . xxi- . . xxii exexts -c.c. S.: ES s pa,38 press s r E. is as: &k. 30. & 25% 3.s is $ 20% s S.E. 5 & s 68 s 8 e

N RN 3 s ... awsawassasaasaias as estate 8 site stat-3 if as x2- . xx

3. xxx 8 s so goes 3 8. E.8ayder answei Migratio Assay s 8. 8. s: age. 3: S; imo- ; : 10% ays post-teatient 28% 2 so: St. S.S., 8

& lo St Sayeen Eraswelligratiog assa s - Says Post-treate

acaxxassigg 88 : 2 : 2.88. 83. ;

A. so E Es SS s & RF radiction in Sys s 8:t sisi. 8 : ga? ear presse: . 23.38 2,6 s S.r. ... i2. E. 898 i actic ex

.

Patent Application Publication Jul. 14, 2016 Sheet 64 of 85 US 2016/0200782 A1

Figure 62

Rabbit Weir Graf Erinathickess 28 days Post-ransplant Weir Grafia-Stott peg,835 : Auscle Acti Staining g.883, 99.8;

8

3. R2s. 38&AK2

K

Rabbit vein Graft intina PEA Staining Rai?ei Grafinitia irretti s 5399. Staining gag.02

SP litics, ei, advertis E-R. 2 Patent Application Publication Jul. 14, 2016 Sheet 65 of 85 US 2016/0200782 A1

8 S

s Patent Application Publication Jul. 14, 2016 Sheet 66 of 85 US 2016/0200782 A1

fiSX SS NINS

**

Patent Application Publication Jul. 14, 2016 Sheet 68 of 85 US 2016/0200782 A1

Figure 65 A. 10000

S. 5. w So 100 s to s s s s N : s : ;3 indicates unirada size distristian Miscs:ctoraa S K2-Niage is 2s

-- is S. to s S rarerra------xxx -- s:s was: s s Riaineers

lead K2i-NP. Forriatics:

Electrostatic

compexation is 18.9 it 26 nr. at .9 it 3.8mV CR NH;COO as i:3 wear ratio MK2:PPAA - 8:1 Mass ratio MK2:PPAA is 1:1.2 Patent Application Publication Jul. 14, 2016 Sheet 69 of 85 US 2016/0200782 A1

Figure 66 A p-HSP20-NP library Size and 2eta Potentia 3%

is: s 4:1 3:1 2:1 a.1 2. 3. 1A :S :t indicates unimodal size distribution NHalszo;: Oppsa www.www.r---- 8 p-HSP20-NPs diameters 14.1 : Sram 20 ro-Mir-i g : it . s: s

9. Biasetern O lead p-HSP20-NP Formulation PPAA: Polypropylacrylic acid

X x^ Electrostatic copiexation - MY D= 4.0 it 5.8 mm s -7.46 it 2.76 my p-Hsp20 peptide 3, CRNH*:COO is 3:1. Molar ratia p-HSP20:PPAA) is 73: Mass ratio p-SP20:PPAA as 8.8: Patent Application Publication Jul. 14, 2016 Sheet 70 of 85 US 2016/0200782 A1

figure 67 Di Cytotoxicity Assay it 3S 88 85. 88 s:

3%

s

%

23.3

: %.: 82.8 AK2. Reptide -SP20-N p-HSP20 peptide

treatment group Patent Application Publication Jul. 14, 2016 Sheet 71 of 85 US 2016/0200782 A1

Figure 68

A. Kitake & Retention a-SP20 take & Retention 8vK2 uptake day 3 2 MK2, retention day.3 83-HSP20 uptake day ap-HS-30 retention day 3

-

s2

38 is is: as a Wiean Farescence intensity A. Rear Forescence intensity A.J.

2 Sea Frescencestersity A. Patent Application Publication Jul. 14, 2016 Sheet 72 of 85 US 2016/0200782 A1

Figure 69

A 2} resterstrengya, 3 digitoirek ...... 4 Was & sociatics seri-pers eatizatio Ce E Sysis

30 firite treatinent : race Rar y task argaraeses is Faiorescence measurerrent to artify « : intracesiuliar peptide distrit

8 EXf Cytosolic rearkers f : GAd

Endosora flyscosora EEA. rarkers is 8C AK2 tracesar Peattice Eistrioticors 500 - 8 tota gs soooo 3 Cytosaic is 4tate e i 30000 & 8 zoooo

... x------& s wks K. + saferrycinAykai-NP. F. ---.S. trace air Peptide distriation s e as 3: s s a Cytosaic 4ts3poo 2coo :

r wn aa p-S2-N L - - ...... S2 --S-20-N P -- afiorycin

Patent Application Publication Jul. 14, 2016 Sheet 73 of 85 US 2016/0200782 A1

Figure ?

% -

s 100% 3. Lugin. : 8 8% i Estig. . Sigf. S Sigfri. 20 agfin 25 gf K30 uglimt. 20% S35 g. Patent Application Publication Jul. 14, 2016 Sheet 74 of 85 US 2016/0200782 A1

Figure 7 Actin Stress Fiber artificatio Contros E. M. 325 85.

88.888&

s NT MKai-NP M82i p-HSP20-NP p-HSP20 J As intensity

starsity 28-...-... ::::::::...... : '..... -...-

S&-E:::::::::::::::::::::::::::::::::::::::::::::::::::::::------'.sy.:

$xxx3-...... Patent Application Publication Jul. 14, 2016 Sheet 75 of 85 US 2016/0200782 A1

Figure 72 A MK2-NPF-Actin Stress Fiber Assay Controls 2S 85 *&888. % tly 8% ; : 5% 40% - 20% - %

Patent Application Publication Jul. 14, 2016 Sheet 76 of 85 US 2016/0200782 A1

Figure: 73

------p-HSP20-NPF-Actin Stress Fiber Assay Controls is :25 is % -: &:8:38.

p-HSP20-NP

Patent Application Publication Jul. 14, 2016 Sheet 77 of 85 US 2016/0200782 A1

F. 7. NHBION OF CONRACON ERICE WASRELAXAN FE CON PREREAE r / a'* \ I. NT oo: ^s. x: TIME ME PPEFEATENT s : - \ 83, | x: ww. T E PE: PHENY EPRNE. ASOCONSRECOR SNP; SODM ITROPRUSSIDE. ASCDAOR E Ki-N BION OF SY CONRACON Ki-NP SY ASOREAXAN th 8 & 0 a 10 23 0

MK-AP PAA ** F C p-HSP20-NP INHIBITION OF HSY CONTRACTION p-HSP20-NPHSY VASORELAXATION 80 25 N 8 25

--K exxx to NT p-HSP20 p-HSP20-NP p-HSP20 Patent Application Publication Jul. 14, 2016 Sheet 78 of 85 US 2016/0200782 A1

Patent Application Publication Jul. 14, 2016 Sheet 79 of 85 US 2016/0200782 A1

Figure 75

Celuia StreSS

Wascar Snooth scie Cei

i:

* x - Wasacc striction x & migration

Y.

Polymerization

Patent Application Publication Jul. 14, 2016 Sheet 80 of 85 US 2016/0200782 A1

ww. herapeutic 88.8. peptide

Endocytic uptake

Endosonolytic 2. Endosorial Acidification

polymer & Polymer Activation

Complexation 3) Endosoma Disruption & Peptide Release & Wasorelaxation

s Wascia Sooth Muscle Cet

Patent Application Publication Jul. 14, 2016 Sheet 81 of 85 US 2016/0200782 A1

Sigure ?? Patent Application Publication Jul. 14, 2016 Sheet 82 of 85 US 2016/0200782 A1

Figure 73

8 i:: ::::: - y - 0800x52,339 :::::: * R: .9996

i i ; 8 (383. Rigiri. Patent Application Publication Jul. 14, 2016 Sheet 83 of 85 US 2016/0200782 A1

Figure 79

pi vs% Recovery at 25°C 101

93

8. w-r-7 days 3i *::::::::::: ays

pit vs. Rate at 25°C s

six3. ays

s

iš o ox...'...... 3. 2. s 3. ry s: Patent Application Publication Jul. 14, 2016 Sheet 84 of 85 US 2016/0200782 A1

Figure 80

05: p vs. Recovery 4

9 5

90

S s

pH vs.Rate at 48°C ::::::::: isy : x2 issys 6 : x7 days : *:::::::::::::::: 3. says

Patent Application Publication Jul. 14, 2016 Sheet 85 of 85 US 2016/0200782 A1

Figure 81. pH vs% Recovery at 60°C OS : 100

p vs Rate at 60°C

O

w8xi sys

& *

US 2016/0200782 A1 Jul. 14, 2016

FORMULATION OF MIK2 INHIBITOR 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase PEPTIDES (EC 2.7.1.103); hydroxymethylglutaryl-CoA reductase (NADPH2) kinase (EC 2.7.1.109); protein-tyrosine kinase CROSS-REFERENCE TO RELATED (EC 2.7.1.112); isocitrate dehydrogenase (NADP+) kinase APPLICATIONS (EC 2.7.1.116); myosin light-chain kinase (EC 2.7.1.117): 0001. This application claims the benefit of priority to hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin U.S. provisional patent application Ser. No. 62/101,190, filed dependent protein kinase (EC 2.7.1.123); rhodopsin kinase Jan. 8, 2015, entitled “FORMULATION OF MK2 INHIBI (EC 2.7.1.125); beta-adrenergic-receptor kinase (EC 2.7.1. TOR PEPTIDES', the content of which is incorporated by 126); myosin heavy-chain kinase (EC 2.7.1.129); Tau pro tein kinase (EC 2.7.1.135); macrollide 2'-kinase (EC 2.7.1. reference herein in its entirety. 136); I-phosphatidylinositol 3-kinase (EC 2.7.1.137); RNA FIELD OF INVENTION polymerase-subunit kinase (EC 2.7.1.141); phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 0002. The described invention relates to the fields of cell 2.7.1.153); and phosphatidylinositol-4-phosphate 3-kinase and molecular biology, polypeptides, pharmaceutical formu (EC 2.7.1.154). Group I further comprises the lipid kinase lations and therapeutic methods of use. family (321 sequences). These kinases include: I-phosphati dylinositol-4-phosphate 5-kinase (EC 2.7.1.68); I D-myo BACKGROUND inositol-triphosphate 3-kinase (EC 2.7.1.127); inositol-tet rakisphosphate 5-kinase (EC 2.7.1.140); Kinases I-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149): 0003 Kinases are a ubiquitous group of enzymes that I-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150); catalyze the phosphoryl transfer reaction from a phosphate inositol-polyphosphate multikinase (EC 2.7.1.151); and donor (usually adenosine-5'-triphosphate (ATP)) to a recep inositol-hexakiphosphate kinase (EC 2.7.4.21). Group I fur tor Substrate. Although all kinases catalyze essentially the ther comprises the ATP-grasp kinases (729 sequences) which same phosphoryl transfer reaction, they display remarkable include inositol-tetrakisphosphate I-kinase (EC 2.7.1.134): diversity in their Substrate specificity, structure, and the path pyruvate, phosphate dikinase (EC 2.7.9.1); and pyruvate, ways in which they participate. A recent classification of all water dikinase (EC 2.7.9.2). available kinase sequences (approximately 60,000 0005 Group II (17,071 sequences) kinases incorporate the sequences) indicates kinases can be grouped into 25 families Rossman-like kinases. Group II comprises the P-loop kinase of homologous (meaning derived from a common ancestor) family (7,732 sequences). These include gluconokinase (EC proteins. These kinase families are assembled into 12 fold 2.7.1.12); phosphoribulokinase (EC 2.7.1.19); thymidine groups based on similarity of structural fold. Further, 22 of kinase (EC 2.7.1.21); ribosylnicotinamide kinase (EC 2.7.1. the 25 families (approximately 98.8% of all sequences) 22); dephospho-CoA kinase (EC 2.7.1.24); adenylylsulfate belong to 10 fold groups for which the structural fold is kinase (EC 2.7.1.25); pantothenate kinase (EC 2.7.1.33); pro known. Of the other3 families, polyphosphate kinase forms a tein kinase (bacterial) (EC 2.7.1.37); uridine kinase (EC 2.7. distinct fold group, and the 2 remaining families are both 1.48); shikimate kinase (EC 2.7.1.71); deoxycytidine kinase integral membrane kinases and comprise the final fold group. (EC 2.7.1.74); deoxyadenosine kinase (EC 2.7.1.76); poly These fold groups not only include some of the most widely nucleotide 5'-hydroxyl-kinase (EC 2.7.1.78); 6-phosphof spread protein folds, such as Rossmann-like fold (three or ructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC more parallel B strands linked by two a helices in the topo 2.7.1.113); tetraacyldisaccharide 4'-kinase (EC 2.7.1.130): logical order B-O-B-C-3), ferredoxin-like fold (a common deoxynucleoside kinase (EC 2.7.1.145); adenosylcobina C+B protein fold with a signature BC, BBC.f3 secondary struc mide kinase (EC 2.7.1.156); polyphosphate kinase (EC 2.7. ture along its backbone), TIM-barrel fold (meaning a con 4.1), phosphomevalonate kinase (EC 2.7.4.2); adenylate served protein fold consisting of eight C-helices and eight kinase (EC 2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4. parallel B-strands that alternate along the peptide backbone), 4); guanylate kinase (EC 2.7.4.8); thymidylate kinase (EC and antiparallel 3-barrel fold (a beta barrel is a large beta 2.7.4.9); nucleoside-triphosphate-adenylate kinase (EC 2.7. sheet that twists and coils to form a closed structure in which 4.10); (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13): the first strand is hydrogen bonded to the last), but also all cytidylate kinase (EC 2.7.4.14); and uridylate kinase (EC major classes (all C, all B, C+B, C/B) of protein structures. 2.7.4.22). Group II further comprises the phosphoenolpyru Within a fold group, the core of the nucleotide-binding vate carboxykinase family (815 sequences). These enzymes domain of each family has the same architecture, and the include protein kinase (HPr kinase/phosphatase) (EC 2.7.1. topology of the protein core is either identical or related by 37); phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1. circular permutation. Homology between the families within 32); and phosphoenolpyruvate carboxykinase (ATP) (EC 4.1. a fold group is not implied. 1.49). Group II further comprises the phosphoglycerate 0004 Group I (23,124 sequences) kinases incorporate kinase (1.351 sequences) family. These enzymes include protein S/T-Y kinase, atypical protein kinase, lipid kinase, phosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate and ATP grasp enzymes and further comprise the protein kinase (GTP) (EC 2.7.2.10). Group II further comprises the S/T-Y kinase, and atypical protein kinase family (22,074 aspartokinase family (2,171 sequences). These enzymes sequences). These kinases include: choline kinase (EC 2.7.1. include carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 32); protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.2.4); acetylglutamate kinase (EC 2.7.2.8 1); glutamate 2.7.1.38); homoserine kinase (EC 2.7.1.39); I-phosphatidyli 5-kinase (EC 2.7.2.1) and uridylate kinase (EC 2.7.4.). Group nositol 4-kinase (EC 2.7.1.67); streptomycin 6-kinase (EC II further comprises the phosphofructokinase-like kinase 2.7.1.72); ethanolamine kinase (EC 2.7.1.82); streptomycin family (1998 sequences). These enzymes include 6-phos 3'-kinase (EC 2.7.1.87); kanamycin kinase (EC 2.7.1.95); phofrutokinase (EC 2.7.1.11); NAD(+) kinase (EC 2.7.1.23); US 2016/0200782 A1 Jul. 14, 2016

I-phosphofructokinase (EC 2.7.1.56); diphosphate-fructose dine 5'-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7. 6-phosphate I-phosphotransferase (EC 2.7.1.90); sphinga 1.148); and phosphomevalonate kinase (EC 2.7.4.2). nine kinase (EC 2.7.1.91); diacylglycerol kinase (EC 2.7.1. 0010 Group VII kinases (1,843 sequences) incorporate 107); and ceramide kinase (EC 2.7.1.138). Group II further AIR synthetase-like kinases. These enzymes include thia comprises the ribokinase-like family (2.722 sequences). mine-phosphate kinase (EC 2.7.4.16) and selenide, water These enzymes include: glucokinase (EC 2.7.1.2); ketohex dikinase (EC 2.7.9.3). okinase (EC 2.7.1.3); fructokinase (EC 2.7.1.4); 6-phospho 0011 Group VIII kinases (565 sequences) incorporate fructokinase (EC 2.7.1.11); ribokinase (EC 2.7.1.15): riboflavin kinases (565 sequences). These enzymes include adenosine kinase (EC 2.7.1.20): pyridoxal kinase (EC 2.7.1. riboflavin kinase (EC 2.7.1.26). 35): 2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); 0012 Group IX kinases (197 sequences) incorporate dihy hydroxymethylpyrimidine kinase (EC 2.7.1.49); hydroxyeth droxyacetone kinases. These enzymes include glycerone ylthiazole kinase (EC 2.7.1.50); I-phosphofructokinase (EC kinase (EC 2.7.1.29). 2.7.1.56); inosine kinase (EC 2.7.1.73); 5-dehydro-2-deoxy 0013 Group X kinases (148 sequences) incorporate puta gluconokinase (EC 2.7.1.92); tagatose-6-phosphate kinase tive glycerate kinases. These enzymes include glycerate (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC kinase (EC 2.7.1.3.1). 2.7.1.146); ADP-dependent glucokinase (EC 2.7.1.147); and 0014 Group XI kinases (446 sequences) incorporate poly phosphomethylpyrimidine kinase (EC 2.7.4.7). Group II fur phosphate kinases. These enzymes include polyphosphate ther comprises the thiamin pyrophosphokinase family (175 kinases (EC 2.7.4.1). sequences) which includes thiamin pyrophosphokinase (EC 00.15 Group XII kinases (263 sequences) incorporate 2.7.6.2). Group II further comprises the glycerate kinase fam integral membrane kinases. Group XII comprises the dolichol ily (107 sequences) which includes glycerate kinase (EC kinase family. These enzymes include dolichol kinases (EC 2.7.1.3.1). 2.7.1.108). Group XII further comprises the undecaprenol 0006 Group III kinases (10,973 sequences) comprise the kinase family. These enzymes include undecaprenol kinases ferredoxin-like fold kinases. Group III further comprises the (EC 2.7.1.66). nucleoside-diphosphate kinase family (923 sequences). 0016 Kinases play indispensable roles in numerous cel These enzymes include nucleoside-diphosphate kinase (EC lular metabolic and signaling pathways, and are among the 2.7.4.6). Group III further comprises the HPPK kinase family best-studied enzymes at the structural, biochemical, and cel (609 sequences). These enzymes include 2-amino-4-hy lular level. Despite the fact that all kinases use the same droxy-6-hydroxymethyldihydropteridine pyrophosphoki phosphate donor (in most cases, ATP) and catalyze apparently nase (EC 2.7.6.3). Group III further comprises the guanido the same phosphoryl transfer reaction, they display remark kinase family (324 sequences). These enzymes include able diversity in their structural folds and substrate recogni guanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7. tion mechanisms. This probably is due largely to the diverse 3.2); arginine kinase (EC 2.7.3.3); and lombricine kinase (EC nature of the structures and properties of their substrates. 2.7.3.5). Group III further comprises the histidine kinase (0017 Mitogen-Activated Protein Kinase (MAPK)-Acti family (9,117 sequences). These enzymes include protein vated Protein Kinases (MK2 and MK3) kinase (histidine kinase) (EC 2.7.1.37); pyruvate dehydro 0018. Different groups of MAPK-activated protein genase (lipoamide) kinase (EC 2.7.1.99); and 3-methyl-2- kinases (MAP-KAPKs) have been defined downstream of oxybutanoate dehydrogenase(lipoamide) kinase (EC 2.7.1. mitogen-activated protein kinases (MAPKs). These enzymes 115). transduce signals to target proteins that are not direct Sub 0007 Group IV kinases (2,768 sequences) incorporate strates of the MAPKs and, therefore, serve to relay phospho ribonuclease H-like kinases. These enzymes include hexoki rylation-dependent signaling with MAPK cascades to diverse nase (EC 2.7.1.1); glucokinase (EC 2.7.1.2); fructokinase cellular functions. One of these groups is formed by the three (EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5); mannokinase MAPKAPKs: MK2, MK3 (also known as 3pK), and MK5 (EC 2.7.1.7); gluconokinase (EC 2.7.1.12); L-ribulokinase (also designated PRAK). Mitogen-activated protein kinase (EC 2.7.1.16); xylulokinase (EC 2.7.1.17); erythritol kinase activated protein kinase 2 (also referred to as “MAPKAPK2. (EC 2.7.1.27); glycerol kinase (EC 2.7.1.30); pantothenate “MAPKAP-K2”, “MK2) is a kinase of the serine/threonine kinase (EC 2.7.1.33); D-ribulokinase (EC 2.7.1.47); L-fu (Ser/Thr) protein kinase family. MK2 is highly homologous colokinase (EC 2.7.1.51); L-xylulokinase (EC 2.7.1.53); to MK3 (approximately 75% amino acid identity). The kinase allose kinase (EC 2.7.1.55): 2-dehydro-3-deoxygalactonoki domains of MK2 and MK3 are most similar (approximately nase (EC 2.7.1.58); N-acetylglucosamine kinase (EC 2.7.1. 35% to 40% identity) to calcium/calmodulin-dependent pro 59); N-acylmannosamine kinase (EC 2.7.1.60); polyphos tein kinase (CaMK), phosphorylase b kinase, and the C-ter phate-glucose phosphotransferase (EC 2.7.1.63); beta minal kinase domain (CTKD) of the ribosomal S6 kinase glucoside kinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1); (RSK) isoforms. The MK2 gene encodes two alternatively butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid spliced transcripts of 370 amino acids (MK2A) and 400 kinase (EC 2.7.2.14); and propionate kinase (EC 2.7.2.15). amino acids (MK2B). The MK3 gene encodes one transcript of 382 amino acids. The MK2- and MK3 proteins are highly 0008 Group V kinases (1,119 sequences) incorporate homologous, yet MK2A possesses a shorter C-terminal TIM B-barrel kinases. These enzymes include pyruvate region. The C-terminus of MK2B contains a functional bipar kinase (EC 2.7.1.40). tite nuclear localization sequence (NLS) (Lys-Lys-Xaa-Xaa 0009 Group VI kinases (885 sequences) incorporate Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Arg-Arg-Lys GHMP kinases. These enzymes include galactokinase (EC Lys: SEQID NO: 21) that is not present in the shorter MK2A 2.7.1.6); mevalonate kinase (EC 2.7.1.36); homoserine isoform, indicating that alternative splicing determines the kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46); fucoki cellular localization of the MK2 isoforms. MK3 possesses a nase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71): 4-(cyti similar nuclear localization sequence. The nuclear localiza US 2016/0200782 A1 Jul. 14, 2016

tion sequence found in both MK2B and MK3 encompasses a 0024 Substrates and Functions D domain (Leu-Leu-Lys-Arg-Arg-Lys-Lys; SEQID NO: 22), (0025 MK2 shares many substrates with MK3. Both which was shown to mediate the specific interaction of enzymes have comparable Substrate preferences and phos MK2B and MK3 with p38C. and p38 B. MK2B and MK3 also phorylate peptide Substrates with similar kinetic constants. possess a functional nuclear export signal (NES) located The minimum sequence required for efficient phosphoryla N-terminal to the NLS and D domain. The NES in MK2B is tion by MK2 was found to be Hyd-Xaa-Arg-Xaa-Xaa-pSer/ Sufficient to trigger nuclear export following stimulation, a pThr (SEQID NO: 22), where Hyd is a bulky, hydrophobic process which may be inhibited by leptomycin B. The residue. sequence N-terminal to the catalytic domain in MK2 and 0026. Accumulating studies have shown that MK2 pho MK3 is proline rich and contains one (MK3) or two (MK2) phorylates a variety of proteins, which include, but are not putative Src homology 3 (SH3) domain-binding sites, which limited to, 5-Lipooxygenase (ALOX5), Cell Division Cycle studies have shown, for MK2, to mediate binding to the SH3 25 Homolog B (CDC25B), Cell Division Cycle 25 Homolog domain of c-Abl in vitro. Recent studies suggest that this C (CDC25C), Embryonic Lethal, Abnormal Vision, Droso domain is involved in MK2-mediated cell migration. phila-Like 1 (ELAVL1), Heterogeneous Nuclear Ribonucle 0019 MK2B and MK3 are located predominantly in the oprotein AO (HNRNPAO), Heat Shock Factor protein 1 nucleus of quiescent cells while MK2A is present in the (HSF1), Heat Shock Protein Beta-1 (HSPB1), Keratin 18 cytoplasm. Both MK2B and MK3 are rapidly exported to the (KRT18), Keratin 20 (KRT20), LIM domain kinase 1 cytoplasm via a chromosome region maintenance protein (LIMK1), Lymphocyte-specific protein 1 (LSP1), Polyade (CRM1)-dependent mechanism upon stress stimulation. nylate-Binding Protein 1 (PABPC1), Poly(A)-specific Ribo Nuclear export of MK2B appears to be mediated by kinase nuclease (PARN), CAMP-specific 3',5'-cyclic Phosphodi activation, as phosphomimetic mutation of Thr334 within the esterase 4A (PDE4A), RCSD domain containing 1 (RCSD1), activation loop of the kinase enhances the cytoplasmic local Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 ization of MK2B. Without being limited by theory, it is (RPS6KA3), TGF-beta activated kinase 1/MAP3K7 binding thought that MK2B and MK3 may contain a constitutively protein 3 (TAB3), and Tristetraprolin (TTP/ZFP36). active nuclear localization signal (NLS) and a phosphoryla (0027 Heat-Shock Protein Beta-1 (also termed HSPB1 or tion-regulated nuclear export signal (NES). HSP27) is a stress-inducible cytosolic protein that is ubiqui 0020 MK2 and MK3 appear to be expressed ubiquitously, tously present in normal cells and is a member of the Small with increased relative expression in the heart, lungs, kidney, heat-shock protein family. The synthesis of HSPB1 is reproductive organs (mammary and testis), skin and skeletal induced by heat shock and other environmental or patho muscle tissues, as well as in immune-related cells such as physiologic stresses, such as UV radiation, hypoxia and white blood cells/leukocytes and dendritic cells. ischemia. Besides its putative role in thermoresistance, 0021 Activation HSPB1 is involved in the survival and recovery of cells 0022 Various activators of p38C. and p38B potently stimu exposed to stressful conditions. late MK2 and MK3 activity. p38 mediates the in vitro and in 0028 Experimental evidence supports a role for p38 in the vivo phosphorylation of MK2 on four proline-directed sites: regulation of cytokine biosynthesis and cell migration. The Thr25, Thr222, Ser272, and Thr334. Of these sites, only targeted deletion of the mk2 gene in mice Suggested that Thr25 is not conserved in MK3. Without being limited by although p38 mediates the activation of many similar kinases, theory, while the function of phosphorylated Thr25 is MK2 seems to be the key kinase responsible for these p38 unknown, its location between the two SH3 domain-binding dependent biological processes. Loss of MK2 leads (i) to a sites Suggests that it may regulate protein-protein interac defect in lipopolysaccharide (LPS)-induced synthesis of tions. Thr222 in MK2 (Thr201 in MK3) is located in the cytokines Such as tumor necrosis factor alpha (TNF-C.), inter activation loop of the kinase domain and has been shown to be leukin-6 (IL-6), and gamma interferon (IFN-Y) and (ii) to essential for MK2 and MK3 kinase activity. Thr334 in MK2 changes in the migration of mouse embryonic fibroblasts, (Thr313 in MK3) is located C-terminal to the catalytic Smooth muscle cells, and neutrophils. domain and is essential for kinase activity. The crystal struc 0029 Consistent with a role for MK2 in inflammatory and ture of MK2 has been resolved and, without being limited by immune responses, MK2-deficient mice showed increased theory, Suggests that Thr334 phosphorylation may serve as a Susceptibility to Listeria monocytogenes infection and switch for MK2 nuclear import and export. Phosphorylation reduced inflammation-mediated neuronal death following of Thr334 also may weaken or interrupt binding of the C focal ischemia. Since the levels of p38 protein also are terminus of MK2 to the catalytic domain, exposing the NES reduced significantly in MK2-deficient cells, it was necessary and promoting nuclear export. to distinguish whether these phenotypes were due solely to 0023 Studies have shown that while p38 is capable of the loss of MK2. To achieve this, MK2 mutants were activating MK2 and MK3 in the nucleus, experimental evi expressed in MK2-deficient cells, and the results indicated dence Suggests that activation and nuclear export of MK2 and that the catalytic activity of MK2 was not necessary to restore MK3 are coupled by a phosphorylation-dependent conforma p38 levels but was required to regulate cytokine biosynthesis. tional switch that also dictates p38 stabilization and localiza 0030. Knockout or knockdown studies of MK2 provide tion, and the cellular location of p38 itself is controlled by strong support that activated MK2 enhances stability of IL-6 MK2 and possibly MK3. Additional studies have shown that mRNA through phosphorylation of proteins interacting with nuclear p38 is exported to the cytoplasm in a complex with the AU-rich 3' untranslated region of IL-6 mRNA. In particu MK2 following phosphorylation and activation of MK2. The lar, it has been shown that MK2 is principally responsible for interaction between p38 and MK2 may be important for p38 phosphorylation of hnRNPAO, an mRNA-binding protein stabilization since studies indicate that p38 levels are low in that stabilizes IL-6 RNA. In addition, several additional stud MK2-deficient cells and expression of a catalytically inactive ies investigating diverse inflammatory diseases have found MK2 protein restores p38 levels. that levels of pro-inflammatory cytokines, such as IL-6, US 2016/0200782 A1 Jul. 14, 2016

IL-1B, TNF-C. and IL-8, are increased in induced sputum and transcription factors. Additional studies have shown that from patients with stable chronic obstructive pulmonary dis MK2-mediated phosphorylation of 14-3-3 on Ser58 compro ease (COPD) or from the alveolar macrophages of cigarette mises its binding activity, Suggesting that MK2 may affect the smokers (Keatings V. et al. Am J Resp. Crit Care Med, 1996, regulation of several signaling molecules normally regulated 153:530-534; Lim, S. et al., J Respir Crit Care Med, 2000, by 14-3-3. 162:1355-1360). 0038. Additional studies have shown that MK2 also inter 0031 Regulation of mRNA Translation. acts with and phosphorylates the p16 subunit of the seven 0032 Previous studies using MK2 knockout mice or member Arp2 and Arp3 complex (p16-Arc) on Ser77. p16 MK2-deficient cells have shown that MK2 increases the pro Arc has roles in regulating the actin cytoskeleton, Suggesting duction of inflammatory cytokines, including TNF-C., IL-1, that MK2 may be involved in this process. Further studies and IL-6, by increasing the rate of translation of its mRNA. have shown that the small heat shock protein HSPB1, lym No significant reductions in the transcription, processing, and phocyte-specific protein LSP-1, and vimentin are phospho shedding of TNF-C. could be detected in MK2-deficient mice. rylated by MK2. HSPB1 is of particular interest because it The p38 pathway is known to play an important role in regu forms large oligomers which may act as molecular chaper lating mRNA stability, and MK2 represents a likely target by ones and protect cells from heat shock and oxidative stress. which p38 mediates this function. Studies utilizing MK2 Upon phosphorylation, HSPB1 loses its ability to form large deficient mice indicated that the catalytic activity of MK2 is oligomers and is unable to block actin polymerization, Sug necessary for its effects on cytokine production and migra gesting that MK2-mediated phosphorylation of HSPB1 tion, Suggesting that, without being limited by theory, MK2 serves a homeostatic function aimed at regulating actin phosphorylates targets involved in mRNA stability. Consis dynamics that otherwise would be destabilized during stress. tent with this, MK2 has been shown to bind and/or phospho MK3 also was shown to phosphorylate HSPB1 in vitro and in rylate the heterogeneous nuclear ribonucleoprotein (hnRNP) vivo, but its role during stressful conditions has not yet been AO, tristetraprolin (TTP), the poly(A)-binding protein elucidated. PABP1, and HuR, a ubiquitously expressed member of the 0039. It was also shown that HSPB1 binds to polyubiq ELAV (Embryonic-Lethal Abnormal Visual in Drosophila uitin chains and to the 26S proteasome in vitro and in vivo. melanogaster) family of RNA-binding protein. These sub The ubiquitin-proteasome pathway is involved in the activa strates are known to bind or copurify with mRNAs that con tion of transcription factor NF-kappa B (NF-kB) by degrad tain AU-rich elements in the 3' untranslated region, Suggest ing its main inhibitor, I kappa B-alpha (IKB-alpha), and it was ing that MK2 may regulate the stability of AU-rich mRNAs shown that overexpression of HSPB1 increases NF-kappaB such as TNF-C. It currently is unknown whether MK3 plays (NF-kB) nuclear relocalization, DNA binding, and transcrip a similar role, but LPS treatment of MK2-deficient fibroblasts tional activity induced by etoposide, TNF-alpha, and Inter completely abolished hnRNPAO phosphorylation, suggest leukin-1 beta (IL-1B). Additionally, previous studies have ing that MK3 is not able to compensate for the loss of MK2. suggested that HSPB1, under stress conditions, favors the 0033 MK3 participates with MK2 in phosphorylation of degradation of ubiquitinated proteins. Such as phosphory the eukaryotic elongation factor 2 (eEF2) kinase. eEF2 kinase lated I kappa B-alpha (IKB-alpha); and that this function of phosphorylates and inactivates eEF2. eEF2 activity is critical HSPB1 accounts for its anti-apoptotic properties through the for the elongation of mRNA during translation, and phospho enhancement of NF-kappa B (NF-kB) activity (Parcellier, A. rylation of eEF2 on Thr56 results in the termination of mRNA et al., Mol Cell Biol, 23(16): 5790-5802, 2003). translation. MK2 and MK3 phosphorylation of eEF2 kinase 0040 MK2 and MK3 also may phosphorylate 5-lipoxy on Ser377 suggests that these enzymes may modulate eEF2 genase. 5-lipoxygenase catalyzes the initial steps in the for kinase activity and thereby regulate mRNA translation elon mation of the inflammatory mediators, leukotrienes. Tyrosine gation. hydroxylase, glycogen synthase, and Akt also were shown to 0034) Transcriptional Regulation by MK2 and MK3 be phosphorylated by MK2. Finally, MK2 phosphorylates the 0035 Nuclear MK2, similar to many MKs, contributes to tumor Suppressor protein tuberin on Ser1210, creating a the phosphorylation of cAMP response element binding docking site for 14-3-3. Tuberin and hamartin normally (CREB), Activating Transcription Factor-1 (ATF-1), serum form a functional complex that negatively regulates cell response factor (SRF), and transcription factor ER81. Com growth by antagonizing mTOR-dependent signaling, Sug parison of wild-type and MK2-deficient cells revealed that gesting that p38-mediated activation of MK2 may regulate MK2 is the major SRF kinase induced by stress, suggesting a cell growth by increasing 14-3 binding to tuberin. role for MK2 in the stress-mediated immediate-early 0041 Accumulating studies have Suggested that the recip response. Both MK2 and MK3 interact with basic helix-loop rocal crosstalk between the p38 MAPK-pathway and signal helix transcription factor E47 in vivo and phosphorylate E47 transducer and activator of transcription 3 (STAT3)-mediated in vitro. MK2-mediated phosphorylation of E47 was found to signal-transduction forms a critical axis successively acti repress the transcriptional activity of E47 and thereby inhibit vated in lipopolysaccharide (LPS) challenge models. It was E47-dependent gene expression, Suggesting that MK2 and shown that the balanced activation of this axis is essential for MK3 may regulate tissue-specific gene expression and cell both induction and propagation of the inflammatory mac differentiation. rophage response as well as for the control of the resolution 0036). Other Targets of MK2 and MK3 phase, which is largely driven by IL-10 and sustained STAT3 0037. Several other MK2 and MK3 Substrates also have activation (Bode, J. et al., Cellular Signalling, 24: 1185-1194, been identified, reflective of the diverse functions of MK2 and 2012). In addition, another study has shown that MK2 con MK3 in several biological processes. The scaffolding protein trols LPS-inducible IFNB gene expression and subsequent 14-3-3 is a physiological MK2 substrate. Studies indicate that IFNB-mediated activation of STAT3 by neutralizing negative 14-3-3 interacts with a number of components of cell sig regulatory effects of MK3 on LPS-induced p65 and IRF3 naling pathways, including protein kinases, phosphatases, mediated signaling. The study further showed that in mk2/3 US 2016/0200782 A1 Jul. 14, 2016 knockout macrophages, IFNB-dependent STAT3 activation Anti-inflammatory Drug Discovery. In Levin, J and Laufer, S occurs independently from IL-10, because, in contrast to (Ed.), RSC Drug Discovery Series No. 26, p. 158-175, the IFNB-, impaired IL-10 expression is not restored upon addi Royal Society of Chemistry, 2012; incorporated by reference tional deletion of MK3 in mk2/3 knockout macrophages in its entirety). (Ehlting, C. et al., J. Biol. Chem., 285 (27): 241 13-24124). 0.042 Kinase Inhibition 0051. The majority of disclosed MK2 inhibitors are clas 0043. The eukaryotic protein kinases constitute one of the sical type I inhibitors as revealed by crystallographic or bio largest Superfamilies of homologous proteins that are related chemical studies. As such, they bind to the ATP site of the by virtue of their catalytic domains. Most related protein kinase and thus compete with intra-cellular ATP (estimated kinases are specific for either serine/threonine or tyrosine concentration 1 mM-5 mM) to inhibit phosphorylation and phosphorylation. Protein kinases play an integral role in the activation of the kinase. Representative examples of Small cellular response to extracellular stimuli. Thus, stimulation of molecule MK2 inhibitors include, but are not limited to, 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 NH2 protein kinases, and a considerable amount of information on primary sequence of the catalytic domains of various protein CN kinases has been published. These sequences share a large number of residues involved in ATP binding, catalysis, and CN r 2 maintenance of structural integrity. Most protein kinases pos n-1No O N NH2: sess a well conserved 30-32 kDa catalytic domain. 0044 Studies have attempted to identify and utilize regu N-1 latory elements of protein kinases. These regulatory elements include inhibitors, antibodies, and blocking peptides. HN 0045. Inhibitors 0046 Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. The binding of s^ an inhibitor may stop a substrate from entering the active site of the enzyme and/or hinder the enzyme from catalyzing its Ol reaction (as in inhibitors directed at the ATP biding site of the kinase). Inhibitor binding is either reversible or irreversible. O O Irreversible inhibitors usually react with the enzyme and change it chemically (e.g., by modifying key amino acid % \ s residues needed for enzymatic activity) so that it no longer is capable of catalyzing its reaction. In contrast, reversible S. H H inhibitors bind non-covalently and different types of inhibi tion are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both. F 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-depen dent (Cdk) kinase, MAP kinase, serine/threonine kinase, Src Family protein tyrosine kinase, tyrosine kinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase (Chkl), gly cogen 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 COH: kinase, Rafkinase, and Rho kinase. N 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 NH N-N have advanced to in vivo pre-clinical studies (Edmunds, J. O and Talanian, MAPKAP Kinase 2 (MK2) as a Target for US 2016/0200782 A1 Jul. 14, 2016

-continued -continued

US 2016/0200782 A1 Jul. 14, 2016 7

-continued -continued H N O

N

IIH

O S

NHMe HN o1 s

US 2016/0200782 A1 Jul. 14, 2016

-continued germinal center kinase (GCK), SAPK (stress-activated pro tein kinase), SEK1 (stress signaling kinase), and focal adhe sion kinase (FAK). w NH. HN 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 rol effects due to serious limitations in its specificity. The low 1N1N specificity of these inhibitors is due to the fact that the ATP ser 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 Blocking Peptides more specific as the Substrate binding sites have a certain 0052 A peptide is a chemical compound that is composed degree of variability among the various protein kinases. of a chain of two or more amino acids whereby the carboxyl 0.058 Although substrate competitive inhibitors usually group of one amino acid in the chain is linked to the amino have a weak binding interaction with the target enzyme in group of the other via a peptide bond. Peptides have been used vitro, studies have shown that chemical modifications can interalia in the study of protein structure and function. Syn improve the specific biding affinity and the in vivo efficacy of thetic peptides may be used interalia as probes to see where substrate inhibitors (Eldar-Finkelman, H. et al., Biochim, protein-peptide interactions occur. Inhibitory peptides may Biophys. Acta, 1804(3):598-603, 2010). In addition, sub be used interalia in clinical research to examine the effects of strate competitive inhibitors show better efficacy in cells than peptides on the inhibition of protein kinases, cancer proteins in cell-free conditions in many cases (van Es, J. et al., Curr. and other disorders. Opin. Gent. Dev. 13:28-33, 2003). 0059. In an effort to enhance specificity and potency in 0053. The use of several blocking peptides has been stud protein kinase inhibition, bisubstrate inhibitors also have ied. For example, extracellular signal-regulated kinase been developed. Bisubstrate inhibitors, which consist of two (ERK), a MAPK protein kinase, is essential for cellular pro conjugated fragments, each targeted to a different binding site liferation and differentiation. The activation of MAPKs of a bisubstrate enzyme, form a special group of protein requires a cascade mechanism whereby MAPK is phospho kinase inhibitors that mimic two natural Substrates/ligands rylated by an upstream MAPKK (MEK) which then, in turn, and that simultaneously associate with two regions of given is phosphorylated by a third kinase MAPKKK (MEKK). The kinases. The principle advantage of bisubstrate inhibitors is ERK inhibitory peptide functions as a MEK decoy by binding their ability to generate more interactions with the target to ERK. enzyme that could result in improved affinity and selectivity 0054) Other blocking peptides include autocamtide-2 of the conjugates, when compared with single-site inhibitors. related inhibitory peptide (AIP). This synthetic peptide is a Examples of bisubstrate inhibitors include, but are not limited highly specific and potent inhibitor of Ca"/calmodulin-de to, nucleotide-peptide conjugates, adenosine derivative-pep pendent protein kinase II (CaMKII). AIP is a non-phospho tide conjugates, and conjugates of peptides with potent ATP rylatable analog of autocamtide-2, a highly selective peptide Substrate for CaMKII. AIP inhibits CaMKII with an IC50 of competitive inhibitors. 100 nM (IC50 is the concentration of an inhibitor required to Protein Transduction Domains obtain 50% inhibition). The AIP inhibition is non-competi (PTD)/Cell Permeable tive with respect to syntide-2 (CaMKII peptide substrate) and Proteins (CPP) ATP but competitive with respect to autocamtide-2. The inhi 0060. The plasma membrane presents a formidable barrier bition is unaffected by the presence or absence of Ca"/calm to the introduction of macromolecules into cells. For nearly odulin. CaMKII activity is inhibited completely by AIP (1 all therapeutics to exert their effects, at least one cellular uM) while PKA, PKC and CaMKIV are not affected. membrane must be traversed. Traditional small molecule 0055. Other blocking peptides include cell division pro pharmaceutical development relies on the chance discovery tein kinase 5 (Cdks) inhibitory peptide (CIP). Cdk5 phospho of membrane permeable molecules with the ability to modu rylates the microtubule protein tau at Alzheimer's Disease late protein function. Although Small molecules remain the specific phospho-epitopes when it associates with p25.p25 is dominant therapeutic paradigm, many of these molecules a truncated activator, which is produced from the physiologi suffer from lack of specificity, side effects, and toxicity. Infor cal Cdk5 activator p35 upon exposure to amyloid f peptides. mation-rich macromolecules, which have protein modulatory Upon neuronal infections with CIP. CIPs selectively inhibit functions far Superior to those of Small molecules, can be p25/Cdk5 activity and suppress the aberrant tau phosphory created using rational drug design based on molecular, cellu lation in cortical neurons. The reasons for the specificity lar, and structural data. However, the plasma membrane is demonstrated by CIP are not fully understood. impermeable to most molecules of size greater than 500 Da. 0056. Additional blocking peptides have been studied for Therefore, the ability of cell penetrating peptides, such as the extracellular-regulated kinase 2 (ERK2), ERK3, p38/HOG1, basic domain of Trans-Activator of Transcription (Tat), to protein kinase C, casein kinase II, Ca"/calmodulin kinase IV. cross the cell membrane and deliver macromolecular cargo in casein kinase II, Cdk4, Cdk5, DNA-dependent protein kinase Vivo, can greatly facilitate the rational design of therapeutic (DNA-PK), serine/threonine-protein kinase PAK3, phos proteins, peptides, and nucleic acids. phoinositide (PI)-3 kinase, PI-5 kinase, PSTAIRE (the cdk 0061 Protein transduction domains (PTDs) are a class of highly conserved sequence), ribosomal S6 kinase, GSK-4. peptides capable of penetrating the plasma membrane of US 2016/0200782 A1 Jul. 14, 2016 mammalian cells and of transporting compounds of many of Tat and other PTDs occurs by way of macropinocytosis, a types and molecular weights across the membrane. These specialized form of endocytosis, has created a new paradigm compounds include effector molecules, such as proteins, in the study of these peptides. Enhanced knowledge of the DNA, conjugated peptides, oligonucleotides, and Small par mechanism of transduction helped improve transduction effi ticles such as liposomes. When PTDs are chemically linked or ciency with the ultimate goal of clinical Success (Snyder E. fused to other proteins, the resulting fusion peptides still are and Dowdy, S., Pharm Res., 21(3):389-393, 2004). able to enter cells. Although the exact mechanism of trans 0066. The current model for Tat-mediated protein trans duction is unknown, internalization of these proteins is not duction is a multistep process that involves binding of Tatto believed to be receptor-mediated or transporter-mediated. the cell Surface, stimulation of macropinocytosis, uptake of PTDs are generally 10-16 amino acids in length and may be Tat and cargo into macropinosomes, and endosomal escape grouped according to their composition, such as, for example, into the cytoplasm. The first step, binding to the cell Surface, peptides rich in arginine and/or lysine. is thought to be through ubiquitous glycan chains on the cell 0062. The use of PTDs capable of transporting effector Surface. Stimulation of macropinocytosis by Tat occurs by an molecules into cells has become increasingly attractive in the unknown mechanism that might include binding to a cell design of drugs as they promote the cellular uptake of cargo Surface protein or occur by way of proteoglycans or glycolip molecules. These cell-penetrating peptides, generally catego ids. Uptake by way of macropinocytosis, a form offluid phase rized as amphipathic (meaning having both a polar and a endocytosis used by all cell types, is required for Tat and nonpolar end) or cationic (meaning of or relating to contain polyarginine transduction. The final step in Tattransduction is ing net positively charged atoms) depending on their escape from macropinosomes into the cytoplasm; this pro sequence, provide a non-invasive delivery technology for cess is likely to be dependent on the pH drop in endoSomes macromolecules. PTDs often are referred to as “Trojan pep that, along with other factors, facilitates a perturbation of the tides”, “membrane translocating sequences', or 'cell perme membrane by Tatand release of Tat and its cargo (i.e. peptide, able proteins” (CPPs). PTDs also may be used to assist novel protein or drug etc.) to the cytoplasm (Snyder E. and Dowdy, HSPB 1 kinase inhibitors to penetrate cell membranes. (see S., Pharm Res., 21(3):389-393, 2004). U.S. application Ser. No. 11/972,459, entitled “Polypeptide 0067 VP22 is the HSV-1 tegument protein, a structural Inhibitors of HSPB1 Kinase and Uses Therefor filed Jan. 10, part of the HSV virion. VP22 is capable of receptor indepen 2008, and Ser. No. 12/188,109, entitled “Kinase Inhibitors dent translocation and accumulates in the nucleus. This prop and Uses Thereof filed Aug. 7, 2008, the contents of each erty of VP22 classifies the protein as a PTD containing pep application are incorporated by reference in their entirety tide. Fusion proteins comprising full length VP22 have been herein). translocated efficiently across the plasma membrane. Homeoproteins with Intercellular Translocation Properties Viral PTD Containing Proteins 0068 Homeoproteins are highly conserved, transactivat ing transcription factors involved in morphological pro 0063. The first proteins to be described as having trans cesses. They bind to DNA through a specific sequence of 60 duction properties were of viral origin. These proteins still are amino acids. The DNA-binding homeodomain is the most the most commonly accepted models for PTD action. The highly conserved sequence of the homeoprotein. Several HIV-1 Transactivator of Transcription (Tat) and HSV-1 VP22 homeoproteins have been described as exhibiting PTD-like protein are the best characterized viral PTD containing pro activity; they are capable of efficient translocation across cell teins. membranes in an energy-independent and endocytosis-inde 0064. Tat (HIV-1 trans-activator gene product) is an 86-amino acid polypeptide, which acts as a powerful tran pendent manner without cell type specificity. scription factor of the integrated HIV-1 genome. Tat acts on 0069. The Antennapedia protein (Antp) is a trans-activat the viral genome, stimulating viral replication in latently ing factor capable of translocation across cell membranes; the infected cells. The translocation properties of the Tat protein minimal sequence capable of translocation is a 16 amino acid enable it to activate quiescent infected cells, and it may be peptide corresponding to the third helix of the protein's involved in priming of uninfected cells for Subsequent infec homeodomain (HD). The internalization of this helix occurs tion by regulating many cellular genes, including cytokines. at 4°C., Suggesting that this process is not endocytosis depen The minimal PTD of Tat is the 9 amino acid protein sequence dent. Peptides of up to 100 amino acids produced as fusion RKKRRQRRR (TAT49-57; SEQID NO: 20). Studies utiliz proteins with AntpHD penetrate cell membranes. ing a longer fragment of Tat demonstrated Successful trans 0070. Other homeodomains capable of translocation duction of fusion proteins up to 120 kDa. The addition of include Fushitarazu (Ftz) and Engrailed (En) homeodomain. multiple Tat-PTDs as well as synthetic Tat derivatives has Many homeodomains share a highly conserved third helix. been demonstrated to mediate membrane translocation. Tat PTD containing fusion proteins have been used as therapeutic Human PTDS moieties in experiments involving cancer, transporting a 0071 Human PTDs may circumvent potential immunoge death-protein into cells, and disease models of neurodegen nicity issues upon introduction into a human patient. Peptides erative disorders. with PTD sequences include: Hoxa-5, Hox-A4, Hox-B5, 0065. The mechanism used by transducing peptides to Hox-B6, Hox-B7, HOX-D3, GAX, MOX-2, and FitzPTD. permeate cell membranes has been the Subject of consider These proteins all share the sequence found in AntpPTD. able interest in recent years, as researchers have sought to Other PTDs include Islet-1, Interleukin-1 (IL-1), Tumor understand the biology behind transduction. Early reports Necrosis Factor (TNF), and the hydrophobic sequence from that Tat transduction occurred by a nonendocytic mechanism Kaposi-fibroblast growth factor or Fibroblast Growth Fac have largely been dismissed as artifactual though other cell tor-4 (FGF-4) signal peptide, which is capable of energy-, penetrating peptides might be taken up by way of direct receptor-, and endocytosis-independent translocation. membrane disruption. The recent findings that transduction Unconfirmed PTDs include members of the Fibroblast US 2016/0200782 A1 Jul. 14, 2016

Growth Factor (FGF) family. FGFs are polypeptide growth sequester reactive groups, the side chains of nearly all resi factors that regulate proliferation and differentiation of a wide dues in a peptide are fully solvent exposed, and can exhibit variety of cells. Several publications have reported that basic chemical degradation through hydrolytic reactions, for fibroblast growth factor (FGF-2) exhibits an unconventional example, oxidation and deamidation. Second, the conforma internalization similar to that of VP-22, Tat, and home tion in aqueous Solution may have little similarity to the odomains. It has also been reported that acidic FGF (FGF-1) structure found when bound to a receptor. Third, many pep translocated cell membranes at temperatures as low as 4°C. tides tend to be monomeric at very low concentration, but However, no conclusive evidence exists about the domain may self-assemble as the concentration is increased and responsible for internalization or the translocation properties behave as if in a highly associated State, but these structures of fusion proteins (Beerens, A. et al., Curr Gene Ther., 3(5): are too transient or fluxional to provide any increase in long 486-494, 2003). term stability. Fourth, the propensity of peptides to self-asso ciate is connected with their physical instablity, meaning their Synthetic PTDs likelihood of forming aggregates. Moreover, excipients present in a peptide formulation can chemically degrade, 0072. Several peptides have been synthesized in an interact with various Surfaces during manufacturing, interact attempt to create more potent PTDs and to elucidate the with the container or closure, or interact with the peptide mechanisms by which PTDs transport proteins across cell itself, thereby negatively affecting critical properties of the membranes. Many of these synthetic PTDs are based on preparation (Lars Hovgaard, and Sven Frokjaer, "Pharmac existing and well documented peptides, while others are uetical Formulation Development of Peptides and Proteins, selected for their basic residues and/or positive charges, 2' Ed., CRC Press (2012) pp. 212-213). which are thought to be crucial for PTD function. A few of (0075. The described invention provides effective formu these synthetic PTDs showed better translocation properties lations comprising a cell-penetrating peptide fused to a pep than the existing ones (Beerens, A. et al., Curr Gene Ther. tide-based inhibitor of MK2. 3(5):486-494, 2003). Exemplary Tat-derived synthetic PTDs include, for example, but are not limited to, WLRRIKAWL SUMMARY OF THE INVENTION RRIKA (SEQ ID NO: 12); WLRRIKA (SEQ ID NO: 13): YGRKKRRQRRR (SEQ ID NO: 14); WLRRIKAWLRRI 0076 According to one aspect, the described invention (SEQID NO: 15); FAKLAARLYR (SEQID NO: 16); KAF provides a pharmaceutical formulation comprising a thera AKLAARLYR (SEQ ID NO: 17); and HRRIKAWLKKI peutic amount of a polypeptide of amino acid sequence (SEQ ID NO: 18). YARAAARQARAKALARQLGVAA; SEQ ID NO: 1 or a functional equivalent thereof, wherein the formulation is Compositions Comprising PTDs Fused to MK2 Inhibitor characterized by preservation of stability and bioavailability Peptide Therapeutic Domains (TD) of the polypeptide. 0077 According to one embodiment, the pharmaceutical 0073. Several MK2 inhibitor peptides (TD) have been formulation is a particulate pharmaceutical formulation. synthesized, fused to synthetic PTDs and the use of compo According to another embodiment, the pharmaceutical for sitions comprising these fused polypeptides has been studied. mulation is an aerosolized pharmaceutical formulation. These polypeptides include, but are not limited to, According to another embodiment, the formulation is pre YARAAARQARAKALARQLGVAA (SEQ ID NO: 1; pared by a process of spray drying. According to another MMI-0100), YARAAARQARAKALNRQLGVA (SEQ ID embodiment, the pharmaceutical formulation comprises 1% NO: 19; MMI-0200), FAKLAARLYRKALARQLGVAA W/w Solids. According to another embodiment, the pharma (SEQ ID NO:3: MMI-0300), KAFAKLAARLYRKALAR ceutical formulation comprises 5% w/w solids. According to QLGVAA (SEQ ID NO: 4; MMI-0400), HRRIKAWLK another embodiment, the pharmaceutical formulation further KIKALARQLGVAA (SEQ ID NO: 7; MMI-0500), comprises trehalose. According to another embodiment, the YARAAARDARAKALNRQLAVAA (SEQ ID NO. 23; polypeptide of amino acid sequence YARAA ARQAR MMI-0600), and YARAAARQARAKALNRQLAVA (SEQ AKALARQLGVAA; SEQID NO: 1 or the functional equiva ID NO: 24; MMI-0600-2). Both in vitro and in vivo studies lent thereof and the trehalose are in a ratio of 80/20 respec have shown that these polypeptides can be useful in the treat tively. According to another embodiment, the MMI-0100 ment of various diseases, disorders and conditions. These (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or include, without limitation, hyperplasia and neoplasm (U.S. the functional equivalent thereof and the trehalose are in a Pat. Nos. 8,536,303 and 8,741,849) inflammatory disorders ratio of 92.5/7.5 respectively. According to another embodi (U.S. application Ser. No. 12/634,476 and U.S. application ment, the pharmaceutical formulation is delivered to a subject Ser. No. 13/934,933), adhesions (U.S. application Ser. No. via a dry powder inhalation device (DPI). 12/582.516), failure of a vascular graft due to neospasm (U.S. 0078. According to one embodiment, the pharmaceutical application Ser. No. 13/114,872), improving neurite out formulation further comprises saline. According to another growth (U.S. application Ser. No. 12/844,815), a cutaneous embodiment, the the saline is NaCl. According to another scar (U.S. application Ser. No. 13/829,876), failure of a coro embodiment, the polypeptide of amino acid sequence nary artery bypass vascular graft (U.S. application Ser. No. YARAAARQARAKALARQLGVAA; SEQID NO: 1 or the 13/700,087) and interstitial lung disease and pulmonary functional equivalent thereof is at a concentration of 0.7 fibrosis (U.S. application Ser. No. 13/445,759). mg/mL. According to another embodiment, the polypeptide 0074 Peptide compositions present a number of particular of amino acid sequence YARAAARQARAKALAR challenges to formulation scientists (R. W. Payne and M. C. QLGVAA; SEQID NO: 1 or the functional equivalent thereof Manning, "Peptide formulation: challenges and strategies.” is at a concentration of 7.0 mg/mL. According to another Innovations in Pharmaceutical Technology, 64-68 (2009)). embodiment, the pharmaceutical formulation is delivered to a First, since peptides do not have a globular structure that can subject via a nebulizer. US 2016/0200782 A1 Jul. 14, 2016

0079 According to one embodiment, the pharmaceutical 0087 FIG. 5 shows an EPIC inhaler device. On the left is formulation comprises an ionic complex of a polypeptide of an assembled device (base unit with attached flow channel). amino acid sequence YARAAARQARAKALARQLGVAA; The inhaler is tethered to an external drive box (pictured on SEQID NO: 1 or a functional equivalent thereof and a nano the right) which contains the electronics. polyplex polymer, the ionic complex being characterized by I0088 FIG. 6 shows a particle size distribution plot of dissociation of the ionic complex in intracellular compart initial aerosol performance results for a MMI-01.005% for ments selected by intracellular pH conditions such that bio mulation at 1 mg and 2 mg. activity and stability of the peptide is preserved. I0089 FIG. 7 shows a particle size distribution plot of fill 0080 According to another aspect, the described inven weights up to 10 mg for MMI-0100 1% solids formulation tion provides a method for treating a vascular graft-induced (after optimization). intimal hyperplasia in a Subject in need of such treatment, the (0090 FIG. 8 shows a linearity plot of fine particle dose method comprising administering the pharmaceutical formu (FPD) from 5 to 10 mg of MMI-01001% solids formulation. lation comprising an ionic complex of a polypeptide of amino (0091 FIG. 9 shows a particle size distribution plot of acid sequence YARAAARQARAKALARQLGVAA; SEQ MMI-0100/Trehalose variant formulations. ID NO: 1 or a functional equivalent thereof and a nano 0092 FIG. 10 shows a particle size distribution plot of polyplex polymer, the ionic complex being characterized by MMI-0100 1% solids formulation after 4 weeks storage in dissociation of the ionic complex in intracellular compart blisters at 40° C. (75% RH. ments selected by intracellular pH conditions such that bio (0093 FIG. 11 shows a particle size distribution plot of activity and stability of the peptide is preserved, comprising a recovered drug at 40°C./75% relative humidity (RH) for the therapeutic amount of a polypeptide of amino sequence MMI-01.001% Solids formulation. YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a 0094 FIG. 12 shows a particle size distribution plot of functional equivalent thereof, and a nano-polyplex polymer, recovered drug at 25° C./60% RH for the MMI-0100 1% wherein the therapeutic amount is effective to inhibit MK2: solids formulation. and to treat a vascular graft-induced intimal hyperplasia. (0095 FIG. 13 shows a particle size distribution plot of 0081. According to one embodiment, the nano-polyplex recovered drug at 4 weeks for the MMI-0100 1% solids polymer is anionic and endosomolytic. According to another formulation. embodiment, the nano-polyplex polymer is poly(propy 0096 FIG. 14 shows a particle size distribution plot of lacrylic acid) (PPAA). According to another embodiment, the recovered drug at 40°C./75% relative humidity (RH) for the nano-polyplex polymer is poly(acrylic acid) (PAA). Accord MMI-01005% solids formulation. ing to another embodiment, the pharmaceutical formulation (0097 FIG. 15 shows a particle size distribution plot of comprises a charge ratio (CR) of the polypeptide of amino recovered drug at 25° C./60% RH for the MMI-01.005% acid sequence YARAAARQARAKALARQLGVAA; SEQ solids formulation. ID NO: 1 or a functional equivalent thereof to PPAA selected (0098 FIG. 16 shows a particle size distribution plot of from the group consisting of 10:1, 9:1.8:1, 7:1, 6:1, 5:1, 4:1, recovered drug at 4 weeks for the MMI-0100 5% solids 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 formulation. 1:10. According to another embodiment, the the charge ratio (0099 FIG. 17 shows a particle size distribution plot of (CR) is 1:3. According to another embodiment, the pharma recovered drug at 40°C./75% relative humidity (RH) for the ceutical formulation is delivered to a Subject via an implan MMI-0100 1% solids, 7.5% Trehalose formulation. tation device. According to another embodiment, the pharma 0100 FIG. 18 shows a particle size distribution plot of ceutical formulation is delivered to a subject topically. recovered drug at 25° C./60% RH for the MMI-0100 1% According to another embodiment, the pharmaceutical for solids, 7.5% Trehalose formulation. mulation is delivered to a subject parenterally. 0101 FIG. 19 shows a particle size distribution plot of 0082. According to one embodiment, the functional recovered drug at 4 weeks for the MMI-01.001% solids, 7.5% equivalent is made from a fusion between a first polypeptide Trehalose formulation. that is a protein transduction domain (PTD) and a second polypeptide that is a therapeutic domain (TD). According to 0102 FIG. 20 shows a particle size distribution plot of another embodiment, the protein transduction domain (PTD) recovered drug at 40°C./75% relative humidity (RH) for the is selected from the group consisting of a polypeptide of MMI-0100 1% solids, 20% Trehalose formulation. amino acid sequence YARAAARQARA (SEQ ID NO: 11), 0103 FIG. 21 shows a particle size distribution plot of FAKLAARLYR (SEQID NO: 16), and KAFAKLAARLYR recovered drug at 25° C./60% RH for the MMI-0100 1% (SEQ ID NO: 17), and a second polypeptide that is a thera solids, 20% Trehalose formulation. peutic domain (TD) of amino acid sequence KALAR 0104 FIG. 22 shows a particle size distribution plot of recovered drug at 4 weeks for the MMI-01.001% solids, 20% QLGVAA (SEQID NO: 2). Trehalose formulation. BRIEF DESCRIPTION OF THE DRAWINGS 0105 FIG. 23 shows a chromatogram of the sample sol Vent. 0.083 FIG. 1 shows the technical characteristics of the 0106 FIG. 24 shows a chromatogram of the limit of quan blister lidding push through. titation (LOQ). 008.4 FIG. 2 shows the technical characteristics of the 0107 FIG. 25 shows a chromatogram of the 11 lug/mL Formpack(R)- 4PLY. working standard (full scale). 0085 FIG.3 shows a dynamic vapor sorption isotherm for 0.108 FIG. 26 shows a chromatogram of the 11 lug/mL a MMI-01.005% solids formulation. working standard (expanded scale). I0086 FIG. 4 shows a chromatogram of an MMI-0100 0109 FIG. 27 shows a schematic of a laser diffraction working standard. device. US 2016/0200782 A1 Jul. 14, 2016

0110 FIG. 28 shows a bar graph representing percent Representative microscopy images of VerhoeffVan-Gieson recovery of MMI-0100 after extraction times of 0.5, 1, 2, 3 (VVG) stained human saphenous vein sections that were and 4 hours. treated for 2 hours and maintained in organ culture for 14 0111 FIG. 29 shows the linear correlation between the days. MK2i-NPs potently blocked neointima formation. Red filled drug amount and the delivered dose (DD) (respirable bars demarcate intimal thickness. Scale bars=100 um. c) dose <5um) nebulized using Nebulizer Type 1. Quantification of intimal thickness from VVG stained histo 0112 FIG. 30 shows the linear correlation between the logical sections; measurements are average of 6-12 radially filled drug amount and the delivered dose (DD) (respirable parallel measurements from at least 3 vein rings from separate dose <5um) nebulized using Nebulizer Type 2. donors. d) Representative western blots showing the phos 0113 FIG.31 shows a bar graph representing nebulization phorylation of MK2 substrates hnRNP AO, CREB, and time of different fill volumes and concentrations nebulized HSP27. e-g) Quantification of western blot analysis from na3 using Nebulizer Type 1 and Nebulizer Type 2. separate donors demonstrating that MK2i-NPs enhanced 0114 FIG. 32 shows a bar graph representing delivered MK2i mediated inhibition of several factors activated down dose of different fill Volumes and concentrations nebulized stream of MK2 that are implicated in migration and inflam using Nebulizer Type 1 and Nebulizer Type 2. mation. 0115 FIG. 33 shows a bar graph representing respirable I0120 FIG.38 shows MMI-0100 (MK2i)-NP formulation dose <5um of different fill volumes and concentrations nebu enhances MMI-0100 (MK2i) bioactivity in HCAVSMCs. a) lized using Nebulizer Type 1 and Nebulizer Type 2. MK2i-NP treatment blocked TNFC. production in 0116 FIG. 34 shows a schematic of the p38-MK2 path HCAVSMCs stimulated with ANG II. All data is normalized way. to cell number (data shown in supplementary FIG. 11). 0117 FIG. 35 shows MMI-0100 (MK2i)-NP synthesis NT=no treatment, n=4. b) MK2i-NP treatment blocked and characterization. a) MK2i-NP synthesis scheme. b) migration in human coronary artery vascular Smooth muscle MK2i-NPs were designed and optimized to mediate endo cells (HCAVSMCs) stimulated with the chemoattractant Some escape and release peptide therapeutics intracellularly. PDGF-BB (50 ng/mL) 24 hours after formation of a scratch c) Treatment comparison summary: MK2i-NPs were formu wound, n=3, c) MK2i-NPs inhibited cell migration towards lated with an endosomolytic PPAA polymer whereas the the chemoattractant PDGF-BB in a Boyden Chamber assay 8 NE-MK2i-NPs were formulated with a PAA polymer that is hours after seeding onto the membrane, n=7. d) Representa structurally similar to PPAA but is not endosomolytic due to tive microscopy images of stained transwell insert mem its lower pKa. Both the MK2i-NPs and NE-MK2i-NPs are branes for each treatment group. made with the MK2i peptide with the sequence shown I0121 FIG. 39 shows intraoperative treatment with MMI (red-modified TAT mimetic cell penetrating peptide 01.00 (MK2i)-NPs reduces neointima formation and mac sequence, green MK2 inhibitory sequence). d) Zeta poten rophage persistence in vivo in transplanted vein grafts. a) tial of polyplexes prepared at different charge ratios (NH3+]/ MK2i-NP treatment reduced neointima formation as shown COO ). For imaging and uptake studies, Alexa NPs were in representative images of VerhoeffVan Gieson stained his formulated from MK2i peptide labeled with an Alexa-488 tological sections of vein grafts. b) Quantification of intimal fluorophore. NE-NPs are formulated with a non-endoso thickness in perfusion fixed jugular vein interposition grafts molytic (NE) PAA polymer. Values shown are an average of 28 days post-op. na7 grafts per treatment group. c) MK2i-NP at least 3 independent measurements. e) MK2i-NPs undergo treatment also reduced persistence of macrophages in the pH-triggered disassembly in the endosomal pH range as dem neointima as shown using RAM-11 immunohistochemsitry onstrated by DLS analysis. on vein grafts. Arrows demarcate positively stained cells. Left 0118 FIG.36 shows MMI-0100 (MK2i)-NP formulations column scale bar 100 um, right column Zoomed view scale increase cellular uptake, extend intracellular retention, and bar-50 um. d) Quantification of RAM-11 positive macroph reduce endo-lysosomal colocalization of MK2i.a) Flow cyto age staining injugular vein graft sections, n=16 histological metric quantification of cellular uptake and retention of fluo images from 4 vein segments per treatment group. rescently labeled MMI-0100 (MK2i), MK2i-NPs, and 0.122 FIG. 40 shows electrospray-ionization mass spec NE-MK2i-NPs. n=3.b) Representative flow histograms dem trometry (ESI-MS) mass spectrum for the HPLC-purified onstrate increased cellular uptake and longer intracellular CPP-MMI-0100 (MK2i) fusion peptide (YARAAARQAR retention of fluorescently labeled MK2i peptide delivered via AKALARQLGVAA (SEQID NO: 1), MW=2283.67 g/mol). MK2i-NPs. c) Red blood cell hemolysis assay shows that The mass spectrum shows three major peaks each corre MK2i-NPs have similar pH-dependent membrane disruptive sponding to the fragmentation of the full peptide sequence. activity to the PPAA polymer while membrane disruption of (0123 FIG. 41 shows "H NMR spectrum of A) poly(acrylic NE-MK2i-NPs and the MK2i peptide is negligible in the acid) (PAA) and B) poly(propylacrylic acid) (PPAA) range tested. d) Representative confocal microscopy images homopolymer in DMSO. Molecular weight was determined of Alexa-488 labeled MK2i colocalization with Lysotracker by comparing the area of peaks associated with the chain red 24 hours after 2 hours of treatment demonstrate that transfer agent (i.e. peaks c.d for PAA and peakb for PPAA) to MK2i-NPs have reduced endo-lysosomal colocalization. peaks associated acrylic acid/propylacrylic acid (i.e. peak a Scale bars-20 lum. e) Quantification of MK2i peptide colo for PAA and peak c for PPAA): PAA degree of polymeriza calization with the endolysosomal dye Lysotracker red 0, 12, tion=106, PPAA degree of polymerization=190. and 24 hours after treatment, ne3 independent images. 0.124 FIG. 42 shows gel permeation chromatography 0119 FIG. 37 shows ex vivo treatment with MK2-NPs (GPC) chromatograms of A) poly(acrylic acid) (PAA): reduces reduces neointima formation and alters phosphory degree of polymerization=150, PDI-1.27, dm/dC=0.09 (mL/ lation of molecules downstream of MK2 in human saphenous g) and B) poly(propylacrylic acid) (PPAA); degree of poly vein. a) MK2i-NP formulation increased delivery of Alexa merization=193, PDI-1.471, dm/dC=0.087 (nL/g) polymers 568-MK2i to HSV tissue ex vivo, scale bars=200 um. b) in DMF. The trace shows UV absorbance at the characteristic US 2016/0200782 A1 Jul. 14, 2016 absorption peak of the trithiocarbonate moiety (310 nm) group. Arrows demarcate positively stained cells. Left col present in the 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfa umn scale bar-100 um, right column Zoomed view scale nylvpentanoic acid (ECT) chain transfer agent utilized in the bar 50 pum. polymerization. 0.136 FIG. 54 shows (A) Flow cytometric quantification 0125 FIG. 43 shows A) Dynamic light scattering analysis of HCAVSMC uptake and retention of fluorescently labeled and B) representative TEM images ofuranyl acetate counter MK2i, MK2i-NPs, and NE-MK2i-NPs. Data are stained MMI-0100 (MK2i)-NPs. Scale bar-100 nm. means-SEM (n-3). P values determined by single factor 0126 FIG. 44 shows a bar graph representing a full data ANOVA. (B) Quantification of intracellular MK2i half-life set for pH-dependent red blood cell membrane disruption. (t1/2) by exponential decay nonlinear regression analysis of Red blood cell hemolysis assay shows that MMI-0100 intracellular peptide fluorescence 0 and 5 days following (MK2i)-NPs have similar pH-dependent and dose-dependent treatment removal. (Cand D) Longitudinal quantification (C) membrane disruptive activity to the PPAA polymer but and representative flow histograms and subsets (D) used to NE-MK2i-NPs and the MK2i peptide alone do not. calculate the percentage of HCAVSMCs positive for MK2i 0127 FIG. 45 shows a bar graph representing average size internalization following removal of treatment with free of intracellular compartments containing MMI-0100 (MK2i) MK2i, MK2i-NPs, or NE-MK2i-NPs. Data are means-SEM 24 hours after treatment with different peptide formulations. (n=3). *P<0.01, **P<0.001 vs. MK2i; IP-0.01, HP<0.001 Compartment area was quantified with Image.J Software. vs. NE-MK2i-NPs; single factor ANOVA. *p-0.001 vs. MK2 and NE-MK2i-NPs, n=50 vesicles from at 0.137 FIG.55 shows (A and B) Flow cytometric quantifi least 3 different images. cation (A) and representative flow histograms (B) of endot 0128 FIG. 46 shows a bar graph representing a full dose helial cell uptake of fluorescently labeled MK2i, MK2i-NPs, response data set of intimal thickness measurements of and NE-MK2i-NPs. Data are means-SEM (n=3). P values human saphenous vein (HSV) explants treated for 2 hours and determined by single factor ANOVA. (C) Quantification and then maintained in organ culture for 14 days, n23 from at least representative images of endothelial cell migration immedi 3 different donors. *ps0.01 compared to no treatment control ately after treatment removal determined by Boyden tran (NT), *ps0.001 compared to NT, p<0.05. swell migration assay. (D) Quantification of MK2i-treated 0129 FIG. 47 shows a bar graph representing tissue VSMC migration in the presence of the chemoattractant viability in HSV rings treated for 2 hours and maintained in PDGF-BB. Migration was determined by calculating percent organ culture for 1 or 14 days as assessed through an MTT wound closure 24 hours after scratch wound application in assay, n>3 vein rings from at least 3 separate donors. vitro. (Cand D) Data are meansitSEM (n-3). P values deter 0130 FIG. 48 shows a bar graph representing TNFC. pro mined by single factor ANOVA. duction in HCAVSMCs stimulated with ANG II for 6 hours, 0138 FIG. 56 shows bar graphs representing MK2i-NP treated for two hours with MMI-0100 (MK2i)-NPs, and MK2i treatment effects on vascular smooth muscle and NE-MK2i-NPs, or the MMI-0100 (MK2i) peptide alone and endothelial monocyte chemoattractant protein-1 (MCP-1) cultured for 24 hours in fresh media. All data is normalized to production over time. Quantification of MCP-1 production cell number. NT=no treatment. *p-0.05 compared to over time relative to untreated controls in both (A) vascular smooth muscle cells (VSMCs) and (B) endothelial cells NT+TNFC. group, "p <0.05 compared to MK2i at the same (ECs). Cells were treated for 2 hours and then cultured in concentration, hip-0.05 compared to NE-MK2i-NPs at the fresh medium afterMK2itreatment removal. After 3 or 5 days same concentration, n=4. cells were stimulated with 20 ng/ml TNFC. for 24 hours and 0131 FIG. 49 shows a bar graph representing MMI-0100 supernatants were collected for cytokine analysis. All treat (MK2i)-NPs partially block TNFC.-induced increase in IL-6 ments used a 10 uM dose of MK2i. Data are meansitSEM production in HCAVSMCs. Cells were stimulated with (n=4). P values determined by single factor ANOVA. TNFO for 6 hours, treated for two hours with MK2i-NPs or 0139 FIG. 57 shows a bar graph representing MK2i-NP MMI-0100 (MK2i) peptide alone, and cultured for 24 hours internalization. MK2i-NP internalization is not affected by in fresh media. All data is normalized to cell number. NT=no membrane bound NPs as shown by minimal differences in treatment. *p-0.05 compared to NT+TNFC. group, p<0.05 MK2i-NP uptake in vascular smooth muscle cells (VSMCs) compared to MK2i at the same concentration, n=4. that either had extracellular fluorescence quenched by trypan (0132 FIG.50 shows a bargraph representing cell viability blue and/or were extensively washed with cell scrub buffer to in HCAVSMCs stimulated with 10 uM ANG II for 6 hours, remove any extracellular NPs following treatment removal. treated for two hours with MMI-0100 (MK2i)-NPs, O140 FIG. 58 shows (A) Quantification of MK2i peptide NE-MK2i-NPs, or the MMI-0100 (MK2i) peptide alone and colocalization with the endolysosomal dye Lysotracker red 0. cultured for 24 hours in fresh media. NT=no treatment, n=4. 12, and 24 hours after treatment, ne3 independent images; (B) 0.133 FIG.51 shows a bargraph representing cell viability average size of intracellular compartments containing MK2i in HCAVSMCs stimulated with TNFC. for 6 hours, treated for 24 hours after treatment with different peptide formulations. two hours with MMI-0100 (MK2i)-NPs or MMI-0100 Compartment area was quantified with Image.J software. (MK2i) peptide alone, and cultured for 24 hours in fresh n=50 vesicles from at least 3 different images. media. n=4. 0141 FIG. 59 shows (a) immunofluorescence microscopy 0134 FIG. 52 shows a bar graph representing cell prolif images of human saphenous vein cross sections treated with eration in HCAVSMCs stimulated treated for 30 minutes with Alexa-568 labeled MK2i, MK2i-NPs, or NE-MK2i-NPs MMI-0100 (MK2i) peptide alone, MK2i-NPs, or NE-MK2i (red) and stained for the vascular smooth muscle marker NPs and cultured for 24 hours in fresh media with (+) or C-Smooth muscle actin (green) showing MK2i-NP colocal without (-) 50 ng/mL PDGF-BB. NT=no treatment, n=4. ization with C-Smooth muscle actin; (b) Zoomed insets from 0135 FIG. 53 shows representative RAM-11 staining images in (a); (c) Zoomed immunofluorescence microscopy images of rabbit jugular vein graft explants for each treatment images of human saphenous vein treated with Alexa-568 US 2016/0200782 A1 Jul. 14, 2016

labeled MK2i, MK2i-NPs, or NE-MK2i-NPs (red) and MW-2731 g/mol). The mass spectra show three major peaks stained for the endothelial marker CD31 (green) demonstrat each corresponding to the fragmentation of the full peptide ing MK2i colocalization with endothelial cells; (d) Zoomed Sequence. insets showing MK2i penetration into the vessel wall for all treatment groups; (e) pixel intensity distribution of the images 0147 FIG. 65 shows (A) Z-average diameter (bars) and shown in (a) demonstrating increased MK2i uptake (red Zeta potential (circles) of MK2i-NPs prepared at a different channel) in vessels treated with MK2i-NPs. charge ratios (CR=NH: COO). Asterisks () 0142 FIG. 60 shows (a-b) immunofluorescence micros denote a unimodal size distribution and the white bar repre copy images of human Saphenous vein treated with Alexa sents the MK2i-NP formulation that yielded a unimodal size 568 labeled MK2i, MK2i-NPs, or NE-MK2i-NPs (red) and distribution with minimal size and polydispersity; (B) repre stained for the vascular Smooth muscle cell marker C-Smooth sentative DLS trace of lead MK2i-NP formulation (CR=1:3); muscle actin (green) showing MK2i-NP colocalization with (C) representative TEM image of uranyl acetate stained C-Smooth muscle actin; (c) immunofluorescence microscopy MK2i-NPs, scale bar 200 nm (D) synthesis and character of demonstrating increased uptake and penetration of MK2i ization summary for lead MK2i-NP formulation. CR-charge NPS into the vessel wall relative to the MK2i and NE-MK2i ratio. D hydrodynamic diameter, Zeta potential. NP treated vessels. 0148 FIG. 66 shows A) Z-average diameter (bars) and 0143 FIG. 61 shows (a) MK2i-NPs inhibited vascular Zeta potential (circles) of p-HSP20-NPs prepared at a differ Smooth muscle cell migration towards the chemoattractant ent charge ratios (CR=NHalseo-COOleet). Aster PDGF-BB in a Boyden Chamber assay 8 hours after seeding isks (*) denote a unimodal size distribution, and the white bar onto the membrane. NT=no treatment; (b) MK2i-NPs inhib represents the p-HSP20-NP formulation that yielded a uni ited endothelial cell migration towards the chemoattractant modal size distribution with minimal size and polydispersity; VEGF in a Boyden Chamber assay 8 hours after seeding onto (B) Representative DLS trace of lead p-HSP20-NP formula the membrane; (c) MK2i-NP treatment blocked TNFC. pro tion (CR-3:1); (C) Representative TEM image of uranyl duction in HCAVSMCs stimulated with ANG II (all data is acetate stained p-HSP20-NPs, scale bar=200 nm (D) Synthe normalized to cell number); (d) MK2i-NP treatment showed sis and characterization summary for lead p-HSP20-NP for sustained inhibition of TNFC. stimulated production of mulation. CR charge ratio, Dihydrodynamic diameter, MCP-1 in both vascular smooth muscle and endothelial cells Zeta potential. whereas treatment with free MK2ior NE-MK2i-NPs did not; 014.9 FIG. 67 shows a bar graph representing NP cyto (e) MK2i-NPs showed sustained inhibition of vascular compatibility. The cytotoxicity of MK2i-NPs and p-HSP20 Smooth muscle cell migration towards the chemoattractant NPs was compared to the corresponding dose of free peptide PDGF-BB 5 days after treatment removal. in HCAVSMCs. Cells were treated for 2 hours and then 014.4 FIG. 62 shows (a) MK2i-NP treatment reduced allowed to incubate in fresh medium for 24 hours prior to neointima formation as shown in representative images of running the cytotoxicity assay. *p-0.05 vs. NT, n=4 Verhoeff Van Gieson stained histological sections of vein mean-SEM. grafts; (b) quantification of intimal thickness in perfusion fixed jugular vein interposition grafts 28 days post-op. na7 (O150 FIG. 68 shows NPuptake and retention. Flow cyto grafts per treatment group; (c) MK2i-NP treatment reduced metric quantification of peptide uptake and retention of (A) proliferation of intimal cells as shown using kió7 immuno MK2i-NPs vs. MK2i and (B)p-HSP20-NPs vs. HSP20 at a 10 histochemistry on vein grafts; (d) quantification of kió7 posi uM dose of peptide after 30 minutes of treatment. MK2i-NPs tive nuclear staining injugular vein graft sections normalized achieved -70 fold increase in peptide uptake at the same to intimal nuclei number; (e) MK2i-NP treatment maintained concentration whereas p-HSP20-NPs achieved a -35 fold higher intimal expression of the contractile marker C-Smooth increase in uptake; (C,D) representative flow histograms of muscle actin, (f) quantification of intimal C-Smooth muscle HCAVSMCs immediately after treatment and (E.F) represen actin positive staining in jugular vein graft sections normal tative flow histograms demonstrating that formulation into ized to intimal nuclei number, (g) MK2i-NP treatment NPs increased peptide cellular retention after 3 days of cul reduced intimal expression of the synthetic vascular Smooth ture in fresh medium post-treatment. The percentages over muscle phenotypic marker vimentin: (h) quantification of written on A-B represent the 96 retention at 3 days relative to intimal vimentin positive staining in jugular vein graft sec 0 days post-treatment. tions normalized to intimal nuclei number. 0151 FIG. 69 shows NPEndosomal Escape and Cytosolic 0145 FIG. 63 shows (a) representative RAM-11 staining Peptide Delivery. (A) Experimental design for separation of images of rabbitjugular vein graft explants for each treatment vascular Smooth muscle cell cytosol and intracellular group. Arrows demarcate positively stained cells. Left col organelles using digitonin semi-permeabilization. Condi umn scale bar 100 um, right column Zoomed view scale tions for semi-permeabilization were optimized as shown in bar-50 um; (b) example images from the color deconvolution FIG.70; (B) Western blot validation of the optimized digito method utilized to quantify positive RAM-11 staining in the nin semi-permeabilization procedure confirmed separation of intima of rabbit jugular vein explants; (c) quantification of the cytosolic proteins mitogen-activated protein kinase intimal RAM-11 positive macrophage staining injugular vein kinase 1/2 (MEK1/2) and glyceraldehyde 3-phosphate dehy graft sections, n=16 histological images from 4 Vein segments drogenase (GAPDH) from the endo-lysosomal markers early per treatment group. endosomal antigen 1 (EEA1) and lysosomal-associated pro 0146 FIG. 64 shows electrospray-ionization mass spec tein 1 (LAMP1); (C and D) comparison of the intracellular trometry (ESI-MS) mass spectra for the HPLC-purified (A) distribution of (C) MK2i and (D) p-HSP20 peptides when MK2i peptide (sequence: YARAAARQARA-KALAR delivered alone or formulated into nano-polyplexes demon QLGVAA, MW=2283.7 g/mol) and (B) p-HSP20 peptide strating increased cytosolic delivery of the NP formulations. (sequence: YARAAARQARA-WLRRASAPLPGLK, Significant inhibition of NP mediated cytosolic peptide deliv