US 2011 02880.11A1 (19) United States (2) Patent Application Publication (10) Pub. No.: US 2011/0288011 A1 Castaigne et al. (43) Pub. Date: Nov. 24, 2011
(54) PEPTIDE THERAPEUTIC CONJUGATES AND A6IP 3/(){} (2006.01) USES THEREOF A6IP 3/08 (2006.01) A6IP 25/22 (2006.01) (76) Inventors: Jean-Paul Castaigne, Mont-Royal A6IP 25/14 (2006.01) (CA); Michel Demeule, A6IP 25/16 (2006.01) Beaconsfield (CA); Catherine A6IP 25/08 (2006.01) Gagnon, Montreal-Nord (CA); A6IP 25/18 (2006.01) Betty Lawrence, Bolton (CA) A6IP 25/20 (2006.01) A6IP 25/00 (2006.01) (21) Appl. No.: 13/133,002 A6 IP I/18 (2006.01) - A6 HP I/16 (2006.01)
- A6 IP I/00 (2006.01) (86) PCT No.: PCT/CA2009/001781 A6IP 13/00 (2006.01) A6IP 9/04 (2006.01) § 371 (Q(t), A6 IP II/00 (2006.01) (2), (4) Date: Aug. 9, 2011 A6IP 3/06 (2006.01) - - - A6IP 29/00 (2006.01) Relatedelate U.S. Applicationpplication DatData A6IP 35/06) (2006.01) (60) Provisional application No. 61/200,947, filed on Dec. A6IP 43/00 (2006.01) 5, 2008. C07K 14/.435 (2006.01) Publication Classification (52) U.S. Cl...... 514/5.3: 530/324; 530/300: 536/23.4; 514/5.8: 514/6.9; 514/16.4: 514/15.7; 514/9.7; (51) Int. Cl. 514/17.8: 514/17.7: 514/21.3; 514/15.4; 514/193; A6 IK 38/22 (2006.01) 514/19.4: 514/19.5: 514/5.5 C07K 19/00 (2006.01) C07K I4/575 (2006.01) (57) ABSTRACT C07H 21/00 (2006.01) A6 HP 3/04 (2006.01) The present invention features a compound having the for A6 HP 3/10 (2006.01) mula A-X-B, where A is peptide vector capable of enhancing A6 HP 9/00 (2006.01) transport of the compound across the blood-brain barrier or A6 HP 9/12 (2006.01) into particular cell types, X is a linker, and B is a peptide A6 IK 38/17 (2006.01) therapeutic. The compounds of the invention can be used to A6 HP 25/28 (2006.01) treat any disease for which the peptide therapeutic is useful. Patent Application Publication Nov. 24, 2011 Sheet 1 of 49 US 2011/0288011 A1
Peptides Amino acid sequence Exendin-4 native HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Exendin-4-Lys(MHA) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPK-(MHA) --- (Cys32)-Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPCSGAPPPS --- —
Exendin-4
1 Lys”-MHA (maleimido hexanoic acid)
Figure 1 Patent Application Publication Nov. 24, 2011 Sheet 2 of 49 US 2011/0288011 A1
Q->-NH
O D-. , - N O + (Cys1)-AN2 O 2% AN2-(Cys20) Exendin-4 NH2 (MHA) AN1-(Cys7)
Buffer PBS 1X, r.t., 1 h
And IC
Series | Qtty (mg) Purity (%) MW (g/mol) Ex-4-AN2 (N-Terminal) 4.9 > 95 6825.45 Ex-4-AN2 (C-Terminal) 5.5 > 95 6825.48 Ex-4-AN1 5.3 > 85 6739.38
Figure 2 Patent Application Publication Nov. 24, 2011 Sheet 3 of 49 US 2011/0288011 A1
Exendin-4-(Cys32) N
(C3) MPA-AN2 (C6) MHA-AN2 (C11) MUA-AN2
Buffer PBS 1X, r.t., 1 h
O O
N N n H
O
(C3) (C6) (C11)
Series Il Qtty (mg) Purity (%) MW (g/mol) Ex-4-(C3)-AN2 13.5 > 98 6656.21 Ex-4-(C6)-AN2 4.8 > 98 6768.43 Ex-4-(C11)-AN2 8.8 > 90 6698.17
Figure 3 Patent Application Publication Nov. 24, 2011 Sheet 4 of 49 US 2011/0288011 A1
1 -Exendin-4Exendin-4-cysAn2 N-terminal Exendin-4-cysAn2 C-terminal 1
| Brain Capillariesarenchyma
Figure 4 Patent Application Publication Nov. 24, 2011 Sheet 5 of 49 US 2011/0288011 A1
Weight
i
Days
--- Control net First treatmenttr —r- Exendin (3 Jg/kg) —y- Exendin (30 Ug/kg) —o- Exen-An2 (4.8 pg/kg) —e— Exen-An2 (48 pg/kg)
Figure 5 Patent Application Publication Nov. 24, 2011 Sheet 6 of 49 US 2011/0288011 A1
Cumulative food intake 80 70 60 50 40 30 20 10
Days
—º-Control —º- Exendin-4 (3 pg/kg) –7– Exendin-4 (30 pg/kg) —o- Exen-An2 (4.8 pg/kg) —e– Exen-An2 (48 pg/kg)
Figure 6 Patent Application Publication Nov. 24, 2011 Sheet 7 of 49 US 2011/0288011 A1
Glycemia
20
10
5
—-- Control —r- Exendin-4 (3 pig?kg) —w- Exendin-4 (30 pg/kg) —c- Exen-An2 (4.8 pg/kg) —e– Exen-An2 (48 pg/kg)
Figure 7
Patent Application Publication Nov. 24, 2011 Sheet 10 of 49 US 2011/0288011 A1
- Exendin-4 (Phoenix Pharm.) C3 T]C6 || Exen-4-An2 C11 | |Exen-4-S4 Exen-An2-An2
30
2 0
1 0
Brain Capillaries Parenchyma
Figure 9 Patent Application Publication Nov. 24, 2011 Sheet 11 of 49 US 2011/0288011 A1
—º- Ctrl -- Exendin-4 (2 pg/kg) -*- Exen-An2-An2 (4.5 pg/kg)
140 § 120 ..º. E 100 3 2, 80 on ..s. 60 3.§ 40 5£ 20 0 {} 30 50 90 Time (min)
Figure 10 Patent Application Publication Nov. 24, 2011 Sheet 12 of 49 US 2011/0288011 A1
Total Brain (15 min after IV bolus)
0.05 5. 0.04 #º 0.03 2 O g |
Exendin-4 Exen-An2 (10 pg/kg) (16 pg/kg)
Figure 11A
Pancreas (15 min after IV bolus)
5 2.0
S. 5 E.
O 69 ..? # Exendin-4 Exen-An2 (10 pg/kg) (16 pg/kg) Figure 11B Patent Application Publication Nov. 24, 2011 Sheet 13 of 49 US 2011/0288011 A1
Dose-response of insulin secretion (RIN-m5F pancreatic cells)
_ Exendin-4 30 _ Exendin-Anz
2 5 20 5.= 15 5 # 1.
0
Concentration (nM) Figure 12 Patent Application Publication Nov. 24, 2011 Sheet 14 of 49 US 2011/0288011 A1
Chromatogram 1 Leptin-C11-AN2 before purification (450 mg of crude ) fintens :
2000
500
{.{j 0.5 3 o 15 2.3 2.5 Time (min)
Figure 13A
Chromatogram 2 Leptin-C11-AN2 after purification
intens. (mAU] f . & | . 2 UV spectrum at 229 mm
600
400 250 mg recovered * > 98% purity
{}_{} 0.5 1..?] 1.5 2.0 2.5 Time [mid]
Figure 13B Patent Application Publication Nov. 24, 2011 Sheet 15 of 49 US 2011/0288011 A1
Chromatogram 3 Purification of Leptin-C11-AN2 on AKTA-explorer On column filled of 95 mL of 30RPC resin
—
fractions } r——,
17 mi /min 15 mi /min 10 mL/min
5 mL/min
Figure 14 Patent Application Publication Nov. 24, 2011 Sheet 16 of 49 US 2011/0288011 A1
D. Leptin 20 - Leptin-An2 (C3) | Leptin-An2 (C6) | Leptin-An2 (c11) 1
| 5 Brain Capillaries Parenchyma
Figure 15 Patent Application Publication Nov. 24, 2011 Sheet 17 of 49 US 2011/0288011 A1
In situ brain perfusion
* 15 [...] Lean mice E | DIOmice Q tº £© 10 E * 5 {} tº:
D.# > 0 Leptin protein Leptinité-130'An2
Figure 16A
30
EO) S. .5 20 3. CD .9 º, 10 E (/) Qº D
O - - - Initial After 3 weeks
Figure 16B Patent Application Publication Nov. 24, 2011 Sheet 18 of 49 US 2011/0288011 A1
Food intake in ob/ob mice after 4 hrs 1.4
1.2
1.0
0.8 –55% 0.6
0.4
0.2
0.0 Control Leptin116-130 Leptin116-130-An2
Figure 17A
Food intake in ob/ob mice after 15 hrs
4 +7%
2 -3.3%
Control Leptin116-130 Leptin116-130-An2
Figure 17B Patent Application Publication Nov. 24, 2011 Sheet 19 of 49 US 2011/0288011 A1
Leptin116-130 (1 mg/mouse) Control Leptin116-130-An2 (2.5mg/mouse) i eq. to 1 mg/mouse
Mice (n=5 per group) received daily IP treatment for 6 days
Figure 18 Patent Application Publication Nov. 24, 2011 Sheet 20 of 49 US 2011/0288011 A1
Effect on oblob mice weight gain Lºpº 16-130 (1 mg/mouse)
Control
i Lepi 15-130-An? (25 mg/mouse)
Treatment daily P
Figure 19 Patent Application Publication Nov. 24, 2011 Sheet 21 of 49 US 2011/0288011 A1
Thrombin
GST-Tag Angiopep-2 GST: glutathion-S-transferase !
Angiopep-2
Figure 20 Patent Application Publication Nov. 24, 2011 Sheet 22 of 49 US 2011/0288011 A1
Thrombin Angiopep-2 Leu Val Pro : Gly Ser?hr Phe Phe Tyr Gly Gly Ser Arg Gly Lys CTG GTT CCG CGTGGA TCC ACC TTT TTC TAT GGC GGC AGC CGT GGC AAA Bamb|I Leptin (116-130) Arg Asn Asn Phe Lys Thr Glu Glu Tyšer Cys Ser Leu Pro Gln Thr CGC AAC AAT TTC AAG ACC GAG GAG TAZNGC TGC TCC CTG CCT CAG ACC Ser Gly Leu Gln Lys Pro Glu Serstop AGT GGC CTG CAG AAG CCA GAG AGKTGA ATT CC EcoRI
Angiopep-2 Leptin (116-130)
Oligo I N. Oligo 3 | ! Oligo 4
Oligo 1 87 bp | 79 bp Oligo 4
Bambi I EcoRI 120 b p
Figure 21 Patent Application Publication Nov. 24, 2011 Sheet 23 of 49 US 2011/0288011 A1
rTAU Wash BDOD sº 5500 5DDD
45UD 4000 3500
3DDD 2500 200D
1500 1000
5DO
Figure 22 Patent Application Publication Nov. 24, 2011 Sheet 24 of 49 US 2011/0288011 A1
Angiopep-2
F 3.7 7.5
1. Gel Tris/Tricine 16.5% Angiopep ? *º º 㺠2. Angiopep-2 Western blot
Figure 23A
Intens. -
?måU] UV spectrum at 229mm 600 pig recovered > 98% purity | |
_––-T T __ –——-º-T ——-T
-- - - r—r g r— r— 1.5 2.0 2.5 Time (min)
Figure 23B
Intens --- - x104 +MS, 0.89-0.96min FE 6
816,07073+(A) Mass spectra 4 2+ (A) 1223.5845
2
4+(A) 612.3440 1631.1062 : O r —r- —l- +---" r—— T – 500 1000 Figure 23c 2000 2500 m/z. Patent Application Publication Nov. 24, 2011 Sheet 25 of 49 US 2011/0288011 A1
Mice in situ brain perfusion
1 5
1 0
5
Synthetic Recombinant Angiopep-2
Figure 24 Patent Application Publication Nov. 24, 2011 Sheet 26 of 49 US 2011/0288011 A1
Parenchyma uptake 1 GST GST-An2 GST-Leptinius-130 I GST-An2-Leptinate-430 |
Figure 25
Patent Application Publication Nov. 24, 2011 Sheet 28 of 49 US 2011/0288011 A1
Hiss-mLeptin Hiss-An?-mLeptin
- H * - , º, kDa . + tºº, + IPTG induction
*— Hiss-An2-mLeptin (MW: 20582 Da) 4— Hiss-mLeptin (MW: 18299 Da)
Inclusion bodies
Figure 27 Patent Application Publication Nov. 24, 2011 Sheet 29 of 49 US 2011/0288011 A1
MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYOOILTSMP SRNVIQISND LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVAISRL QGSLQDMLWQ LDLSPGC
Figure 28 Patent Application Publication Nov. 24, 2011 Sheet 30 of 49 US 2011/0288011 A1
Overview of a purification scheme for mLeptin and An?-mLeptin
Expression in bacteria
Solubilization of inclusion body proteins
Purification by nickel-affinity chromatography
Refolding by dialysis ! Removal of Hiss N-terminal tag by thrombin
Purification by anion-exchange chromatography (Q-Sepharose)
Figure 29A Patent Application Publication Nov. 24, 2011 Sheet 31 of 49 US 2011/0288011 A1
Expression in bacteria
Isolation of inclusion bodies 4. Solubilization of inclusion body proteins (6M Guanidine•HCl)
Purification by nickel-affinity chromatography
Refolding by rapid dilution in the presence of a redox system
Cleavage of the Hisé N-terminal tag by digestion with biotinylated thrombin 4. Removal of thrombin (Streptavidin-agarose) and Removal of cleaved His-tag by nickel-affinity chromatography
Pure, untagged proteins
Figure 29B Patent Application Publication Nov. 24, 2011 Sheet 32 of 49 US 2011/0288011 A1
C ‘E C (D — 5 |
Figure 30 Patent Application Publication Nov. 24, 2011 Sheet 33 of 49 US 2011/0288011 A1
Low-affinity thrombin site 2
MGSSHHHHHHSSGLVPR GSHTFFYGGSRGKRNNFKTEEY-mLeptin |? His-tag Angiopep-2
[Thrombin] —mº
<-Hisé-An2-mLeptin
22°C, 24h
Figure 31 Patent Application Publication Nov. 24, 2011 Sheet 34 of 49 US 2011/0288011 A1
Leptin An2-Leptin
Figure 32 Patent Application Publication Nov. 24, 2011 Sheet 35 of 49 US 2011/0288011 A1
Effect of Leptin recombinant treatment on oblob mice body weight 54
56 Control S s ; º sº - Leptin (0.1 mg/mouse) 42 Leptin (0.25 mg/mouse)
38 -2 : 4 7 10
First treatment Days
Daily IP treatment
Figure 33 Patent Application Publication Nov. 24, 2011 Sheet 36 of 49 US 2011/0288011 A1
1.0 +
~ 0.5 Sº E ..ºf# 0.0 His-mLeptin- - (50 pig) # Control º mLeptin (50 pig) .E -0.5 N. § 3. His-ANG-mLeptin (50 pig) -1.0 1 2 3 4 5 6
Days n=1 4 mice per group Mice received two treatments
Figure 34 Patent Application Publication Nov. 24, 2011 Sheet 37 of 49 US 2011/0288011 A1
Chromatogram 1. EMCS-NT before purification (35 mg of crude)
?mal]] UV spectrum at 100 229nm
Mass 2059 § (EMCS)2-NT
0.0 0.5 1.0 1.5 2.0 2.5 Time (min) Figure 35A
Chromatogram 2. EMCS-NT after purification
UV spectrum at 229mm
> 98% purity
200 al-/ 0.0 0.5 1.0 1.5 2.0 2.5 Time [min] Figure 35B Patent Application Publication Nov. 24, 2011 Sheet 38 of 49 US 2011/0288011 A1
Chromatogram 3. Purification of EMCS-NT on AKTA-explorer With Column filled with 30 ml of 30RPC resin
?º J) *...! I.H.I.:
m 2:0 300 3:0 400 550 500
Figure 36 Patent Application Publication Nov. 24, 2011 Sheet 39 of 49 US 2011/0288011 A1
Chromatogram 4. NT-AN2Cys-NH2 before purification
Im/AU] UV spectrum at :: . . . ; 229mm 150
100
50
y w I y y I * r r y I 0.0 0.5 1.0 1.5 2.0 2.5 Time (min) Figure 37A
Chromatogram 5. NT-AN2Cys-NH2 after purification Intens [mau ; –
* 229mm 80
5.5 mg recovered > 95% purity
r——— r———T----—H---—T-- . 0.0 0.5 1.0 1.5 2.0 2.5 Time (min) Figure 37B Patent Application Publication Nov. 24, 2011 Sheet 40 of 49 US 2011/0288011 A1
Chromatogram 6. Purification of NT-AN2Cys-NH2 on AKTA-explorer with column filled of 30ml of 30RPC resin
3.4 t w 20% B —y
4. - †: -" a ...","..' . . . . A.
#§ ||||||||||||||IHHHHHHIIIllllllllll.
Figure 38 Effect on mice temperature after a single i.v. injection 38 NT (1 mg/kg) 37
36
3 5
NT-An2 (2.5 mg/kg)
0 15 30 45 60 75 go NT-Am2 (5 mg/kg) Minutes
Figure 39 Patent Application Publication Nov. 24, 2011 Sheet 41 of 49 US 2011/0288011 A1
Control MT-An2 (5 mg/kg)
MT-An? (20 mg/kg)
0 30 60 90 120 minutes
Figure 40
—s— Control —NT-An2 (5 mg/kg, PBS) ?º ——NT-An2 (10 mg/kg, PBS)
à ——NT-An2 (20 mg/kg, PBS) —e—NT-An2 (20 mg/kg, saline 0.9%
0 30 60 90 120 150 minutes
Figure 41 Patent Application Publication Nov. 24, 2011 Sheet 42 of 49 US 2011/0288011 A1
Brain uptake of native NT and NT-An2 80
60 e NT-An2
: 20 NT
2 3 Time (min)
Figure 42
Dº Neurotensin Neurotensin-An2
Capillaries Parenchyma
Figure 43 Patent Application Publication Nov. 24, 2011 Sheet 43 of 49 US 2011/0288011 A1
401 i.v. bolus injection (5 mg/kg) a sal' $– *=q, 36 2" # 34 Infusion start (5 mg/kg/30 min * # 32 5 30 Infusion end * * 3> 28 On 26
Hº-r-r-? 0 1 2 3 4 5 6 7 8 20 21 22 23 24 Time (hr)
Figure 44
IV bolus (20 mg/kg) 38 +infusion start (20 mg/kg/hr)
* º infusion stop
Š O2 36 |
(9
H
32 0 30 60 90 120 150 180 210 240 Time (min)
Figure 45 Patent Application Publication Nov. 24, 2011 Sheet 44 of 49 US 2011/0288011 A1
IV bolus (20 *M Ce +infusion start (20 mg/kg/hr) #1.” g 3 o # 3. infusion (40 mg/kg/hr) & 4. # 3 # ,
28 0 60 120 180 240 300 360 Time (min) Figure 46 Rats 38 ANG2002 bolus (20 mg/kg) + infusion start (20 mg/kg/hr)
G 9- 36 sº Eº ?ºl.§ 34 E º £3 32 [?]
30 O 60 120 180 240 300 360 Time (min)
Figure 47 Patent Application Publication Nov. 24, 2011 Sheet 45 of 49 US 2011/0288011 A1
Effect of ANG2002 on body temperature in rat
38 - G. —s— Saline Š- —e—ANG2002 & 3 E s § 3 Bolus injection: 40 mg/kg ; Infusion: 40 mg/kg/hr S. 32 "o O ?m 3 0 2 4 6 8 10 12 14 Time (hrs)
Figure 48
Control ANG-NT (20 mg or 4.6 Umol/kg) 30 º Buprenorphine (1 mg or 2.0 umol/kg) *::: 7 25 * > # 20
tº 15 |E * E 10 3 * 5
Ü 0 15 minutes
Figure 49 Patent Application Publication Nov. 24, 2011 Sheet 46 of 49 US 2011/0288011 A1
40
—º-Control ——NT(8-13) —-Ac-Lys-NT(8-13) —— Ac-Lys-NT(8-13)-D-Tyr11 —e—pClu-NT(8-13) 34
32 0 30 60 90 120 150 Time (min)
Figure 50
—— Control — NT native —— NTnative-An2 CT-36-84 —NT(8-13)-An2 CT-36-78 —— D-tyr(8-13)-An2 CT-36-88
0 30 60 90 120 Time (minutes)
Figure 51 Patent Application Publication Nov. 24, 2011 Sheet 47 of 49 US 2011/0288011 A1
—r-D-tyr(8-13)-An2 (6.25 mg/kg) ——NT(8–13)-D-Tyr11 (1mg/kg)
34.
32 0 30 60 90 120 150 Time (min) Figure 52
Temperature CD-1 mouse
—s—ANG-NT(8-13) D-Tyr (bolus 6.25 mg/kg, infusion at 6.25 mg. 38
3
34 |
32 0 30 60 90 120 150 180 minutes
Figure 53 Patent Application Publication Nov. 24, 2011 Sheet 48 of 49 US 2011/0288011 A1
Binding of Neurotensin-IH' in HT29 cells 4°C vs 37°C
1500
# 1000 9. Cº £ # 500 ?º
0. - + m + NT (40nM) sº 3° Figure 54
1500
# 1000 92. sº ?º E 500 ºl tº
O 0.1 1 10 100 Concentration (nM)
Figure 55 Patent Application Publication Nov. 24, 2011 Sheet 49 of 49 US 2011/0288011 A1
NT vs Dtyr 2000
sº in NT (S12-05-09) # A NT(8-13)Dtyr 9. g 1000 #5 £ ?m
0 0.1 1 10 100 1000 10000 100000 Concentration (nM)
Figure 56
US 2011/0288011 A1 Nov. 24, 2011 impaired glucose tolerance (IGT), diabetic dyslipidemia, subject a compound of the first aspect (e.g., where the peptide hyperlipidemia, a cardiovascular disease, or hypertension. therapeutic can be used to treat the disease or disorder) in an [0012] In another aspect, the invention features a method of amount sufficient to treat the disease or disorder. reducing food intake by, or reducing body weight of a sub [0017] In any of the methods involving administration of a ject. The method includes administering a compound of the compound to a subject, the amount sufficient may be less than first aspect of the invention (e.g., where the peptide therapeu 90%, 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%. 3%, tic that reduces food intake) to a subject in an amount suffi 2%, 1%, or 0.1% of the amount required for an equivalent cient to reduce food intake or reduce body weight. The subject dose of the peptide therapeutic (e.g., any described herein) may be overweight, obese, or bulimic. when not conjugated to the peptide vector. The amount suf [0013] In another aspect, the invention features a method of ficient may reduce a side effect (e.g., vomiting, nausea, or treating (e.g., prophylactically) a disorder selected from the diarrhea) as compared to administration of an effective group consisting of anxiety, movement disorder, aggression, amount of the peptide therapeutic when not conjugated to the psychosis, seizures, panic attacks, hysteria, sleep disorders, peptide vector. The subject may be a mammal such as a Alzheimer’s disease, and Parkinson’s disease. The method human. includes administering a compound of the first aspect of the [0018] In any of the above aspects, the peptide vector may invention to a subject in an amount sufficient to treat or be a polypeptide substantially identical to any of the prevent the disorder. sequences set Table 1, or a fragment thereof. In certain [0014] The invention also features a method of increasing embodiments, the peptide vector has a sequence of Angio neurogenesis in a subject. The method includes administering pep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97), a compound of the first aspect to a subject. The subject may Angiopep-3 (SEQ ID NO:107), Angiopep-4a (SEQ ID desire, or may be in need of neurogenesis. In certain embodi NO:108), Angiopep-4-b (SEQ ID NO:109), Angiopep-5 ments, the subject may be suffering from a disease or disorder (SEQID NO:110), Angiopep-6 (SEQID NO: 111), or Angio of the central nervous system such as Parkinson’s Disease, pep-7 (SEQ ID NO:112)). The peptide vector or conjugate Alzheimer’s Disease, Huntington’s Disease, ALS, stroke, may be efficiently transported into a particular cell type (e.g., ADD, and neuropsychiatric syndromes. In other embodi any one, two, three, four, or five of liver, lung, kidney, spleen, ments, the increase in neurogenesis can improve learning or and muscle) or may cross the mammalian BBB efficiently enhance neuroprotection. (e.g., Angiopep-1, -2, -3, -4-a, -4-b, -5, and -6). In another [0015] In another aspect, the invention features a method embodiment, the peptide vector or conjugate is able to enter a for converting liver stem/progenitor cells into functional pan particular cell type (e.g., any one, two, three, four, or five of creatic cells; preventing beta-cell deterioration and stimula liver, lung, kidney, spleen, and muscle) but does not cross the tion of fl-cell proliferation; treating obesity; suppressing BBB efficiently (e.g., a conjugate including Angiopep-7). appetite and inducing satiety; treating irritable bowel syn The peptide vector may be of any length, for example, at least drome; reducing the morbidity and/or mortality associated 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 35, with myocardial infarction and stroke; treating acute coro 50, 75, 100, 200, or 500 amino acids, or any range between nary syndrome characterized by an absence of Q-wave myo these numbers. In certain embodiments, the peptide vector is cardial infarction; attenuating post-surgical catabolic 10 to 50 amino acids in length. The polypeptide may be changes; treating hibernating myocardium or diabetic cardi produced by recombinant genetic technology or chemical omyopathy; suppressing plasma blood levels of norepi synthesis. nepherine; increasing urinary sodium excretion, decreasing urinary potassium concentration; treating conditions or dis TABLE 1. orders associated with toxic hypervolemia, e.g., renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pul Exemplary Peptide Vectors monary edema, and hypertension; inducing an inotropic SEQ response and increasing cardiac contractility; treating poly ID cystic ovary syndrome; treating respiratory distress; improv NO : ing nutrition via a non-alimentary route, i.e., via intravenous, subcutaneous, intramuscular, peritoneal, or other injection or 1 T F W Y G G C R A K R N N F K S A. E. D infusion; treating nephropathy; treating left ventricular sys 2 T F Q Y G G C M G N G N N F V T E K E tolic dysfunction (e.g., with abnormal left ventricular ejection fraction); inhibiting antro-duodenal motility (e.g., for the treatment or prevention of gastrointestinal disorders such as diarrhea, postoperative dumping syndrome and irritable bowel syndrome, and as premedication in endoscopic proce dures; treating critical illness polyneuropathy (CIPN) and systemic inflammatory response syndrome (SIRS; modulat ing triglyceride levels and treating dyslipidemia; treating organ tissue injury caused by reperfusion of blood flow fol lowing ischemia; or treating coronary heart disease risk factor (CHDRF) syndrome in a subject by administering and effec tive amount of a compound of the first aspect. [0016] In another aspect, the invention features a method of treating (e.g., prophylactically) a cancer, a neurodegenerative disease, or a lysosomal storage disorder (e.g., any disease described herein). The method includes administering to a
US 2011/0288011 A1 Nov. 24, 2011
TABLE 1 – continued TABLE 1 – continued Exemplary Peptide Vectors Exemplary Peptide Vectors
SEQ SEQ ID ID NO : NO :
32 T E F Y G G K. R. G. K R N N F K. R. A. K. Y. 115 C T F. F. Y. G. G. S R G R R N N F R T E E Y
33 T. F. F. Y G G C L G N R N N F K. T E E Y 116 T E F Y G G S R G R. R. N. N. F. R T E E Y C
34 T E F Y G C G R G K R N N F K. T E E Y Polypeptides Nos. 5, 67, 76, and 91, include the sequences of SEQ ID NOS : 5, 67, 76, and 91, respectively, and are amidated at the C-terminus. 35 T. F. F. Y G G R C G K R N N F K. T E E Y Polypeptides Nos. 107, 109, and 110 include the sequences of SEQ ID NOS: 97, 109, and 110, respectively, and are acetylated at 36 T E F Y G G C L G N G N N F D T E E E the N-terminus. [0019. In any of the above aspects, the peptide vector may 87 T F Q Y G G C R G K R N N F K T E E Y include an amino acid sequence having the formula: 38 Y IN K E F G T E N T K G C E R G Y R. F
39 R. F. K. Y G G C L G N M N N F E T L E E
9 O R. F. K. Y G G C L G N K. N N F L R L. K. Y. 91 R F K. Y G G C L G N K. N N Y L. R. L. K. Y. where each of X1-X19 (e.g., X1-X6, X8, X9, X11-X14, and X16-X19) is, independently, any amino acid (e.g., a naturally 92 K. T. K R K R K K Q R V K I A Y E E I F K N Y occurring amino acid such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, 93 K T K R K R K K Q R V K I A Y and Val) or absent and at least one (e.g., 2 or 3) of X1, X10, 94 R G G R L S Y S R R F S T S T G R. and X15 is arginine. In some embodiments, X7 is Seror Cys; or X10 and X15 each are independently Arg or Lys. In some 95 R. R. L. S. Y S R R. R. F embodiments, the residues from X1 through X19, inclusive, are substantially identical to any of the amino acid sequences 96 Q I K I W F Q N R R M K W K. K. of any one of SEQ ID NOS:1-105 and 107-116 (e.g., Angio pep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, 97 T F. F. Y G G S R G K R N N F K T E E Y Angiopep-5, Angiopep-6, and Angiopep-7). In some embodi 98 M R P D F C L E P P Y T G P. C. V. A. R. I ments, at least one (e.g., 2, 3, 4, or 5) of the amino acids I R Y F Y N A K A G L C Q T F V Y G G X1-X19 is Arg. In some embodiments, the polypeptide has C R A K R N N F K S A. E D C M R T C G G A one or more additional cysteine residues at the N-terminal of the polypeptide, the C-terminal of the polypeptide, or both. 99 T F. F. Y. G. G. C. R. G. K R N N F K. T. K E Y [0020] In any of the above aspects, the peptide therapeutic OO R. F. K. Y G G C L G N K. N N Y L. R. L. K. Y. may be selected from the group consisting of antimicrobial or antibiotic peptides, gastrointestinal peptides, pancreatic pep 01 T. F. F. Y G G C R A K R N N F K. R. A. K. Y. tides, peptide hormones, hypothalamic hormones, pituitary O2 N A K A G L C Q T F V Y G G C L A K R N N F hormones, and neuropeptides. The gastrointestinal or pancre E S A E D C M R T C G G A atic peptide may be a cholecystokinin, gastrin, glucagon, epidermal growth factor, vasoactive intestinal peptide (VIP), O3 Y G G C R A K R N N F K S A E D C M R T C G insulin, or a GLP-1 agonist. The hypothalamic or pituitary G A hormone may be a pituitary hormone-releasing hormone O4 G L C Q T F V Y G G C R A K R N N F K S A E (e.g., corticotropin-releasing hormone, gonadotropin-releas ing hormone, growth hormone-releasing hormone, and thy O5 L C Q T F V Y G G C E A K R N N F K S A rotropin-releasing hormone (TRH), a pituitary hormone 07 T E F Y G G S R G K R N N F K. E. E. Y. release inhibiting hormone (e.g., MSH release inhibiting hor mone and somatostatin), pro-opiomelanocortin or an analog 08 R F F Y G G S R G K R N N F K. E. E. Y. orderivative (e.g., cleavage product) thereof (e.g., adrenocor ticotropic hormone (ACTH), Cº-endorphin, fl-endorphin, 09 R F F Y G G S R G K R N N F K. E. E. Y. Y-endorphin, fl-lipotropin, Y-lipotropin, and melanocyte stimulating hormone), growth hormones, thyrotropin, vaso 10 R F F Y G G S R G K R N N F R. E. E. Y. tocin, and oxytocin. The neuropeptide may be any of angio 11 T. F. F. Y G G S R G K R N N F R. E. E. Y. tensin, bombesin, bradykinin, calcitonin, a cholecystokinin, delta sleep inducing peptide, galanin, gastric inhibitory 12 T E F Y G G S R G R R N N F R. E. E. Y. polypeptide, gastrin, neuropeptide Y, neurotensin, an opioid peptide (e.g., a dynorphin, an endorphin, an enkephalin, and 13 C T F. F. Y. G. G. S R G K R N N F K. T. E. E. Y. a nociceptin), vasoactive intestinal peptides, secretin, tachy 14 T E F Y G G S R G K R N N F K. E E Y C kinin, and vasopressin. Other peptide hormones include adi ponectins, adrenomedullins, ghrelin, gonadotropins, inhib ins, matriuretic peptides, parathyroid hormone (PTH) and
US 2011/0288011 A1 Nov. 24, 2011
occurrence of (e.g., preventing) a disease, disorder or condi 38.5% that of Angiopep-1 (250 nM) in the in situ brain per tion by administering a therapeutic agent to the subject. fusion assay described in U.S. patent application Ser. No. [0032] In one example, a subject who is being treated for a 11/807,597, filed May 29, 2007, hereby incorporated by ref metabolic disorder is one who a medical practitioner has erence). Accordingly, a polypeptide which is “not efficiently diagnosed as having such a condition. Diagnosis may be transported across the BBB” is transported to the brain at performed by any suitable means, such as those described lower levels (e.g., transported less efficiently than Angiopep herein. A subject in whom the development of diabetes or obesity is being treated prophylactically may or may not have 6). received such a diagnosis. One in the art will understand that [0039]. By a polypeptide or compound which is “efficiently subject of the invention may have been subjected to standard transported to a particular cell type” is meant that the tests or may have been identified, without examination, as one polypeptide or compound is able to accumulate (e.g., either at high risk due to the presence of one or more risk factors, due to increased transport into the cell, decreased efflux from such as family history, obesity, particular ethnicity (e.g., Afri the cell, or a combination thereof) in that cell type to at least can Americans and Hispanic Americans), gestational diabe a 10% (e.g., 25%, 50%, 100%, 200%, 500%. 1,000%, tes or delivering a baby that weighs more than nine pounds, 5,000%, or 10,000%) greater extent than either a control hypertension, having a pathological condition predisposing substance, or, in the case of a conjugate, as compared to the to obesity or diabetes, high blood levels of triglycerides, high unconjugated agent. Such activities are described in detail in blood levels of cholesterol, presence of molecular markers International Application Publication No. WO 2007/009229, (e.g., presence of autoantibodies), and age (over 45 years of hereby incorporated by reference. age). An individual is considered obese when their weight is [0040] Other features and advantages of the invention will 20% (25% in women) or more over the maximum weight be apparent from the following Detailed Description, the desirable for their height. An adult who is more than 100 drawings, and the claims. pounds overweight, is considered to be morbidly obese. Obe sity is also defined as a body mass index (BMI) over 30kg/m3. [0033] By “a metabolic disorder” is meant any pathological BRIEF DESCRIPTION OF THE DRAWINGS condition resulting from an alteration in a subject’s metabo lism. Such disorders include those resulting from an alter [0041] FIG. 1 is table and schematic diagram showing ation in glucose homeostasis resulting, for example, in hyper exendin-4 and the exendin-4 analogs used in experiments glycemia. According to this invention, an alteration in described herein. glucose levels is typically an increase in glucose levels by at [0042] FIG. 2 is a schematic diagram of the synthetic least 5%, 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. scheme used to conjugate Cys-AngioPep2, Angiopep-2-Cys or even 100% relative to such levels in a healthy individual. NH2, and Angiopep-1 to [Lys”-MHA]exendin-4. Metabolic disorders include obesity and diabetes (e.g., dia betes type I, diabetes type II, MODY, and gestational diabe [0043] FIG. 3 is a schematic diagram of the synthetic tes), satiety, and endocrine deficiencies of aging. scheme used to conjugate [Cys’lexendin-4 to (maleimido [0034] By “reducing glucose levels” is meant reducing the propionic acid (MPA))-Angiopep-2, (maleimido hexamoic level of glucose by at least 10%, 20%, 30%, 40%, 50%, 60%, acid (MHA))-Angiopep-2, and (maleimido undecanoic acid 70%, 80%, 90%. 95%, or 100% relative to an untreated con (MUA))-Angiopep-2. trol. Desirably, glucose levels are reduced to normoglycemic [0044] FIG. 4 is a graph showing transport of exendin-4 and levels, i.e., between 150 to 60 mg/dL, between 140 to 70 exendin-4/Angiopep-2 across the BBB. The total amount in mg/dL, between 130 to 70 mg/dL, between 125 to 80 mg/dL, the brain, along with the amounts in the capillaries and the and preferably between 120 to 80 mg/dL. Such reduction in parenchyma are shown. glucose levels may be obtained by increasing any one of the [0045] FIG. 5 is a graph showing increase in weight of biological activities associated with the clearance of glucose (ob?ob) mice following administration of a control, exendin from the blood (e.g., increase insulin production, secretion, or 4, or the [Lys”-MHA]exendin-4/Angiopep-2-Cys-NH2 con action). jugate (Exen-Anz). Both exendin-4 and Ex-An2 were [0035] By “subject” is meant a human or non-human ani observed to reduce weight gain as compared to the animals mal (e.g., a mammal). receiving the control. [0036] By “increasing GLP-1 receptor activity” is meant increasing the level of receptor activation measured using [0046] FIG. 6 is a graph showing total food consumption by standard techniques (e.g., cAMP activation) by, for example, (ob/ob) mice, where the mice were administered a control, at least 10%, 20%, 50%, 75%, 100%, 200%, or 500% as exendin-4, or the Exen-Anz. Both exendin-4 and Exen-An2 compared to an untreated control. were observed to reduce food intake as compared to the [0037] By “equivalent dosage” is meant the amount of a animals receiving the control. compound of the invention required to achieve the same [0047] FIG. 7 is a graph showing reduction in glycemia molar amount of the peptide therapeutic (e.g., a GLP-1 ago following administration of two doses of exendin-4 (3 plg/kg nist) in the compound of the invention, as compared to the and 30 pig?kg) and equivalent doses of Exen-Anz (4.8 pig?kg unconjugated peptide therapeutic. For example, the equiva and 48 pg/kg). A similar reduction in glycemia at the lower lent dosage of 1.0 mg exendin-4 is about 1.6 pg of the [Lys” dose of Exen-Anz, as compared to the higherdose of exendin MHA]exendin-4/Angiopep-2-Cys-NH2 conjugate described 4, was observed. During this experiment, one mouse in the herein. control group died at day 12. [0038] By a polypeptide which is “efficiently transported [0048] FIG. 8A is a schematic diagram showing the struc across the BBB” is meant a polypeptide that is able to cross ture of an Exendin-4-Angiopep-2 dimer conjugate (Ex4 the BBB at least as efficiently as Angiopep-6 (i.e., greater than (Lys39(MHA))-AN2-AN2). The compound has the structure US 2011/0288011 A1 Nov. 24, 2011
[0064] FIGS. 23A-23C show a western blot (FIG. 23A), a UV spectrum from a liquid chromatography experiment HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPP (FIG. 23B), and a mass spectrum (FIG. 23C) of the recombi nant Angiopep-2 peptide. K (MHA) - TFFYGGSRGKRNNFKTEEYC- (MPA) – TFFY [0065] FIG. 24 is a graph showing uptake of the synthetic and recombinant forms of Angiopep-2 in the in situ brain perfusion assay. where MHA is maleimido hexanoic acid and MPA is male [0066] FIG. 25 is a graph showing uptake of GST, GST imido propionic acid. Angiopep-2, GST-lepting so, and GST-Angiopep-2-lep [0049] FIG. 8B is a schematic structure of an Exendin-4 tin, 16-1so into the parenchyma in the in situ brain perfusion scramble-Angiopep-2 (Ex4(Cys32)-ANS4 (N-Term) or assay. Exen-S4) that was used a control. This compound has the [0067] FIG. 26 is a schematic diagram showing the His Structure tagged-mouse leptin and His-tagged-Angiopep-2-mouse lep tin fusion protein. [0068] FIG. 27 is an image of a gel showing purification of HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPCSGAPPPS – (MHA) – the His-tagged mouse leptin and the human leptin sequence. [0069] FIG. 28 is the sequence of human leptin precursor. GYKGERYRGFKETNFNTFS-OH, Amino acids 22-167 of this sequence form the mature leptin where MHA is maleimido hexanoic acid. peptide. [0050] FIG. 9 is a graph showing the ability of Exendin-4, [0070 FIGS. 29A and 29B are exemplary purification Exendin-4-Angiopep-2 conjugates, the Exen-S4, and Exen schemes for His-tagged leptin (mouse) and the His-tagged din-4 when conjugated conjugated to a dimeric form of Angiopep-2-leptin conjugate. Angiopep-2, to crosses the BBB. [0071] FIG. 30 is photograph of a gel showing successful [0051] FIG. 10 is a graph showing the ability of Exendin-4 small-scale expression of the leptin and Angiopep-2-leptin and Exen-Anz-An2 to reduce glycemia in mice. conjugate. [0072] FIG. 31 is a schematic diagram and picture of a gel [0052] FIGS. 11A and 11B are graphs showing tissue con showing that two products resulted from thrombing cleavage centration in brain (FIG. 11A) and in pancreas (FIG. 11B) of of the His-tagged conjugate. Exendin-4 and Exen-4-An-2. [0073] FIG. 32 is a graph showing uptake of leptin and the [0053] FIG. 12 is a graph showing dose-response of insulin Angiopep-2-leptin fusion protein into the parenchyma of secretion in response to either Exendin-4 or Exen-An2 in DIO mice. RIN-m$F pancreas cells. [0074] FIG.33 is a graph showing the effect of recombinant [0054] FIGS. 13A and 13B are chromatograms showing the leptin on the weight of ob/ob mice. Leptin-AN2 (C11) conjugate before (FIG. 13A) and after [0075] FIG. 34 is a graph showing the change in weight in (FIG. 13B) purification. DIO mice receiving a control, leptin, His-tagged mouse lep [0055] FIG. 14 is a chromatogram showing the results of tin, or the His-tagged Angiopep-2-leptin conjugate. purification of the Leptin-AN2 (C11) conjugate. [0076] FIGS. 35A and 35B are chromatograms showing the [0056] FIG. 15 is a graph showing uptake of the C3, C6, and ECMS-Neurotensin compound (ECMS-NT) before (FIG. C11 Leptin-AN2 conjugates into the brain, capillaries, and 35A) and after (FIG. 35B) purification using the analytical parenchyma using the in situ brain perfusion assay. method described in the examples. [0057] FIGS. 16A and 16B are graphs showing in situ brain [0077|| FIG. 36 is a chromatogram showing purification of perfusion of the leptin, 16-1so and the Leptin-AN2 (C11) con ECMS-NT on an AKTA-explorer with column filled with 30 jugate in lean mice and diet induced obese (DIO) mice (FIG. ml of 30RPC resin. 16A) and plasma levels of leptin in lean mice and DIO mice [0078] FIGS. 37A and 37B are chromatograms showing (FIG. 16B). Neurotensin Angiopep-2-Cys amide conjugate (NT [0058] FIGS. 17A and 17B are graphs showing food intake AN2Cys-NH2 or NT-An2) before (FIG. 37A) and after (FIG. in mice receiving a control injection (saline), leptin, 16-1so, or 37B) purification using the analytical method described in the the Leptin-AN2 (C11) conjugate after either four hours (FIG. examples. 17A) or 15 hours (FIG. 17B). [0079] FIG. 38 is a chromatogram showing purification of [0059] FIG. 18 is a graph showing weight gain over a six NT-Anz on an AKTA-explorer with column filled with 30 ml day period in mice receiving a control, leptin, 16-1so, or the of 30RPC resin. Leptin-AN2 (C11) conjugate. [0080] FIG. 39 is a graph showing hypothermia induction by NT-Anz. Mice received saline (control), NT (1 mg/kg) or [0060] FIG. 19 is a graph showing weight gain over a ten NT-An2 at 2.5 mg/kg or 5.0 mg/kg (equivalent to 1 and 2 day period in ob/ob mice receiving a control, leptin, 16-1so, or mg/kg doses of NT). Rectal temperature was monitored 90 the leptin-AN2 (C11) conjugate by daily IP injection over a minutes following intravenous injection. period of six days. [0081] FIG. 40 is a graph showing the effect of body tem [0061] FIG. 20 is a schematic diagram showing the GST perature in mice upon administration of 5, 15, or 20 mg/kg of tagged Angiopep construct. NT-Anz. [0062] FIG. 21 is a schematic diagram showing the PCR [0082] FIG. 41 is a graph showing the effect of body tem strategy used to generate the Angiopep-2-leptin, 16-1so fusion perature in mice upon administration of 5, 10, or 20 mg/kg of protein. a different preparation of NT-An2. [0063] FIG. 22 is a chromatogram showing purification of [0083] FIG. 42 is a graph showing in situ brain perfusion of the GST-Angiopep2 on a GSH-sepharose column NT and NT-An2. Following iodination, mice brains were US 2011/0288011 A1 Nov. 24, 2011 perfused in the carotid artery with either [**I]-NT or the [0095] FIG. 54 is a graph showing binding of radiolabeled [*I]-NT-An2 derivative in Krebs buffer for the indicated NT ([*H]-NT) to HT29 cells that express the NTSR1 in the times. After the indicated times, brains were further perfused presence or absence of 40 nM of NT at 4°C. or 37° C. for 30 sec to washout the excess of both compound. Both [0096] FIG. 55 is a graph showing binding of [*H]-NT to [*I]-NT or [**I]-NT-An2 derivative in brain were quanti HT29 cells in the presence of NT at concentrations ranging fied using a beta counter. Results are expressed in terms of from 0.4 nM to 40 nM. brain volume of distribution (ml/100 g) as a function of time. [0097] FIG. 56 is graph showing binding of [*H]-NT to [0084] FIG. 43 is a graph showing brain compartmentation HT29 cells in the presence of NT or Ac-Lys-D-Tyr"|NT(8 of NT and NT-An2 after in situ brain perfusion as described 13). for FIG. 40. Brain capillary depletion was performed using Dextran following standard procedures. Both [**I]-NT or DETAILED DESCRIPTION [*I]-NT-An2 derivative present in brain, capillaries, and [0098] We have developed peptide therapeutic conjugates parenchyma were quantified and volume of distribution (ml/ having an enhanced ability to cross the blood-brain barrier 100 g/2 min) is reported. (BBB) or to enter particular cell type(s) (e.g., liver, lung, [0085] FIG. 44 is a graph showing body temperature of kidney, spleen, and muscle) as exemplified by conjugates of mice receiving a bolus 5 mg/kg injection of the NT-An2, peptide vectors to the exemplary peptide therapeutics, exen followed one hour later by a 2.5 hour infusion of NT-An2 at a din-4, leptin, and neurotensin, and analogs thereof. The pep rate of 5 mg/kg/30 min (i.e., 10 mg/kg/hr). tide conjugates of the invention thus include a therapeutic peptide and a peptide vector that enhance transport across the [0086] FIG.45 is a graph showing body temperature of a rat BBB. receiving an intravenous bolus injection of 20 mg/kg NT [0099] Surprisingly, we have also shown that lower doses An2, followed immediately by a 20 mg/kg/hr infusion of of the compounds of the invention, as compared to unconju NT-An2 for 3.5 hours. gated GLP-1 agonists, are effective in treating GLP-1 related [0087] FIG. 46 is a graph showing body temperature of disorders, including a reduction in glycemia. By administer mice receiving an intravenous bolus injection of 20 mg/kg ing lower doses of the conjugated peptides, side effects such NT-Anz, followed immediately by a 20 mg/kg/hr infusion of as vomiting, nausea, and diarrhea observed with the uncon NT-Anz, which was increased to 40 mg/kg/hr after 2.5 hours. jugated agonists can be reduced or eliminated. Alternatively, [0088] FIG. 47 is a graph showing body temperature of rats increased efficacy at higher doses may be obtained. receiving an intravenous bolus injection of 20 mg/kg NT [0100] The peptide therapeutic can be any peptide having An2, followed immediately by a 20 mg/kg/hr infusion of biological known in the art and including peptides such as NT-Anz. those described below. Particular GLP-1 agonists include [0089] FIG. 48 is a graph showing body temperature of exendin-4, GLP-1, and exendin-3 fragments, substitutions ratings receiving an intravenous bolus injection of 40 mg/kg (e.g., conservative or nonconservative substitutions, or sub NT-Anz, followed immediately by a 40 mg/kg/hr infusion of stitutions of non-naturally occurring amino acids), and NT-Anz. This resulted in sustained reduction in body tem chemical modifications to the amino acid sequences (e.g., perature for the 12 hour duration of the experiment. those described herein). Peptide therapuetics, including [0090] FIG. 49 is a graph showing latency in the hot plate GLP-1 agonists, are described in detail below. test in mice of the paw licking response in control mice (left), mice receiving 20 mg/kg NT-An2 (center), and mice receiv Peptide Therapeutics ing 1 mg/kg buprenorphine (right) just prior to and 15 minutes [0101] Any peptide known in the art may be conjugated to following administration of the compound. a peptide vector of the invention. The peptide may be a mam [0091] FIG. 50 is a graph showing body temperature of malian peptide such as mouse, rat, or human peptide, or may mice receiving a bolus intravenous 7.5 mg/kg injection of be a nonmammalian peptide. Exemplary peptides are NT(8-13), Ac-Lys-NT(8-13), Ac-Lys-D-Tyr"|NT(8-13), described below. pGlu-NT(8-13), or a control. From among these analogs, Ac-Lys-D-Tyr"|NT(8-13) was observed to produce the Antimicrobial or Antibiotic Peptides greatest reduction in body temperature. [0102] In certain embodiments, the peptide therapeutic is [0092] FIG. 51 is a graph showing body temperature of an antimicrobial or antibiotic peptide. The conjugate may be mice receiving a bolus intravenous injection of a control, NT, used to treat an infection such as a bacterial infection (e.g., NT-An2, NT(8-13)-An2, and Ac-Lys-D-Tyr"|NT(8-13) any known in the art). Antimicrobial peptides include (KI An2. The greatest reduction in body temperature was AGKIA)s peptide, Apis mellifera abaecin protein, Alal.9-ma observed for NT-An2 and Ac-Lys-D-Tyr"|NT(8-13)-An2 gainin 2 amide, Ala?&13,18)-magainin 2 amide, planto-thio conjugates. nin protein, wheat O.1-purothionin protein, anoplin, [0093] FIG. 52 is a graph showing body temperature of antimicrobial hybrid peptide CM15, antimicrobial peptide mice receiving a bolus intravenous injection of Ac-Lys-ID ESF39A, antimicrobial peptide LL-37, antimicrobial peptide Tyr"|NT(8-13) (1 mg/kg) or Ac-Lys-D-Tyr"|NT(8-13) V4, apidaecin, apoR(133-162), Hyasaraneus arasin 1, aurein An2 (6.25 mg/kg). The An2 conjugated molecule was 1.2 peptide, aurein 2.2 peptide, aurein 2.3 peptide, Bac?/(1 observed to reduce body temperature to a greater extent that 35) peptide, bactericidal permeability increasing protein, f: the unconjugated molecule. lysin, Bombina orientalis BLP-7 protein, bombinin H2, [0094] FIG. 53 is a graph showing body temperature of a BTM-P1 peptide, Anura caerin 1.1, Cavia CAP11 protein, mouse receiving a 6.25 mg/kg bolus intravenous injection of CAP18 lipopolysaccharide-binding protein, cathelicidin the Ac-Lys-[D-Tyr"|NT(8-13)-An2 conjugate followed 60 antimicrobial peptide, cathelicidin, cationic antimicrobial minutes later by a 6.25 mg/kg/hr infusion of the conjugate. protein 57, cationic antimicrobial protein CAP 37, CEC(dir) US 2011/0288011 A1 Nov. 24, 2011
CEC(ret) protein, cecropin A, cecropin A(1-7)melittin(2-9), bial peptide, P1908) antimicrobial peptide, P1909-B) antimi cecropin A(1-8)magainin 201-12), cecropin C, Cecropins, crobial peptide, paracelsin, paracelsin E, parasin I, Litope chrysophsin, chicken CMAP27 protein, D-V13A(D) peptide, naeus setiferus penaeidin-4 protein, peptide 399, peptide D-V13K(D) peptide, DC-1 peptide, DC-2 peptide, DC-2R Gly-Leu-amide, Peptite 2000, pexiganan, polymyxins, peptide, Aesculus hippocastanum Ah-AMP1 protein, human colistin, colbiocin, colistimethate, colistin heptapeptide, Cº-defensin 5, human O-defensin 6, mouse cryptdin 4, colistin nonapeptide, Eu?;1, deacylpolymyxin B, lubasporin, defensin NP-1, defensin NP-3a, human DEFT1P protein, paenimyxin, pelargonoyl cyclic decapeptide polymyxin human neutrophil peptide 1, human neutrophil peptide 2, M(1), polymagma, polymyxin B, auricularum, corti-biciron, human neutrophil peptide 3, human neutrophil peptide 4, rat cortisporin, cyclo[diaminobutyryl-diaminobutyryl-phenyla neutrophil peptide 3, neutrophil peptide 5, Oryctolagus lanyl-leucyl-diaminobutyryl-diaminobutyryl-threonyl), dex cuniculus NP-1 protein, RK-1 peptide, mouse BD-6 protein, apolyspectran, diaminobutyryl-cyclo[diaminobutyryl-di rat B defensin-1 protein, fl-Defensin, human fl-defensin 28, aminobutyryl-phenylalanyl-leucyl-diaminobutyryl human fl-defensin 3, mouse [3-defensin-12 protein, human diaminobutyryl-threonyl), maxitrol, panotile, pelargonoyl fl-defensin-27, human fl-defensin-5 protein, human fl-de cyclic decapeptide polymyxin B(1), polydexa, polymyxin B fensin-6 protein, rat Bin 16 protein, bovine neutrophil [3-de nonapeptide, polyspectran OS, pulpomixine, septomixine, fensin 12, human DEFB-109 peptide, human DEFB1 protein, sP-B compound, sp-Bpy compound, sp-Bw compound, mouse Defb1 protein, mouse Defb14 protein, mouse Defb2 Uniroid, polymyxin B(1), polymyxin S(1), polymyxin T(1), protein, human DEFB4 protein, mouse Defb-9 protein, mouse polypeptin, UCB 630, polyphemusin I, polyphemusin II, por Defb5 protein, mouse Defb7 protein, mouse Defb8 protein, cine myeloid antibacterial peptide 23, PR 11 proline-argin mouse Defr1 protein, chicken gallinacin-8 protein, chicken ine-rich peptide, PR 26, PR 39, prohepcidin, protegrin-1, gallinacin-9 protein, gramicidin, gramicidin A, gramicidin B, protegrin-2, protegrin-3, protegrin-4, protegrin-5, Pseudis gramicidin C, gramicidin D, gramicidin S, hPAB-? protein, paradoxa pseudin-2 protein, purothionin, Pyrrhocoris lingual antimicrobial peptide, mouse Spagll protein, mouse apterus pyrrhocoricin protein, RACA 854, RIN 25 peptide, Td1 protein, mouse CRS4C protein, human DEFB118 pro RL-37 peptide, RPRPNYRPRPIYRP peptide, SB 37, SC5 tein, defensin NP-2, Aeshna cyanea defensin protein, deox synthetic antimicrobial peptide, Shiva 11, Shiva 3, Shiva-1, ypheganomycin D, gallerimycin, chicken gallinacin 1 pro stomoxyn, T22 protein, tachystatin A, Pyrularia pubera tein, chicken gallinacin 2 protein, Phaseolus limensis limenin THI1 protein, thionins, bovine tracheal antimicrobial pep protein, peptide NP-3b, peptide NP-5, Phaseolus coccineus tide, wheat TthV protein, WLBU2 peptide, WS22-N-amide, phaseococcin protein, human retrocyclin-2, rhesus theta xenopsin precursor fragment (XPF), antimicrobial peptide defensin-2, rhesus theta defensin-3, Oryctolagus cuniculus IB-367, PGAa antimicrobial peptide, antibacterial polypep RK-1 protein, Macaca mulatta RTD-2 protein, Macaca tide LCI, antibiotic 2928, antibiotic 5590, antibiotic A 19009, mulatta RTD-3 protein, scorpine, Picea abies SPI1 protein, antibiotic AFC-BC11, antibiotic G0069A, ampullosporin, Lycopersicon esculentum tyas118, theta-defensin, dermasep actinomycin HKI 01:55, actinotiocin, antrimycin, aplasmo tin, dermaseptin K4S4(1-16)a, dermaseptin K4S4(1-28), der mycin, aramycin, argimicin A, auromomycin, auromycin, mcidins, desferri-ferricrocin, Epinephelus coioides epineci azinomycin B, azimothricin, azureomycin A, azureomycin B, din-1, eremomycin aglycone, Evonymous europa lectin, bacillomycin D, berninamycin A, berninamycin B, bernina chicken fowlicidin-3, Zea mays Y-zeathionin proteins, mycin C, berninamycin D, biphenomycin A, biphenomycin Ginkgo biloba ginkbilobin-2 protein, gomesin, Staphylococ C, cairomycin A, cairomycin B, cairomycin C, chandramy cus haemolyticus gonococcal growth inhibitor protein, cin, cycloheptamycin, cypermycin, cystaurimycin, diperamy mouse Hamp2 protein, Amblyomma hebraeum hebraein pro cin, 2-imidazoledistamycin, chloroacetyldistamycin, dista tein, Pyrrhocoris apterus hemiptericin protein, hepcidin, mycin-DAPI, distamycin-EDTA-iron(II), hevein, Dhvar-5, dhvara peptide, HTN1 protein, HTN3 pro M-bromoacetyldistamycin, permethyldistamycin A, stalli tein, P-113D, Apis mellifera hymenoptaecin protein, Impa mycin, thioformyldistamycin, duramycin, duramycin B, tiens balsamina Ib-AMP4 peptide, indolicidin, CP10A duramycin C, echomycin A, echomycin B, echomycin C, indolicidin derivative, IsCT-P peptide, Boophilus microplus enomycin, enramycin, ficellomycin, gardimycin, globomy Ixodidin, K4-S4(1-13)a, K6L5WP peptide, Raja kenojei cin, histidinomycin, hodydamycin, janiemycin, janthinocin kenojeinin I, karabemycin, kutapressin, Lachesana tarabaevi A, janthinocin B, janthinocin C, japonicin 1, japonicin 2, latarcin 2a, levitide, liver-expressed antimicrobial peptide 2, kuwaitimycin, lavendomycin, longicatenamycin, macracid Lachesana tarabaevi Ltc.1 peptide, Lachesana tarabaevi mycin, macromomycin B, macromomycin I protein, macro Ltc2a peptide, Litoria maculatin-1.1 protein, magainin A, momycin II protein, macromomycin protein, malioxamycin, magainin B, magainin G, magainin H, magainins, Bombina muraymycin A1, muraymycin A3, muraymycin C1, napsa maxima maximin 9, MBI-27 protein, melitten or analog mycin B, napsamycin C, napsamycin D, neoberninamycin, thereof (e.g., (4-aminobutanoyl)melittin, (5-aminopen nilemycin, pacidamycin 1, pacidamycin 3, pacidamycin 5, tanoyl)melittin, azidosalicylylmelittin, cecropin A(1-8) pantomycin, phenomycin, sideromycins, siomycin, siomycin melittin(1-18), cecropin A(1-8)melittin(1-2), dioleoylmelit A, siomycin D1, Sohbumycin, sporamycin, sporangiomycin, tin, DNC-melitten, glycylmelittin, hecate 1, hecate-chorionic stendomycidin, stendomycin, sulfomycin, syriamycin, gonadotropin fl-subunit conjugate, melittin(8-26), N-methy takaokamycin, telomycin, termicin, thioxamycin, trichos lanthraniloyl melitten, prepromelittin, promelittin, and tet porin B-la, trichosporin B-IIIa, trichosporin B-IIIb, trichos raacetylmelitten), modelin 1, modelin 5, Bombyx mori mori porin B-IIIc, trichosporin B-IIId, trichosporin B-V, trichos cin protein, Myp30 peptide, myticin, mytilin, neuramide, porin B-VIa, tritrypticin, tsushimycin, tyrothricin, neutrophil basic proteins, novispirin G10, octyl-cecropin(1 vancomycin B, yemenimycin, Zelkovamycin, zwittermicin A, 7)melittin(2-9), Odorrama grahami odorranain-NR, omiga 3-(1-methyl-4-(1-methyl-4-(1-methyl-4-(8-(2-carboxami nan pentahydrochloride, Oxyuranus microlepidotus doethyloxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo(2, omwaprin protein, Oncorhyncin III, ovispirin, P18 antimicro 1-c)(1,4)benzodiazepin-5-one)pyrrole-2-carboxamido)pyr US 2011/0288011 A1 Nov. 24, 2011
role-2-carboxamido)pyrrole-2-carboxamido) 167, JMV 170, JMV 179, JMV 180, JMV 182, JMV 236, propionamidine, AR-1-144, dien-microgonotropen-c, JMV 320, JMV 332, JMV 81, N-(4-(4-azido-3'-iodopheny distamin, Distel, distel(2+), FCE 24561, FCE 25450A, FCE lazo)benzoyl)-3-aminopropionyl-CCK-8, propionyl CCK 26644, FCE 27164, FCE 27266, FCE 27784, MEN 10710, octapeptide sulfate, pClu-sincalide, Phe(CH2SO,Na)(2)-sin MEN 10716, microgonotropen L2, microgonotropen pen calide, SNF 8702, SNF 8814, SNF 8906, succinyl-tyrosyl taazapentabutylamine, MT 12, MT 17, N-(2-(diethylamino) methionyl-glycyl-tryptophyl-methionyl-aspartyl-phenethy ethyl)-1-methyl-4-(1-methyl-4-(4-formamido-1-methylimi lamide, SUT 8701, t-butyloxycarbonyl-(sulfo-Tyr)-Met-Gly dazole-2-carboxamido)pyrrole-2-carboxamido)imidazole Trp-Nie-Asp 2-phenylethyl ester, tert 2-carboxamide, PNU 151807, PNU 153429, PNU 157977, butyloxycarbonylcholecystokinin-8, CCK 15, tallimustine, tren-microgonotropen-b, edeine, edeine A, cholecystokinin(1-14), cholecystokinin(10-20), biotinyl edeine B, edeine D, edeine F, and octapeptin antibiotics. Tyr-Gly-(Thr28-Nle31)-cholecystokinin(25-33), Thr28 Nle31-cholecystokinin(25-33), Tyr25-N1e(28,31)-cholecys Gastrointestinal or Pancreatic Peptides and Peptide Hor tokinin(25-33), 2-(4-azidosalicylamido)-1,3 In OIles dithiopropionate(Thr28-Ahx31)-cholecystokinin(25-33), [0103] In certain embodiments, the peptide therapeutic is a indium-DOTA(0)-Asp26-Nle(28,31)-cholecystokinin(26 gastrointestinal or pancreatic hormone. Gastrointestinal hor 33), iodo-Tyr-Gly-Nle(28,31)-Bpa33-cholecystokinin(26 mones include cholecystokinin, gastrin, glucagon, epidermal 33), iodo-Tyr-Gly-N1e(28,31)-Bpa(29-33)-cholecystokinin growth factor, and vasoactive intestinal peptide (VIP). Other (26-33), benzoyloxycarbonyl-cholecystokinin(27-32) gastrointestinal and pancreatic peptides include glucagon and amide, cholecystokinin(27-32)-amide, cholecystokinin(29 glucagon-like peptides. Pancreatic peptides include insulin 33)-amide, butyloxycarbonyl-cholecystokinin(31-33) and somatostatin. Analogs of these peptides are described amide, Thr34-Ahx37-cholecystokinin(31–39), cholecystoki below. Other gastrointestinal and pancreatic hormones nin 10 C-terminal fragment, cholecystokinin 12 C-terminal include pancreastatin, pancreastatin(33-49), pancreastatin fragment, cholecystokinin 21, cholecystokinin 22 C-terminal 16, pancreastatin-29, and pancreastatin-52, pancreatic fragment, cholecystokinin 33(10-20), cholecystokinin 39, polypeptide, pancreatic polypeptide (31-36), Torpedo mar cholecystokinin 5-pentagastrin, cholecystokinin 58, chole morata gut PLY, pancreatic eicosapeptide, avian pancreatic cystokinin 8, cholecystokinin 9, cholecystokinin C-terminal polypeptide, salmon pancreatic polypeptide, human PPY flanking peptide, cholecystokinin precursor C-terminal pen protein, human PPY2 protein, skin peptide tyrosine-tyrosine, tapeptide, Gly-cholecystokinin, cholecystokinin-J, des glicentin, glicentin (1-16), glicentin(62-69), glicentin-related ulfated benzyloxycarbonyl cholecystokinin(26-33), dimyris pancreatic peptide, Glucagon-Like Peptide 2, ALX-0600, toylmercaptoglycero-N(o)maleoyl-[3-alanyl(Thr,N1e)-CCK 9, JMV 176, MP 2286, MP 2288, pHC 264, glucagon-like peptide-2(3-33), glucagon-like-immunoreac preprocholecystokinin, procholecystokinin, Ro 23-7014, tivity, lysyl-arginyl-asparaginyl-lysyl-asparaginyl-aspar SNF 8815, SNF 9007, sulfated cholecystokinin 15, t-buty agine, oxyntomodulin, oxyntomodulin (19-37), and Nle(27) loxycarbonyl-sulfotyrosyl-norleucyl-glycyl-tyrosyl-aspar oxyntomodulin. tyl-2-phenylethyl ester, U 67827E, and V-9-M cholecystoki [0104] Cholecystokinins nin nonapeptide. [0105] In certain embodiments, the gastrointestinal peptide is cholecystokinin oran analog thereof. Cholecystokinin ana [0106] Epidermal Growth Factor logs include cholescystokinin, 4-(biotin-epsilon-(aminohex [0107] In certain embodiments, the peptide peptide thera anoyl)oxymethyl)-3-nitrobenzoyl-glycyl-(propionypornithi peutic is epidermal growth factor (EGF) or an analog thereof. nyl-epsilon-aminohexanoyl-cholecystokinin, Such peptides include "In-DTPA-hEGF, *Ga-DOTA 4-alanyloxymethyl-3-nitrobenzoyl-epsilon-aminohexanoyl hEGF, biotinyl EGF, biregulin, chicken CALEB protein, E cholecystokinin, A 68552, ARL 15849), X, BC 197, BC 264, 6010, E1-INT, EGF-genistein, Mouse Emré protein, EGF(1 benzyloxycarbonyl-glycyl-tryptophyl-methionyl-asparty! 45), EGF(1-48), EGF(1–53), Cys-SOAH(33,42)-EGF(32-48), (OBu-t)-phenylalaninamide, butyloxycarbonyl-tryptophyl EGF(33-42), [Cys(Acm)20,31] epidermal growth factor (20 leucyl-aspartyl-phenylalaninamide, sincalide, (3-azido-4 31), EGF precursor, Lys(3)-Tyr(22)-EGF, EGF-dextran-ty hydroxy-5-iodobenzimidyl)-CCK-8,8-sulfocholecystokinin rosine conjugate, EGF-dextran conjugate, S(1-5) EGF-like octapeptide, acetylcholecystokinin C-terminal heptapeptide, protein, EGF-ricin complex, epigen, epiregulin, C. elegans ARC15849RF, Bolton Hunter-cholecystokinin nonapeptide, fat? protein, human FAT3 protein, rat FAT3 protein, sea Bolton Hunter-cholecystokinin octapeptide, cholecystokinin urchin fibropellin protein, gigantoxin I, Herdmania momus (26-32), rhodamine-Tyr-Gly-Nle(28.31) phenethyl ester HmEGFL-1 protein, C. elegans Lin-3 protein, mouse Ly64 cholecystokinin(26-32), Tyr-Gly-Nle(28,31) phenethyl protein, Drosophila oep protein, peptabody-EGF, Pseudomo ester-cholecystokinin(26–32), cholecystokinin(26–33), nas exotoxin-epidermal growth factor conjugate, Drosophila cholecystokinin(26-33) sulfated amide, I-Tyr-Gly-(Nle(28, spi protein, human TDGF1 protein, mouse TDGF1 protein, 31), 4-NO,-Pheg3-cholecystokinin(26-33), I-Tyr-Gly-Nle ”Tc-HYNIC-human EGF, *"Tc EGF, and Lys-É-urogas (28,31)-cholecystokinin(26–33), N-acetyl-norleucine(28, trone. 31)-cholecystokinin(26-33), N-O-hydroxysulfonyl-Nle(28, [0108) Glucagon 31)-cholecystokinin(26-33), Tyr-Gly-(Nle(28.31), 4-NO2 [0109] In certain embodiments, the peptide therapeutic is Phe?3)cholecystokinin(26-33), cholecystokinin(27-33), glucagon or an analog thereof. Such peptides include proglu t-butyloxycarbonyl-cholecystokinin(27-33), tert-butyloxy cagon, (deshis 1,despheó,Glu%)-glucagon-NH2, Y-L-gluta carbonyl-Nle(28,31)-cholecystokinin(27-33), cholecystoki moyl(Na-hexadecanoyl)-R(34-7)GLP-1 (37), glucagon(1 nin hexapeptide, desMH2-Tyr-cholecystokinin octapeptide, 17), glucagon(1-21), glucagon(1-6), glucagon(19–29), cholecystokinin pentapeptide, Tyr27-cholecystokinin-pan des??is(1)-glucagon amide, 12-(N(6)–(4-azidophenylami creozymin, desaminopancreozymin octapeptide, desulfated dino)Lys)-glucagon, 2-nitro-4-azidophenylsulfenyl-gluca sincalide, FPL 14294, indium DTPA-Glu-G-CCK8, JMV gon, carboxy-Me-Met(27)-glucagon, deshis(1)-(N(e)-phe US 2011/0288011 A1 Nov. 24, 2011 nylthiocarbamoyl-Lys(12))-glucagon, deshis(1)-Tyr(22) peptide, des(1-21)preproinsulin, glucagon, di-(Ö-(5-nitro-2-pyrimidyl)Orn)(17,18)-glucagon, fluorenylmethoxycarbonyl)-3-insulin, 2,4-dinitrophenol fluorescein-Trp.(25) glucagon, homoArg(12)-glucagon, Met insulin A. chain-fluorescein conjugate, 2-(4 sulfoxide(27)-glucagon, N(O)-citraconyl glucagon, N(o) azidosalicylamido)ethyl-1,3-dithiopropionate insulin, malto-Me-Met(27)-glucagon, N(o)-trinitrophenyl-His(1) acetylinsulin, Albulin, O-2-macroglobulin-insulin complex, homo-Arg(12)-glucagon, oxindolyl-Ala(25)-glucagon, amphioxus insulin-like peptide, ATP-insulin conjugates, protamine zinc-glucagon, thiol-Trp.(2) glucagon, Tyr(22) glucagon, deshis(1)-N1e(9)-Ala(11,16)-glucagon-amide, B-insulin, B22 Glu desB30 insulin, B27. Lys destripeptide des??is(1)-Glu(9)-glucagonamide, imidazopropionyl(7) insulin, B29-biotin insulin, basal insulin, benzoylphenylala arginyl(26)-N(e)-octanoyl-lysyl(34)-glucaon-like peptide-1 nine(B25)insulin, bis(9-fluorenylmethoxycarbonyl)insulin, (7–37)-OH, iodoglucagon, N(o)-biotinyl-N-(epsilon)-ace BSA-insulin-chlorin e(6) conjugate, cholera toxin B-insulin timidoglucagon, N(0)-carbamylglucagon, N(o)-e conjugate, colloidal gold-insulin complex, DKP-insulin, DP acetylglucagon, N(o)-maltoglucagon, N(e) 432, Exubera, glargine, glucose-insulin-potassium cardiople acetimidoglucagon, Ala(1)-N(e)-acetimidoglucagon, deshis gic solution, glycerol-insulin, hexyl-insulin monoconjugate (1)-N(e)-acetimidoglucagon, Phe(1)-N(e) 2, Humalog Mix25, C. elegans ins-1 protein, insulin B(20 acetimidoglucagon, N(e)-decanoylglucagon, N(e) 30), insulin B(22–30), insulin B(9–23), insulin B(9–30), insu hexanoylglucagon, N-4-azido-2-nitrophenylglucagon, lin B-chain tetrapeptide amide B22-B25, insulin covalent N-trinitrophenylglucagon, nitroglucagon, proglucagon(111 aggregate, insulin crosslinked to the catalytic fragment A of 160), S 23521, Trp-S-glucagon dimer, and S-methylgluca diphtheria toxin, insulin detemir, insulin dimer, insulin frag ment A(14-21)-B(17-30), insulin glulisine, mouse insulin I, gon. insulin LISPRO, (2,4,6-trinitrophenyl)sulfonyl(A1)-insulin, [0110] Vasoactive Intestinal Peptides (2-nitro-4-azidophenyl)(A1)-insulin, (2-nitro-4-azidophe [0111] In certain embodiments, the peptide therapeutic is nyl)-Gly(B29)-insulin, (2-nitro-4-azidophenylacetyl) (B29) vasoactive intestinal peptide or an analog thereof. Such pep insulin, (2-nitro-4-azidophenylacetyl)(B2)-desphe(B1)-in tides include vasoactive intestinal peptide precursor, (BZ2 sulin, (2-nitro-4-trimethylammoniophenyl)(A1)-insulin, K24)-vasoactive intestinal peptide, (VIP-neurotensin) hybrid (2-nitro-azidophenylacetyl)(B1)-insulin, (B1)-desphe-insu antagonist, Arg(15,2021)-Leu(17)-VIP-Gly-Lys-Arg-NH2, lin, 2,7-diaminosuberoyl-N(o)(A1)-N(e)(B29)-insulin, 3,5 aviptadil, iodinated vasoactive intestinal peptide, peptide his diiodo-Tyr(A19)-insulin, 3-(N-phenylacetyl)-insulin, tidine valine 42, PG 97-269, preprovasoactive intestinal pep 3-iodo-Tyr(A14)-insulin, 4-(azidophenylacetyl)-2,4-diami tide, preprovasoactive intestinal peptide(111-122), Ro nobutyric acid(A1)-insulin, 4-azido-2-nitrophenyl-insulin, 24-9981, Ro 25-1392, Ro 25-1553, stearyl-Nle(17)-neuro 4-azidobenzoyl(B29)-insulin, 4-fluorophenylalanine(A19) tensin(6-11)VIP(7-28), steary 1-norleucine(17)-vasoactive insulin, 4-succinylamidophenylarabinopyranoside-insulin, intestinal peptide, *"Tc tricarbonyl VD5 peptide, *Tc tri 4-succinylamidophenylglucopyranoside-insulin, 4-succiny carbonyl VD4 peptide, *Tc tricarbonyl VP05 peptide, TP lamidophenylmannopyranoside-insulin, 6-(4-fluorobenzoyl) 3654, TP3982, vasoactive intestinal peptide(1-11), vasoac aminohexanoylphenylalanyl(B1)-insulin, A(27)-B-insulin tive intestinal peptide(1-12), vasoactive intestinal peptide(1 like growth factor I insulin, adipoyl-Arg(B22)-insulin, Aib21 16), Lys(15)-Arg(16)-Leu(27)-vasoactive intestinal peptide B-chain-insulin, Aib22 B-chain-insulin, Ala(A1)-insulin, Ala (1-7)-GRF(8-27), lysyl(15)-arginyl(16)-leucyl(27) (B0)-insulin, Ala(B24)-insulin, Arg(B0)-insulin, Arg(B22) vasoactive intestinal peptide(1-7)-growth hormone-releasing insulin, Arg(B29)-insulin, Arg(B31)-insulin, Arg(B31,B32) factor(8–27), vasoactive intestinal peptide(10–28), vasoactive insulin, Asn(21)-diethylamide(A)-insulin, Asn(B10)-insulin, intestinal peptide(11-28)-NH2, vasoactive intestinal peptide Asn(B12)-insulin, AsnnFL.(A21)-insulin, Asp(B10)-insulin, (22-28), vasoactive intestinal peptide(4-11), vasoactive intes Asp(B28)-insulin, Asp(B9)-Glu(B27)-insulin, Asp-imide tinal peptide(6-23), vasoactive intestinal peptide(6-28), vaso (A21)-insulin, azoisobutyryl-insulin, B1-(4-azidosalicy active intestinal peptide(7–11), vasoactive intestinal peptide loyl)-(B1-biocytin,B2-lysine)-insulin, biotinyl-insulin, bis precursor, (N–Ac-His(1)-N1e(17)-Arg(20.21)-Ala(26))-va (tert-butyloxycarbonyl)desoctapeptide-phenylhydrazide soactive intestinal peptide, 17-norleucine-vasoactive intesti insulin, butyrimidylpyridine disulfide-insulin, carbonyl-bis nal peptide, 4-azidobenzoyl-vasoactive intestinal peptide, Met,N(a1),N(epsilon)(B29)-insulin, carboxymethyl-His 4-chloro-Phe(6)-Leu(17)-vasoactive intestinal peptide, 4-Cl insulin, citraconyl-insulin, depot-insulin, des(heptapeptide) Phe-vasoactive intestinal peptide, Ac-(Lys(12,14)-N1e(17) (B24-B30)-insulin, des(hexapeptide)(B25-30)-Ala(B23) Val(26)-Thr(28))-vasoactive intestinal peptide, Cys(2)-vaso insulin, des(pentapeptide)(B1-B5)-insulin, des(tetrapeptide) active intestinal peptide, Gly-vasoactive intestinal peptide, (B1-B4)-insulin, des(tetrapeptide)(B27-B30)-insulin, iodo-Tyr(10)-Met sulfoxide(17)-vasoactive intestinal pep desAla-insulin, des.Asn(21)-Cys(20)-2,2,2-trifluoroethyla tide, Lys(1)-Pro(2,5)-Leu(17)-vasoactive intestinal peptide, mide(A)-insulin, desAsn21-Cys20-ethylamide(A)-insulin, Lys(1)-Pro(2,5)-Arg(3,4)-Tyr(6)-vasoactive intestinal pep des Asn21-Cys20-isopropylamide(A)-insulin, desAsn(21) tide, N-succinimidyl 4-fluorobenzoate-Arg(8,15,21)-Leu CysNH2(20)(A)-insulin, des.Asn(A21)-desAla(B30)-insulin, (17)-vasoactive intestinal peptide, Arg(15.20,21)-Leu(17) desCºly(1A)-N-((trimethylammonio)acetyl)Ile(2A)-insulin, vasoactive intestinal peptide-GRR, vasoactive intestinal desCºly(A1)-insulin, desCºly(A1)-desphe(B1)-insulin, peptide-neurotensin hybrid, and N-hexanoyl-vasoactive desphe?R1)-des,Val(B2)-insulin, desamido (A21)-insulin, intestinal polypeptide. desamido (B3) insulin, desamido-insulin, deshexapeptide [0112] Insulin (B25-B30)-insulin, desoctapeptide-insulin, despentapeptide [0113] In certain embodiments, the peptide therapeutic is (B26-B30)-His NH2(B25)-insulin, despentapeptide (B26 insulin or an analog thereof. Such peptides include proinsu B30)-PhenH2(B25)-insulin, despentapeptide (B26-B30) lin, (A-C-B) human proinsulin, 9-fluorenylmethoxycarbo TyrNH2(B25)-insulin, despentapeptide(B26-B30)-insulin, nyl-arginyl-glycyl-isoleucyl-valyl-glutamyl-glutaminyl-cys dethiobiotinyl-insulin, diacetyl(A1-B29)-insulin, dicarbain teinyl-cysteinyl-threonyl-serine, C-Peptide, des(27-31)-C (A7,B7)-insulin, dicitraconyl Gly(A1)Phe(B1)-insulin, dihy US 2011/0288011 A1 Nov. 24, 2011
droxycyclohexylene-insulin, disulfide-desAla(B30)-insulin, [0119) Exendin analogs are also described in U.S. Pat. No. dodecoy(A1-B29)-insulin, fluorescein-isothiocyanated-in 7,157,555 and include those of the formula: sulin, fluoresceinthiocarbamyl(B29)-insulin, Gln(A8)-insu lin, Gln(B13)-insulin, Gln(B30)-insulin, Glu(21) B-chain insulin, Glu(A8)-insulin, Gly(A1)-insulin, glycosylated insulin, hexamethylester-insulin, His(B16)-insulin, iodo-in sulin, isophane insulin, Leu(A19)-insulin, Leu(A3)-insulin, Leu(B24)-insulin, Leu(B24,B25)-insulin, Leu(B25)-insulin, Leu(B30)-insulin, Long-Acting Insulin, bovine insulatard, human insulatard, porcine isulatard, Lys(B29)-(N(e)-myris toyl)-des(b30) insulin, methoxy-insulin, N(o)(B1)-biotinyl where X, is His, Arg or Tyr; X, is Ser, Gly, Ala or Thr; X, is Asp or Glu, Xa is Phe, Tyr or Nal; Xs is Thr or Ser; Xa is Ser epsilon-aminocaproyl-insulin, N(e)-palmitoyl Lys(B29)-in or Thr; X, is Asp or Glu, Xs is Leu, Ile, Val, p(ily or Met; X, sulin, N,N-bis(methylsulfonylethoxycarbonyl)-insulin, is Leu, Ile, p(\ly, Val or Met; Xuo is Phe, Tyr, or Nal; X11 is Ile, N-Me-Ile(2A)-insulin, N-Me-Val(3A)-insulin, N-methylpy Val, Leu, poly, t-BuG or Met; Xi, is Glu or Asp; Xis is Trp, ridinium insulin, neutral insulin, Nle(A2)-insulin, Nle(B17) Phe, Tyr, or Nal; Xiaº Xis, Xia and X17 are independently Pro, insulin, octadeutero-Phe(B1)-octadeutero-Val(B2)-insulin, HPro, 3Hyp, 4Hyp, TPro, N-alkylglycine, N-alkyl-pCly or Phe(A 14)-insulin, Phe(A19)-insulin, Phe(B1)-insulin, Phe N-alkylalanine; X is is Ser, Thr, or Tyr; and Z is –OH or (B24)-insulin, polyethylene glycol(B1)-insulin, Pro(B21) —NH2 (e.g., with the proviso that the compound is not exen din-3 or exindin-4.) insulin, Sar(9A)-insulin, Ser(A6,A11)-insulin, Ser(B24)-in [0120) Preferred N-alkyl groups for N-alkylglycine, sulin, Ser(B25)-insulin, single chain des(B30)-insulin, N-alkyl-pCly and N-alkylalanine include lower alkyl groups tetrakis(3-nitro-Tyr)-insulin, tri-Lys-insulin, trifluoroacetyl (e.g., C1-6 alkyl or C1 a alkyl). insulin, triphthaloyl-insulin, tris(N-methylpyridinium)-insu [012.1] In certain embodiments, X, is His or Tyr (e.g., His). lin, Trp(A1)-insulin, Trp(A19)-insulin, Trp(B1)-insulin, X2 can be Gly. X., can be Leu, p(ily, or Met. X is can be Trp or Ultratard Insulin, W(B28),P(B29)-insulin, zebrafish insu Phe. Xa can be Phe or Nal; X11 can be Ile or Val, and Xia, Xis. lin-a protein, zebrafish insulin-b protein, insulin-dextran, Xie and X1, can be independently selected from Pro, HPro, Panulirus argus insulin-like protein 6-kDa, insulin-related TPro, or N-alkylalanine (e.g., where N-alkylalanine has a factor, iodo(A14-tyrosyl)insulin, Leydig insulin-like protein, N-alkyl group of 1 to about 6 carbon atoms). In one aspect, Lys(B29)(N-carboxynonadecanoyl)-des(B30) human insu X1s, X16, and X17 are the same amino acid residue. X1s may lin, Lys(B29)-N(e)-lithocholoyl-gamma-Glu des(b30) insu be Ser or Tyr (e.g., Ser). Z can be –NH2. [0122] In other embodiments, X, is His or Tyr (e.g., His); lin, N(e)B29-(4-azidosalicyloyl) insulin, N(e)B29-(4-azidot X, is Gly; Xa is Phe or Nal; X, is Leu, pCily, or Met; Xuois Phe etrafluorobenzoyl-biocytinyl) insulin, NBC-insulin, or Nal; X11 is Ile or Val; Xia, Xis, Xia, and X17 are indepen NBI6024, neutral protamine lispro, NovoSol Basal insulin, dently selected from Pro, HPro, TPro, or N-alkylalanine; and preproinsulin, sulfated beef insulin, thyroxyl-insulin, methio X is is Ser or Tyr, (e.g., Ser). Z can be –NH2. nyl-lysylproinsulin, miniproinsulin, N-(e29),N-(e59)-bis [0123] In other embodiments, X, is His or Arg; X2 is Gly; (methylsulfonylethoxycarbonyl)proinsulin, proinsulin(46 X, is Asp or Glu, Xa is Phe ornapthylalanine; Xs is Thror Ser; 70), des(31.32)-proinsulin, des(Lys(64), Arg(65)) proinsulin, Xa is Seror Thr; X, is Asp or Glu, Xs is Leu or pGly; Xois Leu des(23-63)-proinsulin, and proinsulin-E. coli tryptophan E or pGly; Xio is Phe or Nal; Xu is Ile, Val, ort-butyltylglycine; chimeric polypeptide. X12 is Glu or Asp; X1s is Trp or Phe, X14: Xi's, X1 g, and X17 [0114] GLP-1. Agonists are independently Pro, HPro, TPro, or N-methylalanine; Xis [0115] In particular embodiments, the peptide therapeutic is Ser or Tyr and Z is –OH or –NH2. (e.g., where the is a GLP-1 agonist. Particular GLP-1 agonists include GLP-1, compound is not exendin-3 or exendin-4). Z can be –NH2. [012.4] In another embodiment, X, is Leu, Ile, Val, or pGly exendin-4, and analogs thereof. Exemplary analogs are (e.g., Leu or pGly) and Xis is Phe, Tyr, or Nal (e.g., Phe or described below. Nal). These compounds can exhibit advantageous duration of [0116] Exendin-4 and Exendin-4 Analogs action and be less subject to oxidative degradation, both in [0117] Exendin-4 and exendin-4 analogs can also be used vitro and in vivo, as well as during synthesis of the compound. in the compositions, methods, and kits of the invention. The [0125] Other exendin analogs also described in U.S. Pat. compounds of the invention can include fragments of the Nos. 7,157,555 and 7,223,725, include compounds of the exendin-4 sequence. Exendin-4 has the sequence. formula:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro Pro-Pro-Ser-NH2 where X, is His, Arg, or Tyr; X, is Ser, Gly, Ala, or Thr; X, is Asp or Glu, Xs is Ala or Thr; Xa is Ala, Phe, Tyr, or Nal; X, is [0118] Particular exendin-4 analogs include those having a Thror Ser; Xs is Ala, Ser, or Thr; X, is Asp or Glu, Xio is Ala, cysteine substitution (e.g., [Cys’lexendin-4) or a lysine sub Leu, Ile, Val, p(\ly, or Met; X11 is Ala or Ser; X12 is Ala or Lys; stitution (e.g., [Lys”|exendin-4). Other exendin-4 analogs X is is Ala or Gln; Xia is Ala, Leu, Ile, pCily, Val, or Met; X1s include (2-sulfo-9-fluorenylmethoxycarbonyl)-3-exendin-4 is Ala or Glu, Xia is Ala or Glu; X17 is Ala or Glu; X19 is Ala and fluorescein-Trp25-exendin-4. or Val; X20 is Ala or Arg; X21 is Ala or Leu; X22 is Phe, Tyr, or
US 2011/0288011 A1 Nov. 24, 2011 tions of the wild type sequence (e.g., the human wild type where R is H.N. H.,N-Ser; H.,N-Val-Ser; H.,N-Asp-Val-Ser; sequence), or may have other chemical modifications. GLP-1 H2N-Ser-Asp-Val-Ser; H2N-Thr-Ser-Asp-Val-Ser; H.N agonists can also be non-peptide compounds, for example, as Phe-Thr-Ser-Asp-Val-Ser; H2N-Thr-Phe-Thr-Ser-Asp-Val described in U.S. Pat. No. 6,927,214. Particular analogs Ser; H2N-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser; H2N-Glu-Gly include BIM 51077, LY307161, LYS48806, CJC-1131, Lira Thr-Phe-Thr-Ser-Asp-Val-Ser; or H2N-Ala-Glu-Gly-Thr glutide, glucagon-like peptide 1(1-36)amide, glucagon-like Phe-Thr-Ser-Asp-Val-Ser; X is Lys or Arg; and R2 is NH2, peptide 1(1-37), glucagon-like peptide 1(7-36), Ala”-gluca OH, Gly-NH2, or Gly-OH. [0139| Other GLP-1 analogs, described in U.S. Pat. No. gon-like peptide 1 (7-36), glucagon-like peptide 1 (7-36) 5,118,666, include the sequence His-Ala-Glu-Gly-Thr-Phe amide, Ser(8)-glucagon-like peptide 1 (7-36)amide, gluca Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala gon-like peptide 1(7–37), N-acetyl-glucagon-like peptide-1 Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X, where X is Lys, Lys (7–36)amide, N-pyroglutamyl-glucagon-like peptide-1 (7 Gly, or Lys-Gly-Arg. 36)amide, and glucagon-like peptide-1 (9–36)-amide. [0140] GLP-1 analogs also include peptides of the formula: [0136] The GLP-1 analog can be truncated form of GLP-1. H2N–X—CO—R1, where R is OH, OM, or—NR,Rs; Mis The GLP-1 peptide may be truncated by 1, 2, 3, 4, 5, 6, 7, 8, a pharmaceutically acceptable cation or a lower branched or 9, 10, 11, 12, 13, 15, 20, or more residues from its N-terminus, unbranched alkyl group (e.g., C1-c alkyl); R2 and Rs are inde its C-terminus, or a combination thereof. In certain embodi pendently selected from the group consisting of hydrogen and ments, the truncated GLP-1 analog is the GLP-1 (7-34), GLP a lowerbranched or unbranched alkyl group (e.g., C1-c alkyl); 1(7–35), GLP-1(7-36), or GLP-1(7-37) human peptide or the X is a peptide comprising the sequence His-Ala-Glu-Gly C-terminal amidated forms thereof. Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln [0137] In other embodiments of the invention, modified Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; forms of truncated GLP-1 peptides are used. Exemplary ana NH2 is the amine group of the amino terminus of X; and CO logs are described in U.S. Pat. No. 5,545,618 and have the is the carbonyl group of the carboxy terminus of X; acid amino acid sequence: addition salts thereof; and the protected or partially protected derivatives thereof. These compounds may have insulinotro pic activity exceeding that of GLP-1(1-36) or GLP-1(1-37). His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser [0141] Other GLP-1 analogs are described in U.S. Pat. No. Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys-Glu-Phe-Ile-Ala 5,981,488 and have the formula: Trp-Leu-Val-Lys-(Gly) - (Arg) - (Gly) R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr where (Gly), (Arg), and (Gly) are present or absent depending on indicated chain length, with at least one modification Leu-Y-Gly-Gln-Ala-Ala -Lys- Z-Phe-Ile-Ala-Trp-Leu selected from the group consisting of (a) substitution of a Val-Lys-Gly-Arg-R2 neutral amino acid, Arg, or a D form of Lys for Lys at position 26 and/or 34 and/or a neutral amino acid, Lys, or a D form of where R is His, D-His, desamino-His, 2-amino-His, fl-hy Arg for Arg at position 36; (b) substitution of an oxidation droxy-His, homohistidine, C-fluoromethyl-His, or O-methyl resistant amino acid for Trp at position 31; (c) substitution His; X is Met, Asp, Lys, Thr, Leu, Asm, Gln, Phe, Val, or Tyr; according to at least one of Tyrfor Val at position 16; Lys for Y and Z are independently selected from Glu, Gln, Ala, Thr, Ser at position 18; Asp for Glu at position 21; Ser for Gly at Ser, and Gly; and R2 is selected from NH, and Gly-OH (e.g., position 22; Arg for Gln at position 23; Arg for Ala at position provided that, if R is His, X is Val, Yis Glu, and Zis Glu, then 24; and Gln for Lys at position 26; (d) a substitution compris R2 is NH2). ing at least one of an alternative small neutral amino acid for [0142] Other GLP-1 analogs are described in U.S. Pat. No. Ala at position 8; an alternative acidic amino acid or neutral 5,512,549 and have the formula: amino acid for Glu at position 9; an alternative neutral amino acid for Gly at position 10; and an alternative acidic amino acid for Asp at position 15; and (e) substitution of an alterna R1-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser tive neutral amino acid or the Asp or N-acylated or alkylated Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala form of His for His at position 7. With respect to modifica tions (a), (b), (d), and (e), the substituted amino acids may be Trp-Leu-Val-Lys (R2) -Gly-Arg-R3 in the D form. The amino acids substituted at position 7 can also be the N-acylated or N-alkylated amino acids. Exem where R1 is 4-imidazopropionyl (desamino-histidyl), 4-imi plary GLP-1 analogs include [D-His’|GLP-1(7-37), [Tyr’] dazoacetyl, or 4-imidazo-O, O.dimethyl-acetyl; R2, which is GLP-1(7-37), [N-acetyl-His’|GLP-1(7-37), [N-isopropyl bound to the side chain of the Lys (e.g., through the e amino His’|GLP-1(7–37), [D-Ala"IGLP-1(7-37), [D-Glu’|GLP-1 group), is Cairo unbranched acyl or is absent; Rs is Gly-OH or (7-37), [Asp”|GLP-1(7–37), [D-Asp"|GLP-1(7–37), NH2; and Xaa is Lys or Arg. [0143] Still other GLP-1 analogs are described in U.S. Pat. [D-Phe"|GLP-1(7-37), [Ser”, Arg”,Arg”,Gln”|GLP-1(7 No. 7,084.243. In one embodiment, the GLP-1 analog has the 37), and [Ser”,Gln”,Tyr”,Lys”,Asp"|GLP-1(7–37). formula: [0138] Other GLP-1 fragments are described in U.S. Pat. No. 5,574,008 have the formula: His-Xs-Glu-Gly-X11-X12-Thr-Ser-Asp-X16– Ser-Ser R1-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe Tyr-Leu-Glu-X22-X23-X24-Ala-X26-X27-Phe-Ile-Ala Ile-Ala-Trp-Leu-Val-X-Gly-Arg-R2
US 2011/0288011 A1 Nov. 24, 2011
where each of X1 g is a naturally or nonnaturally occurring amino acid residue;Y and Z are amino acid residues; and one of the substitutions at the O-carbonatoms of Y and Z may each independently be substituted with a primary substituent group selected from the group consisting of hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl and heteroarylalkyl, heterocyclylalkyl said primary substituent optionally being substituted with a secondary substituent selected from a cycloalkyl, heterocyclyl, aryl, or heteroaryl group; any of said primary or secondary substituents may further be substituted with one or more of H, alkyl, cycloalkyl, arylalkyl, aryl, heterocyclyl, heteroaryl, alkenyl, alkynyl, halo, hydroxy, mercapto, nitro, cyano, amino, acy lamino, azido, guanidino, amidino, carboxyl, carboxamido, carboxamido alkyl, formyl, acyl, carboxyl alkyl, alkoxy, ary loxy, arylalkyloxy, heteroaryloxy, heterocycleoxy, acyloxy, mercapto, mercapto alkyl, mercaptoaryl, mercaptoacyl, halo, cyano, nitro, azido, amino, guanidino alkyl, guanidino acyl, sulfonic, sulfonamido, alkyl sulfonyl, aryl sulfonyl or phos X7-Xs-Glu-Gly-Thr-Phe-Thr-Ser-Asp-XIg-Ser-X1s phonic group; wherein, the primary or secondary substi Tyr-Leu-Glu-X22-Gln-Ala-X2s-Lys-Glu-Phe-Ile-Ala tutents may optionally be bridged by covalent bonds to form one or more fused cyclic or heterocyclic systems with each Trp-Leu-X33-Lys-Gly-Arg-X37 other; where, the other substitution at the alpha-carbon of Y wherein: X7 is L-His, D-His, desamino-His, 2-amino-His, may be substituted with H, C1-6 alkyl, aminoalkyl, hydroxy fl-hydroxy-His, homohistidine, C-fluoromethyl-His, or alkyl or carboxyalkyl; where the other substitution at the O-methyl-His; Xs is Gly, Ala, Val, Leu, Ile, Ser, or Thr; Xia is alpha-carbon of Z may be substituted with hydrogen, C1-12 Val, Phe, Tyr, or Trp; Xis is Ser, Tyr, Trp, Phe, Lys, Ile, Leu, alkyl, aminoalkyl, hydroxyalkyl, or carboxyalkyl; or Val; X22 is Gly, Glu, Asp, or Lys; X2s is Ala, Val, Ile, or Leu: [0151] A and B are optionally present, where A is present Xss is Val or Ile; and X37 is Gly, NH2, or is absent (e.g., and A is H, an amino acid or peptide containing from about provided that the GLP-1 compound does not have the 1-15 amino acid residues, an R group, an R-C(O) (amide) sequence of GLP-1(7-37)OH, GLP-1(7-36)-NH2, [Gly°] group, a carbamate group RO–C(O), a urea R4RsN–C(O), GLP-1(7–37)OH, [Gly°]GLP-1(7-36)NH2, [ValèIGLP-1(7 a sulfonamido R-SO2, or RaFsN–SO2; where R is selected 37)OH, [ValèIGLP-1(7-36)NH2. [Leu"IGLP-1(7–37)OH, from the group consisting of hydrogen, C1-12 alkyl, Cs-lo [Leu°|GLP-1(7–36)NH2, [Ile"|GLP-1(7–37)OH, [Ile"IGLP cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocycloalkyl, US 2011/0288011 A1 Nov. 24, 2011
aryl, heteroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl, and one of the substitutions at the O-carbon is a hydroxylalkyl heteroaryloxyalkyl; Ra and Rs are each independently group; Xs is a naturally or nonnaturally occurring amino acid selected from the group consisting of H, alkyl, cycloalkyl, residue in which one of the substitutions at the Cº-carbon is cycloalkylalkyl, heterocyclyl, heterocycloalkyl, aryl, het C1-12 alkyl, hydroxylalkyl, heteroarylalkyl, or carboxami eroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl, and het doalkyl, and the other substitution at the O-carbon is H or eroaryloxyalky; where the O-amino group of X, is substituted alkyl; X9 is a naturally or nonnaturally occurring amino acid with H or an alkyl group, said alkyl group may optionally residue in which one of the substitutions at Cº-carbon is car form a ring with A; where B is present and B is OR1, NR, R2, boxylalkyl, bis-carboxylalkyl, carboxylaryl, sulfonylalkyl, or an amino acid or peptide containing from 1 to 15 amino carboxylamidoalkyl, or heteroarylalkyl; and where A is H, an acid residues (e.g., 1 to 10 or 1 to 5) terminating at the amino acid or peptide containing from about 1 to about 5 C-terminus as a carboxamide, substituted carboxamide, an amino acid residues, an R group, an R-C(O) amide group, a ester, a free carboxylic acid, or an amino-alcohol; where Ri carbamate group RO–C(O), a urea RaRsN–C(O), a sul and R2 are independently chosen from H. C.12 alkyl, Cs to fonamido R-SO2 or a RaFsM-SO2. cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocycloalkyl, [0155] In certain embodiments, X, is His, D-His, N-Me aryl, heteroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl or thyl-His, D-N-Methyl-His, 4-ThiazolylAla, or D-4-Thiaz heteroaryloxyalkyl. olylAla; X2 is Ala, D-Ala, Pro, Gly, D-Ser, D-Asn, Nma, [0152] Exemplary substitutions on the O-carbonatoms of Y D-Nma, 4-ThioPro, 4-Hyp, L-2-Pip, L-2-Azt, Aib, S- or and Z include heteroarylarylmethyl, arylheteroarylmethyl, R-Iva and Acc3; Xa is Glu, N-Methyl-Glu, Asp, D-Asp, His, and biphenylmethylforming biphenylalanine residues, any of Gla, Adp, Cys, or 4-ThiazolyAla; Xa is Gly, His, Lys, or Asp; which is also optionally substituted with one or more, hydro Xs is Thr, D-Thr, Nle, Met, Nva, or L-Aoc; Xa is Phe, Tyr, gen, alkyl, cycloalkyl, arylalkyl, aryl, heterocyclyl, het Tyr(Bzl), Tyr(3-NO2), Nle, Trp, Phe(penta-fluoro), D-Phe eroaryl, alkenyl, alkynyl, halo, hydroxy, mercapto, nitro, (penta-fluoro), Phe(2-fluoro), Phe(3'-fluoro), Phe(4-fluoro), cyano, amino, acylamino, azido, guanidino, amidino, car Phe(2,3-di-fluoro), Phe(3,4-di-fluoro), Phe(3,5-di-fluoro), boxyl, carboxamido, carboxamido alkyl, formyl, acyl, car Phe(2,6-di-fluoro), Phe(3,4,5-tri-fluoro), Phe(2-iodo), Phe(2 boxyl alkyl, alkoxy, aryloxy, arylalkyloxy, heteroaryloxy, OH), Phe(2-OMe), Phe(3'-OMe), Phe(3-cyano), Phe(2 heterocycleoxy, acyloxy, mercapto, mercapto alkyl, mercap chloro), Phe(2-NH2), Phe(3-NH2), Phe(4-NH2), Phe(4 toaryl, mercaptoacyl, halo, cyano, nitro, azido, amino, guani NO2), Phe(4-Me), Phe(4-ally!), Phe(n-butyl), Phe(4 dino alkyl, guanidino acyl, sulfonic, sulfonamido, alkyl sul cyclohexyl), Phe(4-cyclohexyloxy), Phe(4-phenyloxy), fonyl, aryl sulfonyl and phosphoric group. 2-Nal, 2-pyridylAla, 4-thiazolylAla, 2-Thi, O.-Me-Phe, D-O [0153] Other embodiments include isolated polypeptides Me-Phe, cº-Et-Phe, D-O-Et-Phe, cº-Me-Phe(2-fluoro), D-O where the other substitution at the Cº-carbon of Y is substituted Me-Phe(2-fluoro), O.-Me-Phe(2,3-di-fluoro), D-O-Me-Phe(2, with H, methyl, or ethyl; and where the other substitution at 3-di-fluoro), O.-Me-Phe(2,6-di-fluoro), D-O-Me-Phe(2,6-di the O-carbon of Z is substituted with H, methyl, or ethyl. fluoro), Cº-Me-Phe(penta-fluoro) and D-O-Me-Phe(penta [0154] Further embodiments include isolated polypeptides fluoro); X, is Thr, D-Thr, Ser, or hSer; Xs is Ser, hSer, His, as described above where X, is naturally or non-naturally Asm, or O-Me-Ser; and X9 is Asp, Glu, Gla, Adp, Asm, or His. occurring amino acid residue in which one of the substitu [0156] Additional embodiments include those where Y is tions at the O-carbon is a primary substituent selected from Bip, D-Bip, L-Bip(2-Me), D-Bip(2-Me), L-Bip(2'-Me), the group consisting of heterocyclylalkyl, heteroaryl, het L-Bip(2-Et), D-Bip(2-Et), L-Bip(3-Et), L-Bip(4-Et), L-Bip eroarylkalkyl and arylalkyl, said primary substituent option (2-n-propyl), L-Bip(2-n-propyl, 4-OMe), L-Bip(2-n-propyl, ally being substituted with secondary substituent selected 2'-Me), L-Bip(3-Me), L-Bip(4-Me), L-Bip(2,3-di-Me), from heteroaryl or heterocyclyl; and in which the other sub L-Bip(2,4-di-Me), L-Bip(2,6-di-Me), L-Bip(2,4-di-Et), stitution at the Cº-carbon is H or alkyl; X2 is naturally or L-Bip(2-Me, 2-Me), L-Bip(2-Et, 2’-Me), L-Bip(2-Et, 2'-Et), nonnaturally occurring amino acid residue in which one of L-Bip(2-Me,4-OMe), L-Bip(2-Et,4-OMe), D-Bip(2-Et,4 the substitutions at the C-carbon is an alkyl or cycloalkyl OMe), L-Bip(3-OMe), L-Bip(4-OMe), L-Bip(2,4,6-tri-Me), where the alkyl group may optionally form a ring with the L-Bip(2,3-di-OMe), L-Bip(2,4-di-OMe), L-Bip(2,5-di nitrogen of X2; and wherein the other substitution at the OMe), L-Bip(3,4-di-OMe), L-Bip(2-Et,4,5-di-OMe), L-Bip Cº-carbon is H or alkyl; Xs is a naturally or nonnaturally (3,4-Methylene-di-oxy), L-Bip(2-Et, 4.5-Methylene-di occurring amino acid residue in which one of the substitu oxy), L-Bip(2-CH2OH, 4-OMe), L-Bip(2-Ac), L-Bip(3 tions at the Cº-carbon is a carboxyalkyl, bis-carboxyalkyl, NH=Ac), L-Bip(4-NH=Ac), L-Bip(2,3-di-chloro), L-Bip sulfonylalkyl, heteroalkyl, or mercaptoalkyl; and where the (2,4-di-chloro), L-Bip(2,5-di-chloro), L-Bip(3,4-di-chloro), other substitution at the Cº-carbon is hydrogen or alkyl; Xa is L-Bip(4-fluoro), L-Bip(3,4-di-fluoro), L-Bip(2,5-di-fluoro), a naturally or nonnaturally occurring amino acid residue in L-Bip(3-n-propyl), L-Bip(4-n-propyl), L-Bip(2-iso-propyl), which the Cº-carbon is not substituted, or in which one of the L-Bip(3-iso-propyl), L-Bip(4-iso-propyl), L-Bip(4-tert-bu substitutions at the Cº-carbon is aminoalkyl, carboxyalkylhet tyl), L-Bip(3-phenyl), L-Bip(2-chloro), L-Bip(3-chloro), eroarylalkyl, or heterocycylalkyl; Xs is a naturally or non L-Bip(2-fluoro), L-Bip(3-fluoro), L-Bip(2-CFs), L-Bip(3 naturally occurring amino acid residue in which one of the CFs), L-Bip(4-CFs), L-Bip(3-NO2), L-Bip(3-OCFs), L-Bip substitutions at the O-carbon is an alkyl or hydroxyalkyl, and (4-OCFs), L-Bip(2-OEt), L-Bip(3-OEt), L-Bip(4-OEt), in which the other substitution at the C-carbon is hydrogen or L-Bip(4-SMe), L-Bip(2-OH), L-Bip(3-OH), L-Bip(4-OH), alkyl; Xa is a naturally or nonnaturally occurring amino acid L-Bip(2-CH2—COOH), L-Bip(3-CH2—COOH), L-Bip(4 residue in which one of the substitutions at the Cº-carbon is CH2—COOH), L-Bip(2-CH2—NH2), L-Bip(3-CH2—NH2), C1-12 alkyl, aryl, heteroaryl, heterocyclyl, cycloalkylalkyl, L-Bip(4-CH2—NH2), L-Bip(2-CH2-0H), L-Bip(3-CH2— heterocyclylalkyl, arylalkyl, or heteroarylalkyl group, and the OH), L-Bip(4-CH2—OH), L-Phe(4-(1-propargyl)], L-Phe other substitution at the O-carbon is H or alkyl; X, is a natu [4-(1-propenyl)], L-Phe(4-n-butyl], L-Phe(4-cyclohexyl], rally or nonnaturally occurring amino acid residue in which Phe(4-phenyloxy), L-Phe(penta-fluoro), L-2-(9,10-dihydro US 2011/0288011 A1 Nov. 24, 2011
phenanthrenyl)-Ala, 4-(2-benzo(b)furan)-Phe, 4-(4-Diben [0161] t-BuG or =t-butylglycine zofuran)-Phe, 4-(4-phenoxathiin)-Phe, 4-(2-Benzo(b) [0162] TPro=thioproline thiophene)-Phe, 4-(3-thiophene)-Phe, 4-(3-Quinoline)-Phe, [0163] HPro=homoproline 4-(2-naphthyl)-Phe, 4-(1-Naphthyl)-Phe, 4-(4-(3,5-dimeth [0164] NmA=N-methylalanine ylisoxazole)-Phe, 4-(2,4-dimethoxypyrimidine)-Phe, [0165] Cya=cysteic acid homophe, Tyr(Bzl), Phe(3,4-di-chloro), Phe(4-Iodo), [0166] Thi-É2-Thienyl-Ala 2-Naphthyl-Ala, L-o-Me-Bip, or D-O-Me-Bip; Z is L-Bip, [0167] hSer-homoserine D-Bip, L-Bip(2-Me), D-Bip(2-Me), L-Bip(2'-Me), L-Bip(2 (0168] Aib-a-aminoisobutyric acid Et), D-Bip(2-Et), L-Bip(3-Me), L-Bip(4-Me), L-Bip(3 [0169] Bip-biphenylalanine OMe), L-Bip(4-OMe), L-Bip(4-Et), L-Bip(2-n-propyl,2' (0170] Nle=norleucine Me), L-Bip(2,4-di-Me), L-Bip(2-Me, 2-Me), L-Bip(2-Me,4 (0171] Ahx=2-aminohexanoic acid OMe), L-Bip(2-Et,4-OMe), D-Bip(2-Et,4-OMe), L-Bip(2,6 [0172] Nva=norvaline di-Me), L-Bip(2,4,6-tri-Me), L-Bip(2,3,4,5,-tetra-Me), L-Bip(3,4-di-OMe), L-Bip(2,5-di-OMe), L-Bip(3,4-Methyl Hypothalamic and Pituitary Hormones ene-di-oxy), L-Bip(3-NH=Ac), L-Bip(2-iso-propyl), L-Bip [0173] The peptide therapeutic can be a hypothalamic or (4-iso-propyl), L-Bip(2-Phenyl), L-Bip(4-Phenyl), L-Bip(2 pituitary hormone. These hormones include pituitary hor fluoro), L-Bip(4-CFs), L-Bip(4-OCFs), L-Bip(2-OEt), L-Bip mone-releasing hormones, pituitary hormone release inhibit (4-OEt), L-Bip(4-SMe), L-Bip(2-CH2—COOH), D-Bip(2 ing hormones, pro-opiomelanocortin, growth hormones, CH2—COOH), L-Bip(2-CH2—COOH), L-Bip(3-CH2— pituitary gonadotropins (e.g., those described herein), vaso COOH), L-Bip(4-CH2—COOH), L-Bip(2-CH2—NH2), tocin, oxytocin, and vasopressins (e.g., those described L-Bip(3-CH2—NH2), L-Bip(4-CH2—NH2), L-Bip(2 herein). Other hypothalamic or pituitary hormones include CH2—OH), L-Bip(3-CH2—OH), L-Bip(4-CH2—OH), Coturnix japonica gonadotropin-inhibitory hormone, lac L-Phe(3-Phenyl), L-Phe(4-n-Butyl], L-Phe(4-cyclohexyl], totropin, rat luteinizing hormone release-inhibiting factor, Phe(4-Phenyloxy), L-Phe(penta-fluoro), L-2-(9,10-Dihydro melanin concentrating hormone precursor(109-129)-glycyl phenanthrenyl)-Ala, 4-(3-Pyridyl)-Phe, 4-(2-Naphthyl)-Phe, glutamic acid, melanin-concentrating hormone precursor, 4-(1-naphthyl)-Phe, 2-naphthyl-Ala, 2-fluorenyl-Ala, L-O melanin-concentrating-hormone precursor(129-145) Me-Bip, D-O-Me-Bip, L-Phe(4-NO2), or L-Phe(4-Iodo); A is glutamyl-isoleucinamide, neurohormone C, prolactin-releas H, acetyl, fl-Ala, Ah X, Gly, Asp, Glu, Phe, Lys, Nva, Asn, Arg, Ser, Thr, Val, Trp, Tyr, caprolactam, Bip, Ser(Bzl), 3-pyridy ing peptide, human protein, rat DGF protein, melanin-con 1Ala, Phe(4-Me), Phe(penta-fluoro), 4-methylbenzyl, 4-fluo centrating hormone, melanin-concentrating hormone(2-17), robenzyl, n-propyl, n-hexyl, cyclohexylmethyl, 6-hydroxy Phe(13), Tyr(19)-melanin-concentrating hormone, nasohy pentyl, 2-thienylmethyl, 3-thienylmethyl, penta pophysial factor, decidual luteotropin, lysocorticone, sperm fluorobenzyl, 2-naphthylmethyl, 4-biphenylmethyl, releasing substance, Bos taurus TSP 86-84 protein, coherin, 9-anthracenylmethyl, benzyl, (S)-(2-amino-3-phenyl)propyl, hypophysin, hypostin, and fish somatolactin protein. methyl, 2-aminoethyl, or (S)-2-aminopropyl; and B is OH, [0174] Pituitary Hormone-Releasing Hormones NH2, Trp-NH2, 2-naphthylAla-NH2, Phe(penta-fluoro)-NH2, [0175] In certain embodiments, the peptide therapeutic is a Ser(Bzl)-NH2, Phe(4-NO.)—NH2, 3-pyridylAla-NH2, Nva pituitary hormone-releasing hormone or an analog thereof. NH2, Lys-NH2, Asp-NH2, Ser-NH2, His-NH2, Tyr-NH2, Phe Such hormones include corticotropin-releasing hormone, NH2, L-Bip-NH2, D-Ser-NH2, Gly-OH, beta-Ala-OH, gonadotropin-releasing hormone, growth hormone-releasing GABA-OH, or APA-OH. hormone, and thyrotropin-releasing hormone (TRH). [0157] In certain embodiments, when A is not present, and [0176] Analogs of corticotropin-releasing hormone X, is an R group, an R-C(O) (amide) group, a carbamate include O-helical corticotropin-releasing hormone, Tyr(3) group RO–C(O), a urea RaFsM-C(O), a sulfonamido Pro(4)-N1e(18,21)-O-helical corticotropin releasing hormone R—SO2, or a RARsN–SO2; wherein R is H, C, 12 alkyl, (3–41), astressin, astressin B, cortagine, corticorelin ovine, Cs-10 cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocy Nle(21,38)-Arg(36)-corticotropen-releasing hormone, corti cloalkyl, aryl, heteroaryl, arylalkyl, aryloxyalkyl, heteroary cotropin releasing hormone(9-41), biotinyl-Ser(1)-corti lalkyl, heteroaryloxyalkyl, or heteroarylalkoxyalkyl; and cotropin releasing hormone, Phe(12)-N1e(21,38)-corticotro pin-releasing hormone(12–41), Phe(12)-N1e(21,38)-O-Me where Ra and Rs are each independently H, C1-12 alkyl, Cs-lo Leu(37)-corticotropin-releasing hormone(12–41), Glu(20) cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocycloalkyl, corticotropin-releasing hormone, iodo-Tyr(0)-corticotropin aryl, heteroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl, or releasing hormone, Nle(21)-iodo-Tyr(32)-corticotropin heteroaryloxyalky. releasing hormone, Pro(5)-corticotropin-releasing hormone, [0158] In certain embodiments, when B is not present and Z cyclo(31-34)(phenylalanyl(12)-norleucyl(21,28)-glutamyl is OR1, NR, R2, or an amino-alcohol; where R, and R2 are independently H, C1-12 alkyl, Cs to cycloalkyl, cycloalkyla (31)-lysyl(34))acetyl-corticotropin releasing factor(4-41), Phe(12)-Nle(21,38)-C(O-Meleu(37))-H-R corticotropin lkyl, heterocycle, heterocycloalkyl, aryl, heteroaryl, aryla releasing factor(12–41), phenylalanyl corticotropin-releasing lkyl, aryloxyalkyl, heteroarylalkyl, or heteroaryloxyalkyl. In factor, phenylalanyl corticotropin-releasing factor(12–41), certain embodiments, XI (where applicable), X2, and Xs are pro-corticotropin releasing hormone, prolyl-prolylisoleu N–H or N-alkylated, (e.g., N-methylated) amino acid resi cine, human UCN2 protein, human UCN3 protein, mouse dues. The polypeptide may be a 10-merto 15-mer and capable urocortin 2, and rat urocortin 3. of binding to and activating the GLP-1 receptor. [0177] Analogs of gonadotropin-releasing hormone include (ß-Asp(O-DEA))(6),Gln8-GnRH, (lysine(6)(1,3,8 ABBREVIATIONS trihydroxy-6-carboxyanthraquinone))GnRH, (Arg(6)-Trp [0159) Nal-naphthylalanine (7)-Leu(8)-Pro(9)-NEt)GnRH, 9-hydroxyproline-gona [0160] pGly=pentylglycine dorelin, A 76154, AN 207, argtide, azaline, azo-GnRH
US 2011/0288011 A1 Nov. 24, 2011 20
Cys, Ro 23-7861, somatotropin releasing factor 40, Leu(27) 2-fluoromethylimidazole-TRH, 2-picolyl-TRH, 3-Me-TRH, somatotropin releasing factor 40, somatotropin releasing 4(5)-nitroimidazole-TRH, 4-fluoroimidazole-TRH, beta hormone(1-24)amide, somatotropin releasing hormone(1 (pyrazolyl-1)-Ala(2)-TRH, deamido-TRH, Gly TRH, Gly 26)amide, somatotropin releasing hormone(1–29), His(1)-I Lys-Arg-TRH, Leu(2)-Pip(3)-TRH, linear beta-Ala-TRH, Tyr(10)-N1e(27)-somatotropin releasing hormone(1-32) nVal(2)-TRH, Pro-hydrazide-TRH, and 4-azidosalicyla amide, Pro(15)-Leu(27)-somatotropin releasing hormone(1 mide-TRH. 32)NH2, somatotropin releasing hormone(1-37), [0180] Pituitary Hormone Release Inhibiting Hormones somatotropin releasing hormone(1-37)amide, Ala(34)-Ser [0181] In certain embodiments the peptide therapeutic is a (38)-Arg(40)-somatotropin releasing hormone(1-40)-OH, pituitary hormone release inhibiting hormone or an analog Ac-Tyr(1)-somatotropin releasing hormone(1-40)-OH, thereof. Such peptides include MSH release inhibiting hor somatotropin releasing hormone(1-43), Leu(27)-somatotro mones, somatostatin, or analog thereof. pin releasing hormone(1-44)-OH, somatotropin releasing [0182] Exemplary MSH relase inhibitng hormones include hormone(1-44)amide, somatotropin releasing hormone(1 carbobenzoxyprolyl-leucyl-glycinamide, N-acetyl-prolyl 45), 27-Leu-45-Gly-somatotropin releasing hormone(1-45), leucyl-glycinamide, pareptide, prolyl-leucyl-glycine, prolyl Leu(27)-somatotropin releasing hormone(3-29)NH2, N(a) leucyl-thiazolidine-2-carboxamide, tyrosyl-prolyl-leucyl biotinyl(1-44)amide somatotropin releasing hormone, 1-His glycinamide, tyrosyl-prolyl-leucyl-glycine, tyrosyl-prolyl 2-Ala-27-N1e-somatotropin releasing hormone(1–29), 1-N lysyl-glycinamide, and tyrosyl-prolyl-tryptophyl MeTyr-27-Nle-28-Asn-somatotropin releasing hormone(1 glycinamide. 29)NHEt, 2-Ala-somatotropin releasing-hormone(1–29), [0183] Exemplary somatostatin analogs include angiopep 3'-5' somatotropin-releasing hormone(1-23), somatotropin tin, antrin, AOD 9604, ASS 51, ASS 52, BIM 23003, BIM releasing hormone(1-29)-Gly-Gly-Gly-Gly-Cys-NH2, 23034, BIM 23052, BIM 23120, BIM 23206, BIM 23268, somatotropin-releasing hormone(1-30)amide, Ala?2)-Leu BIM 23926, BIM 23A760, CGP 15425, CGP23996, CH 275, (15)-N1e(27)-GABA(30)-somatotropin-releasing hormone CH 288, CMDTPA-Tyr2-octreotate, cyclo(7-aminohep (1-30)amide, Ile(2)-Ser(8)-Ala(15)-Leu(27)-Ser(28)-Hse tanoyl-phenylalanyl-tryptophyl-lysyl-threonyl), cyclo(7 (30)-somatotropin-releasing hormone(1-30)amide, iso.Asp aminoheptanoylphenylalanyl-tryptophyl-lysyl-benzylthreo (8)-Leu(27)-somatotropin-releasing hormone(1-32)amide, nyl), cyclo[aminoheptanoic acid-cyclo[cysteinyl His(1),Nle(27)-somatotropin-releasing hormone(1-32) phenylalanyl-D-tryptophyl-lysyl-threonyl-cysteinyl)), cyclo amide, Ala(15.29)-somatotropin-releasing hormone(4-29) (fl-methyl-N-benzylglycyl-phenylalanyl-tryptophyl-lysyl OH, Leu(27)-somatotropin-releasing hormone(1-32) amide, threonyl-phenylalanyl), cyclo(N-benzylglycyl Ala(2)-N1e(27)-GABA(30)-somatotropin-releasing hor phenylalanyl-tryptophyl-lysyl-threonyl-phenylalanyl), cyclo mone(1-30)-amide, Asp(8)-Leu(27)-somatotropin-releasing (Pro-Phe-Trp-Lys-Thr-Phe), cyclo(prolyl-thiomethyl hormone(1-32)amide, tesamorelin, U 90349E, and lysyl(15) phenylalanyl-tryptophyl-lysyl-threonyl-phenylalanyl), D.D arginyl(16)-leucyl(27)-vasoactive intestinal peptide(1-7) carbasomatostatin, DC 32-87, dihydrosomatostatin, JF-10 growth hormone-releasing factor(8-27). 81, Lan-7, lanreotide, pentetreotide, Phe-Cys-Phe-Trp-Lys [0179| Analogs of TRH include 2-hydroxy-4-carboxybu Thr-Pen-Thr-NH2, phenylalanyl-cyclo[cysteinyl tyrylhistidyl-prolinamide, 3-(aminocarbonyl)-1-(3-(2-(ami tyrosyltryptophyl-lysyl-threonyl-penicillamine) nocarbonyl)pyrrolidin-1-yl)-3-oxo-2-(((5-oxopyrrolidin-2 threoninamide, phenylalanyl-cyclo[cysteinyltyrosyl yl)carbonyl)amino)propyl)pyridinium, 5-oxoprolyl-2,4(5) tryptophyl-ornithyl-threonyl-penicillamine)threoninamide, diiodohistidyl-prolinamide, 5-oxoprolyl-4(5)-iodohistidyl prosomatostatin, prosomatostatin(29-92), prosomatostatin prolinamide, pGlu-His-amphetamine, CG 3509, cryptic peptide, PTR3173, PTR-3205, RC 161, San 201-456, digipramine, DN 1417, fluorescein-TRH, Glp-asparagine sms-D70, SOM-230, desamino-Trp somatostatin(7-10), proline-D-tyrosine-D-tryptophanylamide, glutaminyl-pyro somatostatin 28, Leu(8)-Trp(22)-iodo-Tyr(25)-somatostatin glutamyl-glutamyl-proline amide, JTP 2942, L-pyro 28, Nle(8)-somatostatin 28, Trp(22)-somatostatin 28, Tyr(7) glutamyl-L-histidyl-3,3-dimethylprolinamide, Gly(10)-somatostatin 28, somatostatin 28(1-12), Tyr(11)-so methylpyroglutamyl-histidyl-pipecolate, MK 771, montine matostatin 14, Tyr(7)-Gly(10)-somatostatin 14, somatostatin lin, N-(2-hydroxy-4-(isobutylcarbamoyl)butyryl)histidyl 20, somatostatin 25, somatostatin 25(1-9), somatostatin 26, prolinamide, rat pFQ7 protein, posatirelin, PR 546, prepro somatostatin 34, somatostatin RC 102, somatostatin(3-6), thyrotropin releasing hormone(160-169), prepro-thyrotropin somatostatin(7-10), 4-NH2-Phe(4)-Trp.(8)-somatostatin, releasing hormone(25-50), prepro-thyrotropin releasing hor 5-fluoro-Trp.(8)-somatostatin, 5-methoxy-Trp.(8)-somatosta mone(53-74), prepro-TRH, prepro-TRH(178-199), pro-thy tin, Ala?2)-Trp.(8)-Cys(14)-somatostatin, Ala(3,14)-soma rotropin releasing hormone, Nle(2)-Prot(3)-protirelin, Nve tostatin, Ala(5)-Orn(9)-somatostatin, Ala(5)-Trp.(8)-soma (2)-Prot(3)-protirelin, rat pSE14 protein, pyro(a tostatin, azidonitrobenzoyl-Lys(4)-iodo-Tyr(11) aminoadipyl)-histidyl-prolinamide, pyroglutamyl-((N3) somatostatin, azidonitrobenzoyl-Lys(9)-iodo-Tyr(11) imidazolylmethyl)-histidyl-n-amylprolinamide, somatostatin, cyclic hexapeptide(Phe-Phe-Trp-Lys-Thr pyroglutamyl-glutamyl-proline amide, pyroglutamyl-histi Phe)-somatostatin, cyclo(des.Ala(1)-desCºly(2)-S-COMe dyl-3-methylprolinamide, pyroglutamyl-histidyl-proline homo-CysNH2(3)-Trp.(8)-desCys(14))-somatostatin, cyclo thioamide, pyroglutamyl-histidyl-proline-tyramine, pyro (Pro-dehydro-Phe-Trp-Lys-Thr-Phe) somatostatin, Cys(3) glutamyl-L-histidyl-L-pipecolic acid amide, pyroglutamyl somatostatin, de-Asn(5)-Trp.(8)-Ser(13)-somatostatin, leucyl-prolinamide, pyroglutamyl-tyrosyl-prolylamide, TA desAla(1)-somatostatin, desAla?1)-desCºly(2)-Trp.(8)-Asn(3, 0910, TA 0910 acid-type, thyroliberin N-ethylamide, TRH 14)-somatostatin, desamino acid(1,2,5)-Glu(7)-Trp.(8) 5-fluoroimidazole, TRH chloromethyl ketone, TRH diazom LAmp(9)-m-I-Tyr(11)-hhDys(12)-somatostatin, des.Asn(5) ethyl ketone, CRM45 thyrotropin releasing hormone, somatostatin, iodine-Tyr-somatostatin, iodo-Tyr(1) 1-(methano-Glp(2,3))-TRH, 1-Me-TRH, 2,4-diiodoimida somatostatin, N–Ac-desAla(1)-desCºly(2)-4-Cl-Phe(6)-Trp zole-TRH, 2.4-Mepro(3)-TRH, 2-diazohistidinyl-TRH, (8)-somatostatin, N-Tyr(1)-somatostatin, nonapeptide-D
US 2011/0288011 A1 Nov. 24, 2011 24 kinin, leucosulfakinin II, leucyl-prolyl-prolyl-glycyl-prolyl protein, SADPNFLRFamide, SALMFamide 1, SALMFa leucyl-prolyl-arginyl-prolinamide, LFRFamide, Lom-AG mide 2, SchistoPLRFamide, schistomyotropin-1, schisto myotropin, 6-Phe-Ala(0)-Lom-MT-II, LUQIN, mouse myotropin-2, schistostatin, human SCN11A protein, mouse Lynx1 protein, mouse Lynx2 protein, mandibular organ-in Scn 11a protein, rat Sch11a protein, scorpion toxin AaB III, hibiting hormone 1, mandibular organ-inhibiting hormone 2, scorpion toxin AaB IT1, scorpion toxin AaB IT2, scorpion rat manserin, Mas-MIPI peptide, Mas-MIPII peptide, mel toxin AaB IT4, SDPFLRFamide, SDRNFLRFamide, secre anocyte-stimulating hormone, Met-callatostatin, desCly-de toneurin, SEEPLY peptide, SEPYLRFamide, human SHC3 sPro-Met-callatostatin, Hyp(2)-Met-callatostatin, methion protein, mouse SHC3 protein, rat SHC3 protein, small car ine sulfoxide NPY, motilin, mu-agatoxin I, myokinin, dioactive peptide A, small cardioactive peptide B, sodium myomodulin, myotropin II, N-acetyl-pituitary adenylate influx-stimulating peptide, type I sodium channel SP19 pep cyclase activity peptide 27, N-N-(4-azido-tetrafluoroben tide, stannin, stichopin, SynGAP protein p135, rat TAFA5 zoyl)-biocytinyloxyl-succinimide, neomyosuppressin, neur peptide, Mouse TAT4 peptide, Tem-HrTH, rat TFF3 protein, exophilin, human Neurod2 protein, mouse Neurod2 protein, threonyl-lysyl-glutaminyl-glutamyl-leucyl-glutamic acid, rat Neurod2 protein, chicken NeuroM protein, neuromedin TNRNFLRFamide, triakontatetraneuropeptide, tuberoin N-125, neuromedin S, neuromedin U, neuromedin U 25, fundibular peptide 39, Tx1 neurotoxin, Tx2 neurotoxin, Tx3 neuromedin U 8, neuromedin U 9, neuronal membrane neurotoxin, urechistachykinin I, urechistachykinin II, Vax1 cytoskeletal protein 4.1, neuropeptide B, neuropeptide F. protein, VD1-RPD2 neuropeptideo.1, WD1-RPD2 neuropep human neuropeptide S, neuropeptide SF, neuropeptide VF, tide oz, VD1-RPD2 neuropeptide B, VD1-RPD2 neuropep mouse neuropeptide W, rat neuropeptide W, neurophysin, tideprepro, VFQNQFKGIQGRF.VGF peptide, VGF protein, human AVP protein, VLDV neurophysin, neuroserpin, neu VPNDWAHFRGSWamide, Y-head activator-head activator rostenin, neurotensin-like immunoreactivity, NocII neu bipeptide, and YFAFPRQamide. ropeptide, NocIII neuropeptide, norbin, human NPVF pro (0202) Angiotensin tein, human NPW protein, rat Nrn protein, human NRN1 [0203] The peptide therapeutic can be angiotensin, angio protein, orcokinin, Ala(13)-orcokinin, Ser(9)-orcokinin, Val tensinogen, or analog thereof. Exemplary angiotensin ana (13)-orcokinin, orexins, mouse Pacsin.1 protein, human logs include angiotensin A, angiotensin I, angiotensin I(1-7), PCDHA4 protein, mouse Pcdha4 protein, rat Pcdha4 protein, angiotensin I(1-9), (5-(4-pyridyl-1-oxide)-Ala-1)-angio human PCDHA6 protein, mouse Pcdha6 protein, rat Pcdhaô tensin I, Arg10-angiotensin I, Asn1-Val?-Gly9-angiotensin I, protein, human PCLO protein, mouse Pclo protein, rat Pclo Asn1-Val?-His'9-angiotensin I, des.Asp1-angiotensin I, protein, mouse Pep2 protein, human PCSK1N protein, Pea desDeu10-angiotensin I, Ileš-angiotensin I, Pro11-Alal 2-an CAH-II neuropeptide, Pea-PK-1, Pea-PK-2, pedal peptide, giotensin I, Sarl-angiotensin I, Sar1-(S-Me)Cys8-angio Pej-PDH-I peptide, Pej-PDH-II peptide, Penaeus japonicus tensin, Sarl-Ala?/-angiotensin I, Sar1-Ileš-O-Me-Ala(7)-an Pej-SGP-IV protein, peptide I (Aplysia), peptide II (Aplysia), giotensin I, Sar(1)-Val(5)-N-Me-Ala(7)-angiotensin I, Sar(1, peptide tyrosine phenylalanine, peptide V, periplaneta-DP. 7)-Val(5)-angiotensin I, Val(5)-angiotensin I, Lys(11) periplanetin CC-1, perisulfakinin, periviscerokinin, perivis angiotensinogen(3-11), angiotensinogen (1-13), cerokinin-2, pev-myomodulin, PFR(Tic)amide, phenylala angiotensinogen(1-14), angiotensinogen(6-13), H 142, H nyl-aspartyl-alanyl-phenylalanyl-threonyl-threonyl-glycyl 189, rat proangiotensin-12, D-Pro7-Ang-(1-7), des-angio phenylalanylamide, phenylalanyl-threonyl-arginyl tensin Irenin substrate, des-aspartate-angiotensin I, angio phenylalaninamide, Helicoverpa zea pheromone tensin II, (2,4-dinitrophenyl)aminohexanoylangiotensin II, biosynthesis activating neuropeptide, pheromone biosynthe (6-biotinylamido)hexanoylangiotensin II, 7-Ala-angiotensin sis-activating neuropeptide II, pheromone-biosynthesis-acti (1-7), Ile8-angiotensin I, angiotensin II(1-6), angiotensin vating neuropeptide I, Pseudaletia pheromonotropin, phyl II(1-7), angiotensin II(1-8), angiotensin II(2–7), angiotensin lomedusin, pineal peptide E5, pituitary adenylate cyclase II(3–7), Phe-A-angiotensin II(3–7), angiotensin II amide, Sar1 activating polypeptide, human ADCYAP1 protein, mouse angiotensin II amide(1-7), (O-Me-Tyr)(4)-angiotensin II, Adcyap1 protein, rat Adcyap1 protein, Arg(15.20,21)-Leu 1-(4-azidobenzoic acid)-Ile8-angiotensin II, 1-hydantoic (17)-PACAP-Gly-Lys-Arg-NH2, P66 peptide, PACAP-re acid-Val?-Ala8-angiotensin II, 1-malyl-angiotensin II, 1-ma lated peptide, pig pituitary adenylate cyclase-activating-pep lyl-Leu(8)-angiotensin II, 1-N(4)-dimethyl-Asp-angiotensin tide(1-38), pituitary adenylate-cyclase-activating peptide(6 II, 1-N-Suc-Val(5)-phenyl-Gly(8)-angiotensin II, 4-amino 27), pituitary adenylate-cyclase-activating-peptide(6-38), Phe(6)-angiotensin II, Aib(1)-angiotensin II, Ala(7)-angio plaferon, PnTx4-3, Polyorchispenicillatus pol-RFamide neu tensin II, Ala(7)-N-Me-Phe(8)-angiotensin II, Ala(8)-angio ropeptides protein, postural asymmetry factor, human pre tensin II, Ala-Pro-Gly-angiotensin II, Asn(1)-Val(5) pro-26RFa protein, pro-aldosterone secretion inhibitory fac angiotensin II, Asp(1)-Val(5)-angiotensin II, cyclo[Sar(1) tor, proctolin, human PROK2 protein, mouse Prok2 protein, Cys(3)-Mpt(5))-angiotensin II, cyclo(Sar(1)-HCys(3)-Mpt rat prokineticin 2, prolyl-phenylalanyl-arginyl-phenylalani (5))-angiotensin II, Cys(1,8)-angiotensin II, Cys(8) namide, mouse protocadherin [16, mouse Ptz3 home angiotensin II, des.Asp(1)–(N(2)-(3-carboxy-1-oxypropyl) odomain protein, p(\DPFLRFamide, pyroglutamyl-leucyl Arg)-Ile(5)-angiotensin II, des.Asp(1)-des.Arg(2)-Ile(5) asparaginyl-phenylalanyl-seryl-threonyl-glycyl angiotensin II, desAsp(1)-Me-Tyr(4)-angiotensin II, desphe tryptophanamide, pyroglutamyl-leucyl-glycyl-arginyl (8)-angiotensin II, His(2)-Ile(5)-angiotensin II, Ile(5)-Mepro phenylalaninamide, pyroglutamyl-leucyl-threonyl (7)-angiotensin II, Ile(8)-angiotensin II, iodo-Sar(1)-Tdf(8) phenylalanyl-threonyl-prolyl-asparaginyl-tryptophyl angiotensin II, Leu(8)-angiotensin II, Lys(2)-angiotensin II, glycyl-serinamide, pyroglutamyl-tryptophyl-leucyl-lysyl N,N-dimethyl-Gly(1)-Ile(5)-angiotensin II, N-(1-octanoyl) glycyl-arginyl-phenylalaninamide, pyrokinin, human PYY2 Ile(5)-Leu(8)-angiotensin II, N-a-(N-fluoresceinthiocarbam protein, mouse Rac1 protein, RFamide peptide, mouse oyl)-Asp(1)-Ile(5)-angiotensin II, N-Me-Phe(8)-angiotensin Rgs19ip1 protein, rat Rgs 191p1 protein, mouse Rgs 19ip3 II, Phe(4)-angiotensin II, Sar(1)-angiotensin II, Sar(1)-4 protein, human RHEB protein, mouse Rheb protein, rat Rheb azido-Phe(8)-angiotensin II, Sar(1)-Ala(7)-angiotensin II,
US 2011/0288011 A1 Nov. 24, 2011 26 inducing peptide, Trp(1)-delta sleep-inducing peptide, Ala Nle(4)-neuropeptide Y, Pro(34)-neuropeptide Y, Trp(32) (4)-delta-sleep-inducing peptide amine, cyclo-Gly-delta neuropeptide Y, NPY(28-36), fish pancreatic peptide Y, pre sleep-inducing peptide, deltaran, and Deltran. proneuropeptide Y, proneuropeptide Y, propionyl-neuropep [0212] Galanin tide Y, PYX 1, PYX 2, WRYamide, and YM 42454. [0213] The peptide therapeutic may be galanin oran analog [0220) Neurotensin thereof. Such peptide include gal(1-14)-(Abu 8) scy-I, human [0221] The peptide therapeutic may be neurotensin or ana GAL protein, rat Gal protein, galanin(1-11)-amide, galanin log thereof. Exemplary neurotensin analogs include (VIP (1-13)-spantide amide, galanin(1-15), Thr(6)-Trp.(8,9)-gala neurotensin) hybrid antagonist, acetylneurotensin(8-13), min(1-15)-15-ol, galanin(1-16), Sar(1)-Ala(12)-galanin(1 JMV 1193, KK13 peptide, neuromedin N, neuromedin N 16)-amide, galanin(1-19), galanin(16-29), galanin(17-30), precursor, neurotensin(1-10), neurotensin(1-11), neurotensin galanin(2-11)-amide, galanin(3–30), galanin message-asso (1-13), neurotensin(1-6), neurotensin(1-8), neurotensin(8 ciated peptide, galanin(1-14)-(aminobutyrate)SCY-I, galanin 13), Asp(12)-neurotensin(8-13), Asp(13)-neurotensin(8-13), (1-13)-bradykinin-(2-9)-amide, galparan, M38 peptide, and Lys(8)-neurotensin(8-13), N-methyl-Arg(8)-Lys(9)-neo-Trp M40, and transportan. (11)-neo-Leu(12)-neurotensin(8-13), neurotensin(9-13), [0214] Gastric Inhibitory Polypeptide neurotensin 69L, Arg(9)-neurotensin, azidobenzoyl-Lys(6) [0215] The peptide therapeutic may be gastric inhibitory Trp(11)-neurotensin, Gln(4)-neurotensin, iodo-Tyr(11)-neu polypeptide (GIP) or an analog thereof. GIP analogs include rotensin, iodo-Tyr(3)-neurotensin, N-O-(fluoresceinylthio GIP(1-14), GIP(1-39), GIP(1-42), GIP(3-42), GIP(7-42), carbamyl)glutamyl(1)-neurotensin, Phe(11)-neurotensin, e-palmitoyl-Lys16-GIP, e-palmitoyl-Lys37-GIP.Hyp3-GIP. Ser(7)-neurotensin, Trp(11)-neurotensin, Tyr(11)-neuro Hyp3-palmitoyllys 16-GIP, N-pyroglutamyl-e-palmitoylly tensin, rat NTT7, PD 149163, proneurotensin, stearyl-Nle syl(16)-GIP, N-pyroglutamyl-e-palmitoyllysyl(37)-GIP, Pro (17)-neurotensin(6-11)VIP(7-28), *Tc-NT-XI, TJN 950, (3)-GIP and N—AcGIP(LysPAL37)). and vasoactive intestinal peptide-neurotensin hybrid. [0216] Gastrin [0222] Opioid Peptides [0217] The peptide therapeutic can be gastrin or an analog [0223] The peptide therapeutic can be an opioid peptide. thereof. Exemplary gastrin analogs include 3-(3-iodo-4-hy Exemplary opioid peptides include dynorphins, endorphins, droxyphenyl)propionyl(Leu15)gastrin-(5-17), APH070, big enkephalins, and nociceptins, or an analog thereof. Other gastrin, dansylgastrin, desglugastrin, diiodograstrin, opioids include (F-G)NOC of Q(1-13)-NH2, (Nphe(1), Arg DM-gastrin, DP-gastrin, E1-INT, desulfonated-gastrin(2 (14),Lys615)N-OFQ NH2, acetyl-arginyl-phenylalanyl 17), Leu(15)-gastrin(2-17)-Gly, gastrin(4-17), gastrin 17, tryptophyl-isoleucyl-asparaginyl-lysine, cyclo[Cys(10,14)) gastrin 34(1-14)-IgG hinge protein-gastrin 17(2–17), gastrin nociceptin(1-13) amide, cyclo(Cys(7,14))nociceptin(1-13) desulfonated, Leu 15-gastrin heptadecapeptide, methoxine amide, cyclo[tyrosyl-ornithyl-phenylalanyl-aspartamide), (15)-gastrin heptadecapeptide, Nle15-gastrin heptadecapep deltorphin, deltorphin I, deltorphin II, Ala(2)-deltorphin I, tide, gastrin hexapeptide, gastrin I, gastrin immunogen, Ala(2)-deltorphin II, Ile(5,6)-deltorphin II, Ala(2)-Cys(4) Asp11-gastrin, Asp11-Phel2-gastrin, Phel 2-gastrin, Glu-oc deltorphin, Leu(2)-deltorphin, dermorphin, dermorphin-sa tagastrin, glycine-extended gastrin 17, IgG hinge protein porin, endomorphin 1, endomorphin 2, Dmt(1)-2-Nal(4)-en gastrin 17(2–17), iodogastrin, JMV 209, JMV 97, minigas domorphin-1, Pro(2)-endomorphin-1,1-Nal(4) trin, des?rp 1-Asp5-Leu12-minigastrin, desTrp1-Nle12 endomorphin-2, Dmt(1)-2-Nal(4)-endomorphin-2, prolyl minigastrin, nanogastrin, preprogastrin, progastrin(1-35), (2)-endomorphin-2, FE 200665, FE 200666, nocistatin, ”Tc-HYNIC(0)-Glu1-desGlu(2-6)-minigastrin, and opiomelanin, prepro-orphanin FQ(154-181), prepro-orpha tyrosyl-glycyl-tryptophyl-methionyl-aspartyl-phenylalanyl nin FQ(160-187), proorphanin, Tyr-W-MIF-1, and UFP-102. glycine). [0224] Dynorphin and dynorphin analogs include 3-nitro [0218) Neuropeptide Y 2-pyridinesulfenyl dynorphin derivative, arodyn, dynorphin [0219). In certain embodiments, the peptide therapeutic is a (1-11), Ala(2)-dynorphin(1-11), Pro(10)-dynorphin(1-11), neuropeptide Y or an analog thereof. Such peptides include dynorphin(1-12), dynorphin(1-13), dynorphin(1-24), dynor bis(31-31')((Cys(31),Nva(34))NPY(27-36)-NH2), D-Trp phin(1-32), dynorphin(1-8), dynorphin(2-17), dynorphin(3 (34)-neuropeptide Y, desamido-neuropeptide Y, EXBP 68, 13), dynorphin A, dynorphin A(1-11)-amide, Pro(3)-dynor galanin-NPY chimeric peptide M32, galanin-NPY chimeric phin A(1-11)-amide, Ala(2)-Trp.(4)-dynorphin A(1-13), Asn peptide M88, N(o)-((biotinylamido)hexanoyl)-neuropeptide (2)-Trp.(4)-dynorphin A(1-13), N-Met-Tyr(1)-dynorphin Y, N(O)-biotinyl-neuropeptide Y, neuropeptide Y(1-27), neu A(1-13), Tyr(14)-Leu(15)-Phe(16)-Asn(17)-Gly(18)-Pro ropeptide Y(1-30), neuropeptide Y(13–36), neuropeptide (19)-dynorphin A(1-13), N-Met-Tyr(1)-dynorphin A(1-13) Y(16-36), neuropeptide Y(17-36), neuropeptide Y(18-36), amide, dynorphin A(1-9), dynorphin A(2-12), dynorphin neuropeptideY(2-36), neuropeptideY(20-36), N-acetyl-(Leu A(6-12), 4-aminocyclohexylcarbonyl(2-3)-dynorphin A (28,31))-neuropeptide Y(24-36) amide, Ac-(Leu(28,31)) amide(1-13), biocytin(13)-dynorphin A amide(1-13), Cys neuropeptide Y(24-36), neuropeptide Y(26-36), desasparagi (2)-Cys(5)-MeArg(7)-Leu(8)-dynorphin A amide(1-9), nyl(29),tryptophyl(28.32)-neuropeptide Y(27-36), Tyr(27, N-methyl-Tyr(1)-4-nitro-Phe(4)-N-methyl-Arg(7)-Leu(8) 36)-Thr(32)-neuropeptide Y(27-36), neuropeptide Y(3–36), dynorphin A ethylamide(1-8), MeTyr(1)-MeArg(7)-Leu(8) bis(31-31')(Cys(31)-Trp.(32)-Nva(34))neuropeptide Y(31 dynorphin A ethylamide(1-9), Ala(2)-desCºly(3)-dynorphin 36), Cys-neuropeptide Y(32-36) amide, cyclic (Lys(28)-Glu A, des'?yr(1)-des'? rp(14)-des.Asp(15)-des.Asn(16)-desClu (32))-neuropeptide Y(Ac-25-36), neuropeptide Y C-terminal (17)-dynorphin A, destyr(1)-Gly(2)-dynorphin A, Dmt(1) flanking peptide, N-acetyl-(Leu(17,28,31)Gln(19)Ala(20, Tic(2)-dynorphin A, NO-benzylTyr(1)-cyclo(Asp(5)-Dap 23))-neuropeptide Y(13–36)amide, Ahz(5-17)-neuropeptide (8))-dynorphin A-(1-11)-NH2, cyclo(N,5)(Trp(3)-Trp.(4) Y, Ah X(5–24), Y-Glu(2)-e-Lys(30)-neuropeptide Y, desAA(7 Glu(5))-dynorphin A-(1-11)amide, dynorphin amide(1-10), 24)-(Ala(5)-Aoc(6)-Trp(32))-neuropeptide Y, Leu(31)-Pro Ala(2)-(5-F-Phe)(4)-dynorphin amide(1-13), dynorphin B, (34)-neuropeptide Y, N(e.7)-biotinyl-Lys(7)-neuropeptide Y, dynorphin B(1-13), dynorphin B(1–29), dynorphin B(5-9),
US 2011/0288011 A1 Nov. 24, 2011 30 maleoyl-[3-alanine)) vasopressin, Glu(NHNH2)(4)-Lys(8) relin, des-n-octanoyl-ghrelin, human GHRL protein, vasopressin, Gly-Lys-Arg-8-Lys-vasopressin, N(epsilon) RC-1291, and human exon 3-deleted preproghrelin. tyrosyl-8-lysyl-vasopressin, N–(N-Gly-Gly)-8-Lys [0241] Gonadotropins vasopressin, N-O-Gly-Gly-Gly-8-Lys-9-desGlyNH, [0242] In certain embodiments, the peptide therapeutic is a vasopressin, N-Gly-8-Lys-vasopressin, vasopressin, -(1-(2 gonadotropin. Exemplary gonadotropins include carp gona mercapto)propionic acid)-N(6)-carboxyfluorescein-8-Lys-, dotropin, carp vitellogenic gonadotropin, Gestyl, PMSG Neo-lidocaton, ornipressin, 2-Gly-9-desCly-2-Phe-8-Orn HCG, chorionic gonadotropin, AB1ER-CR-2XY, asialo-hu vasopressin, 2-Gly-9-desCly-4-Val-8-Orn-vasopressin, man chorionic gonadotropin, asialoagalacto-human 9-desCºly-(2-Phe-8-Orn)-vasopressin, Phe(2)-Ile(3)-Orn(8) chorionic gonadotropin, asialogalactochoriongonadotropin, vasopressin, desCºly(NH2)(9)d(CH2)s-Tyr(Me)(2)-Thr(4) human f subunit chorionic gonadotropin, HCG-5(109-145), Orn(8)-vasotocin, fl-mercapto-fi,?}-cyclopentamethyl HCG-B(112-145), HCG-3(123-145), HCG-B(128-145), enepropionic acid-Trp.(2)-Phe(3)-Ile(4)-Arg(8)-oxytocin, HCG-6(Gly(88,90))82-101, hecate-chorionic gonadotropin pitressin tannate, preprovasopressin, pressinamide, pressi fl-subunit conjugate, human chorionic gonadotropin-tetanus noic acid, proAVP hormone, (f-mercapto-(5,5)-cyclopen toxoid, urinary gonadotropin fragment, Xanthomonas malto tamethylenepropionic acid)-Phe(2)-Ile(4)-Ala(9)-vaso philia chorionic gonadotropin, CTP37 peptide, gestagnost, O. pressin, fl-mercapto-fi,?}-cyclopentamethylenepropionic acid subunit glycoprotein hormone, glycosylated HCG, deglyco (1)-O-ethyl-Tyr(2)-Val(4)-Cit(8)-vasopressin, desCly sylated HCG, des(122-145)-HCG-3, hTSHBCTPo protein, vasopressin, e-hydroxy-N1e(8)-vasopressin, homo-N1e(8) human chorionic gonadotropin-cholera toxoid conjugate, vasopressin, and vasopressinase-altered vasopressin. human chorionic gonadotropin-diphtheria toxin fragment A, iodo-chorionic gonadotropin, nymfon-orion, Ovidrel, Other Peptide Hormones PMSG-HCG, Profasi, prostaglandin 600, selenomethionyl [0234] Other peptide hormones include adipokines, choriogonadotropin, recombinant yoked hormone receptor, adrenomedullins, ghrelin, gonadotropins, inhibins, matri prolactin-like protein-K, mouse, prolactin-like protein-N, uretic peptides, parathyroid hormone (PTH) and parathyroid mouse, prolactin-like protein-O, mouse, and salmon gona hormone related peptide (PTHrP), thymosin, relaxins, and dotropin. analogs thereof. Peptide hormones also include BIM28163, [0243] Pituitary gonadotropins include follicle stimulating GKN1 protein, C. elegans Ins-7 protein, mouse Ins15 protein, hormone (FSH), 4-azidobenzoyl-FSH, 4-azidobenzoylgly human intermedin protein, motilin, 13-Leu-motilin, ANQ cyl-FSH, B-subunit FSH, B-subunit(1-15) FSH, human 11125, atilmotin, biotinyl(Cys(23))motilin, Nle(13)-motilin, fl-subunit(33-53) FSH, human f-subunit(33-53)-(81-95) OHM 11526, Leu(13)-pMot(1-14), Prepromotilin, SK896, peptide amide FSH, f subunit(51-65) FSH, human fl-subunit human obestatin, mouse obestatin, rat obestatin, osteocalcin, (81-95) FSH, porcine f subunit precursor FSH, human Ser osteocalcin(37-49), Peptide PHI, Phe(4)-peptide PHI, pep (51)-FSH-B(33-53), human Ser(82,84.87,94)-FSH-B(81 tide PHI-(1-27)-glycine, Gln(24)-PHI peptide, Arabidopsis 95), deglycosylated FSH, DA-3801, human FSH with HCG RALF1 protein, RC-1139, sauvagine, Tremella brasiliensis C-terminal peptide, human chorionic gonadotropin-tetanus tremerogen A-I protein, mouse urotensin II-related peptide, toxoid, Fundulus gonadotropin I fl-subunit, bass gonadotro rat urotensin II-related peptide, urotensin, (Orn&)urotensin pin If-subunit, Katsuwonus gonadotropin I, tuna gonadotro II, preprourotensin II, urotensin I, urotensin II, Pen(5)-Trp pin I, catfish gonadotropin II Cº-subunit, bass gonadotropin II fl-subunit, catfish gonadotropin II fl-subunit, Fundulus gona (7)-Orn(8)-urotensin II(4-11), Cha(6)-urotensin II(4-11), dotropin II fl-subunit, Katsuwonus gonadotropin II, tuna human UTS2D protein, and Xenopus Xen-dorphin prohor gonadotropin II, salmon gonadotropin-pituitary,?}-subunit I, In OIle. luteinizing hormone (LH), big luteinizing hormone, FSH-O, [0235] Adipokines f; subunit LH, hecate-ÉLH, Phor14-6LH, deglycosylated LH, [0236] In certain embodiments, the peptide therapeutic is desialylated LH, diiodo LH, nitroguanidyl LH, plant LH an adipokine or an analog thereof. Adipokines include adi gelonin conjugate protein, menotropin, h!MG-IBSA, ponectin, leptin, and resistin. Adiponectins include human, menogonadyl, urofollitropin, prolactin, alanyl-seryl-(histi mouse, and rat adiponectin. Leptins include leptin(116-130), dy!)6-isoleucyl-glutamyl-glycyl-arginyl-prolactin, mouse leptin(22-56), leptin(57-92), leptin(93-105), LY396623, Dtprp protein, rat Dtprp protein, fluorescein-5-isothiocyan metreleptin, murine leptin analog, pegylated leptin, and ate-prolactin, methionylprolactin, rat PLP-I protein, prepro methionyl human leptin. Resistins include human, mouse, lactin, Oreochromis niloticus PRL177 protein, Oreochromis and rat resistin. niloticus PRL188 protein, human prolactin 16-kDa fragment, [0237] Adrenomedullins Arg(129)-prolactin, Asp(179)-prolactin, A(1-9)-Arg(129) [0238] In certain embodiments, the peptide therapeutic is prolactin, glycosylated prolactin, polymeric prolactin, and an adrenomedullin or an analog thereof. Such peptides prolactin-daunomycin ligand. include adrenomedullin(1-12), adrenomedullin(1-50), [0244] Inhibins adrenomedullin(11-26), adrenomedullin(13-52), adrenom [0245] In certain embodiments, the peptide therapeutic is edullin(15-22), rat adrenomedullin(20-50), adrenomedullin an inhibin. Exemplary inhibins include inhibin, zebrafish (22-52), rat adrenomedullin(24-50), adrenomedullin(27-52), activin B B, C-inhibin-92,3-inhibin(67–94), human inhibin adrenomedullin precursor(45–92), adrenomedullin(16-31), like peptide(1-31), inhibin A, inhibin O. 1-26, inhibin B, adrenotensin, rat intermedin protein, proadrenomedullin, and inhibin-O subunit, inhibin-Osubunit precursor, inhibin-É A prodepin. subunit precursor, inhibin-fi subunit, mouse erythroid differ [0239| Ghrelin entiation and denucleation factor, human INHBB protein, [0240] In certain embodiments, the peptide therapeutic is mouse INHBB protein, rat INHBB protein, human INHBC ghrelin or a ghrelin analog. Exemplary ghrelin analogs protein, mouse INHBC protein, rat INHBC protein, human include Trp3-Arg5-ghrelin(1-5), BIM-281.25, desCºln14-gh INHBE protein, mouse INHBE protein, rat INHBE protein,
US 2011/0288011 A1 Nov. 24, 2011 32
Leu(11)-PTHrP(7–34)amide, Leu(11)-Trp.(12)-PTHrP(7–34) acantoxin IVa, acetyl(leucyl-alanyl-arginyl-leucyl)3-[3-ala amide, PTHrP(1-38), PTHrP(100-114), and human PTHLH nyl-[3-alanine, acetyl-(LSLLLSL), -CONH2, acetyl protein. AAVALLPAVLLALLAP-DEVD-CHO, acetyl-AAVALL [0252) Peptide YY PAVLLALLAP-YVAD-CHO, NC100668, Ac-PEWLR(Aib) [0253] In certain embodiments, the peptide therapeutic is GVTFPGYIT-NH, Ac-WGHGHGHGPGHGHGH-NH2, peptide YY or an analog thereof. Such peptides include pep Ac-WEAQAREALAKEAQARA-NH2, acetylated peptide tide YY(1-36), peptide YY(13–36), peptideYY(22-36), N-o A, acetylcysteine(asparaginyl-alanyl-asparaginyl-proline)s. acetyl-Phe(27)-peptide YY(22-36), peptide YY(3-36), Leu (31)-Pro(34)-peptide YY, and Pro(34)-peptide YY. actinocarcin, actinoxanthine, Ser(3,11)-acyclic sunflower [0254] Thymosin trypsin inhibitor, adipophilin, adolapin, Aek toxin, AF 10847, [0255] In certain embodiments, the peptide therapeutic is AF 12415, AF 12505, AF 18748, AF13948, AF15705, AFT-I thymosin or a thymosin analog. Such peptides include ((n toxin, AFT-II toxin, pig aglycin peptide, AH 111585, AI nitroveratryl)oxy)chlorocarbamate-caged thymosin f{4, (Met 3688, A1409, aibellin, AIP-1-PEO3-ATP. AIP-2-PEO3-ATP, (0)6.Phe(4F)12)deacetyl-thymosin B4, (Met(O)6,Tyr(Me) AIP-3-PEO3-ATP, AIPII peptide, ala inhibitor peptide, Ala 12)deacetyl-thymosin [4, deacetylthymosin f;(10), (0)-actagardine, P+:ISP, Ala-MPSD, alahopcin, albolabrin, deacetylthymosin f{(11), deacetylthymosin f{(12), ALIN protein, ALL1 peptide, Alloferon, allotrap, O.B-poly deacetylthymosin f{(4), deacetylthymosin f{(4)(Xen), ((2-hydroxyethyl)aspartamide-co-(4-hydroxyphenethyl)as deacetylthymosin f{(7), desacetylthymosin O(11), partamide), O.,?}poly((2-hydroxyethyl)aspartamide) desacetylthymosine O(1), parathymosin O., prothymosin O, tyramine, O.f3-poly(3-dimethylaminopropyl-D,L Arg(30)-prothymosin O(1-30), C elegans tetrathymosin?, aspartamide), O.f3-poly((2-hydroxyethyl)-aspartamide), thymalfasin, thymosin O(1), thymosin O(1)(24-28), thymosin Cº-Glu-36 coiled coil, O.1BAla, Otto protein, alveolar mac rophage growth factor, alytesin, amandin, amastatin, AN 3, O.(11), thymosin O(7), thymosin B(1), thymosin B(10), thy AN 7 peptide complex, Anal-R peptide, Anal-S peptide, mosin f{(10)arginine, thymosin f;(11), thymosin f{(12), thy angiohypotensin, Angiopep-2, anguibactin, annexin A1 pep mosin f{(14), rat thymosin f{(15), thymosin f{(4), thymosin tide(1-25), annexin A1 peptide(2–26), antagonist G, antenna f{(4)(11-19), thymosin f;(4) sulfoxide, thymosin f{4)alanine, pedia-CaMKIINtide, anthopleurin B, anthopleurin C, antho thymosin B(8), thymosin B(9), methionine thymosin f{(9), pleurin-A, anthopleurin-Q, anthrax LF protease inhibitor, human thymosin B-NB, rat thymosin [15, thymosin fraction antiamoebin, antiarrhytmic peptide, anticholecystokinin pep 3, thymosin fraction 5, thymosin fraction 7, and timoptin. tide, pineal antigonadotropin, antineoplaston A2, antine [0256] Relaxin oplaston A3, antineoplaston A5, AP-4F peptide, AP1 peptide, [0257] In certain embodiments, the peptide therapeutic is ApC toxin, apstatin, peptide aptamer C1-1, ARAC peptide, relaxin or an analog thereof. Such peptides include N(o) Arg-Gly-Asp-Phe-Gly-Gly-Gly-Gly-AP26, arginine-alanine formyltyrosyl-relaxin, phenylalanyl relaxin, preprorelaxin, copolymer, arginine-serine polymer, Arga-Tyr-Gly-Ser prorelaxin, human relaxin 3, relaxin C-peptide, mouse Args-Tyr, RR-SRC, arginyl-leucyl-cysteinyl-arginyl-isole relaxin-3 protein, rat relaxin-3, human RLN1 protein, mouse ucyl-valyl-valyl-isoleucyl-arginyl-valyl-cysteinyl-arginyl Rln1 protein, human RLN2 protein, human RLN3 protein, aspartyl-aspartyl-aspartyl-aspartyl-glutamyl-glutamic acid and rat RLN3 protein. (3-11)disulfide, arginyl-leucyl-cysteinyl-arginyl-isoleucyl valyl-valyl-isoleucyl-arginyl-valyl-cysteinyl-arginyl-seryl Other Peptides aspartyl-aspartyl-aspartyl-glutamyl-glutamic acid(3-11) [0258] Other peptides that may be used as a peptide thera disulfide, arietin, AS IBBR, ASK 753, TT1272-1284, peautic include disintegrins, endothelins, and secretory pro aspartate carbamoyltransferse regulatory polypeptide, tein inhibitor proteins. Still other peptides include ((GRGDS human-type tridecapeptide, AT 464, AT 744, Staphylococcus GRKKRRQRRRPPQ)2-K-epsilonAhx-C), (asparaginyl aureus aureocin A53, autocamptide-2-related inhibitory pep alanyl-asparaginyl-proline).s. (CICH2CO)4K2Kf8A core tide II, autocamtide 3, autocamtide-2, Ay-AMP peptide, azo peptide, (glycyl-glycyl)GLP-2, (GPGGA) -G, (Lys(40) tobactin, B 43, B2A2 peptide, B2A2-K-NS, Enterococcus (Ahz-DTPA-"In)NH2)exendin-14, (norleucyl-(succinylly faecalis BacA protein, bacteriocin MMFII, bacteriodpsin(34 syl)4)(8)-norleucine, (OHCCO)4K2K5A core peptide, 65) polypeptide, BE 22179, belactosin A, belactosin C, ben (FGE)s-Y-(GEF)2-GD, (POG)(4)POA(POG)(5) peptide, zyloxycarbonyl-(glycyl-prolyl-proline)s-methyl ester, ber (prolyl-hydroxylprolyl-glycine)10, (prolyl-prolyl-glycine) gofungin, fl-adrenergic receptor kinase inhibitory peptide, 10, (S)-alanyl-3-(o-(S)-chloro-3-(S)-hydroxy-2-oxo-3-aze Oryctolagus cuniculus 5-globin readthrough protein, fl-RTX, tidinylmethyl)-(S)-alanine, (T.G)-A-L, 1,3,5-benzene tricar betabellin 12, BGIA protease inhibitor, bibrotoxin, BIM bonyl ((aminoisobutyryl)(4)methyl ester)(3), 1,6-bis(N,N 23454, BIM 23627, BIM 43004-1, BIM 43073D, bitistatin, dimethyl-2',6'-dimethyltyrosyl-1,2,3,4-tetrahydro-3 bivalirudin, Synechocystis blue-colored linker polypeptide isoquinolineamido)hexane, 1-(S)-hydroxy-2-(S,S) L55, Bmk AS polypeptide, BmRIM peptide, Bmp,02 pep valylamidocyclobutane-1-acetic acid, 101.10 peptide, ‘’’I tide, BmRO3 peptide, BMS180742, BMS.184696, K31440 peptide, 27753R.P., 26.12.1 peptide, 3,6-bis(N,N BMS184697, BMS 205820, BMS 214572, BMY 28160, dimethyl-2',6'-dimethyltyrosyl-1,2,3,4-tetrahydro-3 bogorol A, boletusin, bombolitins, BPI peptide, bresein, isoquinolineamidopropyl)-2(1H)-pyrazinone, 3104-V, 3K(I) brevistin, bsp-RGD(15) peptide, butyrivibriocin OR79A, peptide, 4-fluorobenzoyl-TN-14003, 4.2 kDa peptide, 5-fluo Fagopyrum esculentum BWI 3c peptide, BWI 4c peptide, rouracil-poly-C.?3-(2-hydroxyethypasparamide, synthetic C13-24DE peptide, C18G peptide, C28H2 peptide, cAChR 5-helix protein, 61-26, A 10255, A 10947, A 21978C1, ligand, human CADM-140 peptide, calcemic fraction A, A-FF22, A2-binding peptide, AC 413, Ac-(Gly-Pro-Hyp)s human calcitermin peptide, califin, caloxin 1A1, caloxin 1b1, Gly-Pro-Trp-(Gly-Pro-Hyp)a-Gly-Gly-CONH2, Ac-(Gly caloxin 1c3, caloxin 2A1, caloxin 3A1, CALP2 protein, Pro-Hyp)s-Gly-Trp-Hyp-(Gly-Pro-Hyp), -Gly-Gly-CONH2, CaMKII inhibitor AIP carboxypeptidase B activation pep LEHD-CHO, AC133 antigen, AC3-I peptide, Acanthophis tide, carboxypeptidase R intracellular inhibitor, cardiac anti US 2011/0288011 A1 Nov. 24, 2011 secretory peptide, cardiomyopeptidin, carnocin UI49, carzi opeptide B, FLAG peptide, FLPIVGAKL, Fn-23 peptide, nophilin, caseidin, CAT 1.6.1, CL22 cationic peptide, foroxymithine, FR 900490, FRAP-4 peptide, friulimicin A, cavitein LG2, cavitein LG3, CBLB502, CC 1014, CC 1014B, friulimicin B, friulimicin C, friulimicin D, FROPDOTA com CDA antibiotic, CDIP-2 peptide, cecropin P1-LI, cell differ pound, G10KHc peptide, G25 peptide, gaegurin 5, GALA entiation agent II, cementoin, cepacidine A, cephaibol A, peptide, GALAdel3E peptide, gallidermin, Y-Glu-36 coiled cephaibol B, cephaibol C, ceratitin, cerexins, Cerumenex, coil, gangliin, Gap 26 peptide, gastric releasing peptide, gas cervinin, red-Leu O-acetylated derivative of cervinin, CGP trin-releasing peptide precursor, GAT, GD1O-replica peptide, 78850, CGP 85793, ch/Af{30-16 peptide, Charybdotoxin, GE20372 factor A, Streptosporangium cinnabarinum CTX-Clv, choroid plexus peptide, chromatophorotropin, GE82832 peptide, gene-drived bombinin H-like peptide, chrysospermin A, chrysospermin B, chrysospermin C, chry geninthiocin, gilatoxin, gliadin peptide B 3142, glidobactin sospermin D, chymodenin, chymotrypsin inhibitor 2, cic D, glidobactin E, glidobactin F, glidobactin G, globopeptin, adapeptin I, cicadapeptin II, Cicerarin, cinropeptin, citropep glucagon 29, glucagon releasing peptide, glucose utilization tin, CJC 1131, CKS17, CKS 25, clavaspirin, clonostachin, Cn inhibitor, glucose-6-phosphate dehydrogenase inhibition 412, Phaseolus coccineus coccinin protein, COG112 peptide, controlling cofactor, glutamic acid-arginine-alanine polymer, colibiogen, collagen type I trimeric cross-linked peptide, col glutamic acid-lysine-alanine polymer, glutamic acid-lysine lagen-related peptide, colostrinine, colutellin A, comantokin tyrosine terpolymer, glutamyl-isoleucyl-leucyl-isoleucyl L, comantokin-T, connective tissue-activating peptide, copoly phenylalanyl-tryptophyl-seryl-lysyl-aspartyl-isoleucyl-gly (alanine, methionine), copolymer 1, cordialin, cortexin, cyl-tyrosyl-seryl-phenylalanyl-threonine, EFW-NPSF, corticostatin, corticostatin R4, lamprey corticostatin-related glutathione peroxidase-related selenopeptide, Gly14-huma peptide, corticotensin, CP 14, CP 530, CREBtide, crotavirin, nin, glycine-proline-proline polymer, glycothiohexide O. CSI peptide, CS4 peptide, CS5 peptide, cupiennin 1a, human GGGIY’|-speract, GNRH precursor(14-26), goadsporin, CVS995 protein, cyclic chimeric dodecapeptidyl multiple gold keratinate, goniopora toxin, GR 83074, gratisin, GRF antigen peptide, cyclohexylalanine-prolyl-arginyl-up PHI heptacosapeptide amide, griseoviridin, GsMTx-4 toxin, (COCH2S)glycyl-glycyl-glycyl-glycyl-glycyl-aspartyl-ty growth hormone-releasing peptide, growth hormone-releas rosyl-glutamyl-prolyl-isoleucyl-prolyl-glutamyl-glutamyl ing peptide-6, GSP-6 glycosulfopeptide, GTD-C protein, tyrosyl-cyclohexylalanine-glutamic acid, cystargin, C-e/Ahx guamerin, guanylin 16, guanylin 94, GW395058, serine WKK(Cho)—KKK(Cho)—KKKK(Cho)-YKK(Cho)—KK, human angiotensin tetradecapeptide, H2K4bT protein, CYT379, cytomedins, mouse D-JNKI-1, D2A21, D4E1 pep H5WYG peptide, halocidin, halocin S8, hanatoxin, harzianin tide, DAB(389)-GRP fusion protein, DAK 16, DAPD pep HAV, HB-19 peptide, HBY 793, Hel 13-5 peptide, helical tide, Lys(fluorescein)19-DB3 peptide, Geodia cydonium erythrocyte lysing peptide, heliodermin, helodermin(1-28) DD2 protein, debariocidine, DEFB106, deltibant, Dendroas amide, helospectin, helospectin I, helospectin II, helother pis matriuretic peptide, dendrotoxin A, dendrotoxin B, den mine, hemalin, hematide, HepArrest, hepatic stimulator sub drotoxin K, f dendrotoxin, Y dendrotoxin, depelestat, deprim stance, herbicolin B, hexadecalysyl-glycyl-arginyl-glycyl erones, dermcidin, DiaPep 277, diazepam binding inhibitor, aspartyl-serinyl-prolyl-cysteine, hexamethylene diazepam binding inhibitor(33-50), diazepam binding inhibi diisocyanate cross-linked polypeptides, human immunodefi tor(39–75), dicynthaurin, dihydromycoplanecin A, dihydro ciency virus-1 HGP-30 peptide, HI peptide, hirullin P18, pacidamycin D, dimer E.P. peptide, dopuin, Dox-penetratin hirunorm, hirunorm IV. Hisé-tagged elastin-like peptide, conjugate, Dox-SynB1 conjugate, doxorubicin polyaspartic HLP-1 polypeptide, HLP-2 polypeptide, HLP-3 polypeptide, acid conjugate, doxorubicin-conjugated poly(ethylene gly HLP-6 peptide, Hoa-MIH, HRES-1 p.25 protein, human X col)-poly(aspartic acid) block copolymer, drosulfakinin II, polypeptides, Huia versabilis HV-BBI peptide, HXP4 pep DTK1 peptide, DTK2 peptide, DTS-108, dual altered peptide tide, HXR9 peptide, Hym 346, Hym-323 peptide, hypeptin, ligand, duodenin, DUP-1 peptide, dynorphin A-analogue hypertensive factor, hypotensin (snake), iberiotoxin, iberi kappa ligand, E1 E2 peptide, E1K2 peptide, EAA26, EAK otoxin-D19Y-Y36F, IC 101, ideal amphipathic peptide, ima 16-IV peptide, ecallantide, echistatin, EF40 peptide, efrapep cidins, immunopeptide 1, Lactobacillus inducing factor, tin, efrapeptin C, efrapeptin F, efrapeptin G, mouse Egfló INF7 peptide, insulin resistance factor (uremia), insulin-glu protein, elapherine A, elapherine B, elapherine C, elapherine cagon liberin, human insulin-stimulating peptide, fl-Interleu D, elastin polypentapeptide, Ile(1)-elastin polypentapeptide, kin I(163-171), fl-Interleukin II(44–56), Interleukin II (60 human elastin polypeptide 20-24-24, elegantin, embryonal 70), iodo-TyrS-Phe-6-iberiotoxin, IP 20, isariin, JCP 405, carcinoma-derived growth factor, enamidonin, Endo-Porter, JCP 410, Chilobrachys jingzhao jingzhaotoxin XI, Chilo endosulfine, endothelin converting enzyme substrate, entero brachys jingzhao jingzhaotoxin-I, Chilobrachys jingzhao gastrone, eosinophilopoietin, EP-2104R, EP1873, Staphylo jingzhaotoxin-III, Chilobrachys jingzhao jingzhaotoxin-V, coccus epidermidis Epi/A protein, epiactins, epicidin 280, joining segment peptide, K 582 A, K-MLC 11-23, K1E2 epidermin, epilancin 15X, epilancin K7, epiphisan, epith peptide, K1K2 peptide, K4-M2GlyF protein, K5 peptide, alamin, eptifibatide, eristostatin, erythrotropin, esein, estro KAI 9803, KALA amphipathic peptide, kassinakinin S, kas medins, ETR-p1-fl antisense peptide, exchanger inhibitory sinatuerin-1, kawaguchipeptin B, KB-752 peptide, kedarci peptide, Achatina excitatory peptide 2, Achatina excitatory din peptide, KIA7 protein, KID1-1 peptide, KIE 1-1 peptide, peptide 3, Fusinus excitatory peptide 4, exorphins, F2(Pmp) KILT protein, kinetensin, kistamicin A, kistamicin B, kistrin, 2-TAMzeta? peptide, human F2L peptide, factor XIII activa KL4 surfactant, KM 8, KT 199, plant Kunitz-type protease tion peptide, FALL 39, FE 99.9024, FECO peptide, ferrocin inhibitor, L 363714, L 689502, L 694746, L 733560, L-4F A, ferrocin B, ferrocin C, ferrocin D, FGLL peptide, fibrin peptide, L-glutamic acid-L-tyrosine copolymer, L-JNKI-1, self-assembly inhibitor, fibrinogen binding inhibitor peptide, L-T6DP-1 peptide, lac C. peptide, lactivicin, lactocin S, fibrinopeptide A, 5-tyrosine fibrinopeptide A, desaminoty LAGA, LAH(4) protein, lambda Spi-1, lambda Spi-2, rosylfibrinopeptide A, desAlal-fibrinopeptide A, phospho lanthiopeptin, lantibiotic Pep5, LBMP1620 protein, Ser3-fibrinopeptide A, Fibrinopeptide B, des.Arg14-fibrin leiuropeptide II, leiurutoxin III, Leu-Ser-Lys-Leu peptide, US 2011/0288011 A1 Nov. 24, 2011 34
Leu3 blomhotin, leucinostatin A, leucinostatin B, leucinosta metin A, phenylalanyl-glycyl-glycyl-phenylalanyl-threonyl tin C, leucinostatin D, leucinostatin H. leucinostatin K, leu glycyl-O-aminoisobutyryl-arginyl-lysyl-seryl-O-ami kocyte mobilizing factor, leupeptin, leuteonosticon, Limnop noisobutyryl-arginyl-lysyl-leucyl-alanyl-asparagyl erna fortunei LF22 peptide, LH receptor binding inhibitor, qlutaminamide, PHM polymer, phosphofructokinase lichenysin G, linaclotide, lipid mobilizing substance, lipid regulatory factors, phylloxin, PK1M peptide, PKL-1c pep associating peptides, lipopeptin A, lipoprotein lipase activa tide, plasmatocyte-spreading peptide 1, plauracin, plectasin, tors, LIQ 4, live yeast cell derivative, LL AF2836, LL PNV2 peptide, PNV4 peptide, polistes mastoparan, poly AO341?;1, longibrachin LGA I, longibrachin LGB II, longi (RGD), poly(RGDT), Poly 18 antigen, poly(1-benzylhisti brachin LGB III, LR9 peptide, lumbricin I, LY 295337, LY dine), poly(2-sulfoethyl aspartamide)silica, poly(3-hydrox 315902, lymphoguanylin, lys-guanylin, lysoartrosi, lysome ypropyl)aspartamide, poly(3-hydroxypropyl-propyl) tra, M-81, M1557 peptide, M2 delta, maduropeptin A1, asparamide, poly(Ala)-poly(Lys), poly(alanyl-glutamyl maduropeptin A2, maduropeptin B, maduropeptin C, magai tyrosyl-glycine), poly(alanyl-tyrosyl-glutamyl-glycine), nin-PG|La hybrid peptide, magaratensin, magnificalysin I, poly(alanyl-valyl-glycyl-valyl-prolyl), poly(alanylglycine), magnificalysin II, magnificalysin III, malantide, mamba poly(arginyl-histidine), poly(aspartylhydrazide), poly(di intestinal toxin 1, mammastatin, MAP 1987, Mas-DP II, ethylaminoethylglutamine), poly(dimethylaminoethyl Mas?/ protein, mast 21 peptide, mast cell degranulating pep glutamine), poly(ethylene oxide-co-fº-benzyl-L-aspartate), tide, mastoparan, mastoparan B, mastoparan M. mastoparan poly(Y-glutamylcysteinyl)glycine, poly(Y-methylglutamate) X, 11-dansyl-mastoparan, mating factor, Ala8-mating factor, grafted polyallylamine, poly(Glu56-Lys35-Phe9), poly maximin H1, MB21 peptide, MCP-4-EDTA-SH, MCR 14 (glutamyl-alanyl-tyrosyl-glycine), poly(glutamyl-tyrosyl peptide, MCR4 peptide, MDL 27,367, melanophore-dispers alanyl-glycine), poly(glycyl-prolyl-serine), poly(glycyl ing hormone, meliacin, Mer N5075A, mersacidin, methinin, valyl-glycyl-valyl-prolyl), poly(glycyl-valyl methylenomycin A, michicarcin, microbial alkaline protein hydroxyproline), poly(histidyl-aspartyl-seryl-glycine), poly ase inhibitor, microbisporicin, microcin H47, microcin (hydroxybutylglutamine-co-proline), poly SF608, micrococcin, mitochondrial addressing peptide, (hydroxyethylaspartamide-co-aspartic acid), poly mitomalcin, mitoparan, miyakamide A1, miyakamide A2, (hydroxyethylaspartamide-co miyakamide B1, miyakamide B2, MJ347-81F4A, MJ347 dimethylaminopropylaspartamide), poly(hydroxyprolyl 81 F4 B, MLCK peptide, MMK-1 peptide, monocyto-an prolyl-glycine)(10), poly(L-aspartyl-L-phenylalanine), poly giotropin, monoketo-organomycin, motilin-associated pep (L-tyrosyl-L-glutamyl-L-alanyl-glycyl)glycine ethyl ester, tide, MPGo peptide, MS-681a, MS05 peptide, MS09 poly(leucyl-glycyl-glycyl-valyl-glycyl), poly(leucyl-leucyl peptide, MSI 511, MSI 594, MSI-99 peptide, MT-7 mamba phenylalanyl-proline), poly(lysyl-(glutamyl(i)-alanine(m))), toxin, multide, Leu7-multiple antigen peptide, leucyl(8)-ly poly(lysyl-(leucyl-poly-alanine)), poly(lysyl-seryl-glutamic syl(4)-lysyl(2)-lysyl-[3-alanine multiple antigen peptide, acid), poly(lysyl-tyrosyl-tyrosyl-lysine), poly(N(B)-4-(phe muscarinic toxin 3, mutacin 1140, bacteria mutacin II prepep nylazo)benzoyl-Cº.,5-diaminopropionic acid), poly(N(delta), tide, MW167, mycoplanecin A, Myocardial Depressant Fac N(delta),N(delta)-trimethylomithine), poly(N-(3-aminopro tor, myristoylated autocamtide-2-related inhibitory peptide, pyl)glycine), poly(N-hydroxypropylglutamine-leucine), MGAIPAA, myroridin, myroridin K, myxovalargins, poly(O,O?-dicarbobenzoyx-L-6-3,4-dihydroxyphenyl-O N-acetyl-gastrin releasing peptide ethyl ester, N-methyldep alanine), poly(phenylalanyl-alanyl-glutamyl-glycine), poly sipeptide, N-tert-butyloxycarbonyl-valyl-alanyl-leucyl-ami (phenylalanyl-glutamyl-alanyl-glycine), poly(prolyl-nor noisobutyryl-valyl-alanyl-leucyl(valyl-alanyl-leucyl-ami leucyl-glycine), poly(prolylprolylglycine)15, poly(S noisobutyryl)(2) methyl ester, Nano-1 peptide, mouse NBD carboxymethylcysteine), poly(sarcoyl-glycyl-phenylalanyl peptide, neosulfakinin II, NeoTect, neotelomycin, neovirido leucyl-glycyl-aminoethylaminocarbonylmethyl(N-methyl) griseins, NK911, nocathiacin I, novospirin G-10, NSC amino-co-o,omega-bis(oxiranylmethyl)poly(ethylene 710295, NVP PDF 713, NVP-PDF386, octameric glycol)), poly(tyrosyl-alanyl-glutamyl-glycine), poly(ty MYFGGGGG ligand, OS-3256-B, osteoclast stimulating rosyl-glutamic acid), poly(tyrosyl-glutamyl-alanyl-glycyl), factor, ovcNP-39 peptide, ovocystatin, oxaldie 1, oxaldie 2, p poly(tyrosyl-isoleucyl-glycyl-seryl-arginine), poly(unde 230, P 498, P 500, P-LF II D, Ppolypeptide, P596 peptide, canoylvalinate), poly(valyl-glycyl-glycyl-valyl-glycine), P62 peptide, PA22-2, Paim I, Pam(3)CSK(4) peptide, Gila poly-(His-Glu)-poly Ala-poly Lys, poly-fl-benzyl-aspartate, monster venom pancreatic secretory factor, pancreatic spas poly-delta-L-ornithine, poly-DL-succinimide, poly-L-ly molytic polypeptide, pandinin 1, pandinin 2, pantripin, Asp sylphenylalanine, poly-L-lysyltyrosine, poly-N(5)-(2-hy (12),Arg(13)-paotin-lysyl-GRP-27, PC 1038, PD-145065, droxyethyl)glutamine, poly-N(5)-(3-hydroxypropyl)-1 PD 142893, mouse Pdcd.11g1 protein, mouse Pdcd.11g2 pro glutamine, poly-N(5)-(3-hydroxypropylglutamine)-prazosin tein, PEC-60 polypeptide, pedibin, PEG-DAPD peptide, carbamate, poly-O-acetylserine, poly-O-carbobenzoxyser PEG-PAsp(Dox), penaeidin 1, penaeidin 2, penaeidin 3, ine, poly-S-benzylcysteine, poly-S-carbobenzoxycysteine, Pep-1 peptide, pep-1CF peptide, Pep-3 peptide, Pep-9 oli polyalanine, polyarginine, polyasparagine, polyaspartate, godeoxyribonucleotide-peptide conjugate, pepHs 1-Ac pep polyaspartoyl-L-arginine, polyaspartylglutamate, polychlo tide, PEPHC 1 peptide, pepsanurin, DA RT1(A) peptide 1, rosubtilin, polycysteine, polyetherurethaneurea-polypeptide peptide 106, peptide 18A, rat peptide 19, Fagopyrum escu block copolymer, polyethylenimine-N-succinimidyl-3-(2 lentum peptide 4 kDa, peptide 5F, peptide 74, peptide 78, pyridyldithio)propionyl-MC11, Polygeline, polyglutamine, peptide 9M, peptide I, peptide KPR, peptide leucine arginine, polyglycine, polyisoleucine, polyleucine, polymethionine, peptide MB-35, peptide methionine-tyrosine, peptide NK-2, polymethionine sulfoxide, polymorph migration stimulator, peptide P3, peptide Q, peptide S 42, peptide S-8300, peptide polyoma peptide antigen MT162-176, polyornithine, SC-R8A2, peptide SM-BC3, peptide stabilizing factor, pep polypeptide C, polypeptide oleate condensate, polypeptide tide U6, peptidimer-c, peptilose, peptitergent PD1, Peri Coil pineal extract, polypeptide PPA-80, polyphenylalanine, 1, Perinerin, periodontal ligament chemotactic factor, per polyproline, polysarcosine, polyserine, polytryptophan, US 2011/0288011 A1 Nov. 24, 2011 polytyrosine, polyvaline, prelacticin 481, probursin, procam duck pancreas trypsin inhibitor, tuberoinfundibular peptide ine, progressin, proline-rich polypeptide, prolyl-lysyl-leucyl 38, turmerin, tylopeptin A, tylopeptin B, tyrosinase inhibitor, leucyl-lysyl-threonyl-phenylalanyl-leucyl-seryl-lysyl-tryp U 995, UK 156406, vaccinia growth factor, valosin, valyl tophyl-isoleucyl-glycine, prolyl-seryl-glycyl-phenylalanyl prolyl-glycyl-valyl-glycine polypeptide, VAP-map peptide, tyrosyl-leucyl-lysyl-leucyl-aspartyl-prolyl-arginyl vascular factor, vasoactive intestinal constrictor, vasonin, asparaginyl-phenylalanyl-asparagine, promoinducin, ventriculine, vermilat, villikinin, vishnu, Vitaprost, Gram promothiocin, prostalin, rat prostate specific binding protein mostola spatulata VSTX1 protein, VT 5 peptide, Vueffe, polypeptide C3, prostatilen, protein B23 antigenic peptide, Walsh peptide, Wei.Jia, WF 3161, Wheel-FKFE, WR-PAK18 protein kinase inhibitor peptide, prºb peptide, pseudokonin peptide, Xen2174, xenin 25, xenopsin, XK-19-2, XR 586, xylocandin, Y(21) peptide, YALA peptide, YM 170320, YM KL III, pseudokonin KL VI, PTP-7S peptide, pulmolin, 266183, YM 266184, YTA2 peptide, YTA4 peptide, Z2685, pumilacidin, p"WEC peptide, PW2 peptide, PYL(a), pylorici Z28 peptide, zinc finger peptide Xfin-31, ZP10A peptide, din A, pyloricidin B. pyloricidin C, mouse Qdm protein, QK Zwit-1F peptide, human chorionic thyrotropin protein, VEGF mimetic peptide, QRFP peptide, R18 peptide, R6A-1 decidua inhibitory factor, placental antigen X-P2, placental peptide, rab3AL peptide, ranatensin R, ranatuerin, Raytide, lactogen, placental lactogen A-2, rat placental lactogen I, RC 101, RCS-RF, resact, retinalamin, retro-bombolitin I, placental lactogen I-variant, placental lactogen II, human pla retro-bombolitin III, retro-inverso-TATp53C' peptide, retro cental lactogen-3, placental ribonuclease inhibitor, placental nociceptin methylester, Rev peptide, Rev.4 peptide, RH4 pep uterotrophic factor, and prolactin-releasing factor (placenta). tide, RHM1 peptide, RHM2 peptide, rhodostomin, RI-26 peptide, RK 699A, tetrahymena RNA inhibitor, RP 66453, [0259] Disintegrins RS 83277, S862033, S863390, SHt31, S4(13)-PV peptide, [0260] In certain embodiments, the peptide therapeutic is a S4K2K5A core peptide, S597 peptide, Sadat-Habdan mes disintegrin or an analog thereof. Such peptides include accu enchymal stimulating peptide, Samarosporin, Sarafotoxin-c, tin, acostatin, rat Adam!) protein, Agkistrodon halys brevicau saramycetin, saturnisporin SA II, saturnisporin SA IV, SC dus stejnegeradinbitor protein, alternagin-C, bitisgabonin-1, 40476, SC 42619, Sch 40832, Sch 419558, Sch 419559, SCH bitisgabonin-2, Bothrops jararaca bothrostatin, contortrosta 466456, SCH 466457, schistosomin, scotophobin, Dictyos tin, Echis carinatus EC3 protein, Echis carinatus sochureki telium discoideum SDF-2 protein, semparatide acetate, EC6 protein, Eristocophis macmahoni EMF 10 protein, fla SepOvotropin, sertolin, serum sodium transport inhibitor, Vorodin, flavostatin, jarastatin, jerdonin, Drosophila kuzban mouse Sftpc protein, rat Sftpc protein, Shaker B inactivating ian protein, C. elegans MIG-17 protein, ocellatusin, pisci peptide, short ragweed fraction A-D-glutamic acid-D-lysine vostatin, saxatilin, Trimeresurus stejnegeri stejnin protein, polymer, siamycin I, siamycin II, sideromycin No. 216, sifu Trimeresurus flavoviridis trimestatin protein, Gloydius virtide, silaffin 1A, silaffin 1B, sillucin, sinapultide, SM3 ussuriensis ussuristatin 1 protein, and Gloydius ussuriensis MUC1 peptide, SN50 peptide, SNK 863, SNP-1 protein, ussuristatin 2 protein. SNX202, SNX260, SNX325, Conus striatus SO-3 conopep [0261) Endothelins tide, somatostatin-like peptides, sorbin, spasmolytic [0262] In certain embodiments, the peptide therapeutic is polypeptide, sperm acrosomal peptide P23, spinigerin, rat an endothelin or an endothelin analog. Such peptides include Sponf protein, Streptomyces spore pigment, SQ 20858, SR Phe(22)-big endothelin-1 (19-37), Val(22)-big endothelin-1 41476, stearated-Ht31 peptide, Streptococcus streptococcin (16-38), big-endothelin(1-22), big-endothelin(16-32), BQ A M49 protein, Styela clava styelin A peptide, Styela clava 3020, Cys(11)-Cys(15)-endothelin-1 (11-21), endothelin(16 styelin B peptide, sublancin 168, substance P-like peptides, 21), endothelin(16-21) amide, Endothelin-1, (Sec(3)-Sec suzukacillin, sweet arrow peptide, synthetic peptide O27-50, (11)-N1e(7))-endothelin-1, zebrafish edn] protein, endothe syntide-2, systemin, T-activin, T1BP2 peptide, T1BT pep lin-1 (1-21), (Cys,Acm(1,15),Aib(3,11),Leu(7))-endothelin tide, T22 peptide, human TAFII-17 protein, TAT-R7-LV1 1(1-21), endothelin-1 (1-31), (Aib(1,3,11,15),Nle(7)) peptide, TAT-TIJIP, tatumine, TC5b protein, TcapQ647 pep endothelin-1, Aba? 1,15)-endothelin-1, Ala(10)-endothelin-1, tide, *"Tc-AGGCG, *"Tc-AGGCL, °"Tc-ASSCG, Cys(Acm)(1,15)-Ala(3)-Leu(7)-Aib(11)-endothelin-1, ”Tc-labeled o-M2 peptide, tediuglutide, tendamistate, tes formyltrp(21)-endothelin-1, lysyl(-2)-arginyl(-1)-endothe sulin, testilin, Tet-p peptide, Eisenia tetradecapeptide, tex lin-1, Pen(1,11)-N1e(7)-Ala(18)-endothelin-1, Pen(1,11) enomycin A, mouse TFF1 protein, mouse TFF2 protein, Nle(7)-Asn(18)-endothelin-1, Phe(16)-endothelin-1, Thr human TFF3 protein, theonellapeptolide le, theromin, (18)-Cha(19)-endothelin-1, Thr(18)-Leu(19)-endothelin-1, THG113.31 peptide, thioactin, thiocillin, thiopeptin, thiopep ET-1(Cys(Acm)(1,15)-Ala(3)-Leu(7)-dAsp(8)-Aib(11)), tin A, thiopeptin B, Thomsen-Friedeneich antigen-specific proendothel in 1, Endothelin-3, Ala(1,15)-endothelin 1, Ala peptide P-30, Thr-Met-Lys-Ile-Ile-Pro-Phe-Asn-Arg-Thr (1,3,11,15)-endothelin 1, Ala(3,11)-endothelin 1, Dpr(1) Leu-Ile-Gly-Gly, thrombopoietin mimetic peptide, thy Asp(15)-endothelin 1, N1e(7)-endothelin, Ala(9)-endothelin mocyte growth peptide, thymodepressin, thymohemin, thy 1, Pro(12)-endothelin-1, Thr(18)-y-methyl-Leu(19) mone A, thymone B, thymone C, tick anticoagulant peptide, endothelin-1, endothelin-1,2-6-keto-PGF1-C, endothelin'7 TIFI peptide, tigerinin 1, tissue polypeptide antigen, tissue 21(Leu7,Aib11,Cys(Acm)15), Endothelin-2, IRL 1620, IRL polypeptide specific antigen, TL 119, TM11 peptide, 1720, N(e)-9-azidobenzoyliodoendothelin-1, preproendot TN14003, TNYL-RAW peptide, toxin FS2, TP10-PNA con helin 2, preproendothelin-3, proendothelin 2, proendothelin jugate, transfecting peptide I, transporter peptide HGH6, 3, proendothelin-1(22-39), and proendothelin-1 (31–38). trapoxin A, trapoxin B, trefoil factor, trichocellin, trichogin A [0263] Secretory Proteinase Inhibitory Protein IV, tricholongin BI, tricholongin BII, trichopolyn, trichorzin [0264] In certain embodiments, the peptide therapeutic is a PA IV, trichorzin PA V, trichorzin PA VI, trichotoxin, tri secretory proteinase inhibitory protein or an analog thereof. chotoxin A 50E, trichotoxin A40, trichovirin IIB, trichovirin Such peptides include C.1-antichymotrypsin, C.1-antitrypsin, I-4A, tridecaptins, triflavin, trifolitoxin, trigramin, trikonin S. cerevisiae Alpiz protein, C.1-antitrypsin Christchurch, gin KB II, trimucrin, triwaglerin, trofopar, Trp-cage peptide, O.1-antitrypsin Pittsburgh, C.1-antitrypsin Portland, O.1-antit US 2011/0288011 A1 Nov. 24, 2011 36 rypsin QOtrastevere, O.1-antitrypsin Siiyama, O.1-antitrypsin [0269] Primary amines are the principal targets for NHS W(Bethesda), S C.1-antitrypsin, C.1-antitrypsin-leukocyte esters. Accessible O-amine groups present on the N-termini elastase complex, C105Y peptide, human serpin A1 (A1 of proteins and the e-amine of lysine react with NHS esters. C26), human SERPINA1 protein, human SERPINA2 pro An amide bond is formed when the NHS ester conjugation tein, trypsin-2-0.1-antitrypsin, human VIRIP peptide, Elafin, reaction reacts with primary amines releasing N-hydroxysuc Human PI3 protein, zebrafish Hail protein, human ITIH4 cinimide. These succinimide containing reactive groups are protein, human ITIH5 protein, mouse protease inhibitor 16, herein referred to as succinimidyl groups. In certain embodi human secretory leukocyte peptidase inhibitor (SLPI) pro ments of the invention, the functional group on the protein tein, mouse SLPI protein, rat SLPI protein, human SPINK5 will be a thiol group and the chemically reactive group will be protein, human SPINLW1 protein, and human SPINT1 pro a maleimido-containing group such as gamma-maleimide tein. butrylamide (GMBA or MPA). Such maleimide containing groups are referred to herein as maleido groups. Modified Forms of Peptide Therapeutics [0270] The maleimido group is most selective for sulfhy dryl groups on peptides when the pH of the reaction mixture [0265] Any of the peptide therapeutics described herein is 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups (e.g., GLP-1 agonists) may be modified (e.g., as described with sulfhydryls (e.g., thiol groups on proteins such as serum herein or as known in the art). As described in U.S. Pat. No. albumin or IgG) is 1000-fold faster than with amines. Thus, a 6,924,264, the polypeptide can be bound to a polymer to stable thioether linkage between the maleimido group and the increase its molecular weight. Exemplary polymers include sulfhydryl is formed, which cannot be cleaved under physi polyethylene glycol polymers, polyamino acids, albumin, ological conditions. gelatin, succinyl-gelatin, (hydroxypropyl)-methacrylamide, fatty acids, polysaccharides, lipid amino acids, and dextran. Peptide Vectors [0266] In one case, the polypeptide is modified by addition [0271] The compounds of the invention can feature any of of albumin (e.g., human albumin), or an analog or fragment polypeptides described herein, for example, any of the pep thereof, or the Fc portion of an immunoglobulin. Such an tides described in Table 1 (e.g., Angiopep-1 or Angiopep-2), approach is described, for example, in U.S. Pat. No. 7,271, or a fragment or analog thereofas a peptide vector. In certain 149. embodiments, the peptide vector may have at least 35%, 40%, [0267] In one example, the polypeptide is modified by addi 50%. 60%, 70%, 80%, 90%. 95%, 99%, or even 100% iden tion of a lipophilic substituent, as described in PCT Publica tity to a polypeptide described herein. The peptide vector may tion WO 98/08871. The lipophilic substituent may include a have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, partially or completely hydrogenated cyclopentanophenath or 15) substitutions relative to one of the sequences described rene skeleton, a straight-chain or branched alkyl group; the herein. Other modifications are described in greater detail acyl group of a straight-chain or branched fatty acid (e.g., a below. group including CH3(CH2)CO- or HOOC(CH2),CO—, [0272] The invention also features fragments of these where n or m is 4 to 38); an acyl group of a straight-chain or polypeptides (e.g., a functional fragment). In certain embodi branched alkane o.o-dicarboxylic acid; CH3(CH2)((CH3), ments, the fragments are capable of efficiently being trans COOH)CHNH-CO(CH2)2CO-, where p and q are inte ported to or accumulating in a particular cell type (e.g., liver, gers and p-q is 8 to 33; CH3(CH2)CO-NHCH(COOH) eye, lung, kidney, or spleen) or are efficiently transported (CH2)2CO-, where r is 10 to 24, CH3(CH2)CO-NHCH across the BBB. Truncations of the polypeptide may be 1, 2, ((CH2)2COOH)CO-, where s is 8 to 24; COOH(CH2) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from either ,CO—, wheret is 8 to 24; -NHCH(COOH)(CH2),NH–CO the N-terminus of the polypeptide, the C-terminus of the (CH2)CHs, where u is 8 to 18; –NHCH(COOH)(CH2) polypeptide, or a combination thereof. Other fragments a NH-COCH((CH2)2COOH)NH-CO(CH2), CHA, where include sequences where internal portions of the polypeptide w is 10 to 16; NHCH(COOH)(CH2), NH CO(CH2)2CH are deleted. (COOH)NH-CO(CH2), CHA, where x is 10 to 16; or [0273] Additional peptide vectors may be identified by —NHCH(COOH)(CH2)4NH CO(CH2)2CH(COOH) using one of the assays or methods described herein. For NHCO(CH2)CH, where y is 1 to 22. example, a candidate polypeptide may be produced by con [0268] In other embodiments, the peptide therapeuticis ventional peptide synthesis, conjugated with paclitaxel and modified by addition of a chemically reactive group such as a administered to a laboratory animal. A biologically-active maleimide group, as described in U.S. Pat. No. 6,593,295. polypeptide conjugate may be identified, for example, based These groups can react with available reactive functionalities on its ability to increase survival of an animal injected with on blood components to form covalent bonds and can extend tumor cells and treated with the conjugate as compared to a ing the effective therapeutic in vivo half-life of the modified control which has not been treated with a conjugate (e.g., insulinotropic peptides. To form covalent bonds with the treated with the unconjugated agent). For example, a biologi functional group on a protein, one can use as a chemically cally active polypeptide may be identified based on its loca reactive group a wide variety of active carboxyl groups (e.g., tion in the parenchyma in an in situ cerebral perfusion assay. esters) where the hydroxyl moiety is physiologically accept [0274] Assays to determine accumulation in other tissues able at the levels required to modify the peptide. Particular may be performed as well. Labelled conjugates of a polypep agents include N-hydroxysuccinimide (NHS), N-hydroxy tide can be administered to an animal, and accumulation in sulfosuccinimide (sulfo-NHS), maleimide-benzoyl-succin different organs can be measured. For example, a polypeptide imide (MBS), gamma-maleimido-butyryloxy succinimide conjugated to a detectable label (e.g., a near-IR fluorescence ester (GMBS), maleimido propionic acid (MPA) maleimido spectroscopy label such as Cy5.5) allows live in vivo visual hexanoic acid (MHA), and maleimido undecanoic acid ization. Such a polypeptide can be administered to an animal, (MUA). and the presence of the polypeptide in an organ can be US 2011/0288011 A1 Nov. 24, 2011 37 detected, thus allowing determination of the rate and amount ment of a marker (e.g., fluorescent or radioactive), covalent of accumulation of the polypeptide in the desired organ. In attachment of a lipid or lipid derivative, covalent attachment other embodiments, the polypeptide can be labelled with a of phosphatidylinositol, cross-linking, cyclization, disulfide radioactive isotope (e.g., **I). The polypeptide is then bond formation, demethylation, formation of covalent administered to an animal. After a period of time, the animal crosslinks, formation of cystine, formation of pyroglutamate, is sacrificed and the organs are extracted. The amount of formylation, gamma-carboxylation, glycosylation, GPI radioisotope in each organ can then be measured using any anchor formation, hydroxylation, iodination, methylation, means known in the art. By comparing the amount of a myristoylation, oxidation, proteolytic processing, phospho labeled candidate polypeptide in a particular organ relative to rylation, prenylation, racemization, selenoylation, sulfation, the amount of a labeled control polypeptide, the ability of the transfer-RNA mediated addition of amino acids to proteins candidate polypeptide to access and accumulate in a particu such as arginylation and ubiquitination. lar tissue can be ascertained. Appropriate negative controls [0278] A modified polypeptide can also include an amino include any peptide or polypeptide known not to be efficiently acid insertion, deletion, or substitution, either conservative or transported into a particular cell type (e.g., a peptide related to non-conservative (e.g., D-amino acids, desamino acids) in the Angiopep that does not cross the BBB, or any other peptide). polypeptide sequence (e.g., where such changes do not sub [0275] Additional sequences are described in U.S. Pat. stantially alter the biological activity of the polypeptide). In Nos. 5,807,980 (e.g., SEQ ID NO:102 herein), 5,780,265 particular, the addition of one or more cysteine residues to the (e.g., SEQ ID NO:103), 5,118,668 (e.g., SEQ ID NO:105). amino or carboxy terminus of any of the polypeptides of the An exemplary nucleotide sequence encoding an aprotinin invention can facilitate conjugation of these polypeptides by, analog atgagaccag atttctgcct cpagccgc.cg tacactgggc cctg e.g., disulfide bonding. For example, Angiopep-1 (SEQ ID caaag.c togtatcatC C9ttacttct acaatgcaaaggcaggcctgtgtcagacct NO:67), Angiopep-2 (SEQID NO:97), or Angiopep-7 (SEQ togtatacgg C9gctgcaga gctaag.cgta acaacttcaa atcc.gc.ggaa ID NO:112) can be modified to include a single cysteine gactgcatgc gtacttgcgg tygtgcttag, SEQ ID NO:6; Genbank residue at the amino-terminus (SEQ ID NOS: 71, 113, and accession No. X04666). Other examples of aprotinin analogs 115, respectively) or a single cysteine residue at the carboxy may be found by performing a protein BLAST (Genbank. terminus (SEQ ID NOS: 72, 114, and 116, respectively). www.ncbi.nlm.nih.gov/BLAST/) using the synthetic aproti Amino acid substitutions can be conservative (i.e., wherein a nin sequence (or portion thereof) disclosed in International residue is replaced by another of the same general type or Application No. PCT/CA2004/000011. Exemplary aprotinin group) or non-conservative (i.e., wherein a residue is replaced analogs are also found under accession Nos. CAA37967 (GI: by an amino acid of another type). In addition, a non-naturally 58005) and 1405218C (GI:3604747). occurring amino acid can be substituted for a naturally occur ring amino acid (i.e., non-naturally occurring conservative Modified Polypeptides amino acid substitution or a non-naturally occurring non [0276] The peptide vectors and peptide therapeutics used in conservative amino acid substitution). the invention may have a modified amino acid sequence. In [0279] Polypeptides made synthetically can include substi certain embodiments, the modification does not destroy sig tutions of amino acids not naturally encoded by DNA (e.g., nificantly a desired biological activity (e.g., ability to cross non-naturally occurring or unnatural amino acid). Examples the BBB). The modification may reduce (e.g., by at least 5%, of non-naturally occurring amino acids include D-amino 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or acids, an amino acid having an acetylaminomethyl group 95%), may have no effect, or may increase (e.g., by at least attached to a sulfur atom of a cysteine, a pegylated amino 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the acid, the omega amino acids of the formula NH2(CH2) biological activity of the original polypeptide. The modified ,COOH wherein n is 2-6, neutral nonpolar amino acids, such peptide may have or may optimize a characteristic of a as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl iso polypeptide, such as in vivo stability, bioavailability, toxicity, leucine, and norleucine. Phenylglycine may substitute for immunological activity, immunological identity, and conju Trp, Tyr, or Phe, citrulline and methionine sulfoxide are neu gation properties. tral nonpolar, cysteic acid is acidic, and ornithine is basic. [0277] Modifications include those by natural processes, Proline may be substituted with hydroxyproline and retain the such as posttranslational processing, or by chemical modifi conformation conferring properties. cation techniques known in the art. Modifications may occur [0280] Analogs may be generated by substitutional anywhere in a polypeptide including the polypeptide back mutagenesis and retain the biological activity of the original bone, the amino acid side chains and the amino- or carboxy polypeptide. Examples of substitutions identified as “conser terminus. The same type of modification may be present in the vative substitutions” are shown in Table 2. If such substitu same or varying degrees at several sites in a given polypep tions result in a change not desired, then other type of substi tide, and a polypeptide may contain more than one type of tutions, denominated “exemplary substitutions” in Table 3, or modification. Polypeptides may be branched as a result of as further described herein in reference to amino acid classes, ubiquitination, and they may be cyclic, with or without are introduced and the products screened. branching. Cyclic, branched, and branched cyclic polypep [0281| Substantial modifications in function or immuno tides may result from posttranslational natural processes or logical identity are accomplished by selecting substitutions may be made synthetically. Other modifications include that differ significantly in their effect on maintaining (a) the pegylation, acetylation, acylation, addition of acetomidom structure of the polypeptide backbone in the area of the sub ethyl (Acm) group, ADP-ribosylation, alkylation, amidation, stitution, for example, as a sheet or helical conformation. (b) biotinylation, carbamoylation, carboxyethylation, esterifica the charge or hydrophobicity of the molecule at the target site, tion, covalent attachment to flavin, covalent attachment to a or (c) the bulk of the side chain. Naturally occurring residues heme moiety, covalent attachment of a nucleotide or nucle are divided into groups based on common side chain proper otide derivative, covalent attachment of drug, covalent attach ties: US 2011/0288011 A1 Nov. 24, 2011 38
[0282) (1) hydrophobic: norleucine, methionine (Met), —CH=CH- (cis and trans), —CH2SO–, -CH(OH) Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine CH2—, -COCH2— etc., by methods well known in the art (Ile), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), (Spatola, Peptide Backbone Modifications, Vega Data, 1:267, Phenylalanine (Phe), 1983; Spatola et al., Life Sci. 38:1243-1249, 1986; Hudson et [0283) (2) neutral hydrophilic: Cysteine (Cys), Serine al., Int. J. Pept. Res. 14:177-185, 1979; and Weinstein, 1983, (Ser), Threonine (Thr) Chemistry and Biochemistry, of Amino Acids, Peptides and [0284] (3) acidic/negatively charged: Aspartic acid Proteins, Weinstein eds, Marcel Dekker, New York). Such (Asp), Glutamic acid (Glu) polypeptide mimetics may have significant advantages over [0285) (4) basic: Asparagine (Asn), Glutamine (Gln), naturally occurring polypeptides including more economical Histidine (His), Lysine (Lys), Arginine (Arg) production, greater chemical stability, enhanced pharmaco [0286] (5) residues that influence chain orientation: Gly logical properties (e.g., half-life, absorption, potency, effi cine (Gly), Proline (Pro); ciency), reduced antigenicity, and others. [0287] (6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), [0293] While the peptide vectors described herein may effi Phenylalanine (Phe), Histidine (His), ciently cross the BBB or target particular cell types (e.g., [0288] (7) polar; Ser, Thr, Asn., Gln those described herein), their effectiveness may be reduced [0289] (8) basic positively charged: Arg, Lys, His, and; by the presence of proteases. Likewise, the effectiveness of [0290] (9) charged: Asp, Glu, Arg, Lys, His GLP-1 agonists used in the invention may be similarly Other amino acid substitutions are listed in Table 3. reduced. Serum proteases have specific substrate require ments, including L-amino acids and peptide bonds for cleav TABLE 2 age. Furthermore, exopeptidases, which represent the most Amino acid substitutions prominent component of the protease activity in serum, usu ally act on the first peptide bond of the polypeptide and Original Exemplary Conservative require a free N-terminus (Powell et al., Pharm. Res. residue substitution substitution 10:1268-1273, 1993). In light of this, it is often advantageous Ala (A) Val, Leu, Ile Val to use modified versions of polypeptides. The modified Arg (R) Lys, Gln, Asn Lys polypeptides retain the structural characteristics of the origi Asn (N) Gln, His, Lys, Arg Gln nal L-amino acid polypeptides, but advantageously are not Asp (D) Glu Glu Cys (C) Ser Ser readily susceptible to cleavage by protease and/or exopepti Gln (Q) Asn Asn dases. Glu (E) Asp Asp Gly (G) Pro Pro [0294] Systematic substitution of one or more amino acids His (H) Asm, Gln, Lys, Arg Arg of a consensus sequence with D-amino acid of the same type Ile (I) Leu, Val, Met, Ala, Leu (e.g., an enantiomer; D-lysine in place of L-lysine) may be Phe, norleucine used to generate more stable polypeptides. Thus, a polypep Leu (L) Norleucine, Ile, Val, Ile tide derivative or peptidomimetic as described herein may be Met, Ala, Phe Lys (K) Arg, Gln, Asn Arg all L-, all D-, or mixed D, L polypeptides. The presence of an Met (M) Leu, Phe, Ile Leu N-terminal or C-terminal D-amino acid increases the in vivo Phe (F) Leu, Val, Ile, Ala Leu stability of a polypeptide because peptidases cannot utilize a Pro (P) Gly Gly Ser (S) Thr Thr D-amino acid as a substrate (Powell et al., Pharm. Res. Thr (T) Ser Ser 10:1268-1273, 1993). Reverse-D polypeptides are polypep Trp (W) Tyr Tyr tides containing D-amino acids, arranged in a reverse Tyr (Y) Trp, Phe, Thr, Ser Phe sequence relative to a polypeptide containing L-amino acids. Val (V) Ile, Leu, Met, Phe, Leu Thus, the C-terminal residue of an L-amino acid polypeptide Ala, norleucine becomes N-terminal for the D-amino acid polypeptide, and so forth. Reverse D-polypeptides retain the same tertiary con [0291] Polypeptide Derivatives and Peptidomimetics formation and therefore the same activity, as the L-amino acid [0292] In addition to polypeptides consisting of naturally polypeptides, but are more stable to enzymatic degradation in occurring amino acids, peptidomimetics or polypeptide ana vitro and in vivo, and thus have greater therapeutic efficacy logs are also encompassed by the present invention and can than the original polypeptide (Brady and Dodson, Nature form the peptide vectors or peptide therapeutics used in the 368:692-693, 1994 and Jameson et al., Nature 368:744-746, compounds of the invention. Polypeptide analogs are com 1994). In addition to reverse-D-polypeptides, constrained monly used in the pharmaceutical industry as non-peptide polypeptides comprising a consensus sequence or a substan drugs with properties analogous to those of the template tially identical consensus sequence variation may be gener polypeptide. The non-peptide compounds are termed “pep ated by methods well known in the art (Rizo et al., Ann. Rev. tide mimetics” or peptidomimetics (Fauchere et al., Infect. Biochem. 61:387-418, 1992). For example, constrained Immun. 54:283-287, 1986 and Evans et al., J. Med. Chem. polypeptides may be generated by adding cysteine residues 30:1229-1239, 1987). Peptide mimetics that are structurally capable offorming disulfide bridges and, thereby, resulting in related to therapeutically useful peptides or polypeptides may a cyclic polypeptide. Cyclic polypeptides have no free N- or be used to produce an equivalent or enhanced therapeutic or C-termini. Accordingly, they are not susceptible to proteoly prophylactic effect. Generally, peptidomimetics are structur sis by exopeptidases, although they are, of course, susceptible ally similar to the paradigm polypeptide (i.e., a polypeptide to endopeptidases, which do not cleave at polypeptide ter that has a biological or pharmacological activity) such as mini. The amino acid sequences of the polypeptides with naturally-occurring receptor-binding polypeptides, but have N-terminal or C-terminal D-amino acids and of the cyclic one or more peptide linkages optionally replaced by linkages polypeptides are usually identical to the sequences of the such as CH2NH_, CH2S? CH2—CH2—, polypeptides to which they correspond, except for the pres US 2011/0288011 A1 Nov. 24, 2011 39 ence of N-terminal or C-terminal D-amino acid residue, or While polypeptides having a substantial number of additional their circular structure, respectively. amino acids are not excluded, it is recognized that some large [0295] A cyclic derivative containing an intramolecular polypeptides may assume a configuration that masks the disulfide bond may be prepared by conventional solid phase effective sequence, thereby preventing binding to a target synthesis while incorporating suitable S-protected cysteine or (e.g., a member of the LRP receptor family such as LRP or homocysteine residues at the positions selected for cycliza LRP2). These derivatives could act as competitive antago tion such as the amino and carboxy termini (Sah et al., J. nists. Thus, while the present invention encompasses Pharm. Pharmacol. 48:197, 1996). Following completion of polypeptides or derivatives of the polypeptides described the chain assembly, cyclization can be performed either (1) by herein having an extension, desirably the extension does not selective removal of the S-protecting group with a consequent destroy the cell targeting activity of the polypeptides or its on-support oxidation of the corresponding two free SH-func derivatives. tions, to form a S-S bonds, followed by conventional removal of the product from the support and appropriate [0300|| Other derivatives included in the present invention purification procedure or (2) by removal of the polypeptide are dual polypeptides consisting of two of the same, or two from the support along with complete side chain de-protec different polypeptides, as described herein, covalently linked tion, followed by oxidation of the free SH-functions in highly to one another either directly or through a spacer, such as by dilute aqueous solution. a short stretch of alanine residues or by a putative site for [0296] The cyclic derivative containing an intramolecular proteolysis (e.g., by cathepsin, see e.g., U.S. Pat. No. 5,126, amide bond may be prepared by conventional solid phase 249 and European Patent No. 495 049). Multimers of the synthesis while incorporating suitable amino and carboxyl polypeptides described herein consist of a polymer of mol side chain protected amino acid derivatives, at the position ecules formed from the same or different polypeptides or selected for cyclization. The cyclic derivatives containing derivatives thereof. intramolecular –S-alkyl bonds can be prepared by conven [0301] The present invention also encompasses polypep tional solid phase chemistry while incorporating an amino tide derivatives that are chimeric or fusion proteins containing acid residue with a suitable amino-protected side chain, and a a polypeptide described herein, or fragment thereof, linked at suitable S-protected cysteine or homocysteine residue at the its amino- or carboxy-terminal end, or both, to an amino acid position selected for cyclization. sequence of a different protein. Such a chimeric or fusion [0297] Another effective approach to confer resistance to protein may be produced by recombinant expression of a peptidases acting on the N-terminal or C-terminal residues of nucleic acid encoding the protein. For example, a chimeric or a polypeptide is to add chemical groups at the polypeptide fusion protein may contain at least 6 amino acids shared with termini, such that the modified polypeptide is no longer a one of the described polypeptides which desirably results in a substrate for the peptidase. One such chemical modification is chimeric or fusion protein that has an equivalent or greater glycosylation of the polypeptides at either or both termini. functional activity. Certain chemical modifications, in particular N-terminal gly [0302) Assays to Identify Peptidomimetics cosylation, have been shown to increase the stability of [0303] As described above, non-peptidyl compounds gen polypeptides in human serum (Powell et al., Pharm. Res. erated to replicate the backbone geometry and pharmacoph 10:1268-1273, 1993). Other chemical modifications which ore display (peptidomimetics) of the polypeptides described enhance serum stability include, but are not limited to, the herein often possess attributes of greater metabolic stability, addition of an N-terminal alkyl group, consisting of a lower higher potency, longer duration of action, and better bioavail alkyl of from one to twenty carbons, such as an acetyl group, ability. and/or the addition of a C-terminal amide or substituted [0304] Peptidomimetics compounds can be obtained using amide group. In particular, the present invention includes any of the numerous approaches in combinatorial library modified polypeptides consisting of polypeptides bearing an methods known in the art, including biological libraries, spa N-terminal acetyl group and/or a C-terminal amide group. tially addressable parallel solid phase or solution phase librar [0298] Also included by the present invention are other ies, synthetic library methods requiring deconvolution, the types of polypeptide derivatives containing additional chemi ‘one-bead one-compound’ library method, and synthetic cal moieties not normally part of the polypeptide, provided library methods using affinity chromatography selection. The that the derivative retains the desired functional activity of the biological library approach is limited to peptide libraries, polypeptide. Examples of such derivatives include (1) N-acyl while the other four approaches are applicable to peptide, derivatives of the amino terminal or of another free amino non-peptide oligomer, or small molecule libraries of com group, wherein the acyl group may be an alkanoyl group (e.g., pounds (Lam, Anticancer Drug Des. 12:145, 1997). acetyl, hexanoyl, octanoyl) anaroyl group (e.g., benzoyl) or a Examples of methods for the synthesis of molecular libraries blocking group such as F-moc (fluorenylmethyl-O-CO–); can be found in the art, for example, in: DeWitt et al. (Proc. (2) esters of the carboxy terminal or of another free carboxy or Natl. Acad. Sci. USA 90:6909, 1993); Erb et al. (Proc. Natl. hydroxyl group; (3) amide of the carboxy-terminal or of Acad. Sci. USA 91:11422, 1994); Zuckermann et al. (J. Med. another free carboxyl group produced by reaction with Chem. 37:2678, 1994); Cho et al. (Science 261:1303, 1993); ammonia or with a suitable amine; (4) phosphorylated deriva Carell et al. (Angew. Chem., Int. Ed. Engl. 33:2059, 1994 and tives. ibid 2061); and in Gallop et al. (Med. Chem. 37:1233, 1994). [0299| Longer polypeptide sequences which result from Libraries of compounds may be presented in solution (e.g., the addition of additional amino acid residues to the polypep Houghten, Biotechniques 13:412-421, 1992) or on beads tides described herein are also encompassed in the present (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364: invention. Such longer polypeptide sequences can be 555-556, 1993), bacteria or spores (U.S. Pat. No. 5,223,409), expected to have the same biological activity and specificity plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89:1865 (e.g., cell tropism) as the polypeptides described above. 1869, 1992) or on phage (Scott and Smith, Science 249:386 US 2011/0288011 A1 Nov. 24, 2011 40
390, 1990), or luciferase, and the enzymatic label detected by a peptide bond) or may be bound through a linker. Linkers determination of conversion of an appropriate substrate to include chemical linking agents (e.g., cleavable linkers) and product. peptides. [0305] Once a polypeptide as described herein is identified, [0310] In some embodiments, the linker is a chemical link it can be isolated and purified by any number of standard ing agent. The peptide therapeutic and vector peptide may be methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., conjugated through sulfhydryl groups, amino groups affinity, ion exchange, and size exclusion), or by any other (amines), and/or carbohydrates or any appropriate reactive standard techniques used for the purification of peptides, group. Homobifunctional and heterobifunctional cross-link peptidomimetics, or proteins. The functional properties of an ers (conjugation agents) are available from many commercial identified polypeptide of interest may be evaluated using any sources. Regions available for cross-linking may be found on functional assay known in the art. Desirably, assays for evalu the polypeptides of the present invention. The cross-linker ating downstream receptor function in intracellular signaling may comprise a flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, are used (e.g., cell proliferation). 12, 13, 14, or 15 carbon atoms. Exemplary cross-linkers [0306] For example, the peptidomimetics compounds of include BS3 ([Bis(sulfosuccinimidyl)suberate]; BS3 is a the present invention may be obtained using the following homobifunctional N-hydroxysuccinimide ester that targets three-phase process: (1) scanning the polypeptides described accessible primary amines), NHS/EDC(N-hydroxysuccin hereinto identify regions of secondary structure necessary for imide and N-ethyl-(dimethylaminopropyl)carbodimide; targeting the particular cell types described herein; (2) using NHS/EDC allows for the conjugation of primary amine conformationally constrained dipeptide surrogates to refine groups with carboxyl groups), sulfo-EMCS (IN-e-Maleimi the backbone geometry and provide organic platforms corre docaproic acid]hydrazide; sulfo-EMCS are heterobifunc sponding to these surrogates; and (3) using the best organic tional reactive groups (maleimide and NHS-ester) that are platforms to display organic pharmocophores in libraries of reactive toward sulfhydryl and amino groups), hydrazide candidates designed to mimic the desired activity of the native (most proteins contain exposed carbohydrates and hydrazide polypeptide. In more detail the three phases are as follows. In is a useful reagent for linking carboxyl groups to primary phase 1, the lead candidate polypeptides are scanned and their amines), and SATA (N-succinimidyl-S-acetylthioacetate; structure abridged to identify the requirements for their activ SATA is reactive towards amines and adds protected sulfhy ity. A series of polypeptide analogs of the original are syn dryls groups). thesized. In phase 2, the best polypeptide analogs are inves [0311] To form covalent bonds, one can use as a chemically tigated using the conformationally constrained dipeptide reactive group a wide variety of active carboxyl groups (e.g., surrogates. Indolizidin-2-one, indolizidin-9-one and quino esters) where the hydroxyl moiety is physiologically accept lizidinone amino acids (I*aa, I’aa and Qaa respectively) are able at the levels required to modify the peptide. Particular used as platforms for studying backbone geometry of the best agents include N-hydroxysuccinimide (NHS), N-hydroxy peptide candidates. These and related platforms (reviewed in sulfosuccinimide (sulfo-NHS), maleimide-benzoyl-succin Halab et al., Biopolymers 55:101-122, 2000 and Hanessian et imide (MBS), gamma-maleimido-butyryloxy succinimide al., Tetrahedron 53:12789-12854, 1997) may beintroduced at ester (GMBS), maleimido propionic acid (MPA) maleimido specific regions of the polypeptide to orient the pharmacoph hexanoic acid (MHA), and maleimido undecanoic acid ores in different directions. Biological evaluation of these (MUA). analogs identifies improved lead polypeptides that mimic the [0312] Primary amines are the principal targets for NHS geometric requirements for activity. In phase 3, the platforms esters. Accessible O-amine groups present on the N-termini from the most active lead polypeptides are used to display of proteins and the e-amine of lysine react with NHS esters. organic surrogates of the pharmacophores responsible for An amide bond is formed when the NHS ester conjugation activity of the native peptide. The pharmacophores and scaf reaction reacts with primary amines releasing N-hydroxysuc folds are combined in a parallel synthesis format. Derivation cinimide. These succinimide containing reactive groups are of polypeptides and the above phases can be accomplished by herein referred to as succinimidyl groups. In certain embodi other means using methods known in the art. ments of the invention, the functional group on the protein [0307| Structure function relationships determined from will be a thiol group and the chemically reactive group will be the polypeptides, polypeptide derivatives, peptidomimetics a maleimido-containing group such as gamma-maleimide or other small molecules described herein may be used to refine and prepare analogous molecular structures having butrylamide (GMBA or MPA). Such maleimide containing similar or better properties. Accordingly, the compounds of groups are referred to herein as maleido groups. the present invention also include molecules that share the [0313] The maleimido group is most selective for sulfhy structure, polarity, charge characteristics and side chain prop dryl groups on peptides when the pH of the reaction mixture erties of the polypeptides described herein. is 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups [0308] In summary, based on the disclosure herein, those with sulfhydryls (e.g., thiol groups on proteins such as serum skilled in the art can develop peptides and peptidomimetics albumin or IgG) is 1000-fold faster than with amines. Thus, a screening assays which are useful for identifying compounds stable thioether linkage between the maleimido group and the for targeting an agent to particular cell types (e.g., those sulfhydryl can be formed. described herein). The assays of this invention may be devel [0314] In other embodiments, the linker includes at least oped for low-throughput, high-throughput, or ultra-high one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, throughput screening formats. Assays of the present inven 20, 25, 40, or 50 amino acids). In certain embodiments, the tion include assays amenable to automation. linker is a single amino acid (e.g., any naturally occurring amino acid such as Cys). In other embodiments, a glycine Linkers rich peptide such as a peptide having the sequence [Gly-Gly [0309] The peptide therapeutic may be bound to the vector Gly-Gly-Ser], where n is 1, 2, 3, 4, 5 or 6 is used, as described peptide either directly (e.g., through a covalent bond such as in U.S. Pat. No. 7,271,149. In other embodiments, a serine US 2011/0288011 A1 Nov. 24, 2011
rich peptide linker is used, as described in U.S. Pat. No. diluted in buffer 3. GTP was freshly prepared for each experi 5,525,491. Serine rich peptide linkers include those of the ment. The plate was incubated in the dark with slow agitation formula [X-X-X-X-Gly], where up to two of the X are Thr, for three hours at room temperature followed by counting in and the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser the Fusion"M instrument (Perkin Elmer Life Sciences). Con Ser-Gly, where y is greater than 1). In some cases, the linker centration-response curves were plotted for the individual is a single amino acid (e.g., any amino acid, such as Gly or compounds and ECso values estimated using a four-param Cys). eter logistic model with Prism v. 4.0 (Graphpad, Carlsbad, [0315) Examples of suitable linkers are succinic acid, Lys, Calif.). Glu, and Asp, or a dipeptide such as Gly-Lys. When the linker is succinic acid, one carboxyl group thereof may form an Therapeutic Applications amide bond with an amino group of the amino acid residue, [0319| The compounds of the invention can be used in any and the other carboxyl group thereof may, for example, form appropriate therapeutic application where the activity of the an amide bond with an amino group of the peptide or sub peptide therapeutic is beneficial. The compounds of the stituent. When the linker is Lys, Glu, or Asp, the carboxyl invention can be used to treat infections (e.g., where the group thereof may form an amide bond with an amino group peptide therapeutic is antimicrobial or antibiotic peptide), to of the amino acid residue, and the amino group thereof may, treat neoplasms such as a cancer (e.g., using a agent having for example, forman amide bond with a carboxyl group of the antiproliferative activity, such as a tumor antibiotic or thy substituent. When Lys is used as the linker, a further linker rotropin), for treating pain (e.g., using an opioid), to treat may be inserted between the e-amino group of Lys and the metabolic disorders (e.g., using a GLP-1 agonist, gastric substituent. In one particular embodiment, the further linker inhibitory polypeptide, insulin, growth hormone-releasing is succinic acid which, e.g., forms an amide bond with the hormone, oran analog thereof), neurological disorder such as e-amino group of Lys and with an amino group present in the seizures (e.g., using galanin or an analog thereof), for bone substituent. In one embodiment, the further linker is Glu or diseases such as osteoporosis, Paget’s disease (e.g., using Asp (e.g., which forms an amide bond with the e-amino group PTH, PTHrP, calcintonin, or an analog thereof), and hyper of Lys and another amide bond with a carboxyl group present tension (e.g., using bradykinin or an analog thereof). Com in the substituent), that is, the substituent is a Nº-acylated pounds containing any of the human peptides described lysine residue. herein (or analogs or fragments thereof) as a peptide there apeutic may be used to treat a subject suffering a deficiency of GLP-1 Agonist Activity Assay that peptide. Additional indications are described below. [0316) Determination of whether a compound has GLP-1 [0320] Cancer agonist activity can be performed using any method known in [0321] The compounds of the invention can be used to treat the art. Cyclic AMP (cAMP) production from cells express any cancer, but, in the case of conjugates including a vector ing a GLP-1 receptor (e.g., a human receptor) can be mea that is efficiently transported across the BBB, are particularly sured in the presence and in the absence of a compound, useful for the treatment of brain cancers and other cancers where an increase in cAMP production indicates the com protected by the BBB. These cancers include astrocytoma, pound to be a GLP-1 agonist. pilocytic astrocytoma, dysembryoplastic neuroepithelial [0317] In one example described in U.S. Patent Application tumor, oligodendrogliomas, ependymoma, glioblastoma Publication No. 2008/0207507, baby hamster kidney (BHK) multiforme, mixed gliomas, oligoastrocytomas, medullo cells expressing the cloned human GLP-1 receptor (BHK blastoma, retinoblastoma, neuroblastoma, germinoma, and 467-12A) were grown in DMEM media with the addition of teratoma. Other types of cancer include hepatocellular carci 100 IU/ml penicillin, 100 pg/ml streptomycin, 5% fetal calf noma, breast cancer, cancers of the head and neck including serum, and 0.5 mg/mL Geneticin G-418 (Life Technologies). various lymphomas such as mantle cell lymphoma, non The cells were washed twice in phosphate buffered saline and Hodgkins lymphoma, adenoma, squamous cell carcinoma, harvested with Versene. Plasma membranes were prepared laryngeal carcinoma, cancers of the retina, cancers of the from the cells by homogenisation with an Ultraturrax in esophagus, multiple myeloma, ovarian cancer, uterine cancer, buffer 1 (20 mM HEPES-Na, 10 mM EDTA, pH 7.4). The melanoma, colorectal cancer, bladder cancer, prostate cancer, homogenate was centrifuged at 48,000×g for 15 min at 4°C. lung cancer (including non-small cell lung carcinoma), pan The pellet was suspended by homogenization in buffer 2 (20 creatic cancer, cervical cancer, head and neck cancer, skin mM HEPES-Na, 0.1 mM EDTA, pH 7.4), then centrifuged at cancers, nasopharyngeal carcinoma, liposarcoma, epithelial 48,000×g for 15 min at 4°C. The washing procedure was carcinoma, renal cell carcinoma, gallbladder adenocarci repeated one more time. The final pellet was suspended in noma, parotid adenocarcinoma, endometrial sarcoma, multi buffer 2 and used immediately for assays or stored at -80°C. drug resistant cancers; and proliferative diseases and condi [0318] The functional receptor assay was carried out by tions, such as neovascularization associated with tumor measuring cAMP as a response to stimulation by the insuli angiogenesis, macular degeneration (e.g., wet/dry AMD), notropic agent. cAMP formed was quantified by the AlphaS corneal neovascularization, diabetic retinopathy, neovascular creenTMcAMP Kit (PerkinElmer Life Sciences). Incubations glaucoma, myopic degeneration and other proliferative dis were carried out in half-area 96-well microtiter plates in a eases and conditions such as restenosis and polycystic kidney total volume of 50 pull buffer 3 (50 mM Tris-HCl, 5 mM disease. HEPES, 10 mM MgCl2, pH 7.4) and with the following [0322] Neurological Disease additions: 1 mM ATP, 1 puM GTP, 0.5 mM 3-isobutyl-1 [0323] Because polypeptides described herein are capable methylxanthine (IBMX), 0.01% Tween-20, 0.1% BSA, 6 pig of transporting an agent across the BBB, the compounds of membrane preparation, 15 pig?ml acceptor beads, 20 pg/ml the invention are also useful for the treatment of neurological donor beads preincubated with 6 nM biotinyl-cAMP Com diseases such as neurodegenerative diseases or other condi pounds to be tested for agonist activity were dissolved and tions of the central nervous system (CNS), the peripheral US 2011/0288011 A1 Nov. 24, 2011 42 nervous system, or the autonomous nervous system (e.g., [0328] GLP-1 is also has neurological effects including where neurons are lost or deteriorate). Many neurodegenera sedative or anti-anxiolytic effects, as described in U.S. Pat. tive diseases are characterized by ataxia (i.e., uncoordinated No. 5,846,937. Thus, GLP-1 agonists can be used in the muscle movements) and/or memory loss. Neurodegenerative treatment of anxiety, aggression, psychosis, seizures, panic diseases include Alexander disease, Alper disease, Alzhe attacks, hysteria, or sleep disorders. GLP-1 agonists can also imer’s disease, amyotrophic lateral sclerosis (ALS; i.e., Lou be used to treat Alzheimer’s disease, as GLP-1 agonists have Gehrig's disease), ataxia telangiectasia, Batten disease been shown to protect neurons against amyloid-fi peptide and (Spielmeyer-Vogt-Sjogren-Batten disease), bovine spongi glutamate-induced apoptosis (Perry et al., Curr Alzheimer form encephalopathy (BSE), Canavan disease, Cockayne Res 2:377-85, 2005). syndrome, corticobasal degeneration, Creutzfeldt-Jakob dis [0329] Other therapeutic uses for GLP-1 agonists include ease, Huntington’s disease, HIV-associated dementia, improving learning, enhancing neuroprotection, and alleviat Kennedy’s disease, Krabbé disease, Lewy body dementia, ing a symptom of a disease or disorder of the central nervous Machado-Joseph disease (Spinocerebellar ataxia type 3), system, e.g., through modulation of neurogenesis, and e.g., multiple sclerosis, multiple system atrophy, narcolepsy, neu Parkinson’s Disease, Alzheimer’s Disease, Huntington’s Dis roborreliosis, Parkinson’s disease, Pelizaeus-Merzbacher ease, ALS, stroke, ADD, and neuropsychiatric syndromes disease, Pick’s disease, primary lateral sclerosis, prion dis (U.S. Pat. No. 6,969,702 and U.S. Patent Application No. eases, Refsum’s disease, Schilder’s disease (i.e., adrenoleu 2002/0115605). Stimulation of neurogenesis using GLP-1 kodystrophy), schizophrenia, spinocerebellar ataxia, spinal agonists has been described, for example, in Bertilsson et al., muscularatrophy, Steele-Richardson, Olszewski disease, and J Neurosci Res 86:326-338, 2008. tabes dorsalis. [0330] Still other therapeutic uses include converting liver [0324] Lysosomal Storage Disorders stem/progenitor cells into functional pancreatic cells (U.S. [0325] The conjugates of the invention may also be used to Patent Application Publication No. 2005/0053588); prevent treat a lysosomal storage disease or disorder, many of which ing beta-cell deterioration (U.S. Pat. Nos. 7,259,233 and affect the central nervous system (CNS) and cause or exac 6,569,832) and stimulation of beta-cell proliferation (U.S. erbate neurodegenerative disease. Lysosomal storage dis Patent Application Publication No. 2003/0224983); treating eases include any of the mucopolysaccharidoses (MPS: obesity (U.S. Pat. No. 7,211,557); suppressing appetite and including MPS-I (Hurler syndrome, Scheie syndrome), inducing satiety (U.S. Patent Application Publication No. MPS-II (Hunter syndrome), MPS-IIIA (Sanfilippo syndrome 2003/0232754); treating irritable bowel syndrome (U.S. Pat. A), MPS-IIIB (Sanfilippo syndrome B), MPS-IIIC (Sanfil No. 6,348.447); reducing the morbidity and/or mortality ippo syndrome C), MPS-IIID (Sanfilippo syndrome D), associated with myocardial infarction (U.S. Pat. No. 6,747, MPS-IV (Morquio syndrome), MPS-VI (Maroteaux-Lamy 006) and stroke (PCT Publication No. WO 00/16797); treat syndrome), MPS-VII (Sly syndrome), and MPS-IX (hyalu ing acute coronary syndrome characterized by an absence of ronidase deficiency)), lipidoses (including Gaucher’ disease, Q-wave myocardial infarction (U.S. Pat. No. 7,056,887); Niemann-Pick disease, Fabry disease, Farber’s disease, and attenuating post-surgical catabolic changes (U.S. Pat. No. Wolman’s disease), gangliosidoses (including GM1 and 6,006,753); treating hibernating myocardium or diabetic car GM2 gangliosidoses, Tay-Sachs disease, and Sandhoff dis diomyopathy (U.S. Pat. No. 6,894,024); suppressing plasma ease), leukodystrophies (including adrenoleukodystrophy blood levels of norepinepherine (U.S. Pat. No. 6,894,024); (i.e., Schilder’s disease), Alexander disease, metachromatic increasing urinary sodium excretion, decreasing urinary leukodystrophy, Krabbé disease, Pelizaeus-Merzbacher dis potassium concentration (U.S. Pat. No. 6,703,359); treating ease, Canavan disease, childhood ataxia with central hypo conditions or disorders associated with toxic hypervolemia, myelination (CACH), Refsum’s disease, and cerebrotendin e.g., renal failure, congestive heart failure, nephrotic syn eous xanthomatosis), mucolipidoses (ML; including ML-I drome, cirrhosis, pulmonary edema, and hypertension (U.S. (sialidosis), ML-II (I-cell disease), ML-III (pseudo-Hurler Pat. No. 6,703,359); inducing an inotropic response and polydystrophy), and ML-TV), and glycoproteinoses (includ increasing cardiac contractility (U.S. Pat. No. 6,703,359); ing aspartylglucosaminuria, fucosidosis, and mannosidosis). treating polycystic ovary syndrome (U.S. Pat. No. 7,105, [0326] GLP-1 Related Disorders 489); treating respiratory distress (U.S. Patent Application [0327] In certain embodiments, the peptide therapeutic is a Publication No. 2004/0235726); improving nutrition via a GLP-1 agonist. Such compounds can be used in any thera non-alimentary route, i.e., via intravenous, subcutaneous, peutic application where a GLP-1 agonist activity in the intramuscular, peritoneal, or other injection or infusion (U.S. brain, or in particular tissues, is desired. GLP-1 agonist activ Pat. No. 6,852,690); treating nephropathy (U.S. Patent Appli ity is associated with stimulation of insulin secretion (i.e., to cation Publication No. 2004/0209803); treating left ventricu act as an incretin hormone) and inhibition glucagon secretion, lar systolic dysfunction, e.g., with abnormal left ventricular thereby contributing to limit postprandial glucose excursions. ejection fraction (U.S. Pat. No. 7,192,922); inhibiting antro GLP-1 agonists can also inhibit gastrointestinal motility and duodenal motility, e.g., for the treatment or prevention of secretion, thus acting as an enterogastrone and part of the gastrointestinal disorders such as diarrhea, postoperative “ileal brake” mechanism. GLP-1 also appears to be a physi dumping syndrome and irritable bowel syndrome, and as ological regulator of appetite and food intake. Because of premedication in endoscopic procedures (U.S. Pat. No. these actions, GLP-1 and GLP-1 receptor agonists can be 6,579,851); treating critical illness polyneuropathy (CIPN) used for therapy of metabolic disorders, as reviewed in, e.g., and systemic inflammatory response syndrome (SIRS) (U.S. Kinziget al., JNeurosci 23:6163-6170, 2003. Such disorders Patent Application Publication No. 2003/0199445); modulat include obesity, hyperglycemia, dyslipidemia, hypertriglyc ing triglyceride levels and treating dyslipidemia (U.S. Patent eridemia, syndrome X, insulin resistance, IGT, diabetic dys Application Publication Nos. 2003/0036504 and 2003/ lipidemia, hyperlipidemia, a cardiovascular disease, and 0143183); treating organ tissue injury caused by reperfusion hypertension. of blood flow following ischemia (U.S. Pat. No. 6,284,725); US 2011/0288011 A1 Nov. 24, 2011
treating coronary heart disease risk factor (CHDRF) syn units, each containing a fixed amount of the above-mentioned drome (U.S. Pat. No. 6,528,520); and others. agent or agents, such as in a sealed package of tablets or [0331] Additional Indications capsules. The composition in solid form can also be packaged [0332] The conjugates of the invention can also be used to in a container for a flexible quantity, such as in a squeezable treat diseases found in other organs or tissues. For example, tube designed for a topically applicable cream or ointment. Angiopep-7 (SEQ ID NO:112) is efficiently transported into [0336] The compositions containing an effective amount liver, lung, kidney, spleen, and muscle cells, allowing for the can be administered for prophylactic or therapeutic treat preferential treatment of diseases associated with these tis ments. In prophylactic applications, compositions can be sues (e.g., hepatocellular carcinoma and lung cancer). The administered to a subject with a clinically determined predis compounds of the presents invention may also be used to treat position orincreased susceptibility to a metabolic disorder or genetic disorders, such as Down syndrome (i.e., trisomy 21), neurological disease. Compositions of the invention can be where down-regulation of particular gene transcripts may be administered to the subject (e.g., a human) in an amount useful. sufficient to delay, reduce, or preferably prevent the onset of clinical disease. In therapeutic applications, compositions are Administration and Dosage administered to a subject (e.g., a human) already suffering [0333] The present invention also features pharmaceutical from disease (e.g., a metabolic disorder such as those compositions that contain a therapeutically effective amount described herein, or a neurological disease) in an amount of a compound of the invention. The composition can be sufficient to cure or at least partially arrest the symptoms of formulated for use in a variety of drug delivery systems. One the condition and its complications. An amount adequate to or more physiologically acceptable excipients or carriers can accomplish this purpose is defined as a “therapeutically effec also be included in the composition for proper formulation. tive amount,” an amount of a compound sufficient to substan Suitable formulations for use in the present invention are tially improve some symptom associated with a disease or a found in Remington's Pharmaceutical Sciences, Mack Pub medical condition. For example, in the treatment of a meta lishing Company, Philadelphia, Pa., 17th ed., 1985. For a bolic disorder (e.g., those described herein), an agent or com brief review of methods for drug delivery, see, e.g., Langer pound which decreases, prevents, delays, suppresses, or (Science 249:1527-1533, 1990). arrests any symptom of the disease or condition would be [0334] The pharmaceutical compositions are intended for therapeutically effective. A therapeutically effective amount parenteral, intranasal, topical, oral, or local administration, of an agent or compound is not required to cure a disease or such as by a transdermal means, for prophylactic and/or condition but will provide a treatment for a disease or condi therapeutic treatment. The pharmaceutical compositions can tion such that the onset of the disease or condition is delayed, be administered parenterally (e.g., by intravenous, intramus hindered, or prevented, or the disease or condition symptoms cular, or subcutaneous injection), or by oral ingestion, or by are ameliorated, or the term of the disease or condition is topical application orintraarticular injection at areas affected changed or, for example, is less severe or recovery is accel by the vascular or cancer condition. Additional routes of erated in an individual. administration include intravascular, intra-arterial, intratu [0337] The compounds of the invention may be adminis mor, intraperitoneal, intraventricular, intraepidural, as well as tered in equivalent doses of as specified for the unconjugated nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or peptide therapeutic, may be administered in higher equivalent aerosol inhalation administration. Sustained release adminis doses (e.g., 10%, 25%, 50%, 100%, 200%, 500%, 1000% tration is also specifically included in the invention, by such greater doses), or can be administered in lower equivalent means as depot injections or erodible implants or compo doses (e.g., 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, ments. Thus, the invention provides compositions for 10%, 8%, 7%, 6%. 5%, 4%. 3%, 2%, 1%, 0.5%, or 0.1% of the parenteral administration that comprise the above mention equivalent dose). Amounts effective for this use may depend agents dissolved or suspended in an acceptable carrier, pref on the severity of the disease or condition and the weight and erably an aqueous carrier, e.g., water, buffered water, saline, general state of the subject, but generally range from about PBS, and the like. The compositions may contain pharmaceu 0.05 pig to about 10,000 pig (e.g., 0.5-1000 pig) of an equivalent tically acceptable auxiliary substances as required to approxi amount of the peptide therapeutic the agent or agents perdose mate physiological conditions, such as pH adjusting and buff per subject. Suitable regimes for initial administration and ering agents, tonicity adjusting agents, wetting agents, booster administrations are typified by an initial administra detergents and the like. The invention also provides compo tion followed by repeated doses at one or more hourly, daily, sitions for oral delivery, which may contain inert ingredients weekly, or monthly intervals by a subsequent administration. such as binders or fillers for the formulation of a tablet, a The total effective amount of an agent present in the compo capsule, and the like. Furthermore, this invention provides sitions of the invention can be administered to a mammal as a compositions for local administration, which may contain single dose, either as a bolus or by infusion over a relatively inert ingredients such as solvents or emulsifiers for the for short period of time, or can be administered using a fraction mulation of a cream, an ointment, and the like. ated treatment protocol, in which multiple doses are admin [0335] These compositions may be sterilized by conven istered over a more prolonged period of time (e.g., a dose tional sterilization techniques, or may be sterile filtered. The every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 resulting aqueous solutions may be packaged for use as is, or weeks, once a month). Alternatively, continuous intravenous lyophilized, the lyophilized preparation being combined with infusion sufficient to maintain therapeutically effective con a sterile aqueous carrier prior to administration. The pH of the centrations in the blood are contemplated. preparations typically will be between 3 and 11, more pref [0338] The therapeutically effective amount of one or more erably between 5 and 9 or between 6 and 8, and most prefer agents present within the compositions of the invention and ably between 7 and 8, such as 7 to 7.5. The resulting compo used in the methods of this invention applied to mammals sitions in solid form may be packaged in multiple single dose (e.g., humans) can be determined by the ordinarily-skilled US 2011/0288011 A1 Nov. 24, 2011 44 artisan with consideration of individual differences in age, assay, which is described in U.S. Patent Application Publica weight, and the condition of the mammal. Because certain tion No. 2006/0189515, is performed as follows. The uptake compounds of the invention exhibit an enhanced ability to of labeled exendin-4 and the exendin-4/Angiopep-2 conju cross the BBB, the dosage of the compounds of the invention gates was measured using the in situ brain perfusion method can be lower than (e.g., less than or equal to about 90%, 75%, adapted in our laboratory for the study of drug uptake in the 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%. 5%, mouse brain (Dagenais et al., J Cereb Blood Flow Metab. 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose of 20:381-6, 2000; Cisternino et al., Pharm Res 18, 183-190, required for a therapeutic effect of the unconjugated peptide 2001). Briefly, the right common carotid artery of mice anes therapeutic. The agents of the invention are administered to a thetized with ketamine/xylazine (140/8 mg/kg i.p.) was subject (e.g. a mammal, such as a human) in an effective exposed and ligated at the level of the bifurcation of the amount, which is an amount that produces a desirable resultin common carotid, rostral to the occipital artery. The common a treated subject (e.g. reduction in glycemia, reduced weight carotid was then catheterized rostrally with polyethylene tub gain, increased weight loss, and reduced food intake). Thera ing filled with heparin (25 U/ml) and mounted on a 26-gauge peutically effective amounts can also be determined empiri needle. The syringe containing the perfusion fluid (IºI] cally by those of skill in the art. proteins or [*I]-peptides in Krebs/bicarbonate buffer at pH [0339] The subject may also receive an agent in the range of 7.4, gassed with 95% O2 and 5% CO2) was placed in an about 0.05 to 10,000 pig equivalent dose as compared to infusion pump (Harvard pump PHD 2000; Harvard Appara peptide therapeutic perdose one or more times per week (e.g., tus) and connected to the catheter. Prior to the perfusion, the 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 (e.g., contralateral blood flow contribution was eliminated by sev 2,000, 1,500, 1,000, 500, 100, 10, 1, 0.5, or 0.1) pig dose per ering the heart ventricles. The brain was perfused for 5 min at day, more than once per day, or per week. A subject may also a flow rate of 1.15 ml/min. After perfusion of radiolabeled receive an agent of the composition in the range of 0.1 to molecules, the brain was further perfused for 60s with Krebs 3,000 pig per dose once every two or three weeks. buffer, to wash away excess [**I]-proteins. Mice were then [0340] Single or multiple administrations of the composi decapitated to terminate perfusion and the right hemisphere tions of the invention comprising an effective amount can be was isolated on ice before being subjected to capillary deple carried out with dose levels and pattern being selected by the tion. Aliquots of homogenates, supernatants, pellets, and per treating physician. The dose and administration schedule can fusates were taken to measure their contents and to evaluate be determined and adjusted based on the severity of the dis the apparent volume of distribution. ease or condition in the subject, which may be monitored [0346] From these experiments, brain distribution of both throughout the course of treatment according to the methods exendin-4/Angiopep-2 conjugates was increased 15-50 fold commonly practiced by clinicians or those described herein. over that of unconjugated exendin-4. The brain distribution of [0341] The compounds of the present invention may be exendin-4 was observed at 0.2 ml/100 g/2 min, whereas the used in combination with either conventional methods of conjugate modified at its N-terminal was observed at 3 treatment ortherapy or may be used separately from conven ml/100 g/2 min, and the conjugate modified at its C-terminal tional methods of treatment or therapy. was observed at 10 ml/100 g/2 min. Results are shown in FIG. [0342] When the compounds of this invention are admin 4. istered in combination therapies with other agents, they may Example 3 be administered sequentially or concurrently to an individual. Treatment of Obese Mice with Exendin-4/Angio Alternatively, pharmaceutical compositions according to the pep-2 Conjugates present invention may be comprised of a combination of a compound of the present invention in association with a phar [0347] Obese mice (ob/ob mice) were administered the maceutically acceptable excipient, as described herein, and |Lys”-MHA]exendin-4/Angiopep-2-Cys-NH2 conjugate another therapeutic or prophylactic agent known in the art. (Exen-Anz). In Vivo Study to Determine the Efficacy of Exendin-4-An Example 1 giopep-2 Conjugate Synthesizing GLP-1 Agonist-Angiopep Conjugates [0343] The exemplary GLP-1 conjugates, exendin-4 [0348] cysAn2 N-terminal, and Exendin-4-cysAn2 C-terminal, and Angiopep-1/Exendin 4 conjugates were made by conjugating |Lys(maleimido hexanoic acid)”]exendin-4 to the sulfide in DOSe DOSe DOSe mice/ Q1Dx 28 days cys-An? (SEQID NO:113), in An2-cys (SEQID NO:114), or Groups (ug/kg) (nmol/kg) (Ig/mouse) group (Total amount Ig) in Angiopep-1 (SEQ ID NO:67) in 1zPBS buffer for 1 hour. Control O O O 5 O This resulted in production of exendin-4/Angiopep conju Exendin-4 3 0.72 0.18 5 20.16 gates, as shown in FIG. 2. 30 7.2 1.8 5 201.6 [0344] A second set of exendin-4/Angiopep conjugates Exen-Anz 4.8 0.72 0.288 5 32.256 was made by reacting Angiopep-2 having maleimido propi 48 7.2 2.88 5 322.56 onic acid (MPA), maleimido hexanoic acid (MHA), or male imido undecanoic acid (MUA) bound to its N-terminus with [0349] A 1.6 pg/kg dose of Exen-An2 is equivalent to a 1 [Cys”|Exendin-4 to form a conjugate, as shown in FIG. 3. plg/kg dose of exendin-4. The body weight of each mouse was measured daily. Food intake was estimated based on the mean Example 2 values for each group, and glycemia was measured one hour Brain Uptake of Exendin-4/Angiopep-2 Conjugates following treatment. After 10 days of treatment, body weight In Situ gain and food intake of mice treated at the higher doses of [0345] To measure brain uptake of the exendin-4/Angio either exendin-4 or the conjugate are lower than the control pep-2 conjugates, we used an in situ perfusion assay. The (FIG. 5). Food intake was also reduced in the mice receiving US 2011/0288011 A1 Nov. 24, 2011
the higher doses of either exendin-4 or the conjugate (FIG. 6) the exendin-4-Angiopep-2 dimer conjugate. Mice receiving as compared to the control. either exendin-4 or the conjugate exhibited reduced glycemia [0350] Glycemia measurements showed that the lower as compared to mice receiving the control (FIG. 10). dose of the conjugate had the same effect as the higher doses of either exendin-4 or Exen-Anz (FIG. 7). Surprisingly, a Example 6 similar effect of Vío the dosage on glycemia is observed using Characterization of an Exendin-4-Angiopep-2 Dimer the conjugate, as compared to exendin-4. Conjugate Example 4 [0355] Brain uptake of the exemplary GLP-1 agonist, exen Generation of an Exendin-4-Angiopep-2 Dimer Con din-4, was measured in situ when unconjugated, conjugated jugate to a single Angiopep-2, conjugated to S4, or conjugated to a [0351] Using the conjugation chemistry described herein or dimeric form of Angiopep-2. The experiments were per similar chemistry, an Exendin-4-Angiopep-2 dimer was gen formed as described in Example 2 above. erated having the structure shown in FIG. 8A. Briefly, the [0356] From these results, we observed that conjugation of amine group in the C-terminal lysine of [Lys”|Exendin-4 the exendin-4 analog to the dimeric form of Angiopep-2 was conjugated to an Angiopep-2 dimer through an MHA results in a conjugate with a surprisingly greater ability to linker at the N-terminal threonine of the first Angiopep-2 cross the BBB as compared to either the unconjugated exen peptide. A N-Succinimidyl-5-acetylthiopropionate (SATP) din-4 or to the exendin-4 conjugated to a single Angiopep-2 linker was attached to an Angiopep-2-Cyspeptide at its N-ter (FIG. 8). minus. Through this cysteine, the Angiopep-2-Cys peptide [0357] We also tested the ability of the exendin-4-Angio was conjugated to a second Angiopep-2 peptide, which had pep-2 dimer conjugate to reduce glycemia in DIO mice. Mice been modified to contain an MPA linker. The dimer was the were injected with a bolus containing a control, exendin-4, or linked to the [Lys”|Exendin-4 through an MHA linker A the exendin-4-Angiopep-2 dimer conjugate. Mice receiving control molecule (Exen-S4) was also generated using a either exendin-4 or the conjugate exhibited reduced glycemia scrambed form of Angiopep-2 conjugated at its N-terminal to as compared to mice receiving the control (FIG. 9). the cysteine of [Cys”|Exendin-4 through an MHA linker (FIG.8B). These conjugates were prepared as trifluoroacetate Example 7 (TFA) salts. Pancreatic Uptake and Insulin Response of Exen-4 Example 5 An2 Conjugate Characterization of an Exendin-4-Angiopep-2 Dimer [0358] We also tested the brain and pancreatic uptake of Conjugate both Exendin-4 and the Exen-4-An2 conjugate in mice 15 minutes following an intravenous bolus injection of either [0352] Brain uptake of the exemplary GLP-1 agonist, exen compound. As seen in FIG. 11A, brain uptake was enhanced din-4, was measured in situ when unconjugated, conjugated in the Exen-4-An2 as compared to the unconjugated Exen to a single Angiopep-2, conjugated to a scrambled Angio din-4 peptide, whereas similar levels of pancreatic concen pep-2 (S4), or conjugated to a dimeric form of Angiopep-2. tration were observed with both compounds. The experiments were performed as described in Example 2 [0359] The ability of either Exendin-4 or Exen-4-An2 to above. induce an increase in insulation secretion was measured using [0353] From these results, we observed that conjugation of RIN-m$F pancreatic cells. As shown in FIG. 12, the conju the exendin-4 analog to the dimeric form of Angiopep-2 gate, at all concentrations tested, surprisingly exhibited a results in a conjugate with a surprisingly greater ability to stronger level of insulin secretion, as compared to Exendin-4. cross the BBB as compared to either the unconjugated exen din-4 or to the exendin-4 conjugated to a single Angiopep-2 Example 8 (FIG. 9). [0354] We also tested the ability of the exendin-4-Angio Synthesis of a Leptin Conjugate pep-2 dimer conjugate to reduce glycemia in DIO mice. Mice [0360] The following procedure was used to generate a were injected with a bolus containing a control, exendin-4, or Leptin-(C11)-AN2 conjugate.
O O
Leptin(116-130)-NH2 + N N (Cys-117) N H &O PBS 4X pH 6.30 room temperature 1 hour US 2011/0288011 A1 Nov. 24, 2011 46
-continued O O
s / TN AJJF} Leptin(116-130)-NH2 (Cys-117) O
[0361] MUA-AN2 (264.6 mg, 91.5 pmol, 1.2 eq., 82% peptide content) was dissolved in H2O/ACN (9/1) (14 ml) by adjusting pH from 3.9 to 5.00 with addition of a 0.1 NNaOH solution (1.5 ml). This solution was added to a solution of Time Flow Lepting iso-NH2 (156.5 mg, 76.2 pumol 1 eq., 76% peptide (min) (ml/min) % A % B Curve content) in PBS 4× (pH 6.61, 7 mL). Monitoring of the 0.5 90 10 0.4 0.5 90 10 6 reaction was done with the analytical method described 0.7 0.5 70 30 6 below. Results are shown in FIGS. 13A and 13B (chromato 2.2 0.5 30 70 6 grams 1 and 2). 2.4 0.5 10 90 6 [0362] A cloudy suspension was observed as the reaction 2.7 0.5 10 90 6 went to completion. After 1 h at room temperature, the reac 2.8 0.5 90 10 6 tion (3.62 mM) was complete and the mixture was purified 2.81 0.5 90 10 6 immediately by FPLC chromatography (AKTAexplorer, see chromatogram 3, Table 1). Purification was performed on a [0366] From mass spectroscopy (ESI-TOF-MS; Bruker GE Healthcare AKTA explorer column (GE Healthcare) 30 Daltonics): calculated 4125.53, found 4125.06, m/z 1376.01 RPC resin (polystyrene/divinylbenzene), 95 ml, sample load: (+3), 1032.26 (+4), 826.02 (+5), 688.52 (+6). 450 mg in reaction buffer (21 ml), 10% ACN in H2O, 0.05% [0367] The conjugate was stored under nitrogen atmo TFA (60 ml), DMSO.HCl (pH 2.87, 6 ml), Solution A: H2O, sphere, in a dark room, below —20°C. 0.05% TFA, Solution B: ACN, 0.05% TFA, Flow: 5-17 [0368] The leptin conjugate generated using the procedure ml/min, Gradient: 10-30% B. is called Leptin-AN2 (C11), due its 11-carbon linker. Other [0363] Purification results are shown in FIG. 14 (chromato length carbon linker conjugates, were also generated, includ gram 3). The gradient used to purify the compound is shown ing Leptin-AN2 (C3) and Leptin AN2 (C6) using similar in the table below. procedures. Example 9 Volume Column Flow rate (ml) volume (C.V.) (ml/min) % Solvent B In Situ Brain Perfursion of Leptin, i.e., so Angiopep-2 O O 5 10 Conjugates 33.58 0.35 10 10 186.98 1.61 15 10 282.51 1.01 15 15.0 (over 3 min) [0369] To determine which of the leptin conjugates most 346.26 0.67 16 15 effectively crossed the blood–brain barrier, we tested each 366.68 0.21 17 15 conjugate in the in situ brain perfusion assay. This assay is or 625.3 2.72 17 20.0 (over 5 min) a similar assay is described, for example, in U.S. Patent 876.28 2.64 17 22.5 (over 2 min) 1970.49 11.52 17 25.0 (over 1 min) Publication No. 20060189515, which was based on a method 2233.45 2.77 17 30.0 (over 1 min) described in Dagenais et al., 2000, J. Cereb. Blood Flow 2488.68 2.69 17 40.0 (over 0.5 min) Metab. 2002):381-386. The BBB transport constants were 2577.28 0.93 17 95.0 (over 1 min) determined as previously described by Smith (1996, Pharm. 2777.41 2.11 17 10.0 (over 0.5 min) Biotechnol. 8:285-307). From these experiments, Leptin AN2 (C11) exhibed the greatest transport across the BBB as [0364] After evaporation of acetonitrile and lyophilization, compared to the conjugates having C3 or a C6 linker and was a white solid (250 mg, 79%, purity»98%) was obtained. The thus selected for further experimentation (FIG. 15). mass was checked by ESI-TOFMS (Bruker Daltonics). To [0370] Transport of leptin was compared to the Leptin avoid the possibility that some remaining Leptin(116-130) AN2 (C11) conjugate using the in situ perfusion assay in lean NH, might dimerize (<5%, cysteine peptide Mw-3119.44), and diet-induced obese (DIO) mice (available, e.g., from the immediate purification was performed and an 1.2 equivalent Jackson laboratories). From these results, transport of leptin excess of maleimido-(C11)-AN2 was used. across the BBB in DIO mice was reduced as compared to in [0365] To monitor the reaction, the following analytical lean mice. By contrast, the Leptin-AN2 (C11) conjugate method was used. A Waters Acquity HPLC system with a crossed the brain much more efficiently in both lean and DIO Waters Acquity HPLC BEH phenyl column was used (1.7 mice, and no statistically significant difference between the pum, 2.1×50 mm). Detection was performed at 229 mm. Solu lean and DIO mice in transport of the conjugate was observed tion A was H2O, 0.1% FA, and Solution B was acetonitrile (FIG. 16A). Plasma leptin levels were observed to increase (ACN), 0.1% formic acid (FA). Flow and gradient are shown after 3 weeks on a high fat (60%) diet, suggesting that the in the Table below. mice were becoming leptin resistant (FIG. 16B). US 2011/0288011 A1 Nov. 24, 2011 47
Example 10 tion schemes for the fusion protein are shown in FIGS. 29A and 29B. Results from a small scale purification are shown in Effect of Leptin Conjugates on Food Intake and FIG. 30. Weight Gain [0378] The thrombin cleavage step resulted in production [0371] Mice were injected with an intravenous bolus of of two products, suggesting the possibility that the Angio either Leptin-AN2 (C11) (eq. of 1 mg of leptin, 16 so per pep-2 sequence contains a low-affinity thrombin cleavage mouse), leptin, 16-iso (1 mg/mouse), or a control (Saline) (n=5 site, as shown in FIG. 31. As the leptin-Angiopep-2 has a per group). Food intake of the mice was monitored at 4 hours propensity to agregate in solution, purification conditions to (FIG. 17A) and at 15 hours (FIG. 17B). In both cases, the reduce the aggregation and improve yields are being tested. conjugate exhibited significantly greater reduction in food intake, as compared to either the control mice, or mice receiv Example 12 ing leptin, 16.130. [0372] We also compared weight changes in DIO mice Brain Uptake and Activity of Leptin Fusion Proteins receiving the conjugate (2.5 mg/mouse; equivalent of 1 mg [0379] We then examined the ability of the Angiopep-2 leptin, 16-130 mg/mouse), leptin, 1.6-iso (1 mg/mouse), and a leptinfusion protein to be taken up into the parenchyma of the control over a period of six days. Each mouse received daily brain of DIO mice as compared to leptin using the in situ brain treatment by intraperitoneal injection. Mice receiving leptin perfusion assay (FIG. 32). From this experiment, we or the control exhibited similar amounts of weight gain over observed that the fusion protein exhibited increased uptake as the six days, whereas mice receiving the conjugate showed compared to leptin. marked reduction in weight gain (FIG. 18) as compared to the [0380] As a control, we tested the ability of recombinant control mice and mice receiving leptin, 16-1so. leptin to reduce body weight in ob/ob mice using either 0.1 [0373] We further compared weight changes in leptin-de mg/mouse or 0.25 mg/mouse daily. As shown in FIG. 33, ficient ob/ob mice receiving the conjugate (2.5 mg/mouse; leptin did indeed reduce body weight in these mice in a equivalent of 1 mg leptin, 16-1so mg/mouse), leptin, 16-iso (1 dose-dependent manner. mg/mouse), and a control over a period of six days. Each [0381] DIO mice were also treated with a control or with 50 mouse (n=5 per group) received daily treatment by intraperi pig his-tagged fusion protein, leptin, or the his-tagged leptin. toneal injection. The mice receiving the conjugate exhibited Mice received two treatments, on days three and four as lower weight gain than the mice receiving either leptin, 16-1so indicated. Based on these results, the greatest weight loss was or the control (FIG. 19) during the period of administration. observed in mice receiving the fusion protein (FIG. 34). Example 11 Example 13 Development of Recombinant Angiopep-2 and Angiopep-2 Leptin Fusion Proteins Synthesis of a Neurotensin-Angiopep-2 Conjugate [0374] We also developed an Angiopep-2 fusion protein. [0382] An exemplary neurotensin-Angiopep-2 conjugate As an initial step, a cDNA (ACC TTTTTCTAT GGC GGC was synthesized using the scheme described below. As used AGC CGT GGC AAA CGC AACAATTTCAAG ACC GAG in these examples, the abbreviation NT refers to the pH GAGTAT, SEQID NO:117) was created. This sequence was substituted neurotensin peptide described below. inserted into a p(3EX vector system for bacterial expression, and sequence of the insert was verified (FIG. 20). The GST An2-Leptin, 16-1so construct was made using an overlap Fmoc-Xaa —-1) HBTU/NMM extension PCR strategy (FIG. 21). 2) 20% pip. [0375] The recombinant Angiopep-2 was expressed in a SPPS bacterial expression system and purified using a GSH TFA/H2O/TES Sepharose column. A chromatogram from this procedure is pELYENKPRRPYIL –º shown (FIG. 22). The purified Angiopep-2 was analyzed by 61% Western blot using an Angiopep-2 antibody (FIG. 23A), by liquid chromatography (FIG. 23B), and by mass spectros copy (FIG. 23C). [0376] The in situ brain perfusion assay was performed using recombinant Angiopep-2. The results were compared to NH2 synthetic Angiopep-2 (FIG.24). Similar levels of uptake were pELYENKPRRPYIL-OH observed with both forms of Angiopep-2. Uptake into the Sulfo-EMCS parenchyma between GST, GST-Angiopep-2, GST-Lep PBS 4X, pH 7.61 tin, 16-1so, and GST-Angiopep-2-Leptin, 16-1so was compared rt, 2h (FIG. 25). These results show that fusion proteins containing N O the Angiopep-2 sequence are efficiently taken up into the NaO3S parenchyma, whereas proteins lacking the Angiopep-2 sequence are taken up much less efficiently. NH2 N OH [0377] A His-tagged Angiopep-2/mouse leptin fusion pro Neurotensin (NT) tein containing the full length leptin sequence has been gen C78H121N21O20 O erated (FIG. 26). This fusion protein has been expressed in a Mol. Wt:1672.92 bacterial expression system (FIG. 27). Exemplary purifica US 2011/0288011 A1 Nov. 24, 2011 48
ESI-TOF MS (Bruker Daltonics; calculated 1672.92. found -continued 1671.90, m/z 558.31 (+3), 836.96 (+2). AN2-Cys-NH2 –º (0387] EMCS-NT pELYENKPRRPYIL-OH PBS 4X, pH 6.42 [0388] The N-lysine primary amine of NT was activated by rt, 1 h treating a solution of NT (25 mg, 14.9 pumol, 1 eq. in 3.5 ml of 9% over 2 steps PBS 4×, pH 7.64), with a solution of sulfo-EMCS(N-?e maleimidocaproyloxylsulfosuccinimide ester) (Pierce Bio O technology) (6.1 mg, 14.9 pumol, 1 eq. in 1 ml of PBS 4×). HN sº N Monitoring of the reaction was done with the analytical O / method described below (see chromatograms 1-2 in FIGS. O 35A and 35B). The reaction (3.32 mM, pH 7.61) allowed EMCS-NT proceeding at room temperature for 1 h. The modification was C88H133N22O23 repeated once for 1 h with addition of sulfo-EMCS (4.5 mg, 10.9 pmol, 0.73 eq. in 1 ml of PBS 4×). The mixture was Mol. Wt: 1867,13 purified by FPLC chromatography (AKTA explorer, see pELYENKPRRPYIL-OH chromatogram 3 in FIG. 36). Purification of EMCS-NT was performed on a column containing 30 RPC resin (polysty rene/divinyl benzene), 30 ml. Sample was loaded as 35 mg in reaction buffer (4 ml), 10% acetonitrile (ACN) in H2O, 0.05% TFA (200 ml). Solution A was H2O, 0.05%TFA, and Solution B was ACN, 0.05% TFA. Flow rate 5-9 ml/min with a gradi ent of 10–25% of Solution B. [0389] After the acetonitrile was evaporated, the volume of water was reduced to 5 ml for the next step. A colorless solution of the pure EMCS-modified NT (purity-98%) was obtained. The mass was checked by ESI-TOF MS (Bruker Daltonics), was calculated to be 1867.13, and was found to be 1866.00, m/z 623.01 (+3), 934.00 (+2). [0391] Conjugation was performed with the maleimido containing EMCS-NT and the free thiol residue of AN2Cys [0383] Neurotensin Peptide Synthesis NH2. The pH of the solution of EMCS-NT was adjusted from 1.65 to 6.42 by a slow addition of a 0.1N NaOH solution. A [0384] pHLYENKPRRPYIL-OH, where the unusual hydrolysis side reaction can occur during adjustment of pH amino acid L-pyroglutamic acid (pH) is used, was synthe (=5%, hydrolyzed EMCS-NT Mw-1833). A solution of sized using SPPS (Solid phase peptide synthesis). SPPS was AN2Cys-NH2 (46.4 mg, 14.9 pumol, 1 eq. in 2.5 ml of PBS 4×, carried out on a Protein Technologies, Inc. Symphony R pep pH 7.64) was added to the solution of EMCS-NT. The ana tide synthesizer using Fmoc (9-fluorenylmethyloxycarbonyl) lytical method below was used to monitoring the reaction (see amino-terminus protection. The peptide was synthesized on a chromatograms 4-5 in FIGS. 37A and 37B). The reaction (1.9 100 pumol scale using a 5-fold excess of Fmoc-amino acids mM, pH 6.3) was allowed to proceed at room temperature for 30 minutes. The mixture was purified by FPLC chromatog (200 mM) relative to the resin. Coupling was performed by a raphy (AKTA explorer, see chromatogram 6 in FIG. 38). pre-loaded Fmoc-Leu-Wang resin (0.48 mmol/g) for car Purification of NT-AN2Cys-NH2 was performed using a col boxyl-terminus acids using 1:1:2 amino acid/activator/NMM umn (GE Healthcare) containing 30 RPC resin (Polystyrene/ in DMF with HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tet divinyl benzene), 30 ml, Sample was loaded in the amount of ramethyluronium hexafluorophosphate) and NMM (N-meth 74 mg in 4 ml reaction buffer (10% ACN in H2O, 0.05% TFA ylmorpholine). Deprotection was carried out using 20% pip (200 ul)). Solution A was H2O, 0.05% TFA, and Solution B eridine/DMF. The resin-bound product was routinely cleaved was ACN, 0.05%TFA. The flow rate was 5-9 ml/min, using a using a solution comprised of TFA/water/TES:95/2.5/2.5 for gradient of 10% to 25% of Solution B. 2 hours at room temperature. [0392] After evaporation of acetonitrile and lyophilization, [0385) Pre-loaded Fmoc-Leu-Wang resin (0.48 mmol/g) the conjugated NT-AN2Cys-NH2 was obtained as a pure was purchased from ChemPep, Fnoc-amino acids, HBTU white solid (5.5 mg, 9% over 2 steps, purity-95%). The mass was confirmed by ESI-TOFMS (Bruker Daltonics); MW was from Chemimpex, and the unusual L-pyroglutamic acid from calculated to be 4270.76 and was found to be 4269.17 (m/z Sigma-Aldrich. Side protecting groups for amino acids were 712.54 (+6), 854.84 (+5), 1068.29 (+4), 1424.04 (+3)). Trt (trityl)for aspargine, tRu (ter-butyl)for glutamic acid and [0393] The conjugate was stored under nitrogen atmo tyrosine, Pbf (pentamethyldihydrobenzofuran-5-sulfonyl) sphere, below —20° C. for arginine, and tRoc (tRutyloxycarbonyl) for lysine. [0394] Analytical Method [0386] The crude peptide was precipitated using ice-cold [0395] The following method was used as described above. ether, and purified by RP-HPLC chromatography (Waters To analyze samples during purification, a Waters Acquity Delta Prep 4000). Acetonitrile was evaporated from the col HPLC system was employed with a BEH phenyl column, 1.7 lected fractions and lyophilized to give a pure white solid pum, 2.1×50mm Detection was performed at 229 mm. Solution (204 mg, 61%, purity-98%). The mass was confirmed by A was H2O, 0.1% FA, and Solution B was acetonitrile (ACN),
US 2011/0288011 A1 Nov. 24, 2011 50
Mw Qtty Name Sequence (g/mol) (mg) NT native pELYENKPRRPYIL 1672.97 800, ~95% NT(8-13) RRPYIL 816.99 45, -95% Ac-LysNT H 987.20 78, -95% (8-13)- Dr. N RRPYIL O
NH2 Ac-Lys-[D- H 987.20 55, -95% Tyr"INT N RRPD-YIL (8-13) O
NH2 pGlu-LysNT 1056.26 86, =95% 8–13 (8-13) O { RRPYIL N H O
NH2 MHA-NT O 1010.19 55, -95% (8-13) pººr RRPYIL N O O É- O 1088.32 12, 3.95% mercaptoMHA NT(8-13) (desactive) S pººr /~/ ~ O RRPYIL HO O
[0408] NT and the NT(8-13) analogs were synthesized by Fmoc-AA (200 mM) relative to the resin. Coupling was per using a SPPS method on a Protein Technologies, Inc. Sym formed from a pre-loaded Fmoc-Leu-Wang resin (0.48 phony R peptide synthesizer and Fmoc chemistry. Pre-loaded mmol/g) for carboxyl-terminus acids using 1:1:4AA/HBTU/ NMM in DMF. Deprotection was carried out using 20% Fmoc-Leu-Wang resin (0.48 mmol/g) was purchased from piperidine/DMF. The resin-bound product was routinely ChemPep, Frnoc-amino acids, HBTU from Chemimpex, the cleaved using a cocktail solution comprised of TFA/water/ unusual pH from Sigma-Aldrich, unnatural D-Tyrosine from TES:95/2.5/2.5 for 2 h at room temperature. Chemimpex, Sulfo-EMCS from Pierce Biotechnology. Side [0410] The crude peptide was precipitated using ice-cold protecting groups for amino acids were Trt for aspargine, tRu ether and was purified by RP-HPLC chromatography, Waters for glutamic acid and tyrosine, Pbffor arginine, and thoc for Delta Prep 4000, Kromasil 100-10-C18, H2O/ACN with lysine. Mass was confirmed by ESI-TOF MS (MicroT of 0.05% TFA (“Method A"). Acetonitrile was evaporated from Bruker Daltonics). the collected fractions and lyophilized. This resulted in the [0409] General procedure—Synthesis of Neurotensin (NT) formation of a white and fluffy solid, 800 mg, 80% yield, (pHLYENKPRRPYIL-OH). NT was synthesized using the purity HPLC-98%, calc. 1672.92. found 1671.90, m/z 558. unusual L-pyroglutamic acid (pH) and a 5 fold excess of 31 (+3), 836.96 (+2).