RECEPTOR-TARGETING PEPTIDE-DRUG CONJUGATES

Abstract

The present invention refers to NPY Y1 receptor-targeting peptide moieties, and their use for the target-specific treatment of cancers and other diseases.

Claims

1. A compound having the following formula (I):
R.sup.1-Tyr-Pro-Ser-Lys-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Xaa.sup.33-Pro-Xaa.sup.35-Xaa.sup.36-NH.sub.2   (I) wherein R.sup.1 is hydrogen or an acyl group; Xaa.sup.33 is Arg or a group of formula —N(R.sup.2)—CH(R.sup.3)—(CH.sub.2).sub.n—C(═O)—, wherein R.sup.2 is hydrogen or a methyl group, R.sup.3 is hydrogen or a linear or branched C.sub.1-8 alkyl group and n is 0 or 1; Xaa.sup.35 is Arg or a group of formula —N(R.sup.4)—CH(R.sup.5)—(CH.sub.2).sub.m—C(═O)—, wherein R.sup.4 is hydrogen or a methyl group, R.sup.5 is hydrogen or a linear or branched C.sub.1-8 alkyl group and m is 0 or 1; and Xaa.sup.36 is Tyr or a group of formula —N(R.sup.6)—CH(R.sup.7)—(CH.sub.2).sub.p—C(═O)—, wherein R.sup.6 is hydrogen or a methyl group, R.sup.7 is hydrogen or a linear or branched C.sub.1-8 alkyl group and p is 0 or 1; with the proviso that Xaa.sup.33 is not Arg, when Xaa.sup.35 is Arg and Xaa.sup.36 is Tyr; or a salt thereof.

2. The compound according to claim 1 having the following formula (I):
R.sup.1-Tyr-Pro-Ser-Lys-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Xaa.sup.33-Pro-Xaa.sup.35-Xaa.sup.36-NH.sub.2   (I) wherein R.sup.1 is hydrogen or an acyl group; Xaa.sup.33 is a group of formula —N(R.sup.2)—CH(R.sup.3)—(CH.sub.2).sub.n—C(═O)—, wherein R.sup.2 is hydrogen or a methyl group, R.sup.3 is hydrogen or a linear or branched C.sub.1-8 alkyl group and n is 0 or 1; Xaa.sup.35 is a group of formula —N(R.sup.4)—CH(R.sup.5)—(CH.sub.2).sub.m—C(═O)—, wherein R.sup.4 is hydrogen or a methyl group, R.sup.5 is hydrogen or a linear or branched C.sub.1-8 alkyl group and m is 0 or 1; and Xaa.sup.36 is a group of formula —N(R.sup.6)—CH(R.sup.7)—(CH.sub.2).sub.p—C(═O)—, wherein R.sup.6 is hydrogen or a methyl group, R.sup.7 is hydrogen or a linear or branched C.sub.1-8 alkyl group and p is 0 or 1; or a salt thereof.

3. A compound having the following formula (II):
R.sup.1-Tyr-Pro-Ser-Lys(14.sup.8)-Pro-A sp-Phe-Pro-Gly-Glu-A sp-Ala-Pro-Ala-Glu-A sp-Leu-Ala-Arg-Tyr-Tyr-S er-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Xaa.sup.33-Pro-Xaa.sup.35-Xaa.sup.36-NH.sub.2   (II) wherein R.sup.1 is hydrogen or an acyl group; Xaa.sup.33 is Arg or a group of formula —N(R.sup.2)—CH(R.sup.3)—(CH.sub.2).sub.n—C(50 O)—, wherein R.sup.2 is hydrogen or a methyl group, R.sup.3 is hydrogen or a linear or branched C.sub.1-8 alkyl group and n is 0 or 1; Xaa.sup.35 is Arg or a group of formula —N(R.sup.4)—CH(R.sup.5)—(CH.sub.2).sub.m—C(═O)—, wherein R.sup.4 is hydrogen or a methyl group, R.sup.5 is hydrogen or a linear or branched C.sub.1-8 alkyl group and m is 0 or 1; Xaa.sup.36 is Tyr or a group of formula —N(R.sup.6)—CH(R.sup.7)—(CH.sub.2).sub.p—C(═O)—, wherein R.sup.6 is hydrogen or a methyl group, R.sup.7 is hydrogen or a linear or branched C.sub.1-8 alkyl group and p is 0 or 1; and R.sup.8 is bound to the nitrogen atom at the side chain of the lysine (NE) and is selected from the following groups: R.sup.9-Cys- and R.sup.9-Cys-βAla-, wherein R.sup.9 is hydrogen or an acyl group; with the proviso that Xaa.sup.33 is not Arg, when Xaa.sup.35 is Arg and Xaa.sup.36 is Tyr; or a salt thereof.

4. The compound according to claim 3 having the following formula (II):
R.sup.1-Tyr-Pro-Ser-Lys(R.sup.8)-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-S er-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Xaa.sup.33-Pro-Xaa.sup.35-Xaa.sup.36-NH.sub.2   (II) wherein R.sup.1 is hydrogen or an acyl group; Xaa.sup.33 is a group of formula —N(R.sup.2)—CH(R.sup.3)—(CH.sub.2).sub.n—C(═O)—, wherein R.sup.2 is hydrogen or a methyl group, R.sup.3 is hydrogen or a linear or branched C.sub.1-8 alkyl group and n is 0 or 1; Xaa.sup.35 is a group of formula —N(R.sup.4)—CH(R.sup.5)—(CH.sub.2).sub.m—C(═O)—, wherein R.sup.4 is hydrogen or a methyl group, R.sup.5 is hydrogen or a linear or branched C.sub.1-8 alkyl group and m is 0 or 1; Xaa.sup.36 is a group of formula —N(R.sup.6)—CH(R.sup.7)—(CH.sub.2).sub.p—C(═O)—, wherein R.sup.6 is hydrogen or a methyl group, R.sup.7 is hydrogen or a linear or branched C.sub.1-8 alkyl group and p is 0 or 1; and R.sup.8 is bound to the nitrogen atom at the side chain of the lysine (Nε) and is selected from the following groups: R.sup.9-Cys- and R.sup.9-Cys-βAla-, wherein R.sup.9 is hydrogen or an acyl group; or a salt thereof.

5. The compound according to claim 1, wherein R.sup.1 is hydrogen or an acetyl group and Xaa.sup.33, Xaa.sup.35 and Xaa.sup.36 are independently selected from alanine (Ala; A), valine (Val; V), leucine (Leu; L), isoleucine (Ile; I), beta-alanine (βAla; βA), N-methyl-alanine (N-Me-Ala), norvaline (Nva), norleucine (Nle), β-homo-leucine (β-homo-Leu), β-homo-isoleucine β-homo-Ile), N-methyl-isoleucine (N-Me-Ile), and N-methyl-norleucine (N-Me-Nle).

6. A compound according to claim 3, wherein R.sup.9 is selected from the following groups: palmitoyl, tetradecanoyl, dodecanoyl, decanoyl, octadecanoyl or acetyl; preferably from palmitoyl and dodecanoyl; especially preferably, R.sup.9 is palmitoyl.

7. A compound according to claim 1, wherein the compound is:
H-Tyr-Pro-Ser-Lys-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Ala-Pro-Ala-Ala-NH.sub.2; or
Acetyl-Tyr-Pro-Ser-Lys-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Ala-Pro-Ala-Ala-NH.sub.2; or a salt thereof.

8. A compound of formula (III):
Pep-L-Z   (III) wherein Pep is a compound of formula (II′)
R.sup.1-Tyr-Pro-Ser-Lys(R.sup.8)-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Xaa.sup.33-Pro-Xaa.sup.35-Xaa.sup.36-NH.sub.2   (II′) wherein R.sup.1 is hydrogen or an acyl group; Xaa.sup.33 is Arg or a group of formula —N(R.sup.2)—CH(R.sup.3)—(CH.sub.2).sub.n—C(═O)—, wherein R.sup.2 is hydrogen or a methyl group, R.sup.3 is hydrogen or a linear or branched C.sub.1-8 alkyl group and n is 0 or 1; Xaa.sup.35 is Arg or a group of formula —N(R.sup.4)—CH(R.sup.5)—(CH.sub.2).sub.m—C(═O)—, wherein R.sup.4 is hydrogen or a methyl group, R.sup.5 is hydrogen or a linear or branched C.sub.1-8 alkyl group and m is 0 or 1; Xaa.sup.36 is Tyr or a group of formula —N(R.sup.6)—CH(R.sup.7)—(CH.sub.2).sub.p—C(═O)—, wherein R.sup.6 is hydrogen or a methyl group, R.sup.7 is hydrogen or a linear or branched C.sub.1-8 alkyl group and p is 0 or 1; with the proviso that Xaa.sup.33 is not Arg, when Xaa.sup.35 is Arg and Xaa.sup.36 is Tyr; and R.sup.8 is bound to the nitrogen atom at the side chain of the lysine (Nε) and is selected from the following groups: R.sup.9-Cys- and R.sup.9-Cys-βAla-, wherein R.sup.9 is hydrogen or an acyl group; wherein the hydrogen atom at the SH moiety of Cys at group R.sup.8 is replaced by the bond to L; L is a linker between Pep and Z; and Z is a natural or synthetic tubulysin derivative wherein one hydrogen atom or one OH group has been replaced by the bond to L; or a salt thereof.

9. The compound of formula (III) according to claim 8:
Pep-L-Z   (III) wherein Pep is a compound of formula (II′)
R.sup.1-Tyr-Pro-Ser-Lys(R.sup.8)-Pro-A sp-Phe-Pro-Gly-Glu-A sp-Ala-Pro-Ala-Glu-A sp-Leu-Ala-Arg-Tyr-Tyr-S r-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Xaa.sup.33-Pro-Xaa'-Xaa.sup.36-NH.sub.2   (II′) wherein R.sup.1 is hydrogen or an acyl group; Xaa.sup.33 is a group of formula —N(R.sup.2)—CH(R.sup.3)—(CH.sub.2).sub.n—C(═O)—, wherein R.sup.2 is hydrogen or a methyl group, R.sup.3 is hydrogen or a linear or branched C.sub.1-8 alkyl group and n is 0 or 1; Xaa.sup.35 is a group of formula —N(R.sup.4)—CH(R.sup.5)—(CH.sub.2).sub.m—C(═O)—, wherein R.sup.4 is hydrogen or a methyl group, R.sup.5 is hydrogen or a linear or branched C.sub.1-8 alkyl group and m is 0 or 1; Xaa.sup.36 is a group of formula —N(R.sup.6)—CH(R.sup.7)—(CH.sub.2).sub.p—C(═O)—, wherein R.sup.6 is hydrogen or a methyl group, R.sup.7 is hydrogen or a linear or branched C.sub.1-8 alkyl group and p is 0 or 1; and R.sup.8 is bound to the nitrogen atom at the side chain of the lysine (Nε) and is selected from the following groups: R.sup.9-Cys- and R.sup.9-Cys-≢Ala-, wherein R.sup.9 is hydrogen or an acyl group; wherein the hydrogen atom at the SH moiety of Cys at group R.sup.8 is replaced by the bond to L; L is a linker between Pep and Z; and Z is a natural or synthetic tubulysin derivative wherein one hydrogen atom or one OH group has been replaced by the bond to L; or a salt thereof.

10. The compound according to claim 8, wherein L is selected from the following groups:
—CH.sub.2—CH.sub.2——;
—O—CH.sub.2—CH.sub.2—S—;
—NH—CH.sub.2—CH.sub.2—S—; or
—NH—NH—C(═O)—O—CH.sub.2—CH.sub.2—S—; wherein the sulphur of L is bound to the sulphur of the Cys at group R.sup.8.

11. The compound according to claim 8, wherein Z is a compound of formula (IV): ##STR00018## wherein q is 0, 1 or 2; R.sup.1° is an alkyl, acyl or a heteroalkyl group; R.sup.11 is an optionally substituted alkyl, alkenyl, alkinyl, acyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; R.sup.12 is hydrogen or an optionally substituted alkyl, alkenyl, alkinyl, acyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; R.sup.13 is a group of formula -COOH, -CONH2, -CONHNH2 or -CH2OH or a group of the following formula: ##STR00019## wherein r is 0 or 1; R.sup.14 is hydrogen or an optionally substituted C.sub.1-6 alkyl group or an optionally substituted aryl or heteroaryl group; and R.sup.15 is a group of formula —COOH, —CONH.sub.2, —CONHNH.sub.2 or —CH.sub.2OH; and Ar is an optionally substituted arylene or heteroarylene group; wherein one OH group of a COOH group or one hydrogen atom has been replaced by the bond to L.

12. The compound according to claim 8, wherein Z has the following formula: ##STR00020## wherein R11 is hydrogen, a C.sub.1-6 alkyl group, or a group of formula —CH.sub.2—O—C(═O)—R.sup.17; wherein R.sup.17 is a C.sub.1-6 alkyl group or a C.sub.2-6 alkenyl group or an aryl group or a heteroaryl group; R.sup.12 is a C.sub.1-6 alkyl group or an acetyl group; and R.sup.16 is hydrogen, halogen, OH, NO.sub.2, NH.sub.2, CN, C.sub.1-6 alkyl, —O—C.sub.1-6 alkyl, phenyl, -NH-C1-6 alkyl or —N(C.sub.1-6 alkyl).sub.2.

13. The compound according to claim 8, wherein Z has the following formula: ##STR00021## wherein R.sup.17 is hydrogen, or an alkyl, alkenyl, aryl or heteroaryl group and R.sup.16 is hydrogen or a hydroxy group.

14. A pharmaceutical composition containing a compound according to claim 8 and optionally one or more carriers and/or adjuvants.

15. A method of treating a cancer comprising administering a compound according to claim 8 to a subject.

16. The compound according to claim 3, wherein R.sup.1 is hydrogen or an acetyl group and Xaa.sup.33, Xaa.sup.35 and Xaa.sup.36 are independently selected from alanine (Ala; A), valine (Val; V), leucine (Leu; L), isoleucine (Ile; I), beta-alanine (βAla; βA), N-methyl-alanine (N-Me-Ala), norvaline (Nva), norleucine (Nle), β-homo-leucine (β-homo-Leu), β-homo-isoleucine (β-homo-IIe), N-methyl-isoleucine (N-Me-Ile), and N-methyl-norleucine (N-Me-Nle).

17. A compound according to claim 3, which the compound is:
H-Tyr-Pro-Ser-Lys (Palmitoyl-Cys-(3Ala)-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Ala-Pro-Ala-Ala-NH.sub.2; or
Acetyl-Tyr-Pro-Ser-Lys (Palmitoyl-Cys-(βAla)-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-A sn-Leu-Ile-Thr-Ala-Pro-Ala-Ala-NH.sub.2; or a salt thereof.

Description

EXAMPLES

[0062] The following derivatives were synthesized from building blocks Z′, L′ and Pep′. The building blocks were synthesized according to methods known to a person skilled in the art.

Automated Solid Phase Peptide Synthesis of Pep

[0063] The peptide moieties (Pep′) of the peptide-drug conjugates Z-L-Pep (formula III) were synthesized according to the Fmoc/tBu protection strategy using an automated multiple solid-phase peptide synthesizer Syro II (MultiSynTech GmbH, Bochum, Gelmany). To gain C-terminal peptide amides, a Rink amide resin with a loading capacity of 0.63 mmol/g was used.

[0064] The stepwise synthesis of the complete peptide chains from building blocks is a perseverative cycle of few reactions, i.e. N.sup.α-deprotection, amino acid coupling, and some washing steps. In brief, prior to each single amino acid coupling step the base-labile N.sup.α-protecting group Fmoc had to be cleaved off from the building blocks, and in a first step from the Rink amide resin as well. For Fmoc cleavage, 400 μL piperidine in DMF (40% v/v) were added to the resin and incubated for 3 min while stirring. The deprotection was repeated with 400 μL piperidine in DMF (20% v/v) for 10 min. Subsequently, the resin was washed with 433 600 μL DMF.

[0065] Amino acids were coupled by preincubation of the resin with 200 μL amino acid building block solution (0.5 M in DMF) and 100 μL 3 M Oxyma in DMF for 2 min. Subsequently, 100 μL 3.3 M DIC in DMF were added and the reaction was allowed to proceed for 40 min while stirring. After a washing step with 800 μL DMF, the coupling step was repeated once for each amino acid.

[0066] The synthesis of branched peptides was realized by amino acid coupling and sequence elongation, thus sequence branching, at the N.sup.ε of lysine. To allow its selective deprotection, a lysine building block with Dde-protected N.sup.ε was used. For the selective deprotection of a Dde-protected lysine residue, the fully protected, resin-bound peptide was incubated 12×10 min with 1 mL freshly prepared 3% hydrazine in DMF. After each of the 12 steps, the resin was washed with DMF. Finally, the success of the Dde deprotection was checked by measuring the absorption of the removed hydrazine solution at 301 nm against a reference of fresh hydrazine in DMF. The Dde deprotection was completed if the absorption was <0.1. Otherwise, some more cycles of hydrazine treatment and washing had to be conducted.

Analytical and Preparative Peptide (Pep′) Cleavage from Resin

[0067] For analytical purposes, small amounts of newly synthesized peptides were cleaved off from the resin. Therefore, a small amount of peptide-loaded resin was incubated with TFA/thioanisole/1,2-ethanedithiol (900:70:30 v/v) for 3 h at room temperature, removing all acid-labile protecting groups. Subsequently, the peptide was precipitated for 20 min at −20° C. in 1 mL ice cold diethyl ether, collected by centrifugation (2 min at 7,000 g), and washed with ice cold diethyl ether at least five times. The peptide pellet was dried and finally dissolved in 100 μL H.sub.2O/tBuOH (1:3 v/v) for analysis.

[0068] For preparative cleavage, the complete resin was treated as described above. However, precipitation was done in 10 mL ice cold diethyl ether and centrifugation was performed at 4,400 g. The peptide was dried by using a SpeedVac, and finally lyophilized from 1-2 mL H.sub.2O/tBuOH (1:3 v/v).

Analytical RP-HPLC of Pep′

[0069] The synthesized peptides' purity was analyzed by using analytical RP-HPLC on a reversed phase Phenomenex Jupiter Proteo C18 column (4.6 mm×250 mm, 5 μm), and an elution system composed of (A) 0.1% TFA in H.sub.2O and (B) 0.08% TFA in. A linear gradient of 20-70% solvent B in A over 40 min with a flow rate of 0.6 mL min−1 was used. The peptides were detected at 220 nm.

Preparative RP-HPLC of Pep′

[0070] Purification of the synthesized peptides was achieved by preparative RP-HPLC on a Phenomenex Jupiter Proteo C18 column (21.2 mm×250 mm) using an elution system composed of (A) 0.1% TFA in H.sub.2O and (B) 0.08% TFA in ACN, and an appropriate linear gradient of solvent B in A over 40-50 min and a flow rate of 10 mL min−1. For peptide detection, absorption at 220 nm was measured. Fractions were taken and analyzed by MALDI-TOF and/or ESI mass spectrometry and analytical RP-HPLC. Peptide fractions identified to be pure were combined and lyophilized.

MALDI-TOF Mass Spectrometry of Pep′

[0071] For mass analysis using MALDI-TOF mass spectrometry, a matrix consisting of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (10 g/L in ACN/H20/TFA 50:49.7:0.3 v/v) was used. The MALDI measurements were conducted by using a Bruker Daltonis Ultraflex III TOF/TOF.

ESI Ion Trap Mass Spectrometry of Pep′

[0072] For mass analysis using ESI Ion Trap mass spectrometry, the samples were diluted to 20 μM in H.sub.2O (0.1% HCOOH) with ACN (7:3 v/v), injected and analyzed. The ESI measurements were conducted by using a Bruker HCT mass spectrometer.

Commercial Peptide (Pep′) Supply

[0073] Alternatively to the aforementioned described in-house synthesis, processing and analysis of the peptide moieties, these peptides were also purchased from established commercial suppliers (e.g. AmbioPharm Inc., North Augusta, S.C., USA).

Pep1 (OC561): [K4(C-betaA),F7,A33,P34,A35,A36]-pNPY-amide

[0074] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(H-Cys-betaAla)-Pros-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Ala.sup.33-Pro.sup.34-Ala.sup.35-Ala.sup.36-NH.sub.2

[0075] Calculated average molecular mass: 4167.613 Molecular formula: C189H281N49O56S MS-ESI: 1042.8 [M+4H].sup.4+

Pep2 (OC562): [K4(Pam-C-betaA),F7,A33,P34,A35,A36]-pNPY-amide

[0076] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Ala.sup.33-Pro.sup.34-Ala.sup.35-Ala.sup.36-NH.sub.2

[0077] Calculated average molecular mass: 4406.022 Molecular formula: C205H311N49O57S MS-EST: 1100.5 [M−4H].sup.4−

Pep3 (OC575): Ac-[K4 (Pam-C-betaA),F7,A33,P34,A35,A36]-pNPY-amide

[0078] Acetyl-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys -betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Ala.sup.33-Pro.sup.34-Ala.sup.35-Ala.sup.36-NH.sub.2

[0079] Calculated average molecular mass: 4448.059 Molecular formula: C207H313N49O58S MS-ESI: 1112.8 [M+4H].sup.4+

Pep5 (OC577): [K4 (Pam-C-betaA),F7,A33,P34]-pNPY-amide

[0080] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-HiS.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Ala.sup.33-Pro.sup.34-Arg.sup.35-Tyr.sup.36-NH.sub.2

[0081] Calculated average molecular mass: 4583.225 Molecular formula: C214H322N52O58S MS-ESI: 1146.7 [M+4H].sup.4+

Pep6 (OC579): [K4 (Pam-C-betaA),F7,P34,A35]-pNPY-amide

[0082] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Arg.sup.33-Pro.sup.34-Ala.sup.35-Tyr.sup.36-NH.sub.2

[0083] Calculated average molecular mass: 4583.225 Molecular formula: C214H322N52O58S MS-ESI: 1147.1 [M+4H].sup.4+

Pep7 (OC580) : [K4 (Pam-C-betaA),F7,P34,A36]-pNPY-amide

[0084] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Arg.sup.33-Pro.sup.34-Arg.sup.35-Ala.sup.36-NH.sub.2

[0085] Calculated average molecular mass: 4576.238 Molecular formula: C211H325N55O57S MS-ESI: 1144.9 [M+41H].sup.4+

Pep8 (OC581): [K4 (Pam-C-betaA), F7,A33,P34,A35]-pNPY-amide

[0086] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Ala.sup.33-Pro.sup.34-Ala.sup.35-Tyr.sup.36 -NH.sub.2

[0087] Calculated average molecular mass: 4498.117 Molecular formula: C211H315N49O58 S MS-ESI: 1125.2 [M+4H].sup.4+

Pep9 (OC582): [K4 (Pam-C-betaA),F7,Nle33,P34,Nle35,Nle36]-pNPY-amide

[0088] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Nle.sup.33-Pro.sup.34-Nle.sup.35-Nle.sup.36-NH.sub.2

[0089] (Nle=norleucine)

[0090] Calculated average molecular mass: 4532.261 Molecular formula: C214H329N49O57S MS-ESI: 1134.2 [M+4H].sup.4+

Pep10 (OC583): [K4 (Pam-C-betaA),F7,Nva33,P34,Nva35,Nva36]-pNPY-amide

[0091] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.18-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-Nva.sup.33-Pro.sup.34-Nva.sup.35-Nva.sup.36-NH.sub.2

[0092] (Nva=norvaline)

[0093] Calculated average molecular mass: 4490.181 Molecular formula: C211H323N49O57S MS-ESI: 1122.2 [M+4H].sup.4+

Pep11 (OC584): [K4(Pam-C-betaA),F7,NMeA33,P34,NMeA35,NMeA36]-pNPY-amide

[0094] H-Tyr.sup.1-Pro.sup.2-Ser.sup.3-Lys.sup.4(Palmitoyl-Cys-betaAla)-Pro.sup.5-Asp.sup.6-Phe.sup.7-Pro.sup.8-Gly.sup.9-Glu.sup.10-Asp.sup.11-Ala.sup.12-Pro.sup.13-Ala.sup.14-Glu.sup.15-Asp.sup.16-Leu.sup.17-Ala.sup.18-Arg.sup.19-Tyr.sup.20-Tyr.sup.21-Ser.sup.22-Ala.sup.23-Leu.sup.24-Arg.sup.25-His.sup.26-Tyr.sup.27-Ile.sup.28-Asn.sup.29-Leu.sup.30-Ile.sup.31-Thr.sup.32-NMeAla.sup.33-Pro.sup.34-NMeAla.sup.36-NMeAla.sup.36-NH.sub.2

[0095] (NMeA=N-methyl alanine)

[0096] Calculated average molecular mass: 4448.102 Molecular formula: C208H317N49O57S MS-ESI: 1112.3 [M+4H].sup.4+

Payload (Z′) Supply

[0097] Building blocks composed of the payloads (Z′) and the linker structures (L′) of the peptide-drug conjugates Z-L-Pep (formula III) were received from commercial suppliers. For instance, tubulysin derivative building blocks were purchased from TUBE Pharmaceuticals GmbH (Vienna, Austria).

TubA: Tubulysin A Dithiopyridine Linker (N-[2-(pyridine-2-yldisulfanyl)ethyl]-Tubulysin A)

[0098] ##STR00008##

Peptide-Drug Conjugates (Z-L-Pep)

OC563: [K4(Pam-C(TubA)-betaA),F7,A33,P34,A35,A36]-pNPY-amide

[0099] ##STR00009##

[0100] Calculated average molecular mass: 5307.208 Molecular formula: C250H379N55O66S3 MS-ESI: 1327.8 [M+4H].sup.4+; MS-TOF: 5304.3 [M+H].sup.+

OC591: Ac-[K4 (Pam-C (TubA)-betaA),F7,A33,P34,A35,A36]-pNPY-amide

[0101] ##STR00010##

[0102] Calculated average molecular mass: 5371.420 Molecular formula: C252H403N55O67S3 MS-ESI: 1342.4 [M+4H].sup.4+

OC592: [K4(Pam-C(TubA)-betaA),F7,A33,P34]-pNPY-amide

[0103] ##STR00011##

[0104] Calculated average molecular mass: 5506.586 Molecular formula: C259H412N58O67S3 MS-ESI: 1376.3 [M+4H].sup.4+

OC593: [K4(Pam-C(TubA)-betaA),F7,P34,A35]-pNPY-amide

[0105] ##STR00012##

[0106] Calculated average molecular mass: 5506.586 Molecular formula: C259H412N58O67S3 MS-ESI: 1377.0 [M+4H].sup.4+

OC594: [K4(Pam-C(TubA)-betaA),F7,P34,A36]-pNPY-amide

[0107] ##STR00013##

[0108] Calculated average molecular mass: 5499.598 Molecular formula: C256H415N61O66S3 MS-ESI: 1376.0 [M+4H].sup.4+

OC595: [K4(Pam-C(TubA)-betaA),F7,A33,P34,A35]-pNPY-amide

[0109] ##STR00014##

[0110] Calculated average molecular mass: 5421.478 Molecular formula: C256H405N55O67S3 MS-ESI: 1356.0 [M+4H].sup.4+

OC596: [K4 (Pam-C (TubA) -betaA),F7,Nle33,P34,Nle35,Nle36]-pNPY-amide

[0111] ##STR00015##

[0112] (Nle=norleucine)

[0113] Calculated average molecular mass: 5455.622 Molecular formula: C259H419N55O6653 MS-ESI: 1364.7 [M+4H].sup.4+

OC597: [K4(Pam-C(TubA)-betaA),F7,Nva33,P34,Nva35,Nva36]-pNPY-amide

[0114] ##STR00016##

[0115] (Nva=norvaline)

[0116] Calculated average molecular mass: 5413.542 Molecular formula: C256H413N55O66S3 MS-ESI: 1354.4 [M+4H].sup.4+

OC598: [K4(Pam-C(TubA)-betaA),F7,NMeA33,P34,NMeA35,NMeA36]-pNPY-amide

[0117] ##STR00017##

[0118] (NMeA=N-methyl alanine)

[0119] Calculated average molecular mass: 5371.463 Molecular formula: C253H407N55O66S3 MS-ESI: 1344.0 [M+4H].sup.4+

Functional Receptor Activation (Signal Transduction)

[0120] The NPY-derived peptide-drug conjugates' ability to

[0121] functionally activate the human neuropeptide Y Y1 receptor (hY1R; NPY1R) with high specificity was evaluated by using functional reporter gene assays (using cAMP response element—CRE). For these in vitro assays CHO cells were transiently co-transfected with cDNA encoding human Y1, Y2, Y4, and Y5 receptors, respectively, C-terminally fused to EYFP and the CRE reporter vector pGL4.29 (Promega GmbH, Mannheim, Germany). For this purpose, 2.5.10.sup.6 CHO cells were seeded per 25 cm.sup.2 cell culture flask and allowed to adhere overnight. Subsequently, co-transfection of the cells was done using 10 μg hYxR vector, 2 μg pGL4.29 reporter vector and 25 μL of Metafectene® Pro transfection reagent (Biontex Laboratories GmbH, Martinsried, Germany) per culture flask. After 3 hours transfection in OptiMEM under standard growth conditions, the transfection solution was discarded, transfected cells were detached and seeded in white/clear bottom 96-well plates (50,000 cells/well). The cells were cultured for 48 hours under standard growth conditions to facilitate receptor and reporter gene expression. Subsequently, the transfected cells were co-stimulated with 10.sup.−6 M forskolin (adenylyl cyclase activator for cAMP elevation) and 10.sup.−11-10.sup.−6 M of peptide-drug conjugates under investigation (reduction of cAMP levels by Gαi-mediated signal transduction of activated hYx receptors). After 6 hours stimulation at 37° C., incubation media were removed and 60 μL/96-well of Promega's ONE-Glo™ reagent (1:1 in DMEM/Ham's F-12, v/v) were added. After 10 min incubation at room temperature the reporter gene generated luminescence signal was measured by using a Synergy 2 multiwell plate reader (BioTek, Bad Friedrichshall, Germany).

[0122] FIG. 1 shows EC.sub.50 curves and values of the functional activation of the human NPY Y1 receptor, compared to the human Y4 receptor, by the peptide-drug conjugate OC563 as determined by CRE reporter gene assays.

In Vitro Efficacy

[0123] For early in vitro evaluations of the anti-proliferative and cytotoxic effects, respectively, of the peptide-drug conjugates of formula Z-L-Pep, a fluorometric resazurin-based cell proliferation/viability assay was used. Human cancer cell lines (primarily breast cancer-derived and the Ewing's sarcoma cell line SK-N-MC) and non-cancer cell lines were seeded with low densities into 96-well plates (1,500-20,000 cells per well), and were allowed to adhere for 24 h. Subsequently, the compounds—dissolved to appropriate concentrations in cell line-specific medium—were added to the cells and incubated for 2-72 or 96 h, respectively. In case the initial compound treatment was shorter than 72 or 96 h, respectively, the incubation solution was discarded, cells were rinsed once with cell culture medium and were allowed to proliferate in compound-free medium until 72 or 96 h, respectively, were reached. Subsequently, medium was replaced by 50 μM resazurin in medium, and the cells were incubated for 2 h. Finally, the conversion of resazurin to resorufin by viable, metabolically active cells was measured using a Synergy 2 multiwell plate reader (BioTek, Bad Friedrichshall, Germany) with 540 nm excitation and 590 nm emission filter setting. Dose-response curves were analyzed by using GraphPad Prism 5.04 resulting in IC.sub.50 values.

[0124] FIG. 2 shows the inhibition of the cell proliferation of various breast cancer cell lines (MCF-7, T-47D, MDA-MB-468) and the Ewing's sarcoma cell line SK-N-MC resulting from initial 6 h treatment with peptide-drug conjugate OC563. IC.sub.50 values were calculated by using GraphPad Prism 5.04 based on the depicted dose-response curves. OC563 caused a strong anti-proliferative and cytotoxic effect that correlated very well with the NPY Y1 receptor expression in the different cell lines, since the order of Y1 receptor expression levels was determined by quantitative real-time PCR to be as follows (from high expression to lower expression): SK-N-MC>MCF-7>T-47D>MDA-MB-468.

Peptide-Drug Conjugate (Z-L-Pep)-Induced Receptor Internalization

[0125] As the specific receptor-mediated internalization of the peptide-drug conjugates into the characteristically receptor-(over)expressing diseased cells is the major prerequisite for the aspired targeted therapy, the efficacy of the peptide-drug conjugate-induced receptor internalization was tested by conducting in vitro fluorescence microscopy studies. For that purpose, 2.5.Math.10.sup.6 CHO cells were seeded per 25 cm.sup.2 cell culture flask and allowed to adhere overnight. Then, the cells were transiently transfected with cDNA encoding human NPY Y1 receptor that was C-terminally fused to EYFP. The transfection mix contained 10 μg receptor vector and 25 μL Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific, Waltham, Mass., USA) in 6 mL OptiMEM, and was incubated with the cells for 6 h under standard growth conditions. Subsequently, the transfection solution was discarded, the transfected cells were detached from the culture flask and seeded in Falcon® 8-well chamber slides (Corning, Corning, N.Y., USA) (50,000 cells/well). The cells were cultured for 16 hours in DMEM/Ham's F-12 under standard growth conditions to facilitate receptor expression. Subsequently, the transfected cells were rinsed once with PBS, starved for 30 min with OptiMEM, and then stimulated with 10.sup.−6 M peptide-drug conjugate in OptiMEM for 1 h under standard growth conditions. Subsequently, the cells were rinsed three times with ice-cold PBS, the nuclei were dyed with Hoechst 33342 (0.5 mg/mL), followed by further washing cycles with ice-cold PBS. Finally, the unfixed cells (to avoid fixation artifacts) were covered by Fluoromount-G mounting medium (SouthernBiotech, Birmingham, Ala., USA) and immediately inspected by using a laser scanning microscope LSM 700 or an Axio Observer microscope equipped with an ApoTome imaging system (both: Zeiss, Jena, Germany).

[0126] FIG. 3A illustrates the localization of the majority of NPY Y1 receptors (visualized by its C-terminal EYGP-tag; pseudo-color dark gray) within the plasma membrane in transiently transfected, but unstimulated CHO cells. However, as shown in FIG. 3B, the cells' stimulation with the NPY Y1 receptor-selective peptide-drug conjugate OC563 resulted in substantial peptide-drug conjugate-induced internalization of the Y1 receptors due to the binding and subsequent activation of the receptor by the ligand, as indicated by the loss of receptors (pseudocolor dark gray) in the membrane and increasing intracellular vesicular spots due to endocytotic receptor internalization.

In Vivo Efficacy (Study 1—Breast Cancer)

[0127] The in vivo efficacy of selected peptide-drug conjugates (Z-L-Pep) was tested by using XenTech's patient-derived breast cancer xenograft (PDX) model T272 (XenTech SAS, Evry, France). Female athymic nude-Foxnlnu (outbred) mice (Envigo, Gannat, France) were 6-7 weeks old when the patient-derived tumor specimens of the T272 model, an ER+/PR+ xenograft derived from breast infiltrating ductal adenocarcinoma, were inoculated. For tumor inoculation, the mice were anaesthetized with 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine, then the skin was aseptized with chlorhexidine solution, incised at the level of the interscapular region, and a 20 mm.sup.3 tumor fragment was placed in the subcutaneous tissue. Finally, the skin was closed with clips.

[0128] The mice were housed in groups of a maximum of 5 animals during the experimental phase in individually ventilated cages (IVC) of polysulfone (PSU) plastic (mm 213 W×362 D×185 H; Allentown, USA) with sterilized and dust-free bedding cobs, and under a light-dark cycle (14-hours circadian cycle of artificial light) and controlled room temperature and humidity. Daily, each mouse was offered a complete pellet diet (150-SP-25, SAFE) and filtered, sterilized tap water. T272 tumor-bearing mice received p-estradiol (8.5 mg/L) with the drinking water, from the day of tumor implantation to the end of the study.

[0129] Each study group comprised 10 fit mice, each of them with at least 20 g body weight at the day of randomization and inoculation. Treatment started with mean tumor volumes of 110-120 mm.sup.3 (range 60-200 mm.sup.3). Animals were treated with an application volume of 10 mL/kg by slow i.v. route with 2 mg/kg of the peptide-drug conjugates (Z-L-Pep) tested. As vehicle a physiological (0.9%) NaCl solution with 2.5% ethanol (v/v) was used. Animals were treated three times a week for 3 weeks (D0-D26), followed by a follow-up period of further three weeks (D27-D47). All animals were sacrificed at the end of the experimental phase (D48).

[0130] During the whole experimental period, from grafting day to study termination, the mice were observed daily for physical appearance, behaviour, clinical signs and body weight (BW two times a week during the follow-up period). Tumor growth was measured three times a week during the treatment phase and two times a week during the follow-up period. Tumor growth was monitored by calliper measurement and tumor volume was calculated according to the formula W.sup.2×L/2, where the length (L) and the width (W) were the longest and the shortest diameters of the tumor, respectively.

[0131] FIG. 4 illustrates the in vivo efficacy of the peptide-drug conjugate (Z-L-Pep) OC563, with modified peptide C-terminus in the sense of the present application, compared to two peptide-drug conjugates with the unmodified C-terminus of wild type NPY, OC528 (PCT/EP2013/002790) and OC1508 (PCT/EP2015/000558). The in vivo efficacy was tested in the subcutaneous patient-derived breast cancer xenograft (PDX) model T272 (Xentech SAS, Evry, France). Ten mice per study group were treated by slow i.v. route with 10 mL/kg vehicle (physiological 0.9% NaCl solution with 2.5% ethanol, v/v) and 2 mg/kg of peptide-drug conjugate in vehicle, respectively, three times a week for three weeks (D0-D26), followed by a three weeks follow-up period (D27-D47). The tumor volumes were measured using a caliper and were normalized to the tumor volume at the day of the first treatment (D0), which was set 100, resulting in values of relative tumor volumes (RTVs). FIG. 4A shows the curves of relative tumor volumes for OC563, subject of the present application, compared to the vehicle group as well as groups treated with OC528 and OC1508, respectively. OC563 treatment was significantly more effective than OC528 and OC1508. OC563 reached a T/C % value of 28.3%, which was far better than the best conventional treatment of the T272 model tested so far, according to the supplier's model characterization: a combination of adriamycin (2 mg/kg)/cyclophosphamide (100 mg/kg) with a T/C % value of 42%. FIG. 4B shows the in vivo data as Kaplan-Meier plot representing the median doubling times of the relative tumor volumes. As illustrated, OC563's RTV doubling time is with 44 days more than three times higher than that of untreated tumors (vehicle; 14 days), and more than two and three times higher than the doubling times of OC528 (19.5 days) and OC1508 (13 days), respectively. Furthermore, OC563 effected tumor free survival in 11% of the animals, complete tumor regression (11%), partial tumor regression (22%) and in further 55% of the animals tumor stabilization.

[0132] Hence, OC563, subject of the present application, is significantly more anti-tumor effective in vivo than other peptide-drug conjugates with a peptide C-terminus comparable to wild type NPY, as demonstrated with OC528 (PCT/EP2013/002790) and OC1508 (PCT/EP2015/000558).

In Vivo Efficacy (Study 2—Ewing's Sarcoma)

[0133] The in vivo efficacy of the peptide-drug conjugate OC563 was tested by using a patient-derived Ewing's sarcoma xenograft (PDX) model (EPO GmbH, Berlin-Buch, Germany; model Sarc10228). Female NMRI-nu/nu mice were 6-7 weeks old when the patient-derived tumor specimens of the Sarc10228 model, hY1R-overexpressing Ewing's sarcoma, were inoculated.

[0134] Each study group comprised 3 fit mice, each of them with at least 20 g body weight at the day of randomization and inoculation. Animals were treated with an application volume of 10 mL/kg by slow i.v. route with 2 mg/kg of the peptide-drug conjugate OC563. As vehicle a physiological (0.9%) NaCl solution with 2.5% ethanol (v/v) was used. Animals were treated three times a week for 3 weeks (D0-D18). All animals were sacrificed at the end of the experimental phase.

[0135] During the whole experimental period, from grafting day to study termination, the mice were observed daily for physical appearance, behaviour, clinical signs and body weight. Tumor growth was measured two times a week. Tumor growth was monitored by calliper measurement and tumor volume was calculated according to the formula W.sup.2×L/2, where the length (L) and the width (W) were the longest and the shortest diameters of the tumor, respectively.

[0136] FIG. 5 illustrates the in vivo efficacy of the peptide-drug conjugate (Z-L-Pep) OC563, with modified peptide C-terminus in the sense of the present application. The in vivo efficacy was tested in the subcutaneous patient-derived Ewing's sarcoma xenograft (PDX) model Sarc10228 (EPO GmbH, Berlin-Buch, Germany). Three mice per study group were treated by slow i.v. route with 10 mL/kg vehicle (physiological 0.9% NaCl solution with 2.5% ethanol, v/v) and 2 mg/kg of OC563 in vehicle, respectively, three times a week for three weeks (D0-D18). The tumor volumes were measured using a caliper and were normalized to the tumor volume at the day of the first treatment (D0), which was set 100%, resulting in values of relative tumor volumes (RTVs). FIG. 5A shows the curves of relative tumor volumes for OC563, subject of the present application, compared to the vehicle group. OC563 reached a T/C % value of ˜50%. FIG. 5B shows the in vivo data as Kaplan-Meier plot representing the median doubling times of the relative tumor volumes. As illustrated, OC563's RTV doubling time of 24 days is around two times higher than that of untreated tumors (vehicle; 13 days).

Data Analysis

[0137] For data analysis GraphPad Prism 5.04 and LibreOffice Calc 5.3.3.2 were used.

[0138] Surprisingly, as exemplified by compound OC563, a peptide-toxin conjugate comprising a peptide moiety of the present invention permitted good functional hY1R activation and hY1R-mediated internalization in vitro; against all scientific conviction of the NPY receptor community as aforementioned.

[0139] Even more surprisingly, PDCs comprising these novel artificially modified peptide moieties with its strongly atypical C-terminus permitted in vitro anti-tumor efficacies in a hY1R expression-level dependent manner with IC50 values in the low nanomolar range.

[0140] Very surprisingly, PDCs comprising these novel artificially modified peptide moieties with its strongly atypical C-terminus permitted potent in vivo anti-tumor efficacy in a patient-derived breast cancer xenograft (breast cancer PDX) as well. Most surprisingly, and contrarily to all established conviction on prerequisites for a potent hY1R-addressing peptide, PDCs comprising these novel artificially modified peptide moieties with its strongly atypical C-terminus were significantly more effective in the breast cancer PDX animal models than PDCs containing the well-established “gold standard” of highly affine hY1R-selective peptides, [F.sup.7,P.sup.34]-pNPY (see FIGS. 4A and 4B, wherein the novel conjugate OC563 claimed herein is compared to the recently disclosed OC528 and OC1508; PCT/EP2013/002790 and PCT/EP2015/000558).