Kv1.3 blockers
11780893 · 2023-10-10
Assignee
Inventors
Cpc classification
C07K1/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides novel blockers of the potassium channel Kv1.3, polynucleotides encoding them, and methods of making and using them.
Claims
1. An ion channel blocker or pharmaceutically acceptable salt comprising a Kv1.3 inhibitor component, wherein the Kv1.3 inhibitor component comprises one of the sequences: TABLE-US-00027 (SEQ ID NO. 1) QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 2) QMDMRCSASVECKQKCLKAIGRGFGKCMNKKCKCYPR, (SEQ ID NO. 3) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 4) NMDMRCSASVECKQKCLKAIGRGFGKCMNKKCKCYPR, (SEQ ID NO. 5) MDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 6) DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 7) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 8) NIDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 9) NMDVRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 10) NMDMRCSASVECKQKCKDAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 11) GGNMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 12) NMEMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 13) SGNMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 14) N[Nle]DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 15) NMD[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 16) NMDMRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 17) NMDMRCSASVECKVKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 18) NMDMRCSASVECKQLCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 19) NMDMRCSASVECKQKCKKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 20) NMDMRCSASVECKQKCLDAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 21) NMDMRCSASVECKQKCLKAIRSIFGKCMNKKCKCYPR, (SEQ ID NO. 22) NMDMRCSASVECKQKCLKAIESIFGKCMNKKCKCYPR, (SEQ ID NO. 23) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPRRRTA, (SEQ ID NO. 24) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPRHRRK, (SEQ ID NO. 25) NMDMRCKASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 26) NMDMRCSISVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 27) NMDMRCSASRECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 28) NMDMRCSASVQCKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 29) NMDMRCSASVECLQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 30) NMDMRCSASVECAQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 31) NMDMRCSASVECKEKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 32) NMDMRCSASVECKLKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 33) NMDMRCSASVECKQKCLKAIHSIFGKCMNKKCKCYPR, (SEQ ID NO. 34) NMDMRCSASVECKQKCLKAIGSKFGKCMNKKCKCYPR, (SEQ ID NO. 35) NMDMRCSASVECKQKCLKAIGSRFGKCMNKKCKCYPR, (SEQ ID NO. 36) NMDMRCSASVECKQKCLKAIGSIFGKCMNGKCKCYPR, (SEQ ID NO. 37) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCHCYPR, (SEQ ID NO. 38) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCVCYPR, (SEQ ID NO. 39) N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 40) N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 41) P[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 42) N[Nle]D[Nle]RCRASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 43) N[Nle]D[Nle]RCSASVECEQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 44) N[Nle]D[Nle]RCSASVECQQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 45) N[Nle]D[Nle]RCSASVECKKKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 46) N[Nle]S[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 47) N[Nle]D[Nle]RCSHSVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 48) N[Nle]D[Nle]RCSASVECKQSCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 49) N[Nle]D[Nle]RCSASVECKQKCKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 50) NMDMRCSASVECKQKCYKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 51) NMDMRCSASVECKQKCRKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 52) NMDMRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 53) NMDMRCSASVECKQKCLYAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 54) NMDMRCSASVECKQKCLAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 55) NMDMRCSASVECKQKCLKYIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 56) NMDMRCSASVECKQKCLKRIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 57) NMDMRCSASVECKQKCLKIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 58) NMDMRCSASVECKQKCLKAYGSIFGKCMNKKCKCYPR, (SEQ ID NO. 59) NMDMRCSASVECKQKCLKAEGSIFGKCMNKKCKCYPR, (SEQ ID NO. 60) NMDMRCSASVECKQKCLKARGSIFGKCMNKKCKCYPR, (SEQ ID NO. 61) NMDMRCSASVECKQKCLKAGSIFGKCMNKKCKCYPR, (SEQ ID NO. 62) N[Nle]D[Nle]RCSASVECKQKCLKAIGSPFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 63) N[Nle]D[Nle]RCSASKECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 64) H[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 65) Y[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 66) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 67) s[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 68) S[Nle]D[Nle]RCSA[Abu]VECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 69) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFG[homo- Lys]CMNKKCKCYPR, (SEQ ID NO. 70) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NH2)]PR, (SEQ ID NO. 71) S[Nle]D[Nle]RCSASVECGQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 72) S[Nle]D[Nle]RCSASVECVQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 73) S[Nle]D[Nle]RCSASVECK[2-Amino-5- carboxypentanol]KCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 74) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYQ, (SEQ ID NO. 75) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCRCYPR, (SEQ ID NO. 76) S[Nle]DERCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 77) S[Nle]D[Nle]RCSASVECKQKCLGAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 78) S[Nle]D[Nle]RCSASVECKQKCLVAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 79) S[Nle]D[Nle]RCSASVECAQSCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 80) S[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 81) S[Nle]D[Nle]RCSASVECAQLCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 82) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 83) P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 84) S[Nle]D[Nle]RCSASVECKEKCLQAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 85) P[Nle]D[Nle]RCSASVECKEKCL[homo- Gln]AIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 86) CSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 87) S[Nle]D[Nle]RCSASVECKQKCLAAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 88) P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 89) S[Nle]D[Nle]RCSALVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 90) S[Nle]D[Nle]RCSAVVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 91) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- F)]PR, (SEQ ID NO. 92) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NO.sub.2)]PR, (SEQ ID NO. 93) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- CH.sub.3)]PR, (SEQ ID NO. 94) [Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 95) NID[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 96) PIE[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 97) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 98) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 99) P[Nle]E[Nle]RCSASVECKQKCLLAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 100) PIDERCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 101) PIE[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 102) P[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 103) P[Nle]E[Nle]RCSASVECAQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 104) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 105) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 106) RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 107) SKCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 108) LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 109) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPS, (SEQ ID NO. 110) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYS, (SEQ ID NO. 111) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYG-NH2, (SEQ ID NO. 112) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCY-NH2, (SEQ ID NO. 113) RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 114) LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 115) LRCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 116) LRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 117) CSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 118) P[Nle]E[Nle]RCSASVECKEKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 119) p[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 120) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYR, (SEQ ID NO. 121) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 122) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRR, (SEQ ID NO. 123) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRY, (SEQ ID NO. 124) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRL, (SEQ ID NO. 125) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRH, (SEQ ID NO. 126) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRE, (SEQ ID NO. 127) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[NlE]NKKCKCYPRK S, (SEQ ID NO. 128) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRFE, (SEQ ID NO. 129) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRH R, (SEQ ID NO. 130) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRA K, (SEQ ID NO. 131) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP- [(4-amino-5-hydroxypentyl)guanidine], (SEQ ID NO. 132) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR- [4-amino-5-hydroxypentanamide], (SEQ ID NO. 133) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR ST, (SEQ ID NO. 134) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR RY, (SEQ ID NO. 135) P[Nle]E[Nle]RCSSSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 136) P[Nle]E[Nle]RCSLSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 137) P[Nle]E[Nle]RCSAPVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 138) P[Nle]E[Nle]RCSASPECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 139) P[Nle]E[Nle]RCSASQECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 140) P[Nle]E[Nle]RCSASVECLQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 141) P[Nle]E[Nle]RCSASVECKQPCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 142) P[Nle]E[Nle]RCEASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 143) P[Nle]E[Nle]RCFASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 144) P[Nle]E[Nle]RCSYSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, or (SEQ ID NO. 145) P[Nle]E[Nle]RCSAFVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR.
2. An ion channel blocker or pharmaceutically acceptable salt according to claim 1 consisting of one of the sequences: TABLE-US-00028 (SEQ ID NO. 3) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 4) NMDMRCSASVECKQKCLKAIGRGFGKCMNKKCKCYPR, (SEQ ID NO. 5) MDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 6) DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 7) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 8) NIDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 9) NMDVRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 10) NMDMRCSASVECKQKCKDAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 12) NMEMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 14) N[Nle]DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 15) NMD[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 16) NMDMRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 17) NMDMRCSASVECKVKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 18) NMDMRCSASVECKQLCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 19) NMDMRCSASVECKQKCKKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 20) NMDMRCSASVECKQKCLDAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 21) NMDMRCSASVECKQKCLKAIRSIFGKCMNKKCKCYPR, (SEQ ID NO. 22) NMDMRCSASVECKQKCLKAIESIFGKCMNKKCKCYPR, (SEQ ID NO. 25) NMDMRCKASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 26) NMDMRCSISVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 27) NMDMRCSASRECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 28) NMDMRCSASVQCKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 29) NMDMRCSASVECLQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 30) NMDMRCSASVECAQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 31) NMDMRCSASVECKEKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 32) NMDMRCSASVECKLKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 33) NMDMRCSASVECKQKCLKAIHSIFGKCMNKKCKCYPR, (SEQ ID NO. 34) NMDMRCSASVECKQKCLKAIGSKFGKCMNKKCKCYPR, (SEQ ID NO. 35) NMDMRCSASVECKQKCLKAIGSRFGKCMNKKCKCYPR, (SEQ ID NO. 36) NMDMRCSASVECKQKCLKAIGSIFGKCMNGKCKCYPR, (SEQ ID NO. 37) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCHCYPR, (SEQ ID NO. 38) NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCVCYPR, (SEQ ID NO. 39) N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 40) N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 41) P[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 42) N[Nle]D[Nle]RCRASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR (SEQ ID NO. 43) N[Nle]D[Nle]RCSASVECEQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 44) N[Nle]D[Nle]RCSASVECQQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 45) N[Nle]D[Nle]RCSASVECKKKCLKAIGSIFGKC[Nle]NKKCKCYPR (SEQ ID NO. 46) N[Nle]S[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 47) N[Nle]D[Nle]RCSHSVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 48) N[Nle]D[Nle]RCSASVECKQSCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 49) N[Nle]D[Nle]RCSASVECKQKCKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 50) NMDMRCSASVECKQKCYKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 51) NMDMRCSASVECKQKCRKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 52) NMDMRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 53) NMDMRCSASVECKQKCLYAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 54) NMDMRCSASVECKQKCLAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 55) NMDMRCSASVECKQKCLKYIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 56) NMDMRCSASVECKQKCLKRIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 57) NMDMRCSASVECKQKCLKIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 58) NMDMRCSASVECKQKCLKAYGSIFGKCMNKKCKCYPR, (SEQ ID NO. 59) NMDMRCSASVECKQKCLKAEGSIFGKCMNKKCKCYPR, (SEQ ID NO. 60) NMDMRCSASVECKQKCLKARGSIFGKCMNKKCKCYPR, (SEQ ID NO. 61) NMDMRCSASVECKQKCLKAGSIFGKCMNKKCKCYPR, (SEQ ID NO. 62) N[Nle]D[Nle]RCSASVECKQKCLKAIGSPFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 63) N[Nle]D[Nle]RCSASKECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 64) H[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 65) Y[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 66) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 67) V[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 68) S[Nle]D[Nle]RCSA[Abu]VECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 69) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFG[homo- Lys]CMNKKCKCYPR, (SEQ ID NO. 70) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NH.sub.2)]PR, (SEQ ID NO. 71) S[Nle]D[Nle]RCSASVECGQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 72) S[Nle]D[Nle]RCSASVECVQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 73) S[Nle]D[Nle]RCSASVECK[2-Amino-5- carboxypentanoyl]KCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 74) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYQ, (SEQ ID NO. 75) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCRCYPR, (SEQ ID NO. 76) S[Nle]DERCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 77) S[Nle]D[Nle]RCSASVECKQKCLGAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 78) S[Nle]D[Nle]RCSASVECKQKCLVAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 79) S[Nle]D[Nle]RCSASVECAQSCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 80) S[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 81) S[Nle]D[Nle]RCSASVECAQLCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 82) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 83) P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 84) S[Nle]D[Nle]RCSASVECKEKCLQAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 85) P[Nle]D[Nle]RCSASVECKEKCL[homo- Gln]AlGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 86) CSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 87) S[Nle]D[Nle]RCSASVECKQKCLAAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 88) P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC, (SEQ ID NO. 89) S[Nle]D[Nle]RCSALVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 90) S[Nle]D[Nle]RCSAVVECKQKCLKAIGSIFGKCMNKKCKC(3)YPR, (SEQ ID NO. 91) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- F)]PR, (SEQ ID NO. 92) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NO.sub.2)]PR, (SEQ ID NO. 93) S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- CH.sub.3)]PR, (SEQ ID NO. 94) [Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 95) NID[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 96) PIE[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 97) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 98) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 99) P[Nle]E[Nle]RCSASVECKQKCLLAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 100) PIDERCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 101) PIE[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 102) P[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 103) P[Nle]E[Nle]RCSASVECAQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 104) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 105) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 106) RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 107) SKCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 108) LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 109) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPS, (SEQ ID NO. 110) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYS, (SEQ ID NO. 111) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYG-NH2, (SEQ ID NO. 112) CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCY-NH2, (SEQ ID NO. 113) RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 114) LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 115) LRCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 116) LRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 117) CSASVECKQKCLKAIGSIFGKCMNKKCKCYPR, (SEQ ID NO. 118) P[Nle]E[Nle]RCSASVECKEKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 119) p[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 120) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYR, (SEQ ID NO. 121) [Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 122) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRR, (SEQ ID NO. 123) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRY, (SEQ ID NO. 124) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRL, (SEQ ID NO. 125) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRH, (SEQ ID NO. 126) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRE, (SEQ ID NO. 127) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRK S, (SEQ ID NO. 128) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRFE, (SEQ ID NO. 129) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRH R, (SEQ ID NO. 130) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRA K, (SEQ ID NO. 131) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP- [(4-amino-5-hydroxypentyl)guanidine], (SEQ ID NO. 132) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR- [4-amino-5-hydroxypentanamide], (SEQ ID NO. 133) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRS T, (SEQ ID NO. 134) P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRR Y, (SEQ ID NO. 135) P[Nle]E[Nle]RCSSSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 136) P[Nle]E[Nle]RCSAPVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 137) P[Nle]E[Nle]RCSAPVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 138) P[Nle]E[Nle]RCSASPECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 139) P[Nle]E[Nle]RCSASQECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 140) P[Nle]E[Nle]RCSASVECLQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 141) P[Nle]E[Nle]RCSASVECKQPCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 142) P[Nle]E[Nle]RCEASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 143) P[Nle]E[Nle]RCFASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, (SEQ ID NO. 144) P[Nle]E[Nle]RCSYSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR, or (SEQ ID NO. 145) P[Nle]E[Nle]RCSAFVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR.
3. An ion channel blocker or pharmaceutically acceptable salt according to claim 1 selected from: TABLE-US-00029 (Cpd. 3) (SEQ ID NO. 3) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 4) (SEQ ID NO. 4) H-NMDMRCSASVECKQKCLKAIGRGFGKCMNKKCKCYPR-OH (Cpd. 5) (SEQ ID NO. 5) H-MDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 6) (SEQ ID NO. 6) H-DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 7) (SEQ ID NO. 7) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 8) (SEQ ID NO. 8) H-NIDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 9) (SEQ ID NO. 9) H-NMDVRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 10) (SEQ ID NO. 10) H-NMDMRCSASVECKQKCKDAIGSIFGKCMNKKCKCYPR-OH (Cpd. 11) (SEQ ID NO. 11) H-GGNMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 12) (SEQ ID NO. 12) H-NMEMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 13) (SEQ ID NO. 13) H-SGNMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 14) (SEQ ID NO. 14) H-N[Nle]DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 15) (SEQ ID NO. 15) H-NMD[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 16) (SEQ ID NO. 16) H-NMDMRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 17) (SEQ ID NO. 17) H-NMDMRCSASVECKVKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 18) (SEQ ID NO. 18) H-NMDMRCSASVECKQLCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 19) (SEQ ID NO. 19) H-NMDMRCSASVECKQKCKKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 20) (SEQ ID NO. 20) H-NMDMRCSASVECKQKCLDAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 21) (SEQ ID NO. 21) H-NMDMRCSASVECKQKCLKAIRSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 22) (SEQ ID NO. 22) H-NMDMRCSASVECKQKCLKAIESIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 23) (SEQ ID NO. 23) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPRRRTA-NH.sub.2 (Cpd. 24) (SEQ ID NO. 24) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPRHRRK-NH.sub.2 (Cpd. 25) (SEQ ID NO. 25) H-NMDMRCKASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 26) (SEQ ID NO. 26) H-NMDMRCSISVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 27) (SEQ ID NO. 27) H-NMDMRCSASRECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 28) (SEQ ID NO. 28) H-NMDMRCSASVQCKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 29) (SEQ ID NO. 29) H-NMDMRCSASVECLQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 30) (SEQ ID NO. 30) H-NMDMRCSASVECAQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 31) (SEQ ID NO. 31) H-NMDMRCSASVECKEKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 32) (SEQ ID NO. 32) H-NMDMRCSASVECKLKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 33) (SEQ ID NO. 33) H-NMDMRCSASVECKQKCLKAIHSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 34) (SEQ ID NO. 34) H-NMDMRCSASVECKQKCLKAIGSKFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 35) (SEQ ID NO. 35) H-NMDMRCSASVECKQKCLKAIGSRFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 36) (SEQ ID NO. 36) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNGKCKCYPR-NH.sub.2 (Cpd. 37) (SEQ ID NO. 37) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCHCYPR-NH.sub.2 (Cpd. 38) (SEQ ID NO. 38) H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCVCYPR-NH.sub.2 (Cpd. 39) (SEQ ID NO. 39) H-N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 40) (SEQ ID NO. 40) H-N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 41) (SEQ ID NO. 41) H-P[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 42) (SEQ ID NO. 42) H-N[Nle]D[Nle]RCRASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 43) (SEQ ID NO. 43) H-N[Nle]D[Nle]RCSASVECEQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 44) (SEQ ID NO. 44) H-N[Nle]D[Nle]RCSASVECQQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 45) (SEQ ID NO. 45) H-N[Nle]D[Nle]RCSASVECKKKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 46) (SEQ ID NO. 46) H-N[Nle]S[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 47) (SEQ ID NO. 47) H-N[Nle]D[Nle]RCSHSVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 48) (SEQ ID NO. 48) H-N[Nle]D[Nle]RCSASVECKQSCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 49) (SEQ ID NO. 49) H-N[Nle]D[Nle]RCSASVECKQKCKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 50) (SEQ ID NO. 50) H-NMDMRCSASVECKQKCYKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 51) (SEQ ID NO. 51) H-NMDMRCSASVECKQKCRKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 52) (SEQ ID NO. 52) H-NMDMRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 53) (SEQ ID NO. 53) H-NMDMRCSASVECKQKCLYAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 54) (SEQ ID NO. 54) H-NMDMRCSASVECKQKCLAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 55) (SEQ ID NO. 55) H-NMDMRCSASVECKQKCLKYIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 56) (SEQ ID NO. 56) H-NMDMRCSASVECKQKCLKRIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 57) (SEQ ID NO. 57) H-NMDMRCSASVECKQKCLKIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 58) (SEQ ID NO. 58) H-NMDMRCSASVECKQKCLKAYGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 59) (SEQ ID NO. 59) H-NMDMRCSASVECKQKCLKAEGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 60) (SEQ ID NO. 60) H-NMDMRCSASVECKQKCLKARGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 61) (SEQ ID NO. 61) H-NMDMRCSASVECKQKCLKAGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 62) (SEQ ID NO. 62) H-N[Nle]D[Nle]RCSASVECKQKCLKAIGSPFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 63) (SEQ ID NO. 63) H-N[Nle]D[Nle]RCSASKECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 64) (SEQ ID NO. 64) H-H[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 65) (SEQ ID NO. 65) H-Y[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 66) (SEQ ID NO. 66) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 67) (SEQ ID NO. 67) H-V[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 68) (SEQ ID NO. 68) H-S[Nle]D[Nle]RCSA[Abu]VECKQKCLKAIGSIFGKCMNKKCKCYP R-NH.sub.2 (Cpd. 69) (SEQ ID NO. 69) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFG[homo- Lys]CMNKKCKCYPR-NH.sub.2 (Cpd. 70) (SEQ ID NO. 70) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NH2)]PR-NH.sub.2 (Cpd. 71) (SEQ ID NO. 71) H-S[Nle]D[Nle]RCSASVECGQKCLKAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 72) (SEQ ID NO. 72) H-S[Nle]D[Nle]RCSASVECVQKCLKAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 73) (SEQ ID NO. 73) H-S[Nle]D[Nle]RCSASVECK[2-Amino-5- carboxypentanoyl]KCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 74) (SEQ ID NO. 74) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYQ-NH.sub.2 (Cpd. 75) (SEQ ID NO. 75) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCRCYPR- NH.sub.2 (Cpd. 76) (SEQ ID NO. 76) H-S[Nle]DERCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (Cpd. 77) (SEQ ID NO. 77) H-S[Nle]D[Nle]RCSASVECKQKCLGAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 78) (SEQ ID NO. 78) H-S[Nle]D[Nle]RCSASVECKQKCLVAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 79) (SEQ ID NO. 79) H-S[Nle]D[Nle]RCSASVECAQSCLKAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 80) (SEQ ID NO. 80) H-S[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 81) (SEQ ID NO. 81) H-S[Nle]D[Nle]RCSASVECAQLCLAAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 82) (SEQ ID NO. 82) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 83) (SEQ ID NO. 83) H-P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYP C-NH.sub.2 (Cpd. 84) (SEQ ID NO. 84) H-S[Nle]D[Nle]RCSASVECKEKCLQAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 85) (SEQ ID NO. 85) H-P[Nle]D[Nle]RCSASVECKEKCL[homo- Gln]AIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 86) (SEQ ID NO. 86) H-CSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC-NH.sub.2 (Cpd. 87) (SEQ ID NO. 87) H-S[Nle]D[Nle]RCSASVECKQKCLAAIGCIFGKC[Nle]NKKCKCYP C-NH.sub.2 (Cpd. 88) (SEQ ID NO. 88) H-P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYP C-OH (Cpd. 89) (SEQ ID NO. 89) H-S[Nle]D[Nle]RCSALVECKQKCLKAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 90) (SEQ ID NO. 90) H-S[Nle]D[Nle]RCSAVVECKQKCLKAIGSIFGKCMNKKCKCYPR- NH.sub.2 (Cpd. 91) (SEQ ID NO. 91) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- F)]PR-NH.sub.2 (Cpd. 92) (SEQ ID NO. 92) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NO2)]PR-NH.sub.2 (Cpd. 93) (SEQ ID NO. 93) H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- CH3)]PR-NH.sub.2 (Cpd. 94) (SEQ ID NO. 94) H-[Nle][Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR- OH (Cpd. 95) (SEQ ID NO. 95) H-NID[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 96) (SEQ ID NO. 96) H-PIE[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 97) (SEQ ID NO. 97) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 98) (SEQ ID NO. 98) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-NH.sub.2 (Cpd. 99) (SEQ ID NO. 99) H-P[Nle]E[Nle]RCSASVECKQKCLLAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 100) (SEQ ID NO. 100) H-PIDERCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 101) (SEQ ID NO. 101) H-PIE[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 102) (SEQ ID NO. 102) H-P[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR-OH (Cpd. 103) (SEQ ID NO. 103) H-P[Nle]E[Nle]RCSASVECAQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 104) (SEQ ID NO. 104) H-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 105) (SEQ ID NO. 105) Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 106) (SEQ ID NO. 106) H-RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 107) (SEQ ID NO. 107) Ac-SKCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 108) (SEQ ID NO. 108) Ac-LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (Cpd. 109) (SEQ ID NO. 109) Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPS-NH.sub.2 (Cpd. 110) (SEQ ID NO. 110) Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYS-NH.sub.2 (Cpd. 111) (SEQ ID NO. 111) Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYG-NH.sub.2 (Cpd. 112) (SEQ ID NO. 112) Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCY-NH.sub.2 (Cpd. 113) (SEQ ID NO. 113) Ac-RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 114) (SEQ ID NO. 114) H-LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 115) (SEQ ID NO. 115) Ac-LRCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (Cpd. 116) (SEQ ID NO. 116) Ac-LRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR-OH (Cpd. 117) (SEQ ID NO. 117) H-CSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (Cpd. 118) (SEQ ID NO. 118) H-P[Nle]E[Nle]RCSASVECKEKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 119) (SEQ ID NO. 119) H-p[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 120) (SEQ ID NO. 120) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCY R-OH (Cpd. 121) (SEQ ID NO. 121) H[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR- OH (Cpd. 122) (SEQ ID NO. 122) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RR-OH (Cpd. 123) (SEQ ID NO. 123) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RY-OH (Cpd. 124) (SEQ ID NO. 124) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RL-OH (Cpd. 125) (SEQ ID NO. 125) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RH-OH (Cpd. 126) (SEQ ID NO. 126) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RE-OH (Cpd. 127) (SEQ ID NO. 127) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RKS-OH (Cpd. 128) (SEQ ID NO. 128) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RFE-OH (Cpd. 129) (SEQ ID NO. 129) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RHR-OH (Cpd. 130) (SEQ ID NO. 130) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RAK-OH (Cpd. 131) (SEQ ID NO. 131) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCY P-[(4-amino-5-hydroxypentyl)guanidine] (Cpd. 132) (SEQ ID NO. 132) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-[4-amino-5-hydroxypentanamide] (Cpd. 133) (SEQ ID NO. 133) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RST-OH (Cpd. 134) (SEQ ID NO. 134) H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP RRY-OH (Cpd. 135) (SEQ ID NO. 135) H-P[Nle]E[Nle]RCSSSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 136) (SEQ ID NO. 136) H-P[Nle]E[Nle]RCSLSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 137) (SEQ ID NO. 137) H-P[Nle]E[Nle]RCSAPVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 138) (SEQ ID NO. 138) H-P[Nle]E[Nle]RCSASPECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 139) (SEQ ID NO. 139) H-P[Nle]E[Nle]RCSASQECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 140) (SEQ ID NO. 140) H-P[Nle]E[Nle]RCSASVECLQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH (Cpd. 141) (SEQ ID NO. 141) H-P[Nle]E[Nle]RCSASVECKQPCLAAIGSIFGKC[Nle]NKKCKCYP, R-OH (Cpd. 142) (SEQ ID NO. 142) H-P[Nle]E[Nle]RCEASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH, (Cpd. 143) (SEQ ID NO. 143) H-P[Nle]E[Nle]RCFASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH, (Cpd. 144) (SEQ ID NO. 144) H-P[Nle]E[Nle]RCSYSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH or (Cpd. 145) (SEQ ID NO. 145) H-P[Nle]E[Nle]RCSAFVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP R-OH.
4. A pharmaceutical composition comprising an ion channel blocker or pharmaceutically acceptable salt according to claim 1, 2 or 3, in admixture with a pharmaceutically acceptable carrier.
5. An ion channel blocker or pharmaceutically acceptable salt according to claim 1 which is a fusion protein comprising the Kv1.3 inhibitor component and one or more heterologous polypeptide sequences.
6. An ion channel blocker or pharmaceutically acceptable salt according to claim 5 wherein the Kv1.3 inhibitor component is inserted within a heterologous scaffold polypeptide.
7. An ion channel blocker or pharmaceutically acceptable salt according to claim 1 wherein the ion channel blocker has a maximum length of 200 amino acids.
8. A method of synthesising an ion channel blocker according to claim 1, 2 or 3, the method comprising: (a) synthesising the ion channel blocker by means of solid-phase or liquid-phase peptide synthesis methodology and recovering the peptide thus obtained; (b) expressing the ion channel blocker from a nucleic acid construct that encodes the ion channel blocker and recovering the expression product; or (c) expressing a precursor peptide from a nucleic acid construct that encodes the precursor peptide sequence, recovering the expression product, and modifying the precursor peptide to yield the ion channel blocker.
9. A method for (i) treating an autoimmune disorder, allergy, hypersensitivity, allograft rejection, or graft versus host disease; (ii) treating hay fever, asthma, anaphylaxis, allergic rhinitis, urticaria, eczema, alopecia areata, dermatomyositis, inclusion body myositis, polymyositis, ankylosing spondylitis, vasculitis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus (SLE), uveitis, inflammatory fibrosis, chronic obstructive pulmonary disease (COPD), hepatitis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, colitis, erythema, thyroiditis, psoriasis, atopic dermatitis, allergic contact dermatitis, scleroderma, glomerulonephritis, inflammatory bone resorption, multiple sclerosis or transplant rejection; (iii) inhibiting weight gain, promoting weight loss, or reducing excess body weight; (iv) treating obesity or obesity linked inflammation, obesity linked gallbladder disease or obesity induced sleep apnoea; (v) treating metabolic syndrome, insulin resistance, glucose intolerance, pre-diabetes, increased fasting glucose or type 2 diabetes; (vi) treating a smooth muscle proliferative disorder; (vii) treating Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease or amyotrophic lateral sclerosis (ALS) or (viii) treating cancer in a subject in need thereof, wherein said method comprising administering the ion channel blocker or pharmaceutically acceptable salt of claim 1, 2 or 3 to said subject.
10. A method according to claim 9 wherein: the smooth muscle proliferative disorder is restenosis.
11. The method according to claim 9 wherein the inflammatory fibrosis is scleroderma, lung fibrosis or cirrhosis.
12. The method according to claim 9 wherein the colitis is Crohn's disease or ulcerative colitis.
13. The method according to claim 9 wherein the lymphoma is non-Hodgkin lymphoma (NHL).
14. The method according to claim 13 wherein the lymphoma is large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, precursor T-lymphoblastic lymphoma or Sezary syndrome.
Description
DETAILED DESCRIPTION OF THE INVENTION
(1) Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.
(2) All patents, published patent applications and non-patent publications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.
(3) Each embodiment of the invention described herein may be taken alone or in combination with one or more other embodiments of the invention.
(4) Unless specified otherwise, the following definitions are provided for specific terms which are used in the present written description.
(5) Throughout this specification, the word “comprise”, and grammatical variants thereof, such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or component, or group of integers or components, but not the exclusion of any other integer or component, or group of integers or components.
(6) The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.
(7) The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” may be used interchangeably.
(8) The terms “patient”, “subject” and “individual” may be used interchangeably. A subject may be a mammal, including a human or a non-human mammal, such as a non-human primate (e.g. ape, Old World monkey or New World monkey), livestock animal (e.g. bovine or porcine), companion animal (e.g. canine or feline) or laboratory animal such as a rodent (e.g. mouse or rat).
(9) Throughout the present description and claims the conventional three-letter and one-letter codes for naturally occurring amino acids are used, i.e.
(10) A (Ala), G (Gly), L (Leu), I (Ile), V (Val), F (Phe), W (Trp), S (Ser), T (Thr), Y (Tyr), N (Asn), Q (Gln), D (Asp), E (Glu), K (Lys), R (Arg), H (His), M (Met), C (Cys) and P (Pro);
(11) as well as generally accepted codes for other α-amino acids, such as norleucine (Nle), sarcosine (Sar), α-aminoisobutyric acid (Aib), 2,3-diaminopropanoic acid (Dap), 2,4-diaminobutanoic acid (Dab), 2,5-diaminopentanoic acid (ornithine; Orn) alpha-aminobutyric acid (Abu, also known as homo-alanine), hK, hLys or homo-Lys (homo-lysine), hQ, hGln or homo-Gln (homo-glutamine, also known as 6-oxolysine, carbarmoylnorvaline, 6-amino-6-oxonorleucine or 5-(aminocarbonyl)norvaline), F(4-F) (4-fluoro-phenylalanine), F(4-NH.sub.2) (4-amino-phenylalanine), F(4-NO.sub.2) (4-nitro-phenylalanine), F(4-CH.sub.3) (4-methyl-phenylalanine).
(12) The designation [2-Amino-5-carboxypentanoyl] indicates a peptide residue of 2-amino-5-carboxypentanoic acid:
(13) ##STR00001## which thus has a side chain similar to that of glutamic acid, but with an additional methylene group.
(14) Such other α-amino acids may be shown in square brackets “[ ]” (e.g. “[Nle]”) when used in a general formula or sequence in the present specification, especially when the rest of the formula or sequence is shown using the single letter code. The 20 “naturally occurring” amino acids listed above are those which are encoded by the standard genetic code, and may also be referred to as “proteinogenic” amino acids.
(15) Unless otherwise specified, amino acid residues in peptides of the invention are of the L-configuration. However, D-configuration amino acids may be incorporated. In the present context, an amino acid code written with a small letter represents the D-configuration of said amino acid, e.g. “k” represents the D-configuration of lysine (K).
(16) A “H” (or “Hy-”) moiety at the N-terminus of the sequence in question indicates a hydrogen atom [i.e. R.sup.1=hydrogen], corresponding to the presence of a free primary or secondary amino group at the N-terminus, while an “—OH” or an “—NH.sub.2” moiety at the C-terminus of the sequence [i.e. R.sup.2═OH or NH.sub.2] indicates the presence of a carboxy (COOH) group or an amido (CONH.sub.2) group at the C-terminus of the molecule. A “CH.sub.2OH” moiety at the C-terminus [i.e. R.sup.2═CH.sub.2OH] indicates the presence of a hydroxyl group linked to an alkyl group at the C-terminus of the molecule. The CH.sub.2OH moiety can be comprised in (4-amino-5-hydroxypentyl)guanidine or 4-amino-5-hydroxypentanamide. Designations of other R.sup.1 and R.sup.2 groups should be interpreted accordingly.
(17) KV1.3 Blockers
(18) The term Kv1.3 is used to refer to potassium voltage-gated channel subfamily A member 3, also referred to as KCNA3, HPCN3, HGK5, HuKIII and HLK3. “Subfamily A” may also be referred to as “shaker-related subfamily”. The human amino acid sequence is provided under UniProt accession number P22001, version P22001.3 (Q5VWN2).
(19) The Kv1.3 channel is expressed on T and B lymphocytes and has been implicated in T cell activation. A number of groups are pursuing development of Kv1.3 blockers for the inhibition of immune responses as well as for various other indications. However, the Kv1.3 channel is part of a complex family of related ion channels, also including the Kv1.1, Kv1.2 and Kv1.6 channels, which have different physiological roles. Consequently it is desirable for Kv1.3 inhibitors to be as selective as possible for Kv1.3 in preference to other ion channels, especially other voltage-gated potassium channels, such as Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv1.7 and Kv1.8.
(20) The term “ion channel blocker” is used simply to denote a compound having inhibitor (or blocking) activity against an ion channel, i.e. capable of inhibiting or eliminating ion flow through the respective ion channel, typically by binding to the ion channel. Similarly, the terms“Kv1.3 inhibitor” and “Kv1.3 inhibitor component” refer to a peptide capable of inhibiting or eliminating ion flow through a Kv1.3 ion channel, typically by binding to the Kv1.3 channel. However, the terms “blocker” and “inhibitor” should not be taken to imply any particular mechanism of action, or any particular mode of interaction with the ion channel itself.
(21) The ion channel blocker (and the Kv1.3 inhibitor component in isolation) of the invention has inhibitor or blocker activity at the Kv1.3 ion channel, i.e. it is capable of inhibiting ion flow through the Kv1.3 channel.
(22) IC.sub.50 values may be used as a measure of inhibitor (or blocker) activity or potency. An IC.sub.50 value is a measure of the concentration of an inhibitor required to achieve half of that compound's maximal inhibition of ion channel activity in a given assay. A compound which has a lower IC.sub.50 at a particular ion channel than a reference compound can be considered to be a more active inhibitor, or a more potent inhibitor, than the reference compound. The terms “activity” and “potency” are used interchangeably.
(23) IC.sub.50 values may be determined using any appropriate assay, such as fluorescence-based assays measuring ion flux (e.g. thallium ion flux) and patch clamp assays. They may be performed as described in the Examples below. Patch clamp assays may be preferred, e.g. using the QPatch® system.
(24) The ion channel blocker (or Kv1.3 inhibitor) of the invention may have an IC.sub.50 below 10 nM, but ideally the IC.sub.50 is below 5 nM, below 2 nM or below 1 nM. In some cases it may be as low as 0.5 nM, 0.1 nM, or even lower.
(25) Ion channel blockers of the invention may include the compounds 1-5, 7, 11-42, 44-48, 50-53, 55-60, 62-68, 70-73, 75-81, 83-88, 90-98, 101, 104-108, 116, 122, 129, 131, 132, 134 and 137 as described herein. These compounds are shown in Example 2 herein to have an IC.sub.50 of 0.3 nM or less.
(26) Ion channel blockers of the invention may include the compounds 7, 14-35, 37, 39, 40-48, 50-53, 55-60, 62-68, 70-73, 75-80, 83-97, 90, 91, 93-98, 101, 104-108, 116, 129 and 131 as described herein. These compounds are shown in Example 2 herein to have an IC.sub.50 of 0.2 nM or less.
(27) Ion channel blockers of the invention may include the compounds 7, 15-17, 19-28, 30-35, 37, 39-42, 44, 46-48, 51-53, 55-60, 62-67, 70, 71, 73, 75-80, 83-85, 87, 91, 93, 96-98, 104, 106, 107, 108 and 129 as described herein. These compounds are shown in Example 2 herein to have an IC.sub.50 of 0.15 nM or less.
(28) Ion channel blockers of the invention may include the compounds 1, 3, 17, 19, 21, 23, 26-33, 37, 41-43, 46-49, 52, 62, 63, 66-68, 70-73, 75, 76, 78, 82-84, 87, 91, 94, 97-102, 105-108, 115 and 116 as described herein. These compounds are shown in
(29) Example 5 herein to have an IC.sub.50 of 1 nM or less for inhibiting activation of T cells in rat whole blood.
(30) The ion channel blockers of the invention are selective for Kv1.3. In an embodiment the ion channel blockers of the invention are selective over Kv1.1, Kv1.2, Kv1.4,
(31) Kv1.5, Kv1.6, Kv1.7 and Kv1.8. In particular, the ion channel blockers of the invention are selective for Kv1.3 over one or more of Kv1.1, Kv1.2 and Kv1.6.
(32) For example, they may be: selective for Kv1.3 over Kv1.1; selective for Kv1.3 over Kv1.2; selective for Kv1.3 over Kv1.6; selective for Kv1.3 over Kv1.1 and Kv1.2; selective for Kv1.3 over Kv1.1 and Kv1.6; selective for Kv1.3 over Kv1.2 and Kv1.6; or selective for Kv1.3 over Kv1.1, Kv1.2 and Kv1.6.
(33) Typically, the ion channel blockers are selective for Kv1.3 over Kv1.1.
(34) They may additionally be selective for Kv1.3 over Kv1.2 and/or Kv1.6.
(35) By “selective” in this context is meant that the ion channel blockers have higher inhibitor activity against Kv1.3 than against the respective ones of Kv1.1, Kv1.2 and Kv1.6. Thus, their IC.sub.50 against Kv1.3 is typically lower than against the respective other ion channel or channels.
(36) Selectivity for Kv1.3 over another ion channel X may therefore be expressed as a ratio of the respective IC.sub.50 values, e.g. as IC.sub.50[X]/IC.sub.50[Kv1.3].
(37) The ion channel blockers of the invention may therefore have a selectivity for Kv1.3 over Kv1.1 of at least 10, at least 100, at least 1000, or at least 10000, and may be up to 100000 or even higher. Typically, they have a selectivity for Kv1.3 over Kv1.1 of at least 100, or at least 1000.
(38) Ion channel blockers of the invention may include compounds 1-3, 5, 8, 12, 16-23, 25-31, 34-37, 40-42, 45-49, 52-54, 56, 58, 62, 63, 70, 71, 76, 79, 83, 94-98, 101, 103-108 and 117 as described herein. These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.1 of at least 1000.
(39) Ion channel blockers of the invention may include compounds 3, 12, 16, 18-22, 25-28, 30, 31, 35, 37, 40-42, 52, 53, 62, 70, 71, 76, 79, 94, 95, 98 and 105 as described herein. These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.1 of at least 10000.
(40) The ion channel blockers of the invention may therefore have a selectivity for Kv1.3 over Kv1.2 of at least 10, at least 100, at least 1000, or at least 10000, and may be up to 100000 or even higher. Typically, they have a selectivity for Kv1.3 over Kv1.2 of at least 10, and preferably at least 50 or at least 100 or at least 1000.
(41) Ion channel blockers of the invention may include compounds 1-3, 5, 8, 16-23, 25-31, 34-37, 40-42, 45-49, 52-54, 56, 58, 62, 63, 70, 71, 76, 79, 83, 94-98, 101, 103-108 and 117 as described herein. These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.2 of at least 50.
(42) Ion channel blockers of the invention may include compounds 1, 3, 5, 8, 12, 18, 25, 29, 30, 36, 37, 41, 46-48, 52, 62, 70, 71, 79, 83, 94-98, 101, 103-106 and 117 as described herein. These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.2 of at least 700.
(43) Ion channel blockers of the invention may include compounds 1, 3, 12, 18, 29, 30, 36, 37, 41, 47, 48, 62, 70, 71, 79, 94-98, 101 and 103-105 as described herein.
(44) These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.2 of at least 1000.
(45) The ion channel blockers of the invention may therefore have a selectivity for Kv1.3 over Kv1.6 of at least 10, at least 100, at least 1000, or at least 10000, and may be up to 100000 or even higher. Typically, they have a selectivity for Kv1.3 over Kv1.6 of at least 100, or at least 400, or at least 1000.
(46) Ion channel blockers of the invention may include compounds 1, 2, 3, 5, 8, 12, 16-23, 25-31, 34-37, 40-42, 45-49, 52-54, 56, 58, 62, 63, 70, 71, 76, 79, 83, 94-98, 101, 103-108 and 117 as described herein. These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.6 of at least 400.
(47) Ion channel blockers of the invention may include compounds 3, 12, 16, 18, 20, 22, 26, 30, 31, 37, 41, 52, 53, 70, 71, 76, 79, 94, 95, 98 and 105 as described herein. These compounds are shown in Example 3 herein to have selectivity for Kv1.3 over Kv1.6 of at least 10000.
(48) The ion channel blockers of the invention may have greater selectivity than known ion channel blockers such as ShK, Mokatoxin (Moka1), Vm24, Odk2 or Osk1. Thus the ion channel blockers of the invention may have higher selectivity for Kv1.3 over ion channel X,
(49) i.e. IC.sub.50[X]/IC.sub.50[Kv1.3],
(50) which is greater than the selectivity of the comparison molecule. The selectivity of the two ion channel blockers will be determined under the same conditions for each ion channel to enable direct comparison. As mentioned above, any appropriate assays may be used, such as fluorescence-based ion flux assays and patch clamp assays.
(51) The ion channel blockers of the invention may have lower absolute inhibitor activity (i.e. higher IC.sub.50) than known ion channel blockers (such as Odk2 or Osk1) at any or all of Kv1.1, Kv1.2 and/or Kv1.6. However, it may be acceptable for them to have higher absolute inhibitor activity at any or all of these ion channels, as long as their selectivity for Kv1.3 is higher than that of the comparison compound. Typically, though, the compounds of the invention combine high specificity for Kv1.3 with high potency.
(52) Synthesis and Recombinant Expression
(53) The ion channel blockers described herein may be synthesised by means of solid-phase or liquid-phase peptide synthesis methodology. In this context, reference may be made to WO 98/11125 and, among many others, Fields, G. B. et al., 2002, “Principles and practice of solid-phase peptide synthesis”. In: Synthetic Peptides (2nd Edition), and the Examples herein.
(54) Alternatively, the ion channel blockers described herein may be synthesised by recombinant techniques, or by a combination of recombinant techniques and peptide chemistry.
(55) For example, an ion channel blocker peptide may be synthesised by a method which comprises (a) synthesising the peptide by means of solid-phase or liquid-phase peptide synthesis methodology and recovering the peptide thus obtained; (b) expressing the peptide from a nucleic acid construct that encodes the peptide and recovering the expression product; or (c) expressing a precursor peptide from a nucleic acid construct that encodes the precursor peptide sequence, recovering the expression product, and modifying the precursor peptide to yield an ion channel blocker of the invention.
(56) The precursor peptide may be modified by introduction of one or more non-proteinogenic amino acids (e.g. Nle), introduction of the appropriate terminal groups R.sup.1 and R.sup.2, etc.
(57) Expression of the peptide or precursor peptide from a nucleic acid encoding the peptide or precursor peptide may be performed in a cell or a cell-free expression system comprising such a nucleic acid. Such expression typically requires that the peptide or precursor peptide is composed entirely of proteinogenic amino acids (i.e. the 20 amino acids encoded by the standard genetic code.)
(58) For recombinant expression, the nucleic acid fragments encoding the precursor peptide will normally be inserted in suitable vectors to form cloning or expression vectors. The vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector. Preferred cloning and expression vectors (plasmid vectors) are capable of autonomous replication, thereby enabling high copy-numbers for the purposes of high-level expression or high-level replication for subsequent cloning.
(59) In general outline, an expression vector comprises the following features in the 5′.fwdarw.3′ direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasm), the nucleic acid fragment encoding the precursor peptide, and optionally a nucleic acid sequence encoding a terminator. They may comprise additional features such as selectable markers and origins of replication. When operating with expression vectors in producer strains or cell lines it may be preferred that the vector is capable of integrating into the host cell genome. The skilled person is very familiar with suitable vectors and is able to design one according to their specific requirements.
(60) The vectors of the invention are used to transform host cells to produce the peptide or precursor peptide. Such transformed cells can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors, and/or used for recombinant production of the precursor peptides.
(61) Preferred transformed cells are micro-organisms such as bacteria [such as the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichia pastoris), and protozoans. Alternatively, the transformed cells may be derived from a multicellular organism, i.e. it may be fungal cell, an insect cell, an algal cell, a plant cell, or an animal cell such as a mammalian cell. For the purposes of cloning and/or optimised expression it is preferred that the transformed cell is capable of replicating the nucleic acid fragment of the invention. Cells expressing the nucleic fragment can be used for small-scale or large-scale preparation of the peptides of the invention.
(62) When producing the peptide or precursor peptide by means of transformed cells, it is convenient, although far from essential, that the expression product is secreted into the culture medium.
(63) Therapeutic Indications
(64) As discussed above, blockers of Kv1.3 have been shown to inhibit proliferation of activated T cells and to have a beneficial effect in various experimental models of disease. Without wishing to be bound by theory, it is believed that cellular efflux of potassium via the Kv1.3 channel is required to sustain calcium influx required for T-cell activation.
(65) Kv1.3 is overexpressed in Gad5/insulin-specific T cells from patients with new onset type 1 diabetes, in myelin-specific T cells from MS patients and in T cells from the synovium of rheumatoid arthritis patients (Beeton et al., Proc Natl Acad Sci USA 103:17414-9, 2006), in breast cancer specimens (Abdul et al., Anticancer Res 23:3347, 2003) and prostate cancer cell lines (Fraser et al., Pflugers Arch 446:559, 2003).
(66) Positive outcomes in animal models with Kv1.3 blockers have been described in hypersensitivity models to ovalbumin and tetanus toxoid (Beeton et al., Mol Pharmacol 67:1369, 2005; Koo et al., Clin Immunol 197:99, 1999), models for multiple sclerosis such as rat adoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE) model (Beeton et al., Proc Natl Acad Sci USA 103:17414-9, 2006), inflammatory bone resorption model (Valverde et al., J Bone Mineral Res 19:155, 2004), models for arthritis (Beeton et al., Proc Natl Acad Sci 103: 17414, 2006; Tarcha et al., J. Pharmacol. Exp. Ther. 342: 642, 2012) and obesity, diabetes and metabolic disorders (Xu et al., Hum Mol Genet 12:551, 2003; Xu et al., Proc Natl Acad Sci 101: 3112, 2004).
(67) Topical application of Kv1.3 blockers has been proposed for the treatment of skin and mucosal inflammation.
(68) Thus, the ion channel blockers described in this specification have considerable potential for use in inhibiting or reducing inflammation, especially in the treatment of an inflammatory condition or disorder, including autoimmune disorders.
(69) An inflammatory condition or disorder may be any condition or disorder in which reduction of inflammation is desirable, e.g. where inflammation contributes to symptoms or pathogenesis.
(70) Such conditions include autoimmune disorders, allergy and hypersensitivity, allograft rejection and graft versus host disease.
(71) More specifically, conditions include hay fever, asthma, anaphylaxis, allergic rhinitis, urticaria, eczema, alopecia areata, dermatomyositis, inclusion body myositis, polymyositis, ankylosing spondylitis, vasculitis, arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Sjogren's syndrome, systemic lupus erythematosus (SLE, or simply “lupus”), and uveitis, inflammatory fibrosis (e.g. scleroderma, lung fibrosis, cirrhosis), chronic obstructive pulmonary disease (COPD), hepatitis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, colitis (including Crohn's disease and ulcerative colitis), erythema, thyroiditis, psoriasis, atopic dermatitis, allergic contact dermatitis, scleroderma, glomerulonephritis, inflammatory bone resorption, multiple sclerosis, type 1 diabetes, transplant rejection and graft-versus-host disease.
(72) Blockers of Kv1.3 may also have beneficial metabolic effects, e.g. in relation to energy homeostasis, body weight regulation, and glucose control.
(73) The ion channel blockers described here may therefore be used for inhibiting weight gain, promoting weight loss, reducing excess body weight or treating obesity (e.g. by control of appetite, feeding, food intake, calorie intake, and/or energy expenditure), as well as in the treatment of associated disorders and health conditions including obesity linked inflammation, obesity linked gallbladder disease and obesity induced sleep apnoea.
(74) An effect on body weight may be therapeutic or cosmetic.
(75) The ion channel blockers may also be used for the treatment of conditions caused by or associated with impaired glucose control, including metabolic syndrome, insulin resistance, glucose intolerance, pre-diabetes, increased fasting glucose and type 2 diabetes. Some of these conditions can be associated with obesity. Their effects on these conditions may be mediated in whole or in part via an effect on body weight, or may be independent thereof.
(76) Kv1.3 is also expressed in proliferating human and mouse smooth muscle cells. Blockers of Kv1.3 may be effective in smooth muscle proliferative disorders such as restenosis, e.g. in patients following vascular surgery (e.g. angioplasty).
(77) Further evidence suggests that Kv1.3 channels are involved in the activation and/or proliferation of many types of cells, including tumor cells (Bielanska et al., Curr. Cancer Drug Targets 9:904-14, 2009), microglia (Khanna et al., Am. J. Physiol. Cell Physiol. 280: C796-806, 2001) and differentiation of neuronal progenitor cells (Wang et al., J. Neurosci. 30:5020-7, 2010). Kv1.3 blockers may therefore be beneficial in the treatment of neuroinflammatory and neurodegenerative disorders such as Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease and amyotrophic lateral sclerosis (ALS) (e.g. following viral infections), and cancers including breast cancer, prostate cancer, and lymphoma, such as non-Hodgkin lymphoma (NHL). Non-Hodgkin lymphomas include T-cell NHL and B-cell NHL. Forms of B-cell NHL include diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and mantle cell lymphoma. Forms of T-cell NHL include mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, precursor T-lymphoblastic lymphoma and Sézary syndrome.
(78) Pharmaceutical Compositions and Administration
(79) An aspect of the present invention relates to a composition comprising an ion channel blocker of the invention, or a salt thereof, together with a carrier, excipient or vehicle. In certain embodiments, the composition is a pharmaceutical composition, any salt is a pharmaceutically acceptable salt, and the carrier is a pharmaceutically acceptable carrier, excipient or vehicle.
(80) Accordingly, the compounds of the present invention, or salts thereof, especially pharmaceutically acceptable salts thereof, may be formulated as compositions or pharmaceutical compositions prepared for storage or administration, and which comprise a therapeutically effective amount of a compound of the invention, or a salt thereof.
(81) Suitable salts formed with bases include metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium salts; ammonia salts and organic amine salts, such as those formed with morpholine, thiomorpholine, piperidine, pyrrolidine, a lower mono-, di- or tri-alkylamine (e.g., ethyl-tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a lower mono-, di- or tri-(hydroxyalkyl)amine (e.g., mono-, di- or triethanolamine). Internal salts may also be formed. Similarly, when a compound of the present invention contains a basic moiety, salts can be formed using organic or inorganic acids. For example, salts can be formed from the following acids: formic, acetic, propionic, butyric, valeric, caproic, oxalic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulphuric, benzoic, carbonic, uric, methanesulphonic, naphthalenesulphonic, benzenesulphonic, toluenesulphonic, p-toluenesulphonic (i.e. 4-methylbenzene-sulphonic), camphorsulphonic, 2-aminoethanesulphonic, aminomethylphosphonic and trifluoromethanesulphonic acid (the latter also being denoted triflic acid), as well as other known pharmaceutically acceptable acids. Amino acid addition salts can also be formed with amino acids, such as lysine, glycine, or phenylalanine.
(82) In some embodiments, a pharmaceutical composition of the invention is one wherein the compound is in the form of a pharmaceutically acceptable acid addition salt.
(83) As will be apparent to one skilled in the medical art, a “therapeutically effective amount” of a compound or pharmaceutical composition of the present invention will vary depending upon, inter alia, the age, weight and/or gender of the subject (patient) to be treated. Other factors that may be of relevance include the physical characteristics of the specific patient under consideration, the patient's diet, the nature of any concurrent medication, the particular compound(s) employed, the particular mode of administration, the desired pharmacological effect(s) and the particular therapeutic indication. Because these factors and their relationship in determining this amount are well known in the medical arts, the determination of therapeutically effective dosage levels to achieve the desired therapeutic effect will be within the ambit of the skilled person.
(84) As used herein, the term “a therapeutically effective amount” refers to an amount which reduces symptoms of a given condition or pathology, and preferably which normalizes physiological responses in an individual with that condition or pathology.
(85) Reduction of symptoms or normalization of physiological responses can be determined using methods routine in the art and may vary with a given condition or pathology. In one aspect, a therapeutically effective amount of a compound of the invention, or a pharmaceutical composition, is an amount which restores a measurable physiological parameter to substantially the same value (preferably to within 30%, more preferably to within 20%, and still more preferably to within 10% of the value) of the parameter in an individual without the condition or pathology in question.
(86) In one embodiment of the invention, administration of a compound or pharmaceutical composition of the present invention is commenced at lower dosage levels, with dosage levels being increased until the desired effect of preventing/treating the relevant medical indication is achieved. This would define a therapeutically effective amount. For the compounds of the present invention, alone or as part of a pharmaceutical composition, such human doses of the active compound may be between about 0.01 pmol/kg and 500 μmol/kg body weight, between about 0.01 pmol/kg and 300 μmol/kg body weight, between 0.01 pmol/kg and 100 μmol/kg body weight, between 0.1 pmol/kg and 50 μmol/kg body weight, between 1 pmol/kg and 10 μmol/kg body weight, between 5 pmol/kg and 5 μmol/kg body weight, between 10 pmol/kg and 1 μmol/kg body weight, between 50 pmol/kg and 0.1 μmol/kg body weight, between 100 pmol/kg and 0.01 μmol/kg body weight, between 0.001 μmol/kg and 0.5 μmol/kg body weight, between 0.05 μmol/kg and 0.1 μmol/kg body weight.
(87) An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.
EXAMPLES
Example 1: General Peptide Synthesis
(88) List of abbreviations and suppliers are provided in the table below
(89) TABLE-US-00016 List of abbreviations and suppliers Abbre- viation Name Brand/Supplier Resins TentaGel ™ PHB AA(Proct)- Rapp Polymere Fmoc TentaGel ™ SRAM Rapp Polymere Amino acids Pseudoprolines (E.g. QT, AT, Jupiter Bioscience FS) Ltd. Fmoc-L-AA-OH Senn Chemicals AG Coupling reagents COMU (1-Cyano-2-ethoxy-2- Watson oxoethylidenaminooxy)dimethy International Ltd. lamino-morpholino-carbenium hexafluorophosphate DIC Diisopropylcarbodiimide Fluka/Sigma Aldrich Co. HATU N-[(dimethylamino)-1 H-1,2,3- ChemPep Inc. triazol[4,5-b]pyridine-1- ylmethylene]-N- methylmethanaminium hexafluorophosphate N-oxide HOBt Hydroxybenzotriazole Sigma-Aldrich Co. Solvents reagents Boc.sub.2O Di-tert-butyl pyrocarbonate Advanced ChemTech DCM Dichloromethane Prolabo (VWR) DIPEA Diisopropylethylamine Fluka/Sigma Aldrich Co. DMF N,N-dimethylformamide Taminco DODT 3,6-dioxa-1,8-octanedithiol Sigma-Aldrich Co. Et.sub.2O Diethyl ether Prolabo (VWR) EtOH Ethanol CCS Healthcare AB Formic acid (HPLC) Sigma-Aldrich Co. H.sub.2O Water, Milli-Q water Millipore MeCN Acetonitrile (HPLC) Sigma-Aldrich Co. NMP N-methylpyrrolidone Sigma-Aldrich Co. Piperidine Jubliant Life Sciences Ltd. TFA Trifluoroacetic acid (HPLC) Chemicals Raw Materials Ltd. TIS Triisopropylsilane Sigma-Aldrich Co. MeOH Methanol Sigma-Aldrich Co.
(90) Apparatus and Synthetic Strategy
(91) Peptides were synthesized batchwise on a peptide synthesiser, such as a CEM Liberty Peptide Synthesizer or a Symphony X Synthesizer, according to solid phase peptide synthetic procedures using 9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting group and suitable common protection groups for side-chain functionalities.
(92) As polymeric support based resins, such as e.g. TentaGel™, was used. The synthesizer was loaded with resin that prior to usage was swelled in DMF.
(93) Coupling
(94) CEM Liberty Peptide Synthesizer
(95) A solution of Fmoc-protected amino acid (4 equiv.) was added to the resin together with a coupling reagent solution (4 equiv.) and a solution of base (8 equiv.). The mixture was either heated by the microwave unit to 70-75° C. and coupled for 5 minutes or coupled with no heat for 60 minutes. During the coupling nitrogen was bubbled through the mixture.
(96) Symphony X Synthesizer
(97) The coupling solutions were transferred to the reaction vessels in the following order: amino acid (4 equiv.), HATU (4 equiv.) and DIPEA (8 equiv.). The coupling time was 10 min at room temperature (RT) unless otherwise stated. The resin was washed with DMF (5×0.5 min). In case of repeated couplings the coupling time was in all cases 45 min at RT.
(98) Deprotection
(99) CEM Liberty Peptide Synthesizer
(100) The Fmoc group was deprotected using piperidine in DMF or other suitable solvents. The deprotection solution was added to the reaction vessel and the mixture was heated for 30 sec. reaching approx. 40° C. The reaction vessel was drained and fresh deprotection solution was added and subsequently heated to 70-75° C. for 3 min. After draining the reaction vessel the resin was washed with DMF or other suitable solvents.
(101) Symphony X Synthesizer
(102) Fmoc deprotection was performed for 2.5 minutes using 40% piperidine in DMF and repeated using the same conditions. The resin was washed with DMF (5×0.5 min).
(103) Cleavage
(104) The dried peptide resin was treated with TFA and suitable scavengers for approximately 2 hours. The volume of the filtrate was reduced and the crude peptide was precipitated after addition of diethylether. The crude peptide precipitate was washed several times with diethylether and finally dried.
(105) HPLC Purification of the Crude Peptide
(106) The crude peptide was purified by preparative reverse phase HPLC using a conventional HPLC apparatus, such as a Gilson GX-281 with 331/332 pump combination’, for binary gradient application equipped with a column, such as 5×25 cm Gemini NX 5u C18 110A column, and a fraction collector using a flow 20-40 ml/min with a suitable gradient of buffer A (0.1% Formic acid, aq.) or A (0.1% TFA, aq.) and buffer B (0.1% Formic acid, 90% MeCN, aq.) or B (0.1% TFA, 90% MeCN, aq.). Fractions were analyzed by analytical HPLC and MS and selected fractions were pooled and lyophilized. The final product was characterized by HPLC and MS.
(107) Disulphide Formation
(108) The crude or partially purified linear peptide with six cysteines was dissolved in a buffer such as sodium hydrogen carbonate (NaHCO.sub.3) or ammonium acetate (NH.sub.4Ac) to give a final concentration of approximate 0.1 mg/ml or 25 μM. The pH of the buffer was adjusted to pH 8.0 and the solution was stirred at room temperature under magnetic stirring and open access to the atmosphere. The progress of the reaction was determined by HPLC and was usually evaluated to be complete overnight. The solution was quenched by reducing the pH of the solution by an organic acid such as acetic acid or trifluoroacetic acid (pH<4). The solution was filtered and loaded directly on a prep-HPLC column for purification.
(109) Analytical HPLC
(110) Final purities were determined by analytic HPLC (Agilent 1100/1200 series) equipped with auto sampler, degasser, 20 μl flow cell and Chromeleon software. The HPLC was operated with a flow of 1.2 ml/min at 40° C. using an analytical column, such as Kinetex 2.6 μm XB-C18 100A 100×4.6 mm column. The compound was detected and quantified at 215 nm. Buffers A (0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.).
(111) Mass Spectroscopy
(112) Final MS analysis was performed on a conventional mass spectrometer, e.g. Waters Xevo G2 Tof, equipped with electrospray detector with lock-mass calibration and MassLynx software. It was operated in positive mode using direct injection and a cone voltage of 15V (1 TOF), 30 V (2 TOF) or 45 V (3 TOF) as specified on the chromatogram. Precision was 5 ppm with a typical resolution of 15,000-20,000.
(113) Compounds synthesized are shown in Table 1.
(114) TABLE-US-00017 TABLE 1 Cmpnd No. Sequence 1 H-QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 1) 2 H-QMDMRCSASVECKQKCLKAIGRGFGKCMNKKCKCYPR-OH (SEQ ID NO. 2) 3 H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 3) 4 H-NMDMRCSASVECKQKCLKAIGRGFGKCMNKKCKCYPR-OH (SEQ ID NO. 4) 5 H-MDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 5) 6 H-DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 6) 7 H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 7) 8 H-NIDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 8) 9 H-NMDVRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 9) 10 H-NMDMRCSASVECKQKCKDAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 10) 11 H-GGNMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 11) 12 H-NMEMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 12) 13 H-SGNMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 13) 14 H-N[Nle]DMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 14) 15 H-NMD[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 15) 16 H-NMDMRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 16) 17 H-NMDMRCSASVECKVKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 17) 18 H-NMDMRCSASVECKQLCLKAIGSIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 18) 19 H-NMDMRCSASVECKQKCKKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 19) 20 H-NMDMRCSASVECKQKCLDAIGSIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 20) 21 H-NMDMRCSASVECKQKCLKAIRSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 21) 22 H-NMDMRCSASVECKQKCLKAIESIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 22) 23 H-NMDMRCSASVECKQKCLKAIGSIFGKDANKKCKCYPRRRTA-NH.sub.2 (SEQ ID NO. 23) 24 H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPRHRRK-NH.sub.2 (SEQ ID NO. 24) 25 H-NMDMRCKASVECKQKCLKAIGSIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 25) 26 H-NMDMRCSISVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 26) 27 H-NMDMRCSASRECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 27) 28 H-NMDMRCSASVC2CKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 28) 29 H-NMDMRCSASVECLQKCLKAIGSIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 29) 30 H-NMDMRCSASVECAQKCLKAIGSIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 30) 31 H-NMDMRCSASVECKEKCLKAIGSIFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 31) 32 H-NMDMRCSASVECKLKOLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 32) 33 H-NMDMRCSASVECKQKCLKAIHSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 33) 34 H-NMDMRCSASVECKQKCLKAIGSKFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 34) 35 H-NMDMRCSASVECKQKCLKAIGSRFGKDANKKCKCYPR-NH.sub.2 (SEQ ID NO. 35) 36 H-NMDMRCSASVECKQKCLKAIGSIFGKCMNGKCKCYPR-NH.sub.2 (SEQ ID NO. 36) 37 H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCHCYPR-NH.sub.2 (SEQ ID NO. 37) 38 H-NMDMRCSASVECKQKCLKAIGSIFGKCMNKKCVCYPR-NH.sub.2 (SEQ ID NO. 38) 39 H-N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 39) 40 H-N[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 40) 41 H-P[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 41) 42 H-N[Nle]D[Nle]RCRASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 42) 43 H-N[Nle]D[Nle]RCSASVECEQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 43) 44 H-N[Nle]D[Nle]RCSASVECQQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 44) 45 H-N[Nle]D[Nle]RCSASVECKKKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 45) 46 H-N[Nle]S[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 46) 47 H-N[Nle]D[Nle]RCSHSVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 47) 48 H-N[Nle]D[Nle]RCSASVECKQSCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 48) 49 H-N[Nle]D[Nle]RCSASVECKQKCKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 49) 50 H-NMDMRCSASVECKQKCYKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 50) 51 H-NMDMRCSASVECKQKCIRKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 51) 52 H-NMDMRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 52) 53 H-NMDMRCSASVECKQKCLYAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 53) 54 H-NMDMRCSASVECKQKCLAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 54) 55 H-NMDMRCSASVECKQKCLKYIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 55) 56 H-NMDMRCSASVECKQKCLKRIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 56) 57 H-NMDMRCSASVECKQKCLKIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 57) 58 H-NMDMRCSASVECKQKCLKAYGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 58) 59 H-NMDMRCSASVECKQKCLKAEGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 59) 60 H-NMDMRCSASVECKQKCLKARGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 60) 61 H-NMDMRCSASVECKQKCLKAGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 61) 62 H-N[Nle]D[Nle]RCSASVECKQKCLKAIGSPFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 62) 63 H-N[Nle]D[Nle]RCSASKECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 63) 64 H-H[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 64) 65 H-Y[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 65) 66 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 66) 67 H-V[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 67) 68 H-S[Nle]D[Nle]RCSA[Abu]VECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 68) 69 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFG[homo- Lys]CMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 69) 70 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NH.sub.2)]PR-NH.sub.2 (SEQ ID NO. 70) 71 H-S[Nle]D[Nle]RCSASVECGQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 71) 72 H-S[Nle]D[Nle]RCSASVECVQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 72) 73 H-S[Nle]D[Nle]RCSASVECK[2-Amino-5- carboxypentanoyl]KCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 73) 74 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKCYQ-NH.sub.2 (SEQ ID NO. 74) 75 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCIRCYPR-NH.sub.2 (SEQ ID NO. 75) 76 H-S[Nle]DERCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 76) 77 H-S[Nle]D[Nle]RCSASVECKQKCLGAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 77) 78 H-S[Nle]D[Nle]RCSASVECKQKCLVAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 78) 79 H-S[Nle]D[Nle]RCSASVECAQSCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 79) 80 H-S[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 80) 81 H-S[Nle]D[Nle]RCSASVECAQLCLAAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 81) 82 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 82) 83 H-P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC-NH.sub.2 (SEQ ID NO. 83) 84 H-S[Nle]D[Nle]RCSASVECKEKCLQAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 84) 85 H-P[Nle]D[Nle]RCSASVECKEKCL[homo-Gln]AlGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 85) 86 H-CSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC-NH.sub.2 (SEQ ID NO. 86) 87 H-S[Nle]D[Nle]RCSASVECKQKCLAAIGCIFGKC[Nle]NKKCKCYPC-NH.sub.2 (SEQ ID NO. 87) 88 H-P[Nle]D[Nle]RCSASVECKQKCLKAIGCIFGKC[Nle]NKKCKCYPC-OH (SEQ ID NO. 88) 89 H-S[Nle]D[Nle]RCSALVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 89) 90 H-S[Nle]D[Nle]RCSAVVECKQKCLKAIGSIFGKCMNKKCKCYPR-NH.sub.2 (SEQ ID NO. 90) 91 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4-F)]PR- NH.sub.2 (SEQ ID NO. 91) 92 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- NO.sub.2)]PR-NH.sub.2 (SEQ ID NO. 92) 93 H-S[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKCMNKKCKC[F(4- CH.sub.3)]PR-NH.sub.2 (SEQ ID NO. 93) 94 H-[Nle]D[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 94) 95 H-NID[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 95) 96 H-PIE[Nle]RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 96) 97 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 97) 98 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 98) 99 H-P[Nle]E[Nle]RCSASVECKQKCLLAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 99) 100 H-PIDERCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 100) 101 H-PIE[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 101) 102 H-P[Nle]D[Nle]RCSASVECAQKCLAAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 102) 103 H-P[Nle]E[Nle]RCSASVECAQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 103) 104 H-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 104) 105 Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 105) 106 H-RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 106) 107 Ac-SKCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 107) 108 Ac-LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-NH.sub.2 (SEQ ID NO. 108) 109 Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPS-NH.sub.2 (SEQ ID NO. 109) 110 Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYS-NH.sub.2 (SEQ ID NO. 110) 111 Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYG-NH.sub.2 (SEQ ID NO. 111) 112 Ac-CSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCY-NH.sub.2 (SEQ ID NO. 112) 113 Ac-RCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 113) 114 H-LRCSASVECKQKCLKAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 114) 115 Ac-LRCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 115) 116 Ac-LRCSASVECKQKCLAAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 116) 117 H-CSASVECKQKCLKAIGSIFGKCMNKKCKCYPR-OH (SEQ ID NO. 117) 118 H-P[Nle]E[Nle]RCSASVECKEKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 118) 119 H-p[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 119) 120 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYR-OH (SEQ ID NO. 120) 121 H-[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 121) 122 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRR-OH (SEQ ID NO. 122) 123 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRY-OH (SEQ ID NO. 123) 124 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRL-OH (SEQ ID NO. 124) 125 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRH-OH (SEQ ID NO. 125) 126 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRE-OH (SEQ ID NO. 126) 127 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRKS- OH (SEQ ID NO. 127) 128 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRFE- OH (SEQ ID NO. 128) 129 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRHR- OH (SEQ ID NO. 129) 130 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRAK- OH (SEQ ID NO. 130) 131 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYP-[(4- amino-5-hydroxypentyl)guanidine] (SEQ ID NO. 131) 132 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-[4- amino-5-hydroxypentanamide] (SEQ ID NO. 132) 133 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRST- OH (SEQ ID NO. 133) 134 H-P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPRRY- OH (SEQ ID NO. 134) 135 H-P[Nle]E[Nle]RCSSSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 135) 136 H-P[Nle]E[Nle]RCSLSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 136) 137 H-P[Nle]E[Nle]RCSAPVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 137) 138 H-P[Nle]E[Nle]RCSASPECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 138) 139 H-P[Nle]E[Nle]RCSASQECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 139) 140 H-P[Nle]E[Nle]RCSASVECLQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 140) 141 H-P[Nle]E[Nle]RCSASVECKQPCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 141) 142 H-P[Nle]E[Nle]RCEASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 142) 143 H-P[Nle]E[Nle]RCFASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 143) 144 H-P[Nle]E[Nle]RCSYSVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 144) 145 H-P[Nle]E[Nle]RCSAFVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR-OH (SEQ ID NO. 145)
(115) The following compounds were also synthesised for use as controls:
(116) TABLE-US-00018 ShK (SEQ ID NO: 177) H-RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC-OH ShK186 (SEQ ID NO: 178) H-[phosphotyrosyl)][8-Amino-3,6-dioxaoctanoyl] RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC-NH.sub.2 Moka1 (SEQ ID NO: 179) H-INVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS-OH Vm24 (SEQ ID NO: 180) H-AAAISCVGSPECPPKCRAQGCKNGKCMNRKCKCYYC-NH.sub.2
Example 2: Kv1.3 Blocker Activity in FLIPR Thallium Assay
(117) A human Kv1.3 voltage-gated K+ channel cell line was purchased from Perkin Elmer (TDS-AX-010-C-1). The cell line is based on CHO-DUKX cells stably transfected with the human Kv1.3 voltage-gated K+ channel.
(118) The cell line was grown in MEMα with nucleotides, GlutaMAX (Gibco Cat #32571028), 10% Foetal Bovine Serum (FBS), 0.4 mg/ml Geneticin, 100 units/ml Penicillin, and 100 μg/ml Streptomycin and seeded at 10.000 cells/well in black poly-D-lysine-coated 96 well plates.
(119) The FIuxOR™ Potassium Ion Channel Assay (Invitrogen Cat #F10016) was used to quantitate flux of thallium ions into the cells as a response to Kv1.3 activation with a stimulus buffer causing depolarization of the cell membrane, generating a fluorescent signal, proportional to channel activity. The assay was performed as described by the assay kit manufacturer. Fluorescence responses were recorded and quantified using the FLIPR® Tetra High Throughput Screening System (Molecular Devices, Inc.).
(120) Data from test compounds eliciting an inhibition of thallium flux into the cell were normalised relative to the positive (ShK) and negative control (vehicle) to calculate the IC.sub.50 from the concentration response curve. Results are shown in Table 2. IC.sub.50 can be regarded as a measure of potency of inhibition for the respective compound. The IC.sub.50 value is a measure of the concentration of an inhibitor required to achieve half of that compound's maximal inhibition of ion channel activity in a given assay. A compound which has a lower IC.sub.50 at a particular ion channel than a reference compound can be considered to be a more active inhibitor, or a more potent inhibitor, than the reference compound.
(121) TABLE-US-00019 TABLE 2 Compound IC50 (nM) 1 0.24 2 0.25 3 0.30 4 0.29 5 0.25 6 0.34 7 0.12 8 0.47 9 0.53 10 0.57 11 0.3 12 0.23 13 0.26 14 0.16 15 0.12 16 0.14 17 0.14 18 0.19 19 0.12 20 0.13 21 0.12 22 0.11 23 0.055 24 0.073 25 0.085 26 0.092 27 0.065 28 0.064 29 0.18 30 0.09 31 0.075 32 0.12 33 0.14 34 0.11 35 0.077 36 0.24 37 0.14 38 0.46 39 0.11 40 0.1 41 0.15 42 0.087 43 0.5 44 0.13 45 0.16 46 0.11 47 0.12 48 0.14 49 0.36 50 0.16 51 0.11 52 0.11 53 0.12 54 0.46 55 0.12 56 0.087 57 0.11 58 0.11 59 0.035 60 0.035 61 0.40 62 0.13 63 0.13 64 0.12 65 0.12 66 0.12 67 0.12 68 0.20 69 0.58 70 0.13 71 0.10 72 0.19 73 0.11 74 0.36 75 0.10 76 0.093 77 0.13 78 0.12 79 0.13 80 0.15 81 0.22 82 0.49 83 0.12 84 0.084 85 0.15 86 0.20 87 0.10 88 0.27 89 2.7 90 0.19 91 0.10 92 0.49 93 0.13 94 0.16 95 0.16 96 0.15 97 0.15 98 0.11 99 0.34 100 0.85 101 0.18 102 0.45 103 0.38 104 0.12 105 0.16 106 0.092 107 0.084 108 0.1 109 0.96 110 2.3 111 2.1 112 1.7 113 0.34 114 0.39 115 0.31 116 0.18 117 1.5 118 1.39 119 0.63 120 0.894 121 0.451 122 0.226 123 1.04 124 1.47 125 0.383 126 6.49 127 0.562 128 7.18 129 0.143 130 0.529 131 0.156 132 0.224 133 1.1 134 0.21 135 0.336 136 6.24 137 0.333 138 0.228 139 0.453 140 3.57 141 1.07 142 1.5 143 0.316 144 0.739 145 8.26
Example 3: Kv1.3 Selectivity in Patch Clamp Assay
(122) Chinese Hamster Ovary (CHO) cell lines stably expressing exogenous human α-subunits of each potassium ion channel were grown and passaged under standard culture conditions.
(123) The automated, chip-based planar patch clamp device QPatch® was used to quantitate the ionic currents. All recordings were made in the conventional whole-cell configuration after establishment of gigaohm seals. External recording solution contained (150 mM NaCl, 10 mM KCl, 10 mM HEPES, 1 mM MgCl.sub.2, 3 mM CaCl.sub.2, 10 mM Glucose, pH adjusted to 7.4 with NaOH) and Internal recording solution (20 mM KCl, 120 mM KF, 10 mM HEPES, 10 mM EGTA, 5 mM NaATP, pH adjusted to 7.2 with KOH). During experiments 0.1% (v/v) BSA was included as a vehicle in all external recording solutions. Currents were elicited from a holding potential of −80 mV using a voltage protocol, which shifted the voltage to 30 mV for 500 ms every 15 s. Concentration-response relationships were established by cumulatively applying seven escalating concentrations of test sample to an individual cell with a recording period of 2 min per compound application.
(124) The efficacy was determined as the mean charge for the last three sweeps at the end of each concentration application period from the cursor positions. The percent inhibition for each test dose application period was calculated as the reduction in mean cursor value (charge) relative to the cursor value measured at the end of the vehicle period and used to calculate the IC.sub.50 from the concentration response curve.
(125) Results are shown in Table 3.
(126) TABLE-US-00020 TABLE 3 Potency IC.sub.50 (nM) Selectivity Compound Kv1.1 Kv1.2 Kv1.3 Kv1.6 Kv1.1/Kv1.3 Kv1.2/Kv1.3 Kv1.6/Kv1.3 1 >3000 518 0.38 >3000 >7900 1364 >7900 2 >3000 97 1.50 1810 >2000 65 1204 3 >3000 409 0.27 >3000 >11300 1540 >11300 5 >3000 285 0.38 >3000 >7900 747 >7900 8 >3000 376 0.44 >3000 >6800 857 >6800 12 >3000 228 0.11 >3000 >27600 2093 >27600 16 >3000 133 0.24 2460 >12600 561 10360 17 >3000 66 0.42 912 >7200 159 2182 18 >3000 256 0.24 >3000 >12700 1080 >12700 19 >3000 45 0.22 435 >13500 202 1955 20 >3000 99 0.21 2527 >14300 471 12045 21 >3000 57 0.22 626 >13700 259 2851 22 >3000 181 0.29 >3000 >10200 615 >10200 23 626 28 0.15 215 4280 191 1472 25 >3000 67 0.09 363 >34200 761 4144 26 >3000 43 0.26 >3000 >11800 169 >11800 27 >3000 11 0.12 206 >25900 94 1778 28 >3000 16 0.20 252 >15200 83 1272 29 >3000 874 0.40 >3000 >7400 2163 >7400 30 >3000 666 0.14 >3000 >22000 4897 >22000 31 >3000 179 0.28 >3000 >10800 648 >10800 32 0.38 34 >3000 100 0.42 207 >7100 237 490 35 >3000 18 0.20 192 >15000 88 963 36 >3000 439 0.31 >3000 >9700 1422 >9700 37 >3000 338 0.19 >3000 >16100 1816 >16100 39 0.15 40 >3000 113 0.26 1829 >11600 436 7073 41 >3000 939 0.18 >3000 >17100 5362 >17100 42 >3000 109 0.16 316 >18600 674 1954 45 >3000 257 0.75 >3000 >4000 343 >4000 46 >3000 302 0.32 1395 >9400 943 4359 47 >3000 772 0.54 609 >5600 1438 1134 48 >3000 >3000 0.37 >3000 >8200 >8200 >8200 49 >3000 164 1.34 946 >2200 123 707 50 0.27 51 0.25 52 >3000 88 0.11 >3000 >27500 803 >27500 53 >3000 149 0.29 >3000 >10300 514 >10300 54 >3000 291 2.41 >3000 >1200 121 >1200 55 0.12 56 >3000 57 0.30 932 >9900 187 3062 58 >3000 145 0.34 >3000 >8800 426 >8800 62 >3000 449 0.26 2349 >11600 1740 9102 63 >3000 184 0.31 1231 >9600 587 3929 64 0.25 65 0.14 66 0.18 67 0.32 70 >3000 423 0.25 >3000 >12200 1720 >12200 71 >3000 373 0.11 >3000 >27700 3438 >27700 75 0.13 76 >3000 122 0.28 >3000 >10600 430 >10600 79 >3000 1149 0.21 >3000 >14600 5596 >14600 80 0.16 83 >3000 624 0.63 1222 >4800 995 1949 94 >3000 1859 0.17 >3000 >17800 11034 >17800 95 >3000 2063 0.27 >3000 >11300 7756 >11300 96 >3000 1758 0.39 >3000 >7800 4561 >7800 97 >3000 1117 0.33 >3000 >9100 3384 >9100 98 >3000 311 0.18 >3000 >16200 1689 >16200 101 >3000 1968 0.35 >3000 >8600 5630 >8600 103 >3000 >3000 0.55 >3000 >5500 5500 >5500 104 >3000 >3000 0.43 >3000 >7000 >7000 >7000 105 >3000 384 0.24 >3000 >12400 1591 >12400 106 >3000 280 0.40 1302 >7600 708 3290 107 >3000 196 1.00 1451 >3000 196 1451 108 >3000 122 0.35 2456 >8700 352 7096 117 >3000 1832 2.09 >3000 >1400 878 >1400 ShK 0.0021 11 0.017 0.19 0.12 644 11 ShK-186 0.31 43 0.095 0.45 4.9 682 7.4 Moka1 >3000 275 9.0 >3000 >300 31 >300 Vm24 0.097 4.7 0.15 21 0.65 31 139
(127) Comparison of the data in Examples 2 and 3 shows that a good correlation exists between the IC.sub.50 values as measured in the two assays.
Example 4a: Inhibitory Activity of Kv1.3 Blockers on Human PBMCs
(128) Human peripheral blood mononuclear cells (PBMCs) were used to assess the effects of Kv1.3 blockers on T-cell activation as determined by IL-2 (cytokine) release after stimulation with anti-CD3.
(129) Human PBMCs were obtained from Precision for Medicine (Frederick, Md.). Cells from 5 donors were used. Plate-bound anti-CD3 was used to stimulate bulk T cells in the PBMC preparations. Briefly, 96-well plates were coated with anti-CD3 antibody for 2 hrs at 37° C., using 50 μL of a 0.5 μg/mL anti-CD3 solution diluted in 1×PBS. Thereafter the plates were washed twice.
(130) Kv1.3 blockers as shown in Table 4a were diluted in medium (RPMI 1640 with Glutamax-I containing 10% v/v Fetal Bovine Serum, 1% v/v penicillin-streptomycin solution) and added in a volume of 100 μL at concentrations ranging from 0.01 μM to 100 nM (tenfold dilutions). Cyclosporin A (1 ug/ml) and Vm24 peptide (100 nM) were used as positive controls. Finally, 1×10.sup.5 PBMCs were added to each well in a volume of 100 μL, giving a final volume of 200 μL per well. The plates were incubated for 20-24 hours in a 37° C./5% CO.sub.2 incubator. After centrifugation of the plates, 25 μl supernatant was transferred to IL-2 detection plates (MSD Human IL-2 Tissue Culture Kit, cat #K151AHB-2) and IL-2 was measures as described by the manufacturer (Meso Scale Discovery, Rockville, Md., USA).
(131) Results are shown in Table 4a as geometric mean of IC50 values obtained from anti-CD3 stimulated human PBMC assays. All values derive from at least 4 replicates.
(132) TABLE-US-00021 TABLE 4a anti-CD3 PBMC/IL-2 Compound IC50 [nM] ShK-186 0.07 16 0.05 17 0.4 20 0.1 35 0.1
(133) Incubation with anti-CD3 antibody activated hPBMC and addition of Kv1.3 blockers resulted in dose-dependent reduction in the IL-2 secretion. On average the IC.sub.50 values (as calculated from IL-2 release) of the test compounds were in the range of 0.05 nM to 0.4 nM. This was comparable to the IC.sub.50 observed with ShK186 (IC.sub.50 is 0.07 nM) and about 10-100 fold lower than the IC.sub.50 of Moka1 which was less potent in inhibiting IL-2 secretion.
(134) There is no significant difference between the test compounds and ShK186. ShK186 and test compounds were all significantly lower than Moka1.
(135) Cyclosporine blocked CD3 induced IL-2 release completely in all experiments.
Example 4b: Inhibitory Activity of Kv1.3 Blockers on Human PBMCs
(136) Human peripheral blood mononuclear cells (PBMCs) were used to assess the effects of Kv1.3 blockers on T-cell activation as determined by IL-2 release after stimulation with anti-CD3.
(137) Human PBMCs were obtained from Precision for Medicine (Frederick, Md.). Cells from 5 donors were used. Plate-bound anti-CD3 was used to stimulate bulk T cells in the PBMC preparations. Briefly, 96-well plates were coated with anti-CD3 antibody for app. 16 hours at 5° C., using 50 μl of a 1 μg/ml anti-CD3 solution diluted in PBS. Thereafter the plates were washed twice.
(138) Kv1.3 blockers were subsequently diluted in medium (RPMI 1640 with Glutamax-I containing, 10% v/v Fetal Bovine Serum, 1% v/v penicillin-streptomycin Solution) and added in a volume of 50 μl. The compounds indicated in Table 4b were used at concentrations ranging from 0.3 μM to 1000 nM (half log dilutions, starting concentrations varying). Cyclosporin A (1 μg/ml) and Vm24 peptide (100 nM) were used as positive controls.
(139) Finally, 50.000 PBMCs in the same medium were added to each well in a volume of 50 μl, giving a final volume of 100 μl per well. The plates were incubated for 20-24 hours in a 37° C./5% CO.sub.2 incubator. After centrifugation of the plates, 25 μl supernatant was transferred to IL-2 detection plates (MSD Human IL-2 Tissue Culture Kit, cat #K151AHB-2) and IL-2 was measures as described by the manufacturer (Meso Scale Discovery, Rockville, Md., USA).
(140) Results are shown in Table 4b as geometric mean of IC50 values obtained from anti-CD3 stimulated human PBMC assays. All values derive from at least 6 replicates.
(141) TABLE-US-00022 TABLE 4b anti-CD3 PBMC/IL-2 Compound IC50 [nM] ShK-186 0.04 3 0.06 1 0.09 41 0.03 23 0.06 48 0.07
(142) Incubation with anti-CD3 antibody activated hPBMC and addition of the Compounds of this invention resulted in dose-dependent reduction in the IL-2 secretion. The average IC.sub.50 values (as calculated from IL-2 release) of the test compounds were in the range of 0.01 nM to 0.09 nM as shown in Table 4b. This was comparable to the IC.sub.50 observed with ShK186 (IC.sub.50 is 0.05 nM). This assay was performed using different donors than those used for Example 4a, so identical values for the Shk-186 in the two sets of experiments are not expected.
(143) Cyclosporine blocked anti-CD3 induced IL-2 release completely in all experiments.
Example 5: Inhibitory Activity of Kv1.3 Blockers in Rat Whole Blood
(144) Rat whole blood was used to assess the potency of Kv1.3 blockers on T-cell activation as determined by IL-17A release after stimulation with thapsigargin. Addition of thapsigargin results in activation of a signalling cascade ending up in activation of T cell proliferation and cytokine production where the Kv1.3 ion channel plays a key role, so activity of Kv1.3 blockers in primary cells can be measured in this experimental system.
(145) Rat whole blood was obtained from healthy, naïve Lewis or Sprague-Dawley rats that were terminally bleed from the heart using Sodium Heparin blood sampling tubes for collection. Test compounds were diluted to 4× final testing concentrations in assay buffer (DMEM+GlutaMAX), GlutaMAX is a medium comprising 3.97 mM L-alanine-L-glutamine (Gibco Cat #61965026) supplemented with 25 mM HEPES buffer, 1 mM Sodium Pyruvate, 100 units/ml Penicillin, 100 μg/ml Streptomycin and 0.05% Casein from bovine milk (Sigma-Aldrich)) and 25 μl was added to wells of a 96 well plate. Then 50 μl whole rat blood was added and incubated for minimum 5 minutes at room temperature to allow compound binding. Then 25 μl 40 μM thapsigargin diluted in assay buffer was added to all wells of the assay plates to activate the cells, followed by incubation for 24 Hr at 37° C./5% CO.sub.2 in a humidified box. The assay plates were centrifuged for 10 min at 300 g at 4° C. and the supernatants were transferred to new plates. The concentrations of IL-17A released to the supernatants were measured using a Rat IL-17A ELISA Kit (Abcam Cat #ab214028) as recommended by the manufacturer. Samples were diluted 2.5-fold by transferring 20 μl of the supernatants to wells on ELISA plates containing 30 μl buffer 75BS from the detection kit. Data from test compounds eliciting an inhibition of IL-17A were normalised relative to full thapsigargin activation (no blocker added) and no activation controls (addition of assay buffer instead of thapsigargin) to calculate the IC.sub.50 from the concentration response curve.
(146) Results are shown in Table 5, expressed as IC.sub.50, with standard deviation (IC.sub.50_SD). All values are derived from at least 2 replicates. The biological effects ex vivo show a correlation with the potency of the compounds.
(147) TABLE-US-00023 TABLE 5 CPD NO IC.sub.50 IC.sub.50_SD 1 0.17 0.036 3 1.0 0.31 17 0.88 0.32 18 3.2 0.017 19 0.56 0.2 20 1.2 0.72 21 0.82 0.81 22 2.8 2.5 23 0.57 0.24 26 0.61 0.33 27 0.68 0.47 28 0.53 0.21 29 0.76 0.23 30 0.41 0.021 31 0.86 0.31 32 0.75 0.24 33 0.70 0.40 34 2.1 0.89 35 2.5 2.1 36 1.2 0.7 37 0.77 0.56 38 2.8 1.8 41 0.98 0.60 42 0.91 0.26 43 0.99 0.24 44 1.1 0.47 45 1.4 0.64 46 0.79 0.28 47 0.83 0.4 48 0.63 0.039 49 1.0 0.65 52 0.69 0.21 62 0.44 0.080 63 0.68 0.19 64 1.2 0.71 65 1.1 0.011 66 0.47 0.35 67 0.98 0.076 68 0.93 0.72 69 2.7 0.54 70 0.79 0.15 71 0.30 0.11 72 0.47 0.086 73 0.52 0.019 74 1.6 1.1 75 0.86 0.13 76 0.55 0.25 78 0.39 0.28 82 0.52 0.059 83 0.81 0.13 84 0.69 0.38 85 1.4 1.1 86 1.1 1.3 87 0.73 0.28 88 1.1 1.2 91 0.34 0.07 92 1.3 1.0 93 0.54 0.31 94 0.55 0.42 95 1.2 0.57 96 1.8 1.2 97 0.43 0.42 98 0.34 0.21 99 0.5 0.37 100 0.87 0.84 101 0.61 0.34 102 0.32 0.22 103 1.2 1.5 104 1.6 0.94 105 0.88 0.011 106 0.52 0.34 107 0.36 0.13 108 0.37 0.049 114 2.1 2.5 115 0.9 0.3 116 0.36 0.24 117 1.9 1
Example 6: Pharmacokinetic Characterisation
(148) Method
(149) Sprague Dawley or Wistar rats (males with a body weight of approximately 250-350 g) were given a single subcutaneous (s.c.) injection of each peptide to be tested.
(150) Following s.c. administration of the selected compounds (dose 70 nmol/kg, dosing volume either 2 or 5 mL/kg), blood samples were drawn at 15 min, 30 min, 45 min, 60 min, 90 min, 2 h, 3 h, 4 h post-dose. At each sampling time point, samples from the rats were drawn by sublingual bleeding or by tail cut. After last sampling the rats were sacrificed by O.sub.2/CO.sub.2 anaesthesia. The dosing vehicle was 10 mM phosphate, 0.8% NaCl, 0.05% Polysorbate 20 (pH 6.0).
(151) Plasma samples were analyzed after solid phase extraction (SPE) by liquid chromatography mass spectrometry (LC-MS/MS). Mean plasma concentrations were used for calculation of the pharmacokinetic parameters using the non-compartmental approach in Phoenix WinNonlin 6.4 or a later version. Plasma terminal elimination half-life (T½) was determined as ln(2)/λz where λz is the magnitude of the slope of the log linear regression of the log concentration versus time profile during the terminal phase. AUC.sub.inf is the area under the plasma concentration—time curve extrapolated to infinity (AUC.sub.inf=AUC.sub.last+C.sub.last/λz, where C.sub.last is the last observed plasma concentration). Cmax is the maximum observed concentration, occurring at Tmax. Results for selected compounds are shown in table 6.
(152) TABLE-US-00024 TABLE 6 Cpd. Dose AUC.sub.INF Cmax T.sub.1/2 No. nmol/kg (hr*nmol/L) (nmol/L) (hr) 3 70 83.9 36.9 0.99 8 70 98.3 76.9 0.62 30 70 46.7 33.3 0.74 52 70 48.5 31.2 0.89 53 70 31.1 50.0 0.87 95 70 85.0 46.5 1.09 97 70 112.0 52.6 1.04 101 70 131.8 58.6 0.91 103 70 176.6 68.4 1.36 5 70 72.4 48.4 0.93
Example 7: Effect of Kv1.3 Blocker Treatment in the Keyhole Limpet Hemocyanin (KLH) Ear Inflammation Model in Rats
(153) A classical delayed-type hypersensitivity (DTH) reaction was elicited in one ear of rats. Briefly, male Lewis rats aged 8-10 weeks were immunized on day −7 with 200 μL keyhole limpet hemocyanin (KLH) (from Sigma, cat. no. H7017) (4 mg/mL) emulsified in complete Freund's adjuvant (CFA) (Difco, cat. no. 263810) subcutaneously (SC) at the base of the tail. On day 0 the rats were challenged intradermally with 40 μL KLH/NaCl 0.9% (2 mg/mL) in the left ear. After the ear challenge the rats develop a T-cell dependent inflammation in the left KLH challenged ear. The right ear remains uninflamed and serves as control.
(154) The ability of Kv1.3 blocker treatment to reduce the DTH ear swelling response was investigated by comparing the response in rats (n=8-10/gr) treated with vehicle to that of rats treated with a Kv1.3 blocker. Vehicle or Kv1.3 blocker dissolved in vehicle was administered SC (2 mL/kg) 24 hrs prior to KLH ear challenge. The test dose of Kv1.3 blocker was 50, 70 or 100 nmol/kg. Test vehicle was 10 mM phosphate, 0.8% w/v NaCl, 0.05% w/v polysorbate20, pH 6. Cyclosporine (CsA) was included as positive study control in all experiments. Cyclosporine (Sandimmune Neooral® 100 mg/mL oral solution, Novartis) was administered per os (10 mg/kg) one hour prior to KLH ear challenge and again 6 hours after KLH ear challenge.
(155) As primary read-out of efficacy, the Area Under Curve (AUC) of Δ ear thickness (mm) was calculated for each animal from 0-48 hours post induction of the ear DTH reaction, where the change (D) was calculated as: Left ear thickness-right ear thickness. These results were then used to calculate % inhibition of ear thickness by Kv1.3 blocker treatment: % inhibition: ((1−(individual Δ AUC Kv1.3 blocker/average ΔAUC vehicle group))×100. Results were calculated as % inhibition+/−standard deviation (SD), and are shown in Table 7 and Table 8.
(156) TABLE-US-00025 TABLE 7 Dose (nmol/ Exp kg) Cpd. 3 Cpd. 41 Cpd. 52 CsA* #1 70 38.8 (+/−9.7) 46.9 (+/−10.9) 71.4(+/−4.8) #2 70 25.0 (+/−12.5) 27.7 (+/−11.5) 76.8(+/−13.4) #3 50 37.3 (+/−13.8) 22.2 (+/−11.1) 65.1(+/−4.5) #4 100 41.9 (+/−12.5) 25.1 63.4 (+/−5.9) (+/−6.3)
(157) TABLE-US-00026 TABLE 8 Dose Exp. (nmol/kg) Cpd.95 Cpd. 97 Cpd. 101 Cpd.103 CsA #5 100 39.6 67.1 (+/−8.3) (+/−7.9) #6 100 42.5 44.5 55.3 (+/−14.0) (+/−5.2) (+/−5.5) #7 100 48.9 67.9 (+/−8.6) (+/−5.4) #8 100 37.7 57.8 (+/−10.0) (+/−2.8)
(158) The work described in this specification has received funding from the European Communities Seventh Framework Program FP7/2007-2013 under grant agreement Venomics_CA_20111021.