WO2006002850A2

WO2006002850A2 - OsK1 DERIVATIVES

Info

Publication number: WO2006002850A2
Application number: PCT/EP2005/006897
Authority: WO
Inventors: Stéphanie Mouhat; Jean-Marc Sabatier; Bonabes Olivier DE ROUGÉ
Original assignee: Cellpep Sa
Priority date: 2004-06-25
Filing date: 2005-06-27
Publication date: 2006-01-12
Also published as: CA2572225A1; AU2005259533B2; EP1758930A2; US7745575B1; JP2008503539A; WO2006002850A3; EP1758930B1; JP4874960B2; AU2005259533A1; GB0414272D0

Abstract

OsK1 is a 38-residue peptide with 3 disulphide bridges and is found in the venom of the scorpion Orthochirus scrobiculosus. It is potently active on voltage-gated K+ channels Kv1.1, Kvl.2 and Kvl.3, and moderately active on the type 1 intermediate-conductance Cat2+-activated channel Kca3.1. Derivatives of OsK1, particularly involving truncation or point mutations, have been developed to enhance the activity against and selectivity for the Kvl.3 channel. This renders the derivatives likely candidates for the treatment of autoimmune diseases, including multiple sclerosis. Such use may be alone or in combination therapy with maurotoxin, another scorpion toxin.

Description

TITLE

OsKl Derivatives

DESCRIPTION

The invention relates to derivatives of OsKl and to pharmaceutical compositions containing them. The derivatives are useful in the treatment of neurological disorders including multiple sclerosis.

Background

OsKl is a 38-residue peptide with 3 disulphide bridges and is found in the venom qf the scorpion Orthochirus scrobiculosus. It is one of a number of short chain scorpion toxins which contain between 29 and 39 amino acid residues, are crosslinked by 3 or 4 disulphide bridges and are active on several potassium channel subtypes, both voltage gated and calcium activated. These short chain scorpion toxins have been classified by Tytgat et al., Trends Pharmacol. ScL, 20(11), 444-447, 1999. OsKl is a group 3 toxin and is shown below in Table 1 along with the other group 3 toxins and one group 6 toxin, Maurotoxin (MTx).

TABLE l

3.1 KTx GVEINVKCSGSPQCLKPCKDA-GMR-FGKCMNR-KCHCTPK

3.2 AgTx2 GVPINVSCTGSPQCIKPCKDA-GMR-FGKCMNR-KCHCTPK

3.3 AgTx3 GVPINVPCTGSPQCIKPCKDA-GMR-FGKCMNR-KCHCTPK

3.4 AgTxI GVPINVKCTGSPQCLKPCKDA-GMR-FGKCING-KCHCTPK

3.5 KTx2 VRIPVSCKHSGQCLKPCKDA-GMR-FGKCMNR-KCDCTPK

3.6 BmKTx VGINVKCKHSGQCLKPCKDA-GMR-FGKCING-KCDCTPK

3.7 OsKl GVIINVKCKISRQCLEPCKKA-GMR-FGKCMNG-KCHCTPK

3.8 Bs6 GVPINVKCRGSPQCIQPCRDA-GMR-FGKCMNG-KCHCTPQ

6.2 MTx VSCTGSKDCYAPCRKQTGCPNA-KCINK-SCKCYGC

Both the mammalian voltage-gated K⁺ channel KvI.3 and the type 1 intermediate- conductance Ca²⁺-activated channel Kc_a3.1 (also referred to as IKl) are expressed in immune T-cells. Their relative abundance at the T-cell surface depends on the state of lymphocyte activation and differentiation. The potassium channel phenotype varies during the progression from the resting to the activated T-cell state, and from naive to effector memory T-cells. Therefore, blockers of these channel types possess potent immunomodulatory properties that could be beneficial in the treatment of human diseases, such as multiple sclerosis. Multiple sclerosis is a chronic inflammatory autoimmune disease of the central nervous system, characterised by progressive demyelination and axonal damage caused by reactive autoimmune lymphocytes. Activation and proliferation of these cells (effector memory T- cells) relies upon Kv 1.3 channels (and possibly IKl channels), and can be blocked by Kv 1.3 channel blockers. Demyelination also involves unmasking of the KvI.1 and KvI.2 subtypes, which provokes leak currents and a decrease in nervous transmission (by altering the action potential).

We have found that OsKl, alone amongst the short chain scorpion toxins, is potently active on KvI.1, Kvl.2 and KvI.3 channels, and moderately active on IKl. It would be desirable to find synthetic peptides which retain or enhance this property -or part of this property- and/or block more potently the IKl subtype.

Such derivatives may also be useful against other human disorders, such as graft rejection, rheumatoid arthritis, bone degradation, and cancer (abnormal cell proliferation).

The Invention

The invention provides an OsKl derivative in which up to seven N-terminal amino acid residues have been omitted and/or in which up to four amino acid residues have been point mutated. Preferably, the replacement amino acid residue(s) are in positions 8 to 38 and occur in the corresponding position in another group 3 short chain scorpion toxin or in maurotoxin.

Table 2 lists the OsKl derivatives which the inventors have studied. Not all of them are within the scope of the invention. Differences from OsKl are shown in bold. It will be understood that Δⁿ indicates that the peptide present in position n in OsKl has been omitted and that Xⁿ indicates that the peptide present in position n in OsKl has been replaced with a peptide X. Ac stands for acetyl and VaI for valeryl.

A preferred peptide is [K¹⁶,D²⁰]-OsKl and this has been abbreviated as AOsKl. The substitution of lysine for glutamic acid at position 16 in OsKl and of aspartic acid for lysine at position 20 in OsKl follow the norms in those positions in kaliotoxins 1 and 2, agitoxins 1, 2 and 3 and Buthus martensi kaliotoxin. AOsKl is about five times more potent on the Kv 1.3 channel than OsKl itself and is the most potent Kv 1.3 channel blocker characterized so far. Since AOsKl also shares the same activity on IKl, it makes this derivative a better lead compound than OsKl.

TABLE 2

OsKl GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-oH

OsKl-NH₂ GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂

Ac-OsKl-NH₂ Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-_NH2

[A¹J-OsKl-NH₂ -VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂

[A³⁴]-OsKl GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-oH

[K^I6]-OsKl GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-oH

[D²⁰]-OsKl GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-oH

AOsKl GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-oH

VaI-AOsKl VaI-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-oH

AOsKl-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-_NH2

[A¹J-AOsKl -VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-oH

[A¹I-AOsKl-NH₂ -VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-_NH2

[A¹J²I-AOsKl -TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-oH

[A¹^]-AOsKl NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-OH

[A¹^]-AOsKl KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-oH

[A¹^]-AOsKl CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-OH

[Δ^36"38]-AOsKl GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHC _OH

[K³]-AOsKl-NH₂ GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2

[P¹²]-A0sKl GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-oH

[N²⁵J-AOsKl GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-OH

[R³¹J-AOsKl GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-oH

[Y³⁶]-AOsKl GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-oH

Ac-[K¹²,R¹⁹]-AOsKl Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-oH

Truncating the N-terminal of OsKl or its derivatives reduces the activity of the resultant peptide against the KvI.2 channel and, to a lesser extent, against the KvI.1 channel, and therefore enhances the selectivity of the resultant peptide for the KvI.3 channel. Further, truncating the N-terminal of OsKl or its derivatives reduces the toxicity of the resultant peptide, possibly due to the decreased affinity towards the KvI.1 and KvI.2 channels. By contrast, truncating the C-terminal caused significant loss of activity against all channels measured.

A point mutation at position 2 of AOsKl, coupled with the omission of the peptide at position 1, gave a peptide

possessing an N-terminal domain (i.e. TIINVK) that is identical to those of margatoxin and noxiustoxin, two scorpion toxins potently active on KvI .3 channel. The peptides truncated at the N-terminal, whether or not mutated at position 2, showed good activity against the Kv3.2 channel.

Docldng studies have indicated that His in position 34 of OsKl appeared to be involved in KvI.2 and KvI.3 docking, so replacement of this peptide, e.g. by Ala, is desirable, leading to higher selectivity for KvI.3. Such studies have also suggested the inclusion of Lys at position 3 in place of He.

Asparagine at position 25 has been reported as important in the affinity of toxins with the KvI.3 channel and arginine at position 31 is homologous with other toxins active on the KvI.3 channel. In fact R at position 31 provides an AOsKl derivative which has strong affinity to all of the Kv 1.1, Kv 1.2 and Kv 1.3 channels.

OsKl derivatives according to the invention may be acetylated at the N-terminal or may be amidated at the C-terminal in conventional manner. Another approach is to terminate the OsKl derivative with a fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon- carbon double bonds, or a derivative thereof such as an co-amino-fatty acid. Either the N- terminal or the C-terminal may be so terminated. This should result in increased water solubility for the derivative, and enhanced bioavailability. The preferred fatty acid is valeric acid (or, for the C-terminal, ω-amino-valeric acid).

The invention further provides a pharmaceutical composition comprising an OsKl derivative as described above in admixture with a pharmaceutically acceptable diluent or carrier. Furthermore, the invention also provides the use of OsKl or of an OsKl derivative as described above for the preparation of a medicament for the treatment of autoimmume diseases, including but not limited to multiple sclerosis.

Maurotoxin is the most potent inhibitor amongst the short chain scorpion toxins of the calcium activated potassium channel subtype IKl . Replacing amino acid residue(s) occurring in the corresponding position(s) in maurotoxin may lead to the derivative having an enhanced activity against IKl although the replacement is tyrosine for threonine at position 36 has not been shown to achieve this. In the absence of a single compound combining high activity on KvI.3 with improved activity on IKl, we have investigated the possibility of combination therapy using an OsKl derivative according to the invention with MTx. As shown below, the use of AOsKl and MTx together does appear to be better than the use of either alone. Such combination therapy is thus included within the invention.

Experimental

OsKl and the OsKl derivatives listed in Table 2 were prepared by chemical synthesis using a peptide synthesizer (Model 433A, Applied Biosystems Inc.). Peptide chains were assembled stepwise on 0.25 mmol of HMP resin (1% cross-linked; 0.65 mmol of amino group/g) using 1 mmol of N^-^-fluorenytymethyloxycarbonyl (Fmoc) L-amino acid derivatives. Side chain- protecting groups for trifunctional residues were: trityl for cysteine, asparagine, histidine and glutamine; t-butyl for serine, threonine, tyrosine, aspartate and glutamate; 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl for arginine; and t-butyloxycarbonyl for lysine. N**- amino groups were deprotected by successively treating with 18 and 20% (v/v) piperidine/Ν- methylpyrrolidone for 3 and 8 min, respectively. After three washes with N- methylpyrrolidone, the Fmoc-amino acid derivatives were coupled (20 min) as their hydroxybenzotriazole active esters in N-methylpyrrolidone (4-fold excess). After peptides were assembled, and removal of Ν-terminal Fmoc groups, the peptide resins (ca. 1.5 g) were treated under stirring for 3 h at 25°C with mixtures of trifluoroacetic acid/H₂O/thioanisole/ethanedithiol (73:11:11:5, v/v) in the presence of crystalline phenol (2.1 g) in final volumes of 30 ml per gram of peptide resins. The peptide mixtures were filtered, precipitated and washed twice with cold diethyloxide. The crude peptides were pelleted by centrifύgation (3,200 x g; 8 min). The crude peptides were then dissolved in H₂O and freeze dried. Reduced AOsKl analogues were solubilized at a concentration of ca. 0.6 niM in 0.2 M Tris-HCl buffer (pH 8.3) for oxidative folding (40 to 120 h depending on the peptide, 20°C). The folded/oxidized peptides were purified to homogeneity by reversed-phase high pressure liquid chromatography (HPLC) (C₁₈ Aquapore ODS, 20 μm, 250 x 10 mm; PerkinElmer Life Sciences) by means of a 60-min linear gradient of 0.08% (v/v) trifluoroacetic acid/UaO (buffer A) with 0 to 40% of 0.1% (v/v) trifluoroacetic acid/acetonitrile (buffer B), at a flow rate of 6 ml/min (λ = 230 nm). The purity and identity of each peptide were assessed by: (i) analytical Ci₈ reversed-phase HPLC (Cj₈ Lichrospher 5 μm, 4 x 200 mm; Merck) using a 60 min linear gradient of buffer A with 0-60% of buffer B, at a flow rate of 1 ml/min; (ii) amino acid analysis after peptide acidolysis [6 M HCl/2% (w/v) phenol, 20 h, 120°C, N₂ atmosphere]; and (iii) molecular mass determination by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry. Some of the MS results are shown in Table 3 below and the some of the HPLC profiles are shown in the accompanying drawing.

TABLE 3 Mass spectra of OsKl and its derivatives

Electrophysiological experiments were carried out at 22-24⁰C using the patch-clamp whole- cell recording mode. Cells were bathed with mammalian Ringer's solution (in mM): 160 NaCl, 4.5 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4 with NaOH), with an osmolarity of 290- 320 mOsm. When AOsKl derivatives were applied, 0.1% bovine serum albumin was added to the Ringer's solution. A syringe-driven perfusion device was used to exchange the external recording bath solution. Two internal pipette solutions were used, one for measuring voltage- gated K⁺ currents that contained (in mM): 155 KF, 2 MgCl₂, 10 HEPES, and 10 EGTA (pH 7.2 with KOH), with an osmolarity of 290-320 mOsm and another for measuring Ca²⁺- activated K⁺ currents that contained (in mM): 135 K-aspartate, 8.7 CaCl₂, 2 MgCl₂, 10 EGTA, 10 HEPES (pH 7.2 with KOH), with an osmolarity of 290-320 mOsm. A free [Ca²⁺J₁ of 1 μM was calculated. All currents through voltage-gated K channels were elicited by 200 ms depolarising voltage steps from -80 to +40 mV. Potassium currents through Kc_al.l, Kc_a2.1 and Kc_a3.1 were elicited by 1 μM internal [Ca²⁺]; and 400 ms voltage ramps from -120 to 0 mV (for Kc_a3.1 channel and Kc_al.l channel) and from -120 to +40 mV (for Kc_a2.1 channel). Electrodes were pulled from glass capillaries (Science Products, Germany), and fire-polished to resistances of 2.5-5 MΩ. Membrane currents were measured with an EPC-9 or EPC-10 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany) interfaced to a Macintosh computer running acquisition and analysis software (Pulse and PulseFit). When voltage-gated K⁺ currents were measured, the capacitive and leak currents were subtracted using a P/ 10 procedure. Series resistance compensation (> 80%) was used for currents above 2 nA. The holding potential was -80 mV in all experiments. Data analyses were performed with IgorPro (WaveMetrics, Oregon, USA), and IC₅₀ values were deduced by fitting a modified Hill equation to the data (Itoxin/Icontroi ⁼ 1/[1 + ([AOsKl analogueJ/ICso)], where I is the peak current (for voltage-gated K⁺ channels), or the slope of the ramp current, i.e. the conductance measured between -100 and -60 mV (for Ca²⁺-activated K⁺ channels) to the normalized data points obtained with four different AOsKl analogue concentrations. The results are shown in Table 4. TABLE 4

No activity was detected at micromolar concentrations for any of the synthesized peptides on voltage-gated channels hKvl.4, KvI.5, hKvl.6, mKvl.7, mKv3.1 and hKvl l.x (also referred to as HERG, human ether-a-go-go related gene) or on Ca²⁺-activated K⁺ channels hKcJ.l (also referred to as BK) and hKc_a2.1 (also referred to as SKl).

The peptides were evaluated for toxicity in vivo by determining the 50% lethal dose (LDs₀) after intracerebroventricular injection into 20 g C57/BL6 mice. Groups of six to eight mice per dose were injected with 5 μl peptide solution containing 0.1% (w/v) bovine serum albumin and 0.9% (w/v) NaCl. The LD₅Q values are set out in Table 5.

Table 5

Peptide LD₅₀ (μg/kg)

OsKl 2

OsKl-NH₂ 3.5

Ac-OsKl-NH₂ 11

[A¹J-OsKl-NH₂ 7

[A³⁴J-OsKl 10

[K¹⁶]-OsKl 3

[D²⁰]-OsKl 4.5

AOsKl 2.5

VaI-AOsKl 16

AOsKl-NH₂ 2.25

[A¹J-AOsKl 3

[A¹J-AOsKl-NH₂ 4.5

[A¹J²J-AOsKl 3

[A^1-4J-AOsKl 5

[A^1-6J-AOsKl 6.5

[A^1-7J-AOsKl 15

[Δ^36-38]-AOsKl 800

[K³J-AOsKl-NH₂ 2.25

[P¹²J-AOsKl 7.5

[N²⁵J-AOsKl 11.5

[R³¹J-AOsKl 5

[Y³⁶J-AOsKl 9

Ac-[K¹²,R¹⁹]-AOsKl 17.5 The effect of co-administering AOsKl and MTx has been tested on Lewis rats immunized with spinal cord homogenate emulsified with complete Freund adjuvant. Each rat received 200 μl of emulsion sc, divided in the rear footpads, on day 0. Eight days later, as their body weight decreased, a precursor sign for clinical EAE, an osmotic pump was implanted IP. The pump delivered drags at 1 μl/h for 8 days. They were filled with the peptide blockers, dissolved in Na Cl 0.15 M containing 2% Lewis serum, as vehicle.

Two additional groups of rats non-immunized were treated with the blockers in the same conditions. Blood samples were collected after 4 days of continuous administration in order to measure the blocker concentration in serum, using the patch-clamp technique.

Clinical evaluation. The severity of the disease was scored on a scale of 0 to 6, with 0.5 points for intermediate clinical findings (0: no clinical signs; 1.0: limp tail; 2.0: mild paraparesis and ataxia; 3.0: moderate paraparesis; 4.0: complete hind limb paralysis; 5.0: paralysis + incontinence; 5.5: tetraplegia; 6.0: moribund or death). Rats were weighed every day. Rats showing no clinical signs of EAE and no weight loss were excluded from the studies. The beneficial effects of K⁺ blockers on EAE were evaluated by a reduction in clinical severity and in duration of the disease and in number of relapses compared with those of rats treated with the vehicle only, 0.15 MNaCl.

Data analysis. Statistical analysis was conducted using the Mann- Whitney U test. Mean differences between groups were considered significant at values of P < 0.05.

Results are shown in Table 6. The combination of 50 nM of MTx and 50 nM of AOsKl proved more effective than 50 nM of MT alone. TABLE 6

MTX and AOsKl treatment of chronic relapsing EAE

First episode ofEAE

Groups Onset Duration Max Clin Sc Incid of rats (day)^a (days)^b (%)

Vehicle

1 d9 10 4 2 d7 13 4 3 d7 chronic 5 0 dll 10 5 5 d8 13 5

Mean±SD 4.6 ± 0.5 100

MTx

5O nM

1 dll chronic 4

2 d8 11 1 3 d7 chronic 1 0 (d) d7 chronic 3

Mean ±SD 2.2 ± 1.5* 100

MTx 5OnM AOsKl 5OnM

1 dl l 4 1

2 dl2 4 1 3 dll 3 0.5

Mean±SD 0.83 ± 0.2* 100

MTx 6.2nM AOsKl 5OnM

1 dlO 10 5

2 dl2 7 5 3^d dl3 2 0.5

Mean ±SD 3.5 ± 2.5 100 a. day when first clinical signs (even scored 0.5) appear. number of days until clinical signs reverse to minimal score (0.5). aggravation : 2 sd relapse : Mean differences between groups were considered significant at values of P < 0.05 (Mann-Whitney U test).

Claims

1. An OsKl derivative in which up to seven N-terminal amino acid residues have been omitted and/or in which up to four amino acid residues have been replaced by other amino acid residues.

2. An OsKl derivative according to claim 1 in which the replacement amino acid residue(s) are in positions 8 to 38 and occur in the corresponding position in another group 3 short chain scorpion toxin or in maurotoxin.

3. An OsKl derivative according to claim 1 or claim 2 in which the glutamic acid residue at position 16 in OsKl has been replaced with a lysine residue.

4. An OsKl derivative according to any preceding claim in which the lysine residue at position 20 in OsKl has been replaced with an aspartic acid residue.

5. Any one of the following OsKl derivatives:

• [A¹J-OsKl

• [A³⁴]-OsKl

• [K¹⁶]-0sKl

• [D²⁰J-OsKl

• [K¹⁶,D²⁰]-OsKl

• [Δ^K^D^-OsKl

• [Δ^.K^.D∞l-OsKl

• [Δ^K^D^-OsKl

• [K³,K¹⁶,D²⁰]-OsKl

• [P¹²,K¹⁶,D²⁰]-OsKl

• [K¹⁶,D²⁰,N²⁵]-OsKl [K¹⁶,D²⁰,R³¹J-OsKl

[K¹⁶,D²⁰,Y³⁶]-OsKl [K¹², K¹⁶,R¹⁹,D²⁰]-OsKl.

6. An OsKl derivative according to any preceding claim which is terminated at the N- terminal with an acetyl group or which is amidated at the C-terminal.

7. An OsKl derivative according to any of claims 1 to 5 which is terminated with an ω- amino-fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon-carbon double bonds.

8. A pharmaceutical composition comprising an OsKl derivative according to any one of the preceding claims in admixture with a pharmaceutically acceptable diluent or carrier.

9. Use of OsKl, OsKl terminated at the N-terminal with an acetyl group, OsKl amidated at the C-terminal, OsKl terminated with an ω-amino-fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon-carbon double bonds, or an OsKl derivative according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of autoimmune diseases, including but not limited to multiple sclerosis.

10. Use of OsKl, OsKl terminated at the N-terminal with an acetyl group, OsKl amidated at the C-terminal, OsKl terminated with an ω-amino-fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon-carbon double bonds, or an OsKl derivative according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of autoimmune diseases, including but not limited to multiple sclerosis, in combination with the prior, concurrent or post-administration of maurotoxin.

11. Use of [K¹⁶,D²⁰]-OsKl for the preparation of a medicament for the treatment of autoimmune diseases, including but not limited to multiple sclerosis, in combination with the prior, concurrent or post-administration of maurotoxin. 1/1

[Kl6]-OsKl [D20]-OsKl [Kl6,D20]-OsKl

,K16,D20]-OsKl [K16,D20,-Y36] -OsKl Valerate- [K16,D20]-OsKl