WO2015104305A1 - Bicyclic peptides as protease inhibitors - Google Patents
Bicyclic peptides as protease inhibitors Download PDFInfo
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- WO2015104305A1 WO2015104305A1 PCT/EP2015/050190 EP2015050190W WO2015104305A1 WO 2015104305 A1 WO2015104305 A1 WO 2015104305A1 EP 2015050190 W EP2015050190 W EP 2015050190W WO 2015104305 A1 WO2015104305 A1 WO 2015104305A1
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- prolyl
- isoleucyl
- cysteinyl
- seryl
- threonyl
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- 0 CC*(C)C([C@](CSSC[C@@](C(I)=O)NC([C@](CCC*)*C)=O)*1(CO)C(C)*1)=O Chemical compound CC*(C)C([C@](CSSC[C@@](C(I)=O)NC([C@](CCC*)*C)=O)*1(CO)C(C)*1)=O 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
Abstract
The present invention is related to novel bicyclic peptides which are protease inhibitors.
Description
BICYCLIC PEPTIDES AS PROTEASE INHIBITORS
TECHNICAL FIELD
Disclosed herein are bicyclic peptides which are protease inhibitors, methods of production and use thereof.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
The Sequence Listing, entitled "SEQUENCE LISTING", is 10 kb, was created on 09 January 2014 and is incorporated herein by reference. BACKGROUND
The oral route is by far the most widely used route for drug administration.
Administration of peptides and proteins is however often limited to parenteral routes rather than the preferred oral administration due to several barriers such as enzymatic degradation in the gastrointestinal (GI) tract and intestinal mucosa, as well as insufficient and variable absorption from the intestinal mucosa.
To overcome this barrier, permeation enhancers and inhibitors of proteolytic enzymes are commonly included in oral formulations. However, it has turned out not to be trivial to develop permeation enhancers and inhibitors of proteolytic enzymes which are suitable for use in oral formulations.
A bicyclic peptide which shows protease inhibition has been prepared by RJ.
Leatherbarrow (PEDS, 2004, 17(9), 681-687).
"Solution structure of a novel C2-symmetrical bifunctional bicyclic inhibitor based on SFTI-1" R. J. Leatherbarrow (J Biomol NMR. 2005, SEP; 33(1)57-62) bifunctional bicyclic inhibitor has been created that combines features both from the Bowman-Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), and is abbreviated BiKK (DOI 10.1007/sl0858-005-1210-9). "Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors", Protein Engineering, Design & Selection vol. 17 no. 9 pp. 681-687, 2004
Published online October 14, 2004 (doi : 10.1093/protein/gzh077). It describes two synthetic inhibitors designed to combine the advantageous properties of Bowman Birk inhibitor (BBI) and sunflower trypsin inhibitor-1 (SFTI-1) and is abbreviated BiKF.
However, there is still a need for novel protease inhibitors which are compatible with oral pharmaceutical compositions for oral delivery of peptides and proteins, which
also exert good solubility properties at physiological pH levels (i.e. wide pH ranges through the oral route) and are potent protease inhibitors.
SUMMARY
The present invention is related to bicyclic peptides which are protease inhibitors.
The bicyclic peptides of the present invention comprise at least two cysteine amino acids and at least one charged or partly charged amino acid which is positioned immediately on the /V-terminal side of a cysteine residue.
In one aspect, the invention is related to bicyclic peptides of the formula
Cysl-Thr-X3-Ser-Ile-X6-X7-X8-Cys9-Thr-Xl l-Ser-Ile-X14-X15-X16
[Formula I]
wherein
Cysl is Cys;
X3 is Phe, Tyr, Trp, Lys, Arg or Ala;
X6 is Pro;
X7 is Pro;
X8 is a charged amino acid, a partly charged amino acid or Gin or He;
Cys9 is Cys;
XI 1 is Phe, Tyr, Lys, Leu, Arg, Val or Ala;
X14 is Pro;
X15 is Pro;
X16 is a charged amino acid or He;
wherein at least one of X8 and X16 is a charged amino acid or partly charged amino acid; Cysl and Cys9 are joined by a disulfide bond between the sulfur atoms of the two cysteines; and
Cysl and X16 are joined by an amide bond between the alpha amine of Cysl and the alpha carboxylic group of X16.
In one aspect, the peptide of the invention inhibits one or more enzymes found in the gastrointestinal tract, such as e.g. trypsin, chymotrypsin and/or elastase.
In one aspect, the peptide of the invention is soluble in aqueous solution at a pH which is between pH 1 and pH 10.
The invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
DESCRIPTION
The present invention is related to bicyclic peptides which are protease inhibitors
The bicyclic peptides of the invention may be described as peptides having two links between amino acids of the peptide which are not present in non-cyclic peptides. The bicyclic peptides may also or alternatively be described as peptide structures with two macrocyclic rings, so-called loops. In one aspect, bicyclic peptides of the invention contain a bridging link between the side chains of two amino acids of the bicyclic peptide and a link between the amino terminus and the carboxyl terminus of the peptide if represented as a linear peptide, i.e. such bicyclic peptide does not contain a free amino terminus or a free carboxyl terminus. In one aspect, a bridge is formed as a disulfide bond between two cysteine residues. In one aspect the bicyclic peptide of the invention comprises a link between the amino terminus and carboxyl terminus.
The bicyclic peptides of the present invention comprise at least two cysteine amino acids and at least one charged or partly charged amino acid which is positioned immediately on the /V-terminal side of a cysteine residue. It was surprisingly found by the inventor of the present bicyclic peptides of this invention, that the bicyclic peptides present good solubility. Further this good solubility has surprisingly been found to be present through a wide range of pH. Good solubility is a key feature for protease inhibitors used in pharmaceutical compositions to protect active ingredients against protease degradation, because good solubility will increase the load of the protease inhibitor into the
pharmaceutical composition and thus provide better protection against protease degradation of the active ingredient. Good solubility over a wide pH range increases the flexibility of the protease inhibitor to be used in different formulations with a variety of pH levels and remains the protease inhibitor soluble throughout the different pH environments in the GI tract when used in compositions for oral administration. The inventor has found that the bicyclic peptides of the invention are particularly suitable as protease inhibitors for use in oral pharmaceutical compositions. Further the inventor found that the bicyclic peptides of this invention are equipotent to BiKK and BiKF beside having a good solubility profile over a wide range of pH .
In one aspect, the bicyclic peptides of the invention are soluble in aqueous solution at a pH ranging from pH 1 to pH 9, such as e.g. from pH 2 to pH 9, from pH 3 to pH 9, from pH 4 to pH 9, from pH 5 to pH 9, from pH 6 to pH 9, from pH 2 to pH 8, from pH 2 to pH 7, from pH 2 to pH 6, from pH 2 to pH 5.
In one aspect, a bicyclic peptide of the invention comprises at least two cysteine amino acids and at least two charged or partly charged amino acids, wherein each of the at
least two charged or partly charged amino acids is positioned immediately on the N- terminal side of a cysteine residue.
In one aspect, a bicyclic peptide of the invention comprises 16 amino acids.
In one aspect, a bicyclic peptide of the invention comprises 8 amino acids in one loop and 8 amino acids in the other loop of the bicyclic peptide.
In one aspect, the bicyclic peptide of the invention inhibits one protease.
In one aspect the bicyclic peptide of the invention inhibits two proteases.
In one aspect the bicyclic peptide of the invention inhibits three proteases.
In one aspect, each loop of a bicyclic peptide of the invention inhibits one protease. In one aspect, one loop of a bicyclic peptide of the invention inhibits two proteases. In one aspect, each loop of a bicyclic peptide of the invention inhibits two proteases. In one aspect, one loop of a bicyclic peptide of the invention inhibits one protease and one loop inhibits two proteases. In one aspect of the invention the proteases inhibited by a bicyclic peptide of the invention are selected from the group consisting of trypsin, chymotrypsin and elastase.
In one aspect, a bicyclic peptide of the invention comprises a charged or partly charged amino acid which is positioned immediately on the /V-terminal side of a cysteine residue in at least one loop of said bicyclic peptide. In one aspect, a bicyclic peptide of the invention comprises a charged or partly charged amino acid which is positioned immediately on the /V-terminal side of a cysteine residue in both loops of said bicyclic peptide. In one aspect, a bicyclic peptide of the invention comprises at least one further charged or partly charged amino acid .
When used herein the term "charged or partly charged amino acid" shall mean an amino acid having a side-chain which is charged or partly charged at physiological pH 7.4. In one aspect, a charged or partly charged amino acid to be used in a bicyclic peptide of the invention is selected from the group consisting of Arg, Lys, Asp, Glu and His. In one aspect, a charged or partly charged amino acid to be used in a bicyclic peptide of the invention is selected from the group consisting of Arg, Lys, Asp or Glu. When used herein the terms "alpha amino group" and "alpha carboxylic group" shall mean the amino group, respectively the carboxylic acid group, attached to the first
(alpha-) carbon atom of an amino acid as illustrated with asterisks in Chem . 1, where R is the amino acid side chain .
Chem. 1
When naming the.bicyclic peptides of the invention we herein list all amino acids of the peptide. The peptide chain is numbered from the /V-terminus and the numbers of the residues involved in ring formation are cited in residue number order in front of the prefix anhydro. For example, the term "x,y-an hydro" is used to indicate an amide bond between amino acids x and y, i.e. amino acid "x" is linked via its alpha amino-group to the alpha carboxylic group of amino acid "y". For disulfide bridges between two cysteine amino acids, the term "Sv,Sz-cyclo" is used, wherein V and Z indicate the positions in the peptide of the respective cysteine amino acids forming said disulfide bond.
For example, the bicyclic peptide of example 1 herein is named
cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L-arginine)
(SEQ ID No: l)
Protease inhibitor
The terms "protease inhibitor" or "enzyme inhibitor" as used herein refer to molecules that inhibit the function of proteases. In one aspect of the invention, the protease inhibitors inhibit proteases from the class of serine proteases (serine protease inhibitors). In one aspect of the invention, the protease inhibitor inhibits pancreatic enzymes found in the gastro intestinal tract in mammals.
Pancreatic enzymes are enzymes present in the pancreatic juice and includes lipases, proteases and amylases such as e.g. trypsin, chymotrypsin, carboxypeptidase, elastase, pancreatic lipase, sterol esterase, phospholipase, various nucleases and pancreatic amylase. Protease inhibitors inhibiting pancreatic enzymes found in the gastro intestinal tract in mammals thus inhibit e.g. the enzymes trypsin, chymotrypsin, carboxypeptidase, elastase, pancreatic lipase, sterol esterase, phospholipase, various nucleases and/or pancreatic amylase.
In one aspect of the invention, protease inhibitor is a compound that binds to proteolytic enzymes in such a way to interfere with degradation of peptides/proteins.
In general, compounds can bind to proteolytic enzymes at many different sites, however, it is only binding that interferes with the function of proteolytic enzymes that is of interest when searching for inhibitors of proteolysis. The best way to look for inhibitors is to examine the effect of the presence of the potential inhibitor on the enzymatic reaction catalyzed by the protease in question. Enzyme kinetics describes several possibilities for a compound to inhibit an enzyme as known to the person skilled in the art. Enzyme inhibition may be, for example, competitive, non-competitive, mixed. Procedures for distinguishing different kinds of enzyme inhibition were previously described in many scientific articles and numerous textbooks, for example, Fundamentals of Enzyme
Kinetics by Athel Cornish-Bowden ISBN-13 : 978-3527330744. In addition to enzyme kinetics, interactions of proteolytic enzymes with their inhibitors are commonly examined by many different methods, for example, x-ray crystallography, NMR spectroscopy, numerous spectroscopy techniques (fluorescence, circular dischroism, UV-VIS), mass spectrometry, calorimetry, etcetera as known to the person skilled in the art.
Compounds may also strongly bind to an enzyme but not affect the rate of the catalyzed reaction.
Solid oral pharmaceutical compositions
A bicyclic peptide of the invention may be used as excipient in a solid oral pharmaceutical composition.
Solid oral pharmaceutical compositions for including a bicyclic peptide of the invention may include encapsulation of the active peptide ingredient into nanoparticles,
microparticles, granules, pellets or other kinds of multiparticulate dosage forms. Above mentioned oral pharmaceutical compositions systems may be formulated into a tablet or filled into a suitable hard-shelled or soft-shelled capsule which may be coated to release the active peptide ingredient in a controlled manner or at a preferred intestinal segment. In one aspect, the solid oral pharmaceutical composition for including a bicyclic peptide of the invention comprises granules. In one aspect the term "granulate" refers to one or more types of granules. In one aspect the term "granule" refers to particles gathered into larger particles.
In one aspect, the solid oral pharmaceutical composition for including a bicyclic peptide of the invention is in the form of a solid dosage form. In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention is in the form of a tablet. In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention is in the form of a capsule. In one aspect the solid oral
pharmaceutical composition for including a bicyclic peptide of the invention is in the form of a sachet.
In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention comprises at least one pharmaceutically acceptable excipient.
The term "excipient" as used herein broadly refers to any component other than the active peptide ingredient(s). The excipient may be an inert substance, which is inert in the sense that it substantially does not have any therapeutic and/or prophylactic effect per se. The excipient may serve various purposes, e.g. as a delivery agent, absorption enhancer, vehicle, solubilizing agent, filler (also known as diluents), binder, lubricant, glidant, disintegrant, crystallization retarders, acidifying agent, alkalizing agent, antioxidant, buffering agent, chelating agent, complexing agents, surfactant agent, emulsifying and/or solubilizing agents, wetting agents stabilizing agent, colouring agent, flavouring agent, and/or to improve administration, and/or absorption of the active peptide ingredient. A person skilled in the art may select one or more of the
aforementioned excipients with respect to the particular desired properties of the solid oral dosage form by routine experimentation and without any undue burden. The amount of each excipient used may vary within ranges conventional in the art. Techniques and excipients which may be used to formulate oral dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal
Pharmaceutical Society of Great Britain (2009); and Remington : the Science and Practice of Pharmacy, 21th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).
In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention comprises a binder. In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention comprises a disintegrant. In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention comprises a lubricant. In one aspect the solid oral pharmaceutical composition for including a bicyclic peptide of the invention comprises one or more excipients selected from crystallization retarders, solubilizing agents (also known as surfactants), wetting agents, colouring agents, and/or pH control agents.
In one aspect the capsule including the solid oral pharmaceutical composition for including a bicyclic peptide of the invention is size 4 to size 000 capsules such as in the range of capsule size 1 to 00, where the size is measured according to standard size definition of two-piece capsules.
The pharmaceutical composition for including a bicyclic peptide of the invention may be in a dosage form of a tablet, particulate, multi-particulate, capsule, pellet, mini-tablets,
encapsulated pellet, encapsulated mini-tablets, encapsulated micro-particulate, or mucoadhesive forms (e.g., tablets or capsules).
In one aspect, the pharmaceutical composition for including a bicyclic peptide of the invention may be in a dosage form (e.g., capsule or tablet) without a coating. In one aspect, the pharmaceutical composition for including a bicyclic peptide of the invention is in a delayed release dosage form which minimizes the release of the active peptide ingredient and the enhancer in the stomach, and hence the dilution of the local enhancer concentration therein, and releases the drug and enhancer in the intestine. In other aspects, the pharmaceutical composition for including a bicyclic peptide of the invention is in a delayed release rapid onset dosage form. Such a dosage form minimizes the release of the active peptide ingredient and enhancer in the stomach, and hence the dilution of the local enhancer concentration therein, but releases the active peptide ingredient and enhancer rapidly once the appropriate site in the intestine has been reached, maximizing the delivery of the poorly permeable active peptide ingredient by maximizing the local concentration of the active peptide ingredient and enhancer at the site of absorption.
In one aspect, the pharmaceutical composition for including a bicyclic peptide of the invention may be in a form of a capsule solid oral dosage form. In one aspect, the capsule dosage form is an enteric coated capsule dosage form. In one aspect, the capsules dosage form is a capsule with enteric properties.
The term "capsule" as used herein includes, but is not limited to a relatively stable shell used for encapsulation of pharmaceutical formulations for oral administration. The two main types of capsules are hard-shelled capsules, which are normally used for dry, powdered ingredients, miniature pellets or mini tablets, and soft-shelled capsules, primarily used for oils and for active ingredients that are dissolved or suspended in oil. Both hard-shelled and soft-shelled capsules may be made from aqueous solutions of gelling agents such as animal protein, e.g. gelatin, or plant polysaccharides or their derivatives, e.g. carrageenans, and modified forms of starch and cellulose. Other ingredients may be added to the gelling agent solution such as plasticizers, e.g. glycerin and/or sorbitol, to decrease the capsule's hardness, coloring agents, preservatives, disintegrants, lubricants and surface treating agents.
Methods of preparation
The bicyclic peptide of the invention may be prepared as is known in the art. In one aspect the solid oral pharmaceutical composition may be prepared as described in the examples herein.
The production of peptides, e.g., bicyclic peptides, is well known in the art. The bicyclic peptide may for instance be produced by classical peptide synthesis, e.g., solid phase peptide synthesis using Boc or Fmoc chemistry or other well established techniques, see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza Dorwald, "Organic Synthesis on solid Phase", Wiley-VCH Veriag GmbH, 2000, and "Fmoc Solid Phase Peptide Synthesis", Edited by W.C. Chan and P.D. White, Oxford University Press, 2000. The bicyclic peptide may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the bicyclic peptide in a suitable nutrient medium under conditions permitting the expression of the bicyclic peptide. Several recombinant methods may be used in the production of bicyclic peptides. Examples of methods which may be used in the production of a bicyclic peptide are, e.g., disclosed in Xu M.-Q. et al J. Biol. Chem. 274, 18359-18363 (1999). Enzyme inhibition
The efficiency of a peptide of the invention in inhibiting proteolytic enzymes may be measured according to methods known by the person skilled in the art.
For example, the use of chromogenic substrates to monitor activity of proteolytic enzymes is known in the field (for example DelMar, E. G., et al., Anal. Biochem., 99, 316-320, (1979)). For example, /V-succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide is commonly used substrate for measuring chymotrypsin activity. Enzymatic cleavage of 4-nitroanilide substrates yields 4-nitroaniline (yellow color under alkaline conditions).
An assay following the increase in absorbance at 395 nm as a function of time is established in 96 well format using Varioskan Flash Multimode Meter (Thermo Scientific). Each well contains 70 μΙ of Dulbecco's phosphate buffer saline (Invitrogen catalogue
# 14190-094), 10 μΙ of /V-succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide (Sigma cat# S 7388) in dimethyl sulfoxide (DMSO, different concentrations are used in order to obtain the inhibition constant), 10 μΙ of sample containing a bicyclic peptide of the invention (for example dissolved solid dosage form, bicyclic peptide solution etc) in varying
concentration and 10 μΙ of a stock solution of chymotrypsin. The incubations are performed at 37°C. Absorbance at 395 nm is measured immediately after addition of the enzyme to the 96 well plate and also every minute for the next 80 minutes. The concentration of the enzyme is optimized to allow determination of slopes for the time course of initial absorbance increase with and without added inhibitors. The slopes are determined by linear regression of the linear part of the fluorescence trace (for example, the first 10 min of the reaction). Each assay is performed in duplicate and average of the
two traces is included in the calculations. The inhibition effect could be expressed as the concentration of the sample at which the slope of the absorbance trace equals to 50% of uninhibited reaction (EC50) . This is done by plotting the slopes achieved with different concentrations of the sample as a function of their concentrations and fitting the experimental results using, for example, sigmoidal logistic regression (2 parameters, Sigma Plot v 11). Inhibition constants for the interaction between the bicyclic peptide and proteolytic enzymes is also obtained by performing the assay described above with varying concentrations of the inhibitor and substrate and analyzing the results, for example, by double reciprocal transformation as known to the person skilled in the art and described for example in Hubalek, F. et al J . Med . Chem. 47, 1760- 1766 (2004) . When replacing /V-succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide with A/-(p-Tosyl)-Gly-Pro-Arg- p-Nitroanilide (Sigma T1637) inhibition of trypsin is measured, and when replacing with /V-succinyl-Ala-Ala-Ala-p-Nitroanilide (Sigma S4760) inhibition of elastase is measured . In one aspect, the peptide of the invention has an in vitro potency against chymotrypsin determined using the method described above corresponding to an EC50 potency of less than 10 μΜ, less than 5 μΜ, less than 3 μΜ, less than 1 μΜ, less than 0.5 μΜ or less than 0.1 μΜ .
In one aspect, the peptide of the invention has an in vitro potency against trypsin determined using the method described above corresponding to an EC50 potency of less than 10 μΜ, less than 5 μΜ, less than 3 μΜ, less than 1 μΜ, less than 0.5 μΜ or less than 0.1 μΜ .
In one aspect, the peptide of the invention has an in vitro potency against elastase determined using the method described above corresponding to an EC50 potency of less than 10 μΜ, less than 5 μΜ, less than 3 μΜ, less than 1 μΜ, less than 0.5 μΜ or less than 0.1 μΜ .
In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in the GI tract at least 2-fold compared to the half-life of said insulin peptide in the GI tract when administered without said bicyclic peptides. In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in the GI tract at least 3-fold . In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in the GI tract at least 5-fold . In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in the GI tract at least 10-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in the GI tract at least 2- fold compared to the half-life of said GLP- 1 peptide in the GI tract when administered without said bicyclic peptide. In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in the GI tract at least 3-fold . In one aspect, a bicyclic peptide
of the invention increases half-life of a GLP- 1 peptide in the GI tract at least 5-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in the GI tract at least 10-fold . In one aspect, a bicyclic peptide of the invention increases half- life of a growth hormone peptide in the GI tract at least 2-fold compared to the half-life of said insulin peptide in the GI tract when administered without said bicyclic peptides. In one aspect, a bicyclic peptide of the invention increases half-life of a growth hormone peptide in the GI tract at least 3-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a growth hormone peptide in the GI tract at least 5-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a growth hormone peptide in the GI tract at least 10-fold .
In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in rat GI juice at least 2-fold compared to the half-life of said insulin peptide in rat GI juice when administered without said bicyclic peptides. In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in rat GI juice at least 3-fold . In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in rat GI juice at least 5-fold . In one aspect, a bicyclic peptide of the invention increases half-life of an insulin peptide in rat GI juice at least 10-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in rat GI juice at least 2- fold compared to the half-life of said GLP- 1 peptide in rat GI juice when administered without said bicyclic peptide. In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in rat GI juice at least 3-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in rat GI juice at least 5-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a GLP- 1 peptide in rat GI juice at least 10-fold . In one aspect, a bicyclic peptide of the invention increases half- life of a growth hormone peptide in rat GI juice at least 2-fold compared to the half-life of said growth hormone peptide in rat GI juice when administered without said bicyclic peptide. In one aspect, a bicyclic peptide of the invention increases half-life of a growth hormone peptide in rat GI juice at least 3-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a growth hormone peptide in rat GI juice at least 5-fold . In one aspect, a bicyclic peptide of the invention increases half-life of a growth hormone peptide in rat GI juice at least 10-fold .
The invention is further described by the following non-limitina embodiments:
1. A bicyclic peptide of the formula
Cysl-Thr-X3-Ser-Ile-X6-X7-X8-Cys9-Thr-Xl l-Ser-Ile-X14-X15-X16
[Formula I]
wherein
Cysl is Cys;
X3 is Phe, Tyr, Trp, Lys, Arg or Ala;
X6 is Pro;
X7 is Pro;
X8 is a charged amino acid, a partly charged amino acid or Gin or He;
Cys9 is Cys;
XI 1 is Phe, Tyr, Lys, Leu, Arg, Val or Ala;
X14 is Pro;
X15 is Pro;
X16 is a charged amino acid or He; wherein at least one of X8 and X16 is a charged amino acid or partly charged amino acid; Cysl and Cys9 are joined by a disulfide bond between the sulfur atoms of the two cysteines; and
Cysl and X16 are joined by an amide bond between the alpha amine of Cysl and the alpha carboxylic group of X16.
2. The peptide according to embodiment 1, wherein X8 is a charged amino acid or partly charged amino acid.
3. The peptide according to embodiment 1 or 2, wherein X8 is a charged amino acid.
4. The peptide according to any one of the preceding embodiments, wherein X8 is selected from the group consisting of Arg, Lys, Asp, Glu and His
5. The peptide according to embodiment 1, wherein X8 is selected from the group consisting of Arg, Lys, He and Gin
6. The peptide according to any one of the preceding embodiments, wherein X16 is a charged amino acid or partly charged amino acid
7. The peptide according to any one of the preceding embodiments, wherein X16 is a charged amino acid
8. The peptide according to any one of the preceding embodiments, wherein X8 is selected from the group consisting of Arg, Lys, Asp, Glu and His
9. The peptide according to any one of the preceding embodiments, wherein X16 is selected from the group consisting of Arg, Lys and He
10. The peptide according to any one of the preceding claims, wherein X8 and X16 are charged amino acids or partly charged amino acids.
11. The peptide according to any one of the preceding embodiments, wherein X8 and X16 are charged amino acids.
12. The peptide according to any one of the preceding embodiments, wherein X8 and X16 are independently selected from the group consisting of Arg, Lys, Asp, Glu, Gin, He, and His.
13. The peptide according to any one of the preceding embodiments, wherein X8 and X16 are independently selected from the group consisting of Arg, Lys, Gin and He.
13a. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is selected from the group consisting of Lys, Ala, Phe or Tyr and XI 1 is Tyr
13b. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Lys and XI 1 is selected from the group consisting of Lys, Ala, Phe and Tyr
13c. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is selected from the group consisting of Lys and Arg and XI 1 is Phe
13d. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Phe and XI 1 is selected from the group consisting of Ala, Phe, Arg, Leu and Lys 133. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Phe and with Xl l is Tyr
13f. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Phe and with Xl l is Arg
13g. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Phe and with Xl l is Ala
13h. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Tyr and with Xl l is Lys
13 i . The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Lys and with Xl l is Phe
13j. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Lys and with Xl l is Tyr
13k. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Lys and with Xl l is Lys
131. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Phe and with Xl l is Leu
13m. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Tyr and with Xl l is Ala
13n. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Trp and with Xl l is Ala
13o. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Arg and with Xl l is Phe
13p. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Ala and with Xl l is Val
13q. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Lys and with Xl l is Ala
13r. The bicyclic peptide according to any one of the preceding embodiments, wherein X3 is Ala and with Xl l is Ala
14. The peptide according to any one of the preceding embodiments which is selected from the group consisting of:
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-L-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-aspartyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-histidyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-histidyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-arginine),
SEl,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-aspartyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tryptophyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-
L-lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-V-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine), l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginine),
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-lysine), and
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine).
14a. The peptide according to any one of the preceding embodiments which is 1,16- anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14b. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysine)
14c. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
14d. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
14e. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-L-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14f. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine),
14g. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14h. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14i. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14j. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14k. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14I. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14m. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14n. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-aspartyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
14o. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucine)
14p. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-histidyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
14q. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
r. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
s. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-histidyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L prolyl-L-isoleucine)
t. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl L-prolyl-L-arginine)
u. The peptide according to any one of the preceding embodiments which is
SEl,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl
L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
v. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
x. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
y. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
z. The peptide according to any one of the preceding embodiments which is
14aa. The peptide according to any one of the preceding embodiments which isl,16 anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14ab. The peptide according to any one of the preceding embodiments which is l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-
L-prolyl-L-isoleucine)
14ac. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
14ad. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14ae. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysine)
14af. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14ag. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-aspartyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
14ah. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine)
14ai. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-arginine)
14aj. The peptide according to any one of the preceding embodiments which isl,16- anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucine)
14ak. The peptide according to any one of the preceding embodiments which is l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
14al. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tryptophyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
14am. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine)
14an. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-arginine)
14ao. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-lysine)
14ap. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine)
14aq. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine)
14ar. The peptide according to any one of the preceding embodiments which is
l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-V-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine),
14as. The peptide according to any one of the preceding embodiments which isl,16- anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-arginine)
14at. The peptide according to any one of the preceding embodiments which isl,16- anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine)
14au. The peptide according to any one of the preceding embodiments which isl,16- anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl- L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L-lysine).
15. The peptide according to any one of the preceding embodiments which is a protease inhibitor
16. The peptide according to any one of the preceding embodiments which inhibits at least one protease enzyme.
17. The peptide according to any one of the preceding embodiments which inhibits at least two protease enzymes.
18. The peptide according to any one of the preceding embodiments which inhibits one or more enzymes found in the gastrointestinal tract.
19. The peptide according to any one of the preceding embodiments which inhibits one or more of the protease enzymes selected from the group consisting of trypsin,
chymotrypsin and elastase.
20. The peptide according to any one of the preceding embodiments which inhibits two or more of the protease enzymes selected from the group consisting of trypsin,
chymotrypsin and elastase.
21. The peptide according to any one of the preceding embodiments which inhibits at least the protease enzymes trypsin, chymotrypsin and elastase.
22. The peptide according to any one of the preceding embodiments which is soluble in aqueous solution at a pH which is between pH 1 and pH 10.23. The peptide according to any one of the preceding embodiments which is soluble in aqueous solution at a pH which is between pH 2 and pH 9.
24. The peptide according to any one of the preceding embodiments which is soluble in aqueous solution at a pH which is between pH 1 and pH 7 or between pH 8 and pH 10. 24a. The peptide according to any one of the preceding embodiments which is soluble in aqueous solution at a pH which is between pH 2 and pH 7 or between pH 8 and pH 9.
25. The peptide according to any one of the preceding embodiments which is soluble in aqueous solution at a pH which is between pH 7 and pH 8.
26. The peptide according wherein said peptide is not SEQ ID NO 12 or SEQ ID No 13.
EXAMPLES
List of Abbreviations
Aib: a-aminoisobutyric acid
API : Active Pharmaceutical Ingredient
ApoA-I: Apolipoprotein AI
AUC: Area Under the Curve
BHK: Baby Hamster Kidney
Boc: t-butyloxycarbonyl
BSA: Bovine serum albumin
CAS : Chemical Abstracts Service
Clt: 2-chlorotrityl
collidine: 2,4,6-trimethylpyridine
DCM : dichloromethane
DesH : des-amino histidine (may also be referred to as imidazopropionic acid,
Imp)
DIC: diisopropylcarbodiimide
DIPEA: diisopropylethylamine
DMEM : Dulbecco's Modified Eagle's Medium (DMEM)
DMF: A/^-dimethylformamide
EDTA: ethylenediaminetetraacetic acid
EGTA: ethylene glycol tetraacetic acid
Fmoc: 9-fluorenylmethyloxycarbonyl
GI juice: gastrointestinal extracts
HATU : (0-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluoro- phosphate)
HBSS : Hanks' balanced salt solution
HBTU : (2-(lH-benzotriazol-l-yl-)-l,l,3,3 tetramethyluronium hexafluoro- phosphate)
HEPES: 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
HFIP: l,l,l,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol
HOAt: l-hydroxy-7-azabenzotriazole
HPLC: High Performance Liquid Chromatography
HSA: Human Serum Albumin
IBMX: 3-isobutyl-l-methylxanthine
Imp: Imidazopropionic acid (also referred to as des-amino histidine, DesH) i.v. : Intravenously
ivDde: l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
LCMS : Liquid Chromatography Mass Spectroscopy
MALDI-MS : See MALDI-TOF MS
MALDI-TOF MS : Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass
Spectroscopy
MeOH : methanol
Mmt: 4-methoxytrityl
Mtt: 4-methyltrityl
NMP: N-methyl pyrrolidone
OEG: 8-amino-3,6-dioxaoctanic acid
OtBu : tert butyl ester
Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PBS: Phosphate Buffered Saline
Pen/Strep: Penicillin/Streptomycin
RP: Reverse Phase
RP-HPLC: Reverse Phase High Performance Liquid Chromatography
RT: Room Temperature
Rt: Retention time
s.c. : Subcutaneously
SBTI: Soybean Trypsin Inhibitor
SEC-HPLC: Size Exclusion High Performance Liquid Chromatography
SPA: Scintillation Proximity Assay
SPPS: Solid Phase Peptide Synthesis
tBu : tert butyl
TFA: trifluoroacetic acid
TIS: triisopropylsilane
Tris: tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethyl- propane-l,3-diol
Trt : Trityl or triphenylmethyl
Tween-20 : polyoxyethylene (20) sorbitan monolaurate
UPLC: Ultra Performance Liquid Chromatography
General Methods of Preparation
This section relates to methods for solid phase peptide synthesis (SPPS methods, including methods for de-protection of amino acids, methods for cleaving the peptide
from the resin, and for its purification), as well as methods for detecting and
characterising the resulting peptide (LCMS, MALDI, and UPLC methods). The solid phase synthesis of peptides may in some cases be improved by the use of di-peptides protected on the di-peptide amide bond with a group that can be cleaved under acidic conditions such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or 2,4,6-trimethoxybenzyl. In cases where a serine or a threonine is present in the peptide, pseudoproline di-peptides may be used (available from, e.g., Novabiochem, see also W.R. Sampson (1999), J. Pep. Sci. 5, 403). The Fmoc-protected amino acid derivatives used were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)- OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc- His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe- OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, or, Fmoc-Val-OH etc. supplied from e.g. Anaspec, Bachem, Iris Biotech, or Novabiochem. Where nothing else is specified the natural L-form of the amino acids are used.
Synthesis of resin bound protected peptide hydrazides
Preparation of hydrazine-resin
2-Chlorotrityl chloride resin (25 g, 1.70 mmol/g) was swollen in DMF (125 ml) for 30 min under nitrogen, and then cooled to 0 °C. A mixture of hydrazine-hydrate (7.7 ml, 160 mmol) and triethylamine (6.7 ml, 48 mmol) in DMF (10 ml) was added dropwise and the suspension was stirred 30 min at 25 °C. The suspension was then cooled to 0 °C followed by dropwise addition of a mixture of hydrazine-hydrate (7.7 ml, 160 mmol) and triethylamine (6.7 ml, 48 mmol) in DMF (10 ml). The suspension was stirred 30 min at 25 °C and then filtered and washed with NMP (6x), water (6x), NMP (6x), methanol (6x) and DCM (6x). The resin was then dried in vacuo overnight to afford 22.60 g light-yellow resin.
Method : SPPS_P
SPPS_P was performed on a Prelude Solid Phase Peptide Synthesizer from
Protein Technologies (Tucson, AZ 85714 U.S.A.) at 250-μιηοΙ or 400-μιηοΙ scale using six fold excess of Fmoc-amino acids (300 mM in NMP with 300 mM Oxyma Pure®) relative to resin loading (typical loading of hydrazine-resin was 0.3 mmol/g). Fmoc-deprotection was performed using 20% piperidine in NMP. Coupling was performed using 3 : 3 : 3 : 4 amino acid/Oxyma Pure®/DIC/collidine in NMP. NMP and DCM top washes (7 ml, 0.5 min, 2 x 2 each) were performed between deprotection and coupling steps. Coupling
times were generally 60 minutes. Some amino acids including, but not limited to Fmoc- Arg(Pbf)-OH, Fmoc-Aib-OH, Fmoc-Cys(Trt)-OH or Boc-His(Trt)-OH were "double coupled", meaning that after the first coupling (e.g. 60 min), the resin was drained and more reagents were added (amino acid, Oxyma Pure®, DIC, and collidine), and the mixture allowed to react again (e.g. 60 min).
Method : SPPS L
SPPS_L was performed on a microwave-based Liberty peptide synthesiser from CEM Corp. (Matthews, NC 28106, U.S.A.) at 250-μιηοΙ scale using six fold excess of Fmoc-amino acids (300 mM in NMP with 300 mM Oxyma Pure®) relative to resin loading . (typical loading of hydrazine-resin was 0.3 mmol/g). Fmoc-deprotection was performed using 5% piperidine in NMP at up to 75°C for 30 seconds where after the resin was drained and washed with NMP and the Fmoc-deprotection was repeated this time for 2 minutes at 75°C. Coupling was performed using 1 : 1 : 1 amino acid/Oxyma Pure®/DIC in NMP. Coupling times and temperatures were generally 5 minutes at up to 75°C.
Longer coupling times were used for larger scale reactions, for example 10 min. Histidine and cysteine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericaiiy hindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 minutes and then heated to 75°C for 5 min. Some amino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at 75°C), the resin was drained and more reagents were added (amino acid, Oxyma Pure® and DIC), and the mixture was heated again (e.g. 5 min at 75°C). NMP washes (5 x 10 ml) were performed between deprotection and coupling steps. Method : SPPS I
SPPS_I was performed on a Multipep RSi synthesizer from Intavis Bioanalytical Instruments AG (Koeln, Germany) at 100-μιηοΙ scale in parallel using 2.5 fold excess of Fmoc-amino acids (300 mM in NMP with 300 mM Oxyma Pure®) relative to resin loading. Coupling of amino acids and Fmoc-deprotection was performed at 45 °C with interval shaking (30 seconds shaking every 2 minutes at 550 rpm). All amino acids were "double coupled", meaning that after the first coupling (60 min), the resin was drained and more reagents were added (amino acid, Oxyma Pure®, DIC, and collidine), and the mixture allowed to react again (60 min).
Clevage of resin bound peptide hydrazides and purification
Method : CP M l
After synthesis the resin was washed with DCM, and the peptide was cleaved from the resin by a 3 hour treatment with TFA/TIS/2-mercaptoethanol/water
(87.5/5/5/2.5) followed by precipitation with diethylether. The peptide was dissolved in a suitable solvent (such as, e.g., 10/90 acetic acid/water) and purified by standard RP- HPLC on a C18, 5μΜ column, using acetonitrile/water/TFA. The fractions were analysed by a combination of UPLC, MALDI and LCMS methods, and the appropriate fractions were pooled.
Method : CP M2
After synthesis the resin was washed with ethanol, and the peptide was cleaved from the resin by a 3 hour treatment with TFA/TIS/2-mercaptoethanol/water
(85/5/7.5/2.5) followed by precipitation with diethylether.
Cyclization of peptide hydrazides, disulfide oxidation and purification
Method : NCL M l
The pooled fractions from RP-HPLC purification of the peptide hydrazides were diluted with water to 80 : 20 water/acetonitrile. Disodium phosphate was added to a final concentration of 0.2 M and the pH was adjusted to 3.0 with concentrated hydrochloric acid (aq). The mixture was cooled to 0 °C and sodium nitrite (10 eq, 0.2 M in water) was added, and the mixture was stirred for 20 minutes at 0 °C. Sodium 2- mercaptoethanesulfonate (20 eq) was added and the pH was adjusted to 7.0 with 1 M NaOH (aq). The reaction mixture was stirred at 25 °C for 16 hours and then purified by standard RP-HPLC on a C18, 5μΜ column, using acetonitrile/water/TFA. The fractions were analysed by a combination of UPLC, MALDI and LCMS methods, and the appropriate fractions were pooled and lyophilized.
Method : NCL M2
The crude peptide hydrazide was dissolved in 0.2 M disodium phosphate (aq, pH
3.0, 7 ml) and the mixture was cooled to 0 °C. Sodium nitrite (2.0 M in water, 10 eq) was added, and the mixture was stirred for 20 minutes at 0 °C. Sodium 2- mercaptoethanesulfonate (2.0 M in water, 20 eq) was added and the pH was adjusted to 7.0 with 1 M NaOH (aq). The mixture was stirred at 25 °C for 16 hours and then loaded onto a Seppak C18 column (500 mg), washed with water (3 ml) and then eluted with a mixture of water/acetonitrile/trifluoroacetic acid 50 : 50 : 0.1 (4 ml). The mixture was
diluted with water (6 ml) and purified by standard RP-HPLC on a C18, ΙΟμΜ column, using acetonitrile/water/TFA. The fractions were analysed by a combination of UPLC, MALDI and LCMS methods, and the appropriate fractions were pooled and lyophilized. General Methods of Detection and Characterisation
LCMS
LC-MS was performed on a setup consisting of Waters Acquity UPLC system and LCT Premier XE mass spectrometer from Micromass. Eluents: A: 0.1% Formic acid in water, B: 0.1% Formic acid in acetonitrile.
The analysis was performed at room temperature (RT) by injecting an appropriate volume of the sample (preferably 2-10μΙ) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass
spectrometer settings were:
Column : Waters Acquity UPLC BEH, C-18, 1.7μιη, 2.1mm x 50mm.
Gradient: Linear 5% - 95% acetonitrile during 4.0 min at 0.4ml/min.
Detection : 214 nm (analogue output from TUV (Tunable UV detector))
MS ionisation mode: API-ES
Scan : 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu. UPLC
The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY UPLC BEH, C18, 1.7 μιτι, 2.1 mm x 150 mm column, 40°C. The UPLC system was connected to two eluent reservoirs containing : A: 99.95% H20, 0.05% TFA; B: 99.95% CH3CN, 0.05% TFA. The following linear gradient was used : 95% A, 5% B to 5% A, 95% B over 16 minutes at a flow-rate of 0.40 ml/min.
Example 1: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-arginine)
SEQ ID No: l :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1755 m/z; found m/1 : 1755, m/2 : 878, m/3 : 586; Rt-UV = 1.8 min
UPLC: Rt = 6.9 min.
Example 2: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1712 m/z; found m/1 : 1712, m/2 : 856, m/3 : 571; Rt-UV = 1.8 min
UPLC: Rt = 7.0 min.
Example 3: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 3 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1712 m/z; found m/1 : 1712, m/2 : 856; Rt- UV = 2.0 min
UPLC: Rt = 7.5 min.
Example 4: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1727 m/z; found m/1 : 1727, m/2 : 864, m/3 : 576; Rt-UV = 2.1 min
UPLC: Rt = 6.8 min.
Example 5: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-L-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
SEQ ID No: 5 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1712 m/z; found m/1 : 1713, m/2 : 856; Rt- UV = 2.3 min
UPLC: Rt = 8.5 min.
Example 6: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-arginine)
SEQ ID No: 6 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1771 m/z; found m/1 : 1771, m/2 : 886, m/3 : 591; Rt-UV = 1.7 min
UPLC: Rt = 6.1 min.
Example 7: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-arginine)
SEQ ID No: 7 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1756 m/z; found m/1 : 1756, m/2 : 878, m/3 : 586; Rt-UV = 1.7 min
UPLC: Rt = 6.3 min.
Example 8: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1743 m/z; found m/2: 872; Rt-UV = 1.7 min
UPLC: Rt = 6.0 min.
Example 9: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
SEQ ID No:9 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1728 m/z; found m/2: 864; Rt-UV = 1.7 min
UPLC: Rt = 6.2 min.
Example 10: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
EQ ID No: 10 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1783 m/z; found m/1 : 1783, m/2 : 892, m/3 : 595; Rt-UV = 1.7 min
UPLC: Rt = 6.8 min.
Example 11: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginine)
SEQ ID No: l l :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1783 m/z; found m/1 : 1784, m/2 : 892, m/3 : 595; Rt-UV = 1.7 min
UPLC: Rt = 6.7 min.
Example 12: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1698 m/z; found m/1 : 1698, m/2 : 850; Rt- UV = 1.9 min
UPLC: Rt = 7.3 min.
Example 13: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
SEQ ID No: 13 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1698 m/z; found m/1 : 1698, m/2 : 850; Rt- UV = 1.9 min
UPLC: Rt = 7.2 min.
Example 14: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-aspartyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 14:
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1699 m/z; found m/1 : 1699, m/2 : 850; Rt- UV = 2.1 min
UPLC: Rt = 7.6 min.
Example 15: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 15 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1713 m/z; found m/1 : 1713, m/2 : 857; Rt- UV = 2.1 min
UPLC: Rt = 7.7 min.
Example 16: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-histidyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SE ID No: 16 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1721 m/z; found m/1 : 1722, m/2 : 861 ; Rt UV = 2.0 min
UPLC: Rt = 7.1 min.
Example 17: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 17 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1740 m/z; found m/1 : 1740, m/2 : 870; Rt- UV = 2.0 min
UPLC: Rt = 7.6 min.
Example 18: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1755 m/z; found m/1 : 1755, m/2 : 878, m/3 : 586; Rt-UV = 1.8 min
UPLC: Rt = 6.9 min.
Example 19: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-histidyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 19 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1721 m/z; found m/1 : 1721, m/2 : 861 ; Rt UV = 2.0 min
UPLC: Rt = 7.6 min.
Example 20: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L prolyl-L-prolyl-L-arginine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1811 m/z; found m/2: 906, m/3 : 604; Rt- UV = 1.7 min
UPLC: Rt = 6.8 min.
Example 21: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-arginine)
SEQ ID No: 21 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1756 m/z; found m/1 : 1756, m/2 : 879; Rt UV = 1.7 min
UPLC: Rt = 6.6 min.
Example 22: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1740 m/z; found m/1 : 1740, m/2 : 870; Rt UV = 2.0 min
UPLC: Rt = 7.6 min.
Example 23: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutaminyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginine)
SEQ ID No: 23 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1783 m/z; found m/2: 892, m/3 : 595; Rt- UV = 1.9 min
UPLC: Rt = 7.0 min.
Example 24: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1727 m/z; found m/1 : 1727, m/2 : 864, m/3 : 577; Rt-UV = 1.7 min
UPLC: Rt = 6.5 min.
Example 25: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-is L-prolyl-L-prolyl-L-arginine)
SEQ ID No: 25 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1784 m/z; found m/1 : 1785, m/2 : 893, m/3 : 596; Rt-UV = 1.8 min
UPLC: Rt = 6.7 min.
Example 26: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L prolyl-L-prolyl-L-isoleucine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein. LCMS (LCMS method described herein) : calc. 1768 m/z; found m/1 : 1769, m/2 : 885; Rt UV = 2.0 min
UPLC: Rt = 7.7 min.
Example 27: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 27 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1740 m/z; found m/1 : 1740, m/2 : 871 ; Rt- UV = 1.9 min
UPLC: Rt = 7.2 min.
Example 28: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl- L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1728 m/z; found m/1 : 1728, m/2 : 865; Rt- UV = 1.7 min
UPLC: Rt = 6.5 min.
Example 29: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
SEQ ID No: 29 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1728 m/z; found m/1 : 1728, m/2 : 864; Rt- UV = 1.8 min
UPLC: Rt = 6.9 min.
Example 30: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-arginyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-is L-prolyl-L-prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1756 m/z; found m/1 : 1756, m/2 : 879; Rt- UV = 1.8 min
UPLC: Rt = 6.6 min.
Example 31: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-aspartyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
SEQ ID No: 31 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1699 m/z; found m/1 : 1699, m/2 : 850; Rt- UV = 2.2 min
UPLC: Rt = 8.0 min.
Example 32: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1743 m/z; found m/1 : 1743, m/2 : 872, m/3 : 582; Rt-UV = 1.5 min
UPLC: Rt = 5.8 min.
Example 33: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-arginyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
SEQ ID No: 33 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1799 m/z; found m/1 : 1800, m/2 : 900, m/3 : 601; Rt-UV = 1.6 min
UPLC: Rt = 6.0 min.
Example 34: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-glutamyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-isoleucine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_I, cleavage method CP_M2 and cyclization method NCL_M2 described herein.
LCMS (LCMS method described herein) : calc. 1713 m/z; found m/1 : 1713, m/2 : 857; Rt- UV = 2.2 min
UPLC: Rt = 8.0 min.
Example 35: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
SEQ ID No: 35 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1670 m/z; found m/1 : 1670, m/2 : 835; Rt- UV = 1.8 min
UPLC: Rt = 7.2 min.
Example 36: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tryptophyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1765 m/z; found m/2: 883; Rt-UV = 1.9 min
UPLC: Rt = 7.4 min.
Example 37: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-lysine)
SEQ ID No: 37 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1686 m/z; found m/1 : 1686, m/2 : 843; Rt- UV = 1.7 min
UPLC: Rt = 6.5 min.
Example 38: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-tyrosyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1742 m/z; found m/2: 871 ; Rt-UV = 1.7 min
UPLC: Rt = 6.6 min.
Example 39: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
SEQ ID No: 39 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1636 m/z; found m/1 : 1636, m/2 : 818; Rt- UV = 1.6 min
UPLC: Rt = 6.1 min.
Example 40: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1651 m/z; found m/1 : 1651, m/2 : 826, m/3 : 551; Rt-UV = 1.4 min
UPLC: Rt = 5.4 min.
Example 41: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
SEQ ID No:41 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1594 m/z; found m/1 : 1594, m/2 : 797; Rt- UV = 1.6 min
UPLC: Rt = 6.2 min.
Example 42: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-V-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L- lysine)
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1622 m/z; found m/1 : 1622, m/2 : 811 ; Rt- UV = 1.7 min
UPLC: Rt = 6.7 min.
Example 42a: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-arginyl-L-cysteinyl-L-threonyl-L-alanyl-L-seryl-L-isoleucyl-L- prolyl-L-prolyl-L-arginine)
SEQ ID No:43 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1726 m/z; found m/2: 863, m/3 : 576; Rt- UV = 1.8 min
UPLC: Rt = 7.4 min.
Example 42b: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
SEQ ID No:44:
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1693 m/z; found m/1 : 1693, m/2 : 847, m/3 : 565; Rt-UV = 1.3 min
UPLC: Rt = 5.1 min.
Example 42c: l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl- L-prolyl-L-prolyl-L-lysyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L-isoleucyl-L-prolyl-L- prolyl-L-lysine)
SEQ ID No:45 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1708 m/z; found m/1 : 1708, m/2 : 854, m/3 : 570; Rt-UV = 1.2 min
UPLC: Rt = 4.7 min.
Example 42d: (BiKF from RJ. Leatherbarrow, Protein Engineering, Design & Selection, 2004, 17(9), 681-687) : l,16-anhydro-S1,S9-cyclo(cysteinyl-L-threonyl-L-phenylalanyl-L- seryl-L-isoleucyl-L-prolyl-L-prolyl-L-isoleucyl-L-cysteinyl-L-threonyl-L-lysyl-L-seryl-L- isoleucyl-L-prolyl-L-prolyl-L-isoleucine)
SEQ ID No:46 :
The bicyclic peptide was prepared according to preparation methods: Synthesis method SPPS_P, cleavage method CP_M 1 and cyclization method NCL_M 1 described herein. LCMS (LCMS method described herein) : calc. 1697 m/z; found m/1 : 1697, m/2 : 849; Rt- UV = 2.2 min
UPLC: Rt = 8.2 min.
Example 43: Enzyme inhibition
/V-succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide was used as substrate for measuring chymotrypsin activity. Enzymatic cleavage of 4-nitroanilide substrates yielded 4- nitroaniline (yellow color under alkaline conditions).
An assay following the increase in absorbance at 395 nm as a function of time was established in 96 well format using Varioskan Flash Multimode Meter (Thermo Scientific). Each well contained 70 μΙ of Dulbecco's phosphate buffer saline (Invitrogen catalogue # 14190-094), 10 μΙ of /V-succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide (Sigma cat# S 7388) in dimethyl sulfoxide (DMSO, different concentrations were used in order to obtain the inhibition constant), 10 μΙ of sample containing a bicyclic peptide of the invention (for example dissolved solid dosage form, bicyclic peptide solution etc) in varying concentration and 10 μΙ of a stock solution of chymotrypsin. The incubations were performed at 37°C. Absorbance at 395 nm was measured immediately after addition of the enzyme to the 96 well plate and also every minute for the next 80 minutes. The concentration of the enzyme was optimized to allow determination of slopes for the time course of initial absorbance increase with and without added inhibitors. The slopes were determined by linear regression of the linear part of the fluorescence trace (for example, the first 10 min of the reaction). Each assay was performed in duplicate and average of the two traces was included in the calculations. The inhibition effect could be expressed as the concentration of the sample at which the slope of the absorbance trace equals to 50% of uninhibited reaction (EC50). This was done by plotting the slopes achieved with different concentrations of the sample as a function of their concentrations and fitting the experimental results using, for example, sigmoidal logistic regression (2 parameters, Sigma Plot v 11). Inhibition constants for the interaction between the bicyclic peptide and proteolytic enzymes was also obtained by performing the assay described above with varying concentrations of the inhibitor and substrate and analyzing the results, for example, by double reciprocal transformation as known to the person skilled in the art and described for example in Hubalek, F. et al J. Med. Chem. 47, 1760- 1766 (2004).
When replacing /V-succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide with A/-(p-Tosyl)-Gly- Pro-Arg-p-Nitroanilide (Sigma T1637) inhibition of trypsin was measured, and when replacing with /V-succinyl-Ala-Ala-Ala-p-Nitroanilide (Sigma S4760) inhibition of elastase was measured.
The results are shown in table 1 and la.
Table 1: Enzyme inhibition
SEQ ID Inhibition chymotrypsin Inhibition trypsin Inhibition elastase
No: (EC50, μΜ) (EC50, μΜ) (EC50, μΜ)
1 0.07 0.07
2 0.13 0.09
SEQ ID Inhibition chymotrypsin Inhibition trypsin Inhibition elastase No: (EC50, μΜ) (EC50, μΜ) (EC50, μΜ)
3 0.07 0.09
4 0.09 0.07
5 0.12 1.22 4.3
6 0.03 0.04
7 0.05 0.05
8 0.08 0.08
9 0.15 0.08
10 0.03 0.02
11 0.06 0.03
12 0.05 1.00 7.2
13 0.08 2.13 5.9
14 0.15 0.09
15 0.11 0.07
16 0.08 0.10
17 0.29 0.12
18 0.07 0.04
19 0.14 0.25
20 0.10 0.08
21 0.07 0.02
22 0.08 0.19
23 0.03 0.02
24 0.04 0.07
25 0.09 0.05
26 0.07 0.10
27 0.06 0.08
28 0.04 0.07
29 0.38 0.42
30 0.04 0.03
31 0.29 0.10
32 0.05 0.09
33 0.02 0.03
34 0.08 0.13
35 0.12 1.2 7.3
36 0.08 > 100 0.69
37 0.07 6.8 9.1
38 0.03 7.3 1.0
39 > 100 0.06 0.95
40 > 100 0.04 54
41 > 100 > 100 9.0
SEQ ID Inhibition chymotrypsin Inhibition trypsin Inhibition elastase No: (EC50, μΜ) (EC50, μΜ) (EC50, μΜ)
42 > 100 > 100 23
Table la: Enzvme inhibition
Example 44: Insulin peptide degradation by gastrointestinal extracts (GI juice) in presence of inhibitor
96-well plates were coated by incubating with 0.4% BSA solution for minimum of 60 min. To each well, 210 μΙ of buffer (Hanks' balanced salt solution buffered with 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid (HBSS-HEPES buffer) with 0.005 % polyoxyethylene (20) sorbitan monolaurate (Tween 20) and 0.005 % BSA, pH 6.5), 30 μΙ of a substrate (100 μΜ insulin peptide in buffer) and 30 μΙ of inhibitor (10 mM) or buffer were added. The plates were pre-incubated for 60 min at 37°C, before adding GI juice. After addition of 30 μΙ of GI juice, the plates were incubated for 60 min/37 °C on shaker. Samples (40μΙ) were taken at 0, 3, 6, 10, 30 and 60 min, stopped with 3 vol. cold 96 % ethanol w. 1 % formic acid and centrifuged (4500 rcf for 10 min at 4°C). The resulting supernatant from the samples were diluted 5 times with the buffer prior to LCMS analysis. Reference samples without inhibitor (0.1, 0.5, 1.0, 5.0, 10.0 μΜ) were prepared and treated as the samples with inhibitor. Standard curve was analysed both at the beginning and the end of the analytical run. Two replicates of each tested condition were included. Intact insulin peptide was determined in each sample. The results were plotted against the incubation time. Half-life of the insulin peptide were determined by nonlinear regression of the results using, for example GraphPad Prism. GI juice was prepared from male Sprague Dawley rats (200-250 g) by excising approximately 20 cm piece of mid jejunum and rinsing the inside with 2.5 ml 0.9% aqueous sodium chloride solution. The sodium chloride solution was collected in a centrifuge tube, pooled from all rats (20) and centrifuged (3200 rcf. for 10 min at 4°C). The supernatant was aliquoted in tubes and stored at -80 °C. The results are listed in table 2 below.
Example 45: GLP-1 peptide degradation by gastrointestinal extracts (GI juice) in presence of inhibitor
96 well plates were coated by incubating with 0.4% BSA solution for a minimum of 60 min. To each well, 210 μΙ of buffer (HBSS-HEPES buffer with 0.005 % Tween 20 and 0.005 % BSA, pH 6.5), 30 μΙ of a substrate (100 μΜ GLP-1 peptide in buffer) and 30 μΙ of buffer or inhibitor (10 mM) were added. The plates were pre-incubated for 60 min at 37°C, before adding GI juice. After addition of 30 μΙ of GI juice, the plates were incubated for 60 min/37°C on shaker. Samples (40μΙ) were taken at 0, 3, 6, 10, 30 and 60 min, stopped with 3 vol. cold 96 % ethanol w. 1 % formic acid and centrifuged (4500 rcf for 10 min at 4°C). The resulting supernatant from the samples were diluted 5 times with the buffer prior to LCMS analysis. Reference samples without inhibitor (0.1, 0.5, 1.0, 5.0, 10.0 μΜ) were prepared and treated as the samples with inhibitor. Standard curve was analysed both at the beginning and the end of the analytical run. Two replicates of each tested conditions were included. Intact GLP-1 peptide was determined in each sample. The results were plotted against the incubation time. Half-life of the GLP-1 peptide was determined by nonlinear regression of the results using for example
GraphPad Prism. GI juice was prepared from male Sprague Dawley rats (200-250 g) by excising approximately 20 cm piece of mid jejunum and rinsing the inside with 2.5 ml 0.9% aqueous sodium chloride solution. The sodium chloride solution was collected in a centrifuge tube, pooled from all rats (20) and centrifuged (3200 rcf. for 10 min at 4°C). The supernatant was aliquoted in tubes and stored at -80°C. The results are listed in table 2.
Table 2
T1/2 insulin peptide T1/2 GLP-1 peptide rat
SEQ ID No:
rat GI juice (min) GI juice (min)
no inhibitor 1.8 <0.5
1 stabile 9.7
2 stabile 5.8
3 445 10.4
4 stabile 6.8
5 57 1.4
6 950 2.8
7 420 3.2
8 stabile 2.4
9 stabile 2.8
"P/2 insulin peptide "Π/2 GLP-1 peptide rat
SEQ ID No:
rat GI juice (min) GI juice (min)
10 230 4.1
11 120 4.5
Insulin peptide: /V{Epsilon-B29}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17- carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy ]acetyl]-[GluA14,HisB25],des-ThrB30-Insulin(human)
GLP-1 peptide: /V-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17- carboxyheptadecanoylamino)-butyrylamino]ethoxy}ethoxy)- acetylamino]ethoxy}ethoxy)acetyl]-[Aib8,Arg34]GLP-l-(7-37)
Example 46: Growth Hormone degradation by gastrointestinal extracts (GI juice) in presence of inhibitor
Stock solutions of human growth hormone (30 mg/ml) and inhibitors (1 mM) were prepared in a 100 mM dipotassium phosphate buffer, pH 8.2. Inhibitor was mixed with human growth hormone in 50 mM ammonium bicarbonate to a final concentration of 0.2 mM and 5 mg/ml respectively. After 10 minutes of pre-incubation at 37°C, samples were added rat GI juice (13.3 μί/100 μΙ_ sample), mixed and incubated at 37°C for 4 hours. At times: 0, 0.5, 1, 2 and 4 h, 5 μΙ sample were taken out and added 245 μΙ 1%
trifluoroacetic acid, ending all further enzymatic degradation. The remaining amount of intact human growth hormone was determined by analysis on a Waters Acquity UPLC system. Separation was achieved using a linear gradient of acetonitrile in 0.1%
trifluoroacetic acid (20-70% over 12 minutes) using BEH C4 column (Waters 1.7μιη, 300A, 1.0x150 mm) with a flow rate of 0.12 ml/min at 60°C. Quantification was performed using Waters Empower 3 software. The results are listed in table 3.
Table 3.
SEQ ID No: T1/2 of GH with 4 T1/2 of GH with 0.2 mM
mg/ml inhibitor (min) inhibitor (min)
SBTI 1.49 1.42
1 2.68 0.24
2 3.95 0.22
3 0.78 0.39
37 0.35 0.08
Example 47: Precipitation screen
Each test sample was mixed 1 : 1 with a buffer ("mixing buffer") strong enough to maintain the pH of the sample to have a final pH within 0.2 pH units of the setpoint. The assay was conducted in a sitting drop crystallisation plate, and mixing buffer was added together with sample buffer to fill up the reservoir.
The mixing was performed with the assistance of a pipetting robot to produce drops of a size of app. 1 μΙ. The presence of precipitates was controlled with the aid of a microscope at t=0 and after 24 hours. Each test sample was checked for crystals, aggregates or phase separation. The results are listed in table 4 (0 means crystals or aggregation and 1 means that the sample is soluble).
Table 4.
Buffer 200 200 mM 200 mM 200 mM 200 mM 200 mM 200 200 mM Na2HPO Na2HPO Na2HP04 Na2HP04 Na2HP04 mM mM Na2H + 100 mM + 100 mM + 100 Na2HP TRIS P04 + 100 + 100 citric acid citric acid mM 04
mM mM citric
citric citric acid
acid acid
PH 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
SEQ ID No : 1 1 1 1 1 1 1 1 1
SEQ ID No : 2 1 1 1 1 1 1 1 1
SEQ ID No : 3 1 1 1 1 1 1 1 1
SEQ ID No : 4 1 1 1 1 1 1 1 1
SEQ ID No : 5 1 1 1 1 1 1 1 1
SEQ ID No : 6 1 1 1 1 1 1 1 1
SEQ ID No : 7 1 1 1 1 1 1 1 1
SEQ ID No : 8 1 1 1 1 1 1 1 1
SEQ ID No : 9 1 1 1 1 1 1 1 1
SEQ ID No : 10 1 1 1 1 1 1 1 1
SEQ ID No : 11 1 1 1 1 1 1 1 1
SEQ ID No : 12 1 1 1 1 1 0 1
SEQ ID No : 13 1 1 1 1 1 0 1
SEQ ID No : 14 1 1 1 1 1 1 1 1
SEQ ID No : 15 1 1 1 1 1 1 1 1
SEQ ID No : 16 1 1 1 1 1 1 1 1
SEQ ID No : 17 1 1 1 1 1 1 1 1
SEQ ID No : 22 1 1 1 1 1 1 1 1
SEQ ID No : 26 1 1 1 1 1 1 1 1
SEQ ID No : 46 1 1 1 0 0 0 0 0
Example 48: Rat intestinal injection model
Hypnorm/Midazolam anaesthetized, fasted, male Sprague-Dawley rats were used to compare the bioavailability and duration of action of a GLP-1 peptide in different formulations after intraintestinal injection.
In brief, the anaesthetized rats were placed on heating blankets and midline incisions were made in the skin and abdominal wall. Polyethylene catheters filled with test compound (1000 nmol/ml GLP-1 peptide, 55 mg/ml sodium decanoate, 6.5 mM inhibitor in 50 mM disodium phosphate buffer) were inserted into mid-jejunum approximately 50 cm from caecum. The abdominal wall was closed with autoclips.
At time 0, rats were dosed via the catheter, 0.1 ml/rat. Blood samples for determination of plasma GLP-1 concentrations were collected regularly until 3 h after dosing.
After analysis of plasma GLP-1 peptide using LOCI assays, AUC is calculated.
Table 5: GLP-1 peptide bioavailability after intraintestinal injection to rats with solutions containing GLP-1 peptide, inhibitor and sodium caprate.
GLP-1 peptide: /V-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17- carboxyheptadecanoylamino)-butyrylamino]ethoxy}ethoxy)- acetylamino]ethoxy}ethoxy)acetyl]-[Aib8,Arg34]GLP-l-(7-37)
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
Claims
1. A bicyclic peptide of the formula
Cysl-Thr-X3-Ser-Ile-X6-X7-X8-Cys9-Thr-Xl l-Ser-Ile-X14-X15-X16
[Formula I]
wherein
Cysl is Cys;
X3 is Phe, Tyr, Trp, Lys, Arg or Ala;
X6 is Pro;
X7 is Pro;
X8 is a charged amino acid, a partly charged amino acid or Gin or He;
Cys9 is Cys;
XI 1 is Phe, Tyr, Lys, Leu, Arg, Val or Ala;
X14 is Pro;
X15 is Pro;
X16 is a charged amino acid or He; wherein at least one of X8 and X16 is a charged amino acid or partly charged amino acid; Cysl and Cys9 are joined by a disulfide bond between the sulfur atoms of the two cysteines; and
Cysl and X16 are joined by an amide bond between the alpha amine of Cysl and the alpha carboxylic group of X16.
2. The peptide according to claim 1, wherein X8 is a charged amino acid or partly charged amino acid.
3. The peptide according to claim 1 or 2, wherein X8 is a charged amino acid.
4. The peptide according to any one of the preceding claims, wherein X8 is selected from the group consisting of Arg, Lys, Asp, Glu and His.
5. The peptide according to any one of the preceding claims, wherein X16 is a charged amino acid or partly charged amino acid
6. The peptide according to any one of the preceding claims, wherein X16 is selected from the group consisting of Arg, Lys and He
7. The peptide according to any one of the preceding claims, wherein X8 and X16 are charged amino acids or partly charged amino acids.
8. The peptide according to any one of the preceding claims, wherein X8 and X16 are independently selected from the group consisting of Arg, Lys, Asp, Glu, Gin, He, and His.
9. The peptide according to any one of the preceding claims which is a protease inhibitor
10. The peptide according to any one of the preceding claims which inhibits at least one protease enzyme.
11. The peptide according to any one of the preceding claims which inhibits at least two protease enzymes.
12. The peptide according to any one of the preceding claims which inhibits one or more enzymes found in the gastrointestinal tract.
13. The peptide according to any one of the preceding claims which inhibits one or more of the protease enzymes selected from the group consisting of trypsin, chymotrypsin and elastase.
14. The peptide according to any one of the preceding claims which inhibits two or more of the protease enzymes selected from the group consisting of trypsin, chymotrypsin and elastase.
15. The peptide according to any one of the preceding claims which is soluble in aqueous solution at a pH which is between pH 1 and pH 10.
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Citations (2)
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|---|---|---|---|---|
| US20050130883A1 (en) * | 2003-09-30 | 2005-06-16 | The Gov. Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services | Serine protease inhibitors |
| WO2012083385A1 (en) * | 2010-12-23 | 2012-06-28 | Queensland University Of Technology | Serine protease inhibitors |
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| US20050130883A1 (en) * | 2003-09-30 | 2005-06-16 | The Gov. Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services | Serine protease inhibitors |
| WO2012083385A1 (en) * | 2010-12-23 | 2012-06-28 | Queensland University Of Technology | Serine protease inhibitors |
Non-Patent Citations (6)
| Title |
|---|
| AGNÈS M JAULENT ET AL: "Solution Structure of a Novel C2-Symmetrical Bifunctional Bicyclic Inhibitor Based on SFTI-1", JOURNAL OF BIOMOLECULAR NMR, KLUWER ACADEMIC PUBLISHERS, DO, vol. 33, no. 1, 1 September 2005 (2005-09-01), pages 57 - 62, XP019249632, ISSN: 1573-5001 * |
| ANNA LEGOWSKA ET AL: "Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing", FEBS JOURNAL, vol. 277, no. 10, 19 May 2010 (2010-05-19), pages 2351 - 2359, XP055112405, ISSN: 1742-464X, DOI: 10.1111/j.1742-4658.2010.07650.x * |
| JAULENT ET AL: "Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors", PROTEIN ENGINEERING, DESIGN AND SELECTION, OXFORD JOURNAL, LONDON, GB, vol. 17, no. 9, 1 September 2004 (2004-09-01), pages 681 - 687, XP002331513, ISSN: 1741-0126, DOI: 10.1093/PROTEIN/GZH077 * |
| JEFFREY AUSTIN ET AL: "In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1", AMINO ACIDS ; THE FORUM FOR AMINO ACID AND PROTEIN RESEARCH, SPRINGER-VERLAG, VI, vol. 38, no. 5, 14 August 2009 (2009-08-14), pages 1313 - 1322, XP019805426, ISSN: 1438-2199 * |
| JI-SHEN ZHENG ET AL: "Synthesis of Cyclic Peptides and Cyclic Proteins via Ligation of Peptide Hydrazides", CHEMBIOCHEM, vol. 13, no. 4, 2 February 2012 (2012-02-02), pages 542 - 546, XP055120720, ISSN: 1439-4227, DOI: 10.1002/cbic.201100580 * |
| KAI HILPERT ET AL: "Complete substitional analysis of a sunflower trypsin inhibotor with different serine proteases", JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOCIETY / OXFORD UNIVERSITY PRESS, TOKYO; JP, vol. 138, no. 4, 5 October 2011 (2011-10-05), pages 383 - 390, XP002665036, ISSN: 0021-924X, DOI: 10.1093/JB/MVI140 * |
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