Patent Application
20160168210
The present invention relates to derivatives of collagen-binding hairpin peptides, particularly peptides for imaging and drug delivery to fibrosing/fibrotic tissues.
Targeted delivery is an actively sought-after strategy for the control of pharmaceutical action of drugs that are either too toxic for long-term administration or chemically unstable, leading to reduced efficacy. Targeted drug delivery employs the conjugation of a small-molecule drug or a protein therapeutic with a targeting moiety such as a polypeptide, an antibody or a polymer to achieve drug accumulation at sites of pathology (Duncan 2003; Majumdar 2012). The pharmaceutical action is thereby concentrated at local tissues and/or activated (or released) by unique enzymes or changes of the physicochemical environment, e.g. for anti-thrombotic drug delivery (Peter 2003; Topcic 2011) or for anti-cancer therapies (Rooseboom 2004).
The requirement for targeted therapies is the availability of targeting moieties (TM) specific for biomarkers of disease-causing cells and/or pathological tissues. In addition to targeting specificity, the TM-biomarker pair must also accumulate in diseased tissues in sufficient concentrations, which may not be possible to achieve due to the often-observed spatiotemporal variations of biomarkers across different tissue locations. The latest research has therefore placed emphasis on the use of peptide ligands specific for components of the extracellular matrix (ECM), especially those that are either unique or exposed in pathological tissues (Rothenfluh 2008; O'Neil 2009; Peters 2009; Muzzard 2009; Chan 2010). Fibrotic/fibrosing tissues are of particular interest in this regard, since fibroproliferative diseases caused by ECM degradation and fibrotic scarring are the underlining causes of lethality associated with many chronic ailments of the heart, kidney, liver, lungs, joints, the skin or the vasculature in general (Wynn 2004). As such, there has been a constant search for more effective and tissue-specific targeting agents to facilitate disease diagnosis and localized drug delivery (Caravan 2007; Rothenfluh 2008; O'Neil 2009; Peters 2009; Muzzard 2009; Helms 2009; Chan 2010).
Published work up to date (Table 1) has reported short peptides ranging from 5-10 residues as specific binders for abundant components of the extracellular matrix, especially fibrin or collagens (Takagi 1992; Vanhoorelbeke 2003; Rothenfluh 2008; Peters 2009; Helms 2009; Chan 2010; Sawada 2011). Some longer peptides (of 12-13 residues) in cyclized forms have been found as specific binders for the most abundant ECM protein, type-I collagen (Caravan 2007; Muzzard 2009). However, these long peptides need to be modified to include Cys residues at locations suitable for cyclization and affinity enhancement. Generally, practical applications of peptide-based targeting agents are often limited by conformational flexibility and a lack of well-defined secondary and tertiary structures of short linear peptides (see, for example, Collier 2011). Introduction of disulfide-forming Cys residues will, on the other hand, make it more difficult to incorporate the collagen-binding functionality into bioactive proteins, e.g. for collagen-based delivery of cytokines or growth factors (Han 2009, Sun 2009).
WREPSFCALS
WYRGRL
Rothenfluh et al (2008) described a 6-residue peptide, WYRGRL, discovered using a phage-displayed library panned against collagen IIα1, a locally-enriched component of the cartilage matrix. A peptide-nanoparticle conjugate was prepared using an acetylated and Cys-modified peptide Ac-WYRGRLC and thiol-reactive conjugation chemistry. The peptide-polymer nanoparticle, WYRGRL-polypropylene sulphide (or PPS), was shown to target and bind to articular cartilage tissue as promising drug delivery vehicles (Setton 2008).
In another publication, a 6-residue peptide KLWLLPK was reported as a specific binder of collagen IV, which is a main component of the vascular basement membrane (Chan 2010). A peptide-conjugated nanoparticule system, called nanoburrs, was prepared from a modified peptide KLWLLPKGGC using thiol-maleimide conjugation chemistry. This peptide-conjugated nanoparticle was shown to enable spatiotemporal controlled delivery to injured vasculatures.
Phage display was also used to discover peptides that bind to tissue grafts composed of chondroitin sulfate and collagen for drug-delivery applications (Sawada, 2011). Some of these phage-derived peptides contained no cysteine residues nor disulfide bonds and were highly enriched with Trp residues. However, none of these peptides was shown to specifically bind collagen or to other components in the artificial tissue graft used for phage panning.
The possibility of targeting the most abundant extracellular protein, type-I collagen, has been demonstrated in vivo by Caravan et al (2007), by Helms et al (2009) and by Muzzard et al (2009) using animal models of various pathological conditions. In all these studies, it was necessary to employ affinity enhancement strategies such as disulfide-mediated cyclization, as in the cyclic peptide moieties KWHCTTKFPHHYCLY (Caravan 2007) and CPGRVMHG-LHLGDDEGPC (Muzzard 2009) or the multivalent conjugation of the linear peptide HVWMQAPGGGK to synthetic dendrimers (Helms 2009).
Earlier work relied on identifying short peptides that can mimic the binding of von Willebrand factor (vWF) to type-I collagen (Takagi 1992; Depraetere 1998; Vanhoorelbake 2003). Therefore, Takagi et al (Takagi 1992) reported a 10-residue peptide fragment, WREPSFCALS, derived from vWF as having the capacities to bind collagen and inhibit the vWF-collagen interaction. Using phage display, Depraetere et al (Depraetere 1998) isolated two 6-residue cyclized peptides, CVWLWEQC and CVWLWENC, as epitopes potentially mimicking vWF, and inhibiting the interactions of vWF with type-I collagen. In a more recent study, the same research group identified a consensus sequence SPWR potentially mimicking a discrete epitope in vWF in the form of representative cyclic peptides, CMTSPWRC, CRTSPWRC and CYRSPWRC (Vanhorredbeke 2003).
Despite attempts at discovery and design of collagen-binding peptides (Table 1), none of the above collagen-specific compounds is known to bind different states of collagen, especially the monomeric versus the more prevalent polymerized collagen fibrils. Regardless, one striking feature of almost all the above peptide ligands, except that of Caravan et al (2007), is the presence of tryptophan residues as one potential determinant for specific binding to collagen. Another important characteristic is the need for peptide cyclization, mostly in the form of a disulfide bond, which reduces the conformational flexibility of linear peptide fragments and which in certain cases may induce the formation of unique three-dimensional structures required for binding specificity.
From a purely structural point of view, cyclization of linear peptides has been shown to invariably induce the formation of a (β)-turn structure and in some cases well-folded β-hairpin structures emerge from cyclic peptides with certain sequence characteristics (Cochran 2001a; Mirassou 2009). A recent study reported that non-covalent indole-indole interactions, as conferred by a Trp-Trp pair, can largely reproduce the native hairpin structure of a bioactive peptide (Mirassou 2009). Such a β-hairpin structure stabilized by a Trp-Trp pair is also known for a class of linear peptides that contain additional Trp residues, referred to as tryptophan zippers or trpzip peptides (Cochran 2001b, Cochran 2005; Cochran 2007). It is not yet known, however, that any such Trp-rich linear hairpin peptide has a binding capacity for abundant components of the ECM, especially not for collagens.
Trpzip peptides, especially trpzip1, trpzip4, trpzip5 and trpzip6 (Cochran 2001b) were employed as heat-sensitive linkers to enable the control of the inhibitory activities of a new class of bivalent thrombin inhibitors (WO/2012/142696). Trpzip linkers in heat-sensitive bivalent thrombin-inhibitors were found to respond to the presence of type-I collagen, the most abundant extracellular protein particularly enriched in inflamed vascular lesions. It was further disclosed that responsiveness to collagen resides within the trpzip linker segments and that the trpzip peptides alone, also respond to type I collagen. A series of new NMR-based binding data revealed that all the trpzip peptides and bivalent thrombin inhibitors containing these hairpin peptides studied respond to and appear to have specific affinity for only unpolymerized or monomeric collagen abundant in fibrotic/fibrosing tissues undergoing active and uncontrolled remodelling. Most importantly, localization of a representative compound in diseased (fibrotic) tissues was determined through fluorescence imaging using both a rat model of venous thrombosis and a mouse model of pulmonary fibrosis. Such biophysical (NMR) and in vivo data show that compounds bearing a collagen-binding linear hairpin have an affinity for inflamed/fibrosing tissues in vivo, very likely due to specific binding to newly-secreted collagens enriched in these tissue environments.
The present invention relates to derivatives of collagen-binding hairpin peptides, particularly peptides for imaging and drug delivery to fibrosing/fibrotic tissues.
The present invention provides a molecule of Formula (I)
(Y)n-(CBLH)-(Z)m (I)
that specifically binds to collagen. In the molecule as described above, Y is a first compound of interest; Z is a second compound of interest; Y and Z may be different or the same, and n and m are independently 0 or 1. At least one of n and m is 1. Additionally, CBLH is a collagen-binding linear hairpin peptide comprising 19 or fewer amino acids and comprising a turn amino acid sequence. The turn amino acid sequence may comprise 4 to 6 amino acid residues providing a stable turn structure; the turn sequence is flanked on one side by a first flanking sequence comprising SEQ ID NO: 1 and flanked on the other side by a second flanking sequence comprising SEQ ID NO: 2. The W residue at position 1 of SEQ ID NO: 1 may form a cross-strand indole-indole or cation-π interaction pair with the amino acid residue at position 3 of SEQ ID NO: 2 without any disulfide bond.
In the molecule as described above, the amino acid residue at position 2 of SEQ ID NO: 1 may be threonine; additionally, the amino acid residue at position 3 of SEQ ID NO: 1 may be tryptophan or tyrosine.
In the molecule as described herein, the amino acid residue at position 2 of SEQ ID NO: 2 may be threonine; additionally, the amino acid residue at position 3 of SEQ ID NO: 2 may be tryptophan or arginine.
In the molecule of the present invention, the collagen-binding linear hairpin peptide may comprise SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. Alternatively, the collagen-binding linear hairpin peptide may comprise SEQ ID NO: 17 SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.
In the molecule as described herein, Y may be KGG, acetyl, SEQ ID NO: 53, SEQ ID NO: 54, CGG, G, alginate-COOH, Dextran-COOH, or Dextran-NH2 or D-α-tocopheryl polyethylene glycol succinate (TPGS). Furthermore, in the molecule as described above, Z may be GGK, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, GGC, G, Fluor750, alginate-COOH, Dextran-COOH, Dextran-NH2, D-α-tocopheryl polyethylene glycol succinate (TPGS), SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:110, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63.
In specific embodiments of the present invention, the molecule of Formula (I) may comprise SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO:111, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 105 or SEQ ID NO:106.
The present invention further provides a method of delivering a compound of interest to a site of interest, the site of interest containing collagen. In the method, a molecule according to the present invention is provided at the site of interest; the collagen-binding linear hairpin peptide then binds to collagen at the site of interest, thereby delivering the compound of interest to the site of interest. The site of interest may be fibrotic or fibrosing tissue.
The present invention further provides a pharmaceutical composition comprising a molecule described herein and a pharmaceutically acceptable carrier, diluent or excipient.
Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:
The present invention relates to derivatives of collagen-binding hairpin peptides, particularly peptides for imaging and drug delivery to fibrosing/fibrotic tissues.
In one aspect of the invention there is provided a molecule of Formula (I),
(Y)n-(CBLH)-(Z)m (I)
Y is a first compound of interest and Z is a second compound of interest, where Y and Z may be the same or different; n and m are independently 0 or 1 with the proviso that at least one of n and m is 1. CBLH is a collagen-binding linear hairpin peptide.
The collagen-binding linear hairpin (CBLH) peptide in the molecule described herein binds to collagen, particularly to type-I collagen, the most abundant extracellular protein in living tissues; type-I collagen is also over-accumulated in fibrotic organs and in inflamed vascular lesions. More specifically, the CBLH peptides have affinity only for unpolymerized or monomeric collagen abundant in fibrotic/fibrosing tissues undergoing active and uncontrolled remodelling. Therefore, the CBLH peptides lack affinity for well-aligned collagen fibrils in healthy tissues, binding only fibrotic/fibrosing tissues, thereby conferring utility for targeting fibrotic tissues and for imaging and localized drug delivery.
The CBLH peptide may comprise 19 or fewer amino acids and a turn amino acid sequence comprising 4 to 6 amino acid residues providing a stable turn structure. The turn sequence may be flanked on one side by a first flanking sequence comprising SEQ ID NO: 1 and on the other side by a second flanking sequence comprising SEQ ID NO: 2. The W residue at position 1 of SEQ ID NO: 1 forms a cross-strand indole-indole or cation-π interaction pair with the amino acid residue at position 3 of SEQ ID NO: 2 without any disulfide bond. The collagen-binding linear hairpin peptide binds to monomeric or unpolymerized collagen at the site of interest thereby delivering the compound of interest to the site of interest.
The collagen-binding linear hairpin (CBLH) peptide comprises less than 20 amino acid residues that possess an autonomously stable three-dimensional structure free from disulfide bonds. The turn amino acid sequence is any amino acid sequence that comprises 4 to 6 amino acid residues and provides a stable turn structure. Some examples of suitable turn sequences are DDATKT (SEQ ID NO: 3), EpNK (SEQ ID NO: 4), ENGK (SEQ ID NO: 5), EGNK (SEQ ID NO: 6), NGSA (SEQ ID NO: 7), NGTN (SEQ ID NO: 8), NGSTA (SEQ ID NO: 9), NDSN (SEQ ID NO: 10), NNSA (SEQ ID NO: 11), NNST (SEQ ID NO: 12), NGSN (SEQ ID NO: 13) and NPATGK (SEQ ID NO: 14).
The first flanking sequence of the CBLH peptide is SEQ ID NO: 1, which is WX1X2 where: X1 is T, R, H, V, I, L, N, K, A, F, Y or W; and, X2 is W, Y, F or K. The second flanking sequence of the CBLH peptide is SEQ ID NO: 2, which is X1X2 X3 where: X1 is W, Y, F or K; X2 is T, R, H, V, I, L, N, K, A, F, Y or W; and, X3 is W or R. The amino acid residue at position 1 of SEQ ID NO: 1 is tryptophan. The amino acid residue at position 2 of SEQ ID NO: 1 is preferably threonine. The amino acid residue at position 3 of SEQ ID NO: 1 is preferably tryptophan or tyrosine. The amino acid residue at position 1 of SEQ ID NO: 2 is preferably tryptophan. The amino acid residue at position 2 of SEQ ID NO: 2 is preferably threonine. The amino acid residue at position 3 of SEQ ID NO: 2 is tryptophan or arginine. Thus, the first flanking sequence preferably has a WT(W/Y) motif. The second flanking sequence preferably has a WT(W/R) motif. When the amino acid residue at position 3 of SEQ ID NO: 2 is tryptophan, the tryptophan residue at position 1 of SEQ ID NO: 1 forms a cross-strand indole-indole bond with the tryptophan at position 3 of SEQ ID NO: 2. When the amino acid residue at position 3 of SEQ ID NO: 2 is arginine, the tryptophan residue at position 1 of SEQ ID NO: 1 forms a cross-strand cation-π interaction pair with the arginine at position 3 of SEQ ID NO: 2.
Some representative examples of collagen-binding linear hairpin (CBLH) peptides are provided in Table 2.
Molecules useful in the present invention comprise at least one compound of interest (Y and/or Z). In some embodiments, where both n and m are 1, the molecule comprises two compounds of interest, i.e. both Y and Z are present. The compounds of interest (Y and Z) may be the same or different. The compounds of interest are covalently linked to the collagen-binding linear hairpin (CBLH) peptide through chemical conjugation. Compounds of interest may be any compound that can be conjugated to a CBLH peptide using standard linkers and conjugation chemistry. Such compounds may be, for example, active pharmaceutical ingredients (APIs), probes or inactive carriers. Particular examples of compounds of interest include imaging probes (e.g. fluorescent probes, magnetic resonance probes, surface plasmon resonance probes), small-molecule drugs, small peptides (e.g. thrombin binding peptides, pharmacologically active peptides), proteins (e.g. antigens, antibodies), polysaccharides, and/or nanoparticles (e.g. polymeric or metallic). Some representative examples of linkers and compounds of interest (i.e. Y and Z moieties) are provided in Table 3.
In Table 3, VHpro10, VHpro5, VHpro10c and VHpro5c are examples of antibody VHH domains that are ligands specific for human prothrombin. The amino acid sequences of these are as follows:
In the molecule described herein, the compound(s) of interest may be linked to the CBLH peptide using a linker; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the molecule(s), but should not hamper the collagen-binding properties of the molecule. For example, and without wishing to be limiting, the linkermay be selected from G, GGK, GGC, CGG, KGG, GGS, GGGSS (SEQ ID NO:107), GGGGSS (SEQ ID NO:108), and GDFEE (SEQ ID NO:109).
The compound(s) of interest in the molecule of the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection tag (for example, but not limited to c-Myc), a purification tag (for example, but not limited to a His5 or His6), or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670. As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags.
In yet another aspect of the present invention, there is provided a pharmaceutical compositions comprising a molecule of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
Pharmaceutical compositions comprise a molecule of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, a collagen-binding hairpin peptide may be covalently coupled to a carrier, diluent or excipient, in which case one of the compounds of interest in the molecule would be the carrier, diluent or excipient. In another embodiment, the pharmaceutical composition is a vaccine in which collagen-binding hairpin peptide is covalently conjugated to an antigen or other immune-response stimulating agent.
Pharmaceutical compositions may be formulated in a dosage form. Dosage forms include powders, tablets, capsules, softgels, solutions, suspensions, emulsions and other forms that are readily appreciated by one skilled in the art. The compositions may be administered orally, parenterally, intravenously or by any other convenient method. Some pharmaceutically acceptable carriers, diluents or excipients include, for example, antiadherents, binders (e.g. starches, sugars, cellulose, hydroxypropyl cellulose, ethyl cellulose, lactose, xylitol, sorbitol and maltitol), coatings (e.g. cellulose, synthetic polymers, corn protein zein and other polysaccharides), disintegrants (e.g. starch, cellulose, cross-linked polyvinyl pyrrolidone, sodium starch glycolate and sodium carboxymethyl cellulose), fillers/diluents (e.g. water, plant cellulose, dibasic calcium phosphate, vegetable fats and oils, lactose, sucrose, glucose, mannitol, sorbitol and calcium carbonate), flavors and colors, glidants, lubricants (e.g. talc, silica, vegetable stearin, magnesium stearate and stearic acid), preservatives (e.g. vitamin A, vitamin E, vitamin C, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben and propyl paraben), antioxidants, sorbents, sweeteners, and mixtures thereof. Molecules or compositions of the present invention are packaged in a commercial package together with instructions for their use. Such packages are known to one skilled in the art and include, for example, bottles, jars, blister packs, boxes, etc.
Molecules and compositions of the present invention are particularly useful in medical applications for diagnosis and treatment of diseases and other conditions in a subject. The subjects may be human or other animals, especially those with blood circulatory systems, particularly mammals, for example, humans, dogs, cats, horses and rodents (e.g. hamsters, mice and rats). Because the CBLH peptides bind to collagen, the invention is particularly effective for delivering a compound of interest to fibrotic or fibrosing tissue.
Some exemplary applications of the method and molecules of the present invention include the following. Fluorescently-labelled CBLH peptides or those conjugated by other imaging probes can localize in fibrotic/fibrosing tissues for diagnosis of diseases involving fibrosis. CBLH peptides can be developed as universal tags for recombinant production of polypeptides and proteins with specificity to inflamed/fibrosing tissues. Conjugates of nanoparticulate carriers or polymers with CBLH peptides can enable localized delivery of pharmaceutical payloads or compositions to disease-specific tissues. CBLH peptides can also be conjugated directly to peptide-based or polysaccharide antigens and small-molecule drugs to achieve tissue-specific accumulation of vaccines and/or release of active drugs for treating a large number of pathologies and diseases, such as infections, atherosclerosis, cancer, arthritis etc., all with aberrant tissue (ECM) remodeling and fibrosis (Wynn 2004; Caravan 2007; Muzzard 2009).
Molecules of the present invention comprising collagen-binding hairpin peptides are of particular utility for imaging and drug delivery to inflamed mucosal surfaces in the nasal, oral and gastrointestinal (GI) cavities or tracks. For example, conventional strategies of immunization through systemic administration have been found to be sub-optimal for the control of bacterial colonization in the oral cavity, such as dental caries (Koga 2002; Chen 2010). The effectiveness of anti-microbial agents therefore require the development of dentotropic (localized) delivery systems, i.e. targeting agents specific to the dental structures, especially the dentin (Chen 2010). Tissue inflammation on mucosal surfaces, such as in the oral and nasal cavities, is known to lead to local accumulation of collagen (Liu 1990; Switalski 1993; Love 1997; Sciotti 1997; Petersen 2001; Koga 2002; Rivas 2004; Nikawa 2006). Molecules of the present invention (i.e. conjugates of collagen-binding hairpin peptides) are therefore expected to render therapeutic agents or vaccines particularly efficacious when the conjugates are delivered through oral or intranasal administration.
In a particularly interesting application adapted from previous work (Abou Neel 2012), therapeutic collagen preparations based on molecules of the present invention may be prepared whereby unpolymerized or monomeric collagen is complexed or blended with one or more active pharmaceutical ingredients conjugated with collagen-binding hairpin peptides to further increase local delivery to a site of interest, especially into fibrotic/fibrosing tissues. Drug carriers are commonly utilized to improve the bioavailability of orally-delivered drugs, specifically through molecular adhesion to the epithelial surfaces of the body (Peppas 2009). Non-covalent blends of collagen with polymer carriers and/or active pharmaceutical ingredients conjugated with collagen-binding hairpin peptides therefore have the potential to increase the retention of drugs by epithelial tissues undergoing inflammation and active fibrosis.
Thus, the present invention further provides a method of delivering a compound of interest to a site of interest, the site of interest containing collagen. The method may comprise providing a molecule as described herein at the site of interest. The collagen-binding linear hairpin peptide binds to collagen at the site of interest, thereby delivering the compound of interest to the site of interest.
The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
Table 2 shows a list of representative peptides that are selected as collagen binders based on their sequence characteristics. Reported collagen binders (Table 1) share two important structural features: (1) almost all (except for one) contain at least one Trp residue and (2) peptide cyclization (mostly via a Cys/Cys disulfide bond) was employed to reduce the conformational flexibility and potentially to increase binding affinity to collagen. We hypothesize that well-structured peptides abundant in Trp residues may also have the capacity to bind to collagen. In this regard, indole-indole interactions between two strategically-located Trp residues have been shown to stabilize the native hairpin structure of a bioactive peptide in place of a covalent disulfide bond between a pair of Cys residues (Mirassou 2009). This invention therefore focuses on well-structured linear hairpin peptides particularly rich in Trp residues (Santiveri 2010) as ligands of collagen (Table 2), in particular of unpolymerized soluble collagen.
As demonstrated in our previous patent application (WO/2012/142696), four (4) trpzip peptides, i.e. trpzip1, trpzip4, trpzip5 and trpzip6 (Table 2), all responded to collagen in terms of differential perturbations including resonance broadening of the peptide proton NMR spectra in the presence of collagen (see Example 4). On the other hand, the gb1 peptide (Table 4) containing the parent sequence of trpzip4, trpzip5 and trpzip6 (Cochran 2001b) showed no response to collagen under a variety of experimental conditions. The best-structured linear hairpin peptide, trpzip4, showed the most pronounced changes in response to the presence of collagen, indicating that two trpzip motifs, i.e. WTW, located on both strands of the hairpin structure may confer specific binding to collagen (see Example 4) in addition to being the structure-stabilizing force for the linear peptide (Cochran 2001b). Three selection criteria are utilized to define the structural characteristics of collagen-binding (responsive) peptides: (1) being linear; (2) having at least two Trp residues; (3) having a significantly folded hairpin structure. Representative peptides satisfying these three criteria are listed in Table 2. All peptides have a WT1W1 motif in the N-terminal region followed by a turn (loop) sequence and in the C-terminal region a second W2T2(W/R) motif potentially stabilizing a well-structured hairpin fold. Therefore, two WTW motifs are found in trpzip1, trpzip2, trpzip3, trpzip4, HP5W4 and in the V4 loop peptide of the HIV envelop protein gp120 (Q77430-9H1V1) (Table 2). Trpzip6 has the WTW motif on the first strand of a putative hairpin structure, while the second strand has the KTW variant of the W2T2(W/R) motif. The trpzip5 peptide has the WTY motif in the N-terminal region and a FTW variant in the C-terminal strand. In trpzip7, trpzip8 and trpzip9, the WTW motifs are substituted by WVW and WHW, respectively. In hairpin sequences of gp120 variants (Table 2), some WTW motifs are substituted by WTR, which may provide stabilization for the hairpin structure through strong cation-π interactions (Santiveri 2010; Chen 2005). A putative protein predicted from the genome sequence of the infectious agent Mycobacterium tuberculosis also contains a hairpin-like sequence rich in Trp, TWRWNGSTAWTWS (Table 2) with the WRW sequence as the first WT1W1 motif. It is important to note that a single WLW motif is already found in a collagen-binding ligand mimicking the vWF protein (Depraetere 1998), i.e. in two peptides called the Q-peptide and the N-peptide (Table 1).
Rat collagen hydrogels were prepared using rat-tail collagen type I (at 4 mg/ml in 0.02 N acetic acid) from BD Bioscience. A volume of 200 μL of the concentrated collagen solution was mixed with an equal volume of a buffer solution that was 400 mM in Tris-HCl and 400 mM in NaCl with a pH of 7.6 and with 50 μL of deuterated water (D2O). The sample mixture was transferred to an NMR tube followed by gentle mixing under agitation using a Thermolyne™ Max Mix-II apparatus. The NMR tube containing the collagen solution was placed within the RF probe housed in a 500 MHz super-conducting magnet (Bruker Avance-500 NMR spectrometer). The probe and sample bulk temperature was kept at 277 K for 3 hours and then elevated to 310 K at a rate of 1 degree/10 min to enable fibril alignment during a slow process of collagen gelation under the influence of the magnetic field (Ma 2008). The degree of fibril alignment of the collagen hydrogels was determined by use of deuterium NMR spectroscopy of the added D2O as described (Ma 2008).
Human collagen hydrogels were prepared using either human placenta collagen type I from BD Bioscience (at 2.23 mg/ml in 2 mM HCl) or the VitroCol™ preparation of human collagen from Advanced BioMatrix (at 2.9 mg/ml in 0.01 N HCl). A volume of 400 μL of the concentrated collagen solution was mixed with 50 μL of the solution of 10×PBS (phosphate-buffered saline) supplemented by Na2PO4 at 500 mM and pH 7.4 and with 50 μL of deuterated water (D2O). The sample mixture was transferred to an NMR tube followed by gentle mixing before being subjected to the same gelation process as described above.
Randomly-deposited collagen (hydrogel) matrix was prepared following the same procedure as above for partially aligned hydrogels, except that the RF probe and the NMR tube (or test tubes) containing the collagen solution were placed outside the magnetic field. Peptides containing collagen-binding hairpins were introduced into the hydrogels in two ways, the first with the collagen stock solution diluted (50:50 in volume ratio) by the buffer of 400 mM in Tris-HCl and 400 mM in NaCl at pH 7.6 containing the peptides of interest (i.e. for
The autonomous or modular nature of collagen-binding linear hairpin (CBLH) peptides was demonstrated by the NMR data for thrombin inhibitors harboring the sequences of these CBLH peptide, in particular MH2-wZIP4, MH2-wZIP5 and MH2-wZIP6 (WO/2012/142696). MH2-wZIP4 exhibited significantly altered properties and/or a differential entrapment in the presence of collagen or collagen hydrogels (
Deuterium NMR spectroscopy studies of collagen responsiveness (and/or binding) for each peptide were carried out using a volume of (400-X) μL of a peptide dissolved in pure water mixed with 50 μL of a 10×PBS (phosphate-buffered saline) supplemented by sodium phosphate to a final concentration of 50 mM, with 50 μL of deuterated water (D2O) to form the reference sample (where X=0), or the peptide-collagen complex with X μL of rat-tail collagen type I (at 3.6 to 4 mg/ml in 0.02 N acetic acid from BD Bioscience) adjusted to achieve the desired final concentration of collagen. The pH value of the reference peptide samples was adjusted to match the pH of the final peptide-collagen complex in order to facilitate NMR spectral comparison. The peptide-collagen solutions were also subjected to gelation under magnetic field guidance followed by the measurement of fibril alignment (Ma 2008) as a semi-quantitative indication of peptide-collagen binding. Deuterium coupling constants measuring the degree of collagen fibril alignment were reduced by more than 1.0 Hz for MH2-wZIP4, by less than 0.5 Hz for MH2-wZIP6 and MH2-wZIP5, which parallel those observed for trpzip4, trpzip6, trpzip5 and the gb1 peptide (see Example 4). As a comparison, a solution of rat collagen at a concentration of 1.8 mg/ml in 200 mM Tris-Cl/200 mM NaCl at pH 7.6 formed a partially-aligned hydrogel with a deuterium coupling constant (Ma, 2008) of 2.5 Hz, while a solution of rat collagen at a concentration of 3 mg/ml in PBS (137 mM NaCl/2.68 mM KCl/10.1 mM Na2HPO4/1.76 mM KH2PO4) supplemented with Na2HPO4 to achieve a final concentration of 50 mM at pH 7.4 formed a partially-aligned hydrogel with a deuterium coupling constant of 4.8 Hz. A solution of human collagen at a concentration of 2.23 mg/ml in PBS supplemented with Na2HPO4 with a final concentration of 50 mM at pH 7.4 formed a partially-aligned hydrogel with a deuterium coupling constant of 1.3 Hz.
The MH2-wZIP4 molecule exhibited a greatly-reduced entrapment when diffused into preparations of collagen hydrogels that are pre-aligned to mimic the collagen matrix in healthy tissues (WO/2012/142696). Looking more closely at
NMR experiments and data shown in
The methods of this invention are illustrated by a sequence dissection of MH2-wZIP4 in relation to the collagen-binding properties of the MH2-wZIP series of peptides containing the Trp-rich hairpin sequences, especially that of trpzip4. Therefore, an N-terminal fragment IRFTDGEWTWDDA of MH2-wZIP4, or MH2-wZIP4-F1 (Table 4) does not respond to collagen binding, as its proton NMR spectrum remains essentially the same free (dotted spectrum of
Table 4 lists some Trp-rich peptides that do not bind collagen are therefore not useful in the present invention.
Table 5 lists some representative molecules of the present invention containing collagen-binding hairpin motifs.
The utility of molecules of the present invention is further illustrated by the following polypeptide from Table 5: IRFTD5GEWTW10DDATK15TWTWT20EGSFN25PRGS (MH2-wZIP4-F4—SEQ NO: 82) with the DFEEIPEEYL (SEQ ID NO: 92) moiety replaced by recognition site. Ser-Phe-Asn-Pro-Arq (or SFNPR (SEQ ID NO: 93)) for thrombin-specific cleavage of peptide substrates, as established in previous studies (Ni 1995). The use of a thrombin-specific sequence such as FNPR (SEQ ID NO: 94) will enable the release of the GS moiety mimicking a drug conjugated to the rest of the peptide at tissue sites with elevated thrombin activity, e.g. within inflamed joints (Gabriela 2009) and under other inflammatory conditions (Morris 1994; Bogatkevich 2011).
The collagen-binding characteristics of MH2-wZIP4-F4 were established using proton NMR spectroscopy (
Collagen-binding (or responsiveness) of the trpzip4 motif is also essentially independent of the flanking sequences, as an N-terminally extended analog of MH2-wZIP4 or FQPRPRFTDGEWTWDDATKTWTWTEGDFEEIPEEYLQ (SEQ ID NO: 90—from WO/2012/142696) exhibits a similar pattern of NMR signal perturbations (
Proton NMR spectroscopy is used to illustrate the folding behavior of the peptides GEWTYDDATKTFTVTE (gb1—SEQ ID NO: 70), GEWTWDDATKTWTVTE (trpzip6—SEQ ID NO: 15), GEWTYDDATKTFTWTE (trpzip5—SEQ ID NO: 16) and GEWTWDDATKTWTWTE (trpzip4—SEQ ID NO: 17) in aqueous solutions. The peptides were synthesized mostly in aminated (R—NH2) forms and some as C-terminal carboxylate (R—COOH) using standard Fmoc chemistry and purified by use of reverse-phase HPLC. Their identity was confirmed by mass-spectroscopy and NMR spectroscopy. The proton NMR spectra of trpzip4-NH2, trpzip5-NH2, trpzip6-NH2, and gb1-NH2 (
Peptide GEWTYDDATKTFTVTE (gb1—SEQ ID NO: 70) also has little binding capacity nor change of its conformation in the collagen hydrogel (
In the short (12 aa) trpzip peptide (such as trpzip2, Table 2), a well-defined hairpin structure is maintained even after two of the (inner) Trp residues are replaced by Tyr, as in the WYYW-trpzip2 peptide, SWTYENGKYTWK (SEQ ID NO: 23—Wu 2010). Titration with collagen (
The putative hairpin sequence, TWRWNGSTAWTWS (i.e. SEQ ID NO: 28—pp-D7EUT8, Table 2) based on a predicted protein in Mycobacterium tuberculosis does not appear to fold into a stable hairpin structure since its proton NMR spectrum is somewhat broad (spectrum not shown). However, some proton NMR perturbations are observed for pp-D7EUT8 after titration with collagen (data not shown). Such less stable hairpin folding is likely due in part to the non-optimal 5-residue turn sequence NGSTA, as compared to the four-residue ones in the V4 hairpin loops of HIV proteins gp120 and gp160 (Table 2). Further stabilization of the hairpin structure may require the addition of N- and C-terminal extensions based on the predicted protein sequence, i.e. from TWRWNGSTAWTWS (SEQ ID NO: 28) to STWRWNGSTAWTWSTAS (SEQ ID NO: 29—Table 2). Such sequence extensions will also be required for hairpin stabilization of the peptides derived from the V4 loops of other variants of the HIV1 envelope protein gp120, i.e. v4-gp120v2, pp-Q77413, pp-Q77425 and pp-Q904S5 (Table 2). In this regard, the two extended and derivatized peptides, acetyl-STWTWNGSAWTWNEGGK (SEQ ID NO: 76—Table 5) and acetyl-STWTWNGTNWTRNDGGK (SEQ ID NO: 77—Table 5) were shown to bind to type-I collagen by use of proton NMR spectroscopy (data not shown), similarly to other CBLH peptides (
The collagen-binding capacity of Trp-rich hairpin peptides was further illustrated through a recombinant protein containing MH2-wZIP4-F6/BRI-TP304 with a sequence of IRFTD5GEWTW10DDATK15TWTWT20EG (SEQ ID NO: 84—Table 5) at the N-terminal region:
Here, VHpro10 is an antibody VHH fragment in the form of a prothrombin-specific ligand. The collagen-binding properties of BRI-TP404 or WZ4-GGS-VHpro10 (Table 5) are characterized by use of NMR spectroscopy similarly to what was carried out with MH2-wZIP4 (
The binding specificity of BRI-TP404 was further determined through the dissolution of the collagen (gel) matrix using enzymes that cleave and convert the triple-helical collagen into small peptides.
Similarly to the CBLH-conjugated protein BRI-TP404, our previous patent publication (Ni 2012) demonstrated the collagen-binding capacity of another antibody VH10 protein conjugated to the peptide IRFTDGEWTWDDATKTWTWTEG (SEQ ID NO: 84) at its N-terminus to form BRI-T404 (SEQ ID NO: 91) (Table 5). The relevant NMR data are reproduced here as
In the absence of cysteine (Cys) residues (or disulfide bonds), collagen-binding hairpin peptides can accommodate the addition of a Cys residue as either an N-terminal or a C-terminal extension. An example is MH2-wZIP4-F5 (Table 5), which contains the collagen binding trpzip4 with an N-terminal extension of IRFTD and with a Cys residue added to the C-terminus through a two-residue (GG) connecting sequence. The corresponding peptide, P4247, was synthesized using standard Fmoc chemistry and purified by use of reverse-phase HPLC. Its identity was confirmed by mass-spectroscopy and NMR spectroscopy. This Cys-containing peptide, i.e. P4247, can be coupled to a fluorescent probe, a small-molecule drug or a polymer support following the well-established thiol-maleimide conjugation chemistry (Ghosh 1990; Brinkley 1992). Therefore, a fluorescent dye such as the HiLyte Fluor-750 C2 maleimide (AnaSpec, California) or DyLight(DL) Fluor-755 maleimide (Thermo Scientific) was first prepared at a concentration of ˜4 μM in 2 mL of degassed ˜0.2 M HEPES, 1 mM EDTA, pH 7.7. To this solution of the fluorescent probe was added ˜0.2 mL of a 0.2 mM solution of the thiolated peptide P4247 (˜40 nmol) in 25 mM sodium borate, pH 9. The reaction mixture was kept under an argon atmosphere and the conjugation was allowed to proceed for 16 h at 23° C. Depending on the nature of the collagen-binding peptide, the fluorescent probe containing the maleimide functional group can also be in molar excess (5-10 fold) as compared to that of the Cys-modified peptide, following essentially the experimental procedure as reported previously (Ghosh 1990; Mushero 2011). The peptide-probe conjugate was separated and purified by use of HPLC and identified by use of mass spectrometry and NMR spectroscopy. The purified material was dried by lyophilization and reconstituted in pure water at a concentration between 0.2 mg/mL and 1 mg/mL before use.
Carboxyl- and amine-terminated solid supports are available commercially and can be activated for coupling to the free —SH group of a Cys-modified CBLH peptide. For example, an NH2-terminated dextran coating can be modified with the N-succinimidyl 3-(2-pyridyldithio)-propionate reagent (SPDP). The SPDP reagent adds disulfide-containing linkages that can be cleaved with reducing agents for final conjugation to sulfhydryl-containing peptides through a disulfide bond. On the other hand, the carboxy-terminated dextran coating can be modified through maleimide coupling with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC) to create a non-labile thioether bond between the peptide and the solid support.
Polymers conjugated to collagen-binding hairpin peptides can generally be synthesized through a selective amine coupling procedure employing water-soluble carbodiimide chemistry (Lee 2008; Lee 2012). Here, collagen-binding peptides are first extended at the N-terminus, e.g. via a four-residue (SEC) ID NO: 96) or G4 linker sequence or alternatively to include the five-residue sequence IRFTD (SEQ ID NO: 53) as the linker as in the case for BRI-TP304 or MH2-wZIP4-F6 (Table 5). Solutions of such modified collagen-binding peptides are mixed with or loaded onto a polymer containing free carboxylic groups, one example of which is the anionic GRAS (Generally-Regarded As Safe) polysaccharide alginate (Lee 2012) or carboxylated dextran matrices. Other examples for conjugation with collagen-binding peptides are lipid micelles decorated with free carboxylic groups, such as the vitamin E derivative D-α-tocopheryl polyethylene glycol succinate (TPGS) (Zhang 2012) or a structurally-related analog D-α-tocopheryl polyethylene glycol sebacate (Lipshutz 2008). To hydrated polymers or lipid micelles is added a freshly mixed solution containing N-ethyl-N′-(3-diethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) with a controlled reaction times for potentially selective coupling (Lee 2008) to the free N-terminal amines of the modified collagen-binding peptide. Alternatively, insoluble polysaccharide polymers can be activated by oxidation with periodate (Sanderson 1971) to produce reactive aldehydes for amine-reactive coupling as utilized for the synthesis of peptide-conjugated polysaccharide vaccines (Lett 1995; Lett 1994) and for the fabrication of biocompatible soft tissue adhesives (Mo 2000; Wang 2007; Mandevi 2008). In this regard, the collagen-binding v4-gp120 and v4-gb160 peptides (Table 2) are of particular utility since the absence of Lys residues enables selective coupling via an added Lys to N-terminally-acetylated peptides. In other words, both v4-gp120 and v4-gp160 can be acetylated at their N-termini and extended at their C-termini to contain an added Lys residue, resulting in the modified CBLH peptides, acetyl-STWTWNGSAWTWNEGGK (SEQ ID NO: 76—Table 5) and acetyl-STWTWNGTNWTRNDGGK (SEQ ID NO: 77—Table 5), which can be used for selective conjugation with GRAS polysaccharides or caboxylated drug delivery vesicles. Such GRAS substances conjugated with collagen-binding hairpin peptides, or CBLH-GRASPs in short, can be used as tissue-specific vehicles for localized drug delivery to fibrotic/fibrosing tissues as previously demonstrated for other collagen-binding peptides (Rothenfluh 2008; Setton 2008; Chan 2010) and for naturally-occurring polysaccharides derivatized with cell-specific binding capacities (Lee 2012). We further envision the fabrication of blends of CBLH-GRASPs with unpolymerized collagen, which may increase the retention and localization of drug delivery vehicles through a “seemless” integration with native collagen accumulated at sites of tissue inflammation and fibrosis.
Collagen-binding peptides can also be conjugated to magnetic nanoparticles through covalent chemistry. Two types of dextran-coated magnetic nanoparticles are used for covalent conjugation, one MNP-CO2H with free carboxylates and the second MNP-NH2 with free amines. 10 mg (300 nmols) (at 10 mg/ml) of MNP-CO2H is activated for 15 minutes by addition of EDC at 0.6 mg (3 μmols)/60 μl H2O and sulfo-NHS at 1.73 mg (15 μmols)/200 μl H2O before addition of the CBLH peptide at 1.5 μmol/150 μl of 25% CH3CN in H2O. The reaction is allowed for 2 hours before the MNP-peptide product is concentrated. 10 mg (300 nmols) (at 10 mg/ml) of MNP-NH2 is activated for 30 minutes by addition of SM(PEG)4 at 4 μl (250 mM)(1 μmol) before addition of the CBLH peptide with a free thiol group at 1 μmol/100 μl in 25% of CH3CN in H2O. The reaction is allowed for 30 minutes before the MNP-peptide product is concentrated.
Collagen-binding hairpin peptides can be used to create hydrogel formulations of drugs, peptides or therapeutic proteins for localization of their pharmaceutical action, such as and especially for tissue engineering applications (Han 2009; Sun 2009). In this regard, collagen-based hydrogels are re-emerging as biomaterials in regenerative medicine due to the abundance of collagen in the animal kingdom and the low antigenicity, biocompatibility and biodegradability of collagen-based biomimetic scaffolds (Abou Neel 2012). Of particular importance is our experimental finding (
Peptide P4247 conjugated to a fluorescent probe, i.e. FL4247 (Table 5), was therefore used together with rat-tail collagen (type I) to assess the binding capacity of CBLH peptides to collagen and to illustrate the potential applications of collagen hydrogels formulated with compounds of this invention. The fluorescent probe, HiLyte Fluor750 (AnaSpec, California) was selected with a maximum absorption at an optical wavelength of 750 nm and with a maximum fluorescence emission at 800 nm. Therefore, the intensity of fluorescence emissions would also be optimal for in vivo imaging of tissue locations with a differential enrichment of the injected fluorescent compound (see Examples 8-10). Here, small disks of collagen hydrogels were formed in a petri dish following the established protocols for making randomly-deposited collagen hydrogels (see Example 2). For this first experiment, FL4247 in a hydrogel solution were loaded onto the pre-formed collagen hydrogels and molecular diffusion off FL4247 was followed by fluorescence imaging (
Immune-compromised nude mice (20-30 g) and normal Sprague-Dawley rats (250-310 g; Charles River Laboratories, St-Constant, QC, Canada) were acclimated for at least 3 days prior to the start of the study. Animals were housed in microisolator cages and were kept on a 12-hr light/dark cycle with constant temperature and humidity. Food and water were provided ad libitum. Stock solutions of various agents and peptides of this invention were diluted in saline (0.9% sodium chloride) prior to use. Saline formulations of FL4247 were prepared in such a way that similar dose levels were achieved based on the body weight of the experimental animals used for the study (i.e. in mg/kg).
Of the different methods available, imaging with fluorescently-labeled test compounds is particularly useful for assessing the in vivo biodistribution in real time with live animals. Therefore, the biodistribution of FL4247 was followed over time in two nude mice each for two routes of administration: 1) intravenous (I.V.); and 2) intraperitoneal (I.P.). Another imaging experiment was carried out using a Sprague-Darley rat (Charles River Laboratories, St-Constant, QC, Canada) through a tail-vein (I.V.) administration of an FL4247 solution.
The I.V. injection route depicted similar results in both studies even though the imaging sequence parameters were different (study 1=1 image/2 minutes over 30 minutes; study 2=1 image/5 minutes over 60 minutes). Immediately following injection, FL4247 accumulated rapidly in the head region followed by a slow decrease of fluorescent intensity over time (
Similar results were obtained in two studies involving the I.P. route of injection. Different imaging parameters were again used (study 1=1 image/2 minutes over 60 minutes; study 2=1 image/5 minutes over 90 minutes). The I.P. injection of FL4247 led to the accumulation of the compound at the level of the head within 5 minutes. However, the total accumulation of the fluorescent compound was far inferior over time compared to the I.V. injection. The distribution of FL4247 over the entire body is complete within 30 minutes accompanied by a slow accumulation of fluorescence in the bladder region. Besides the bladder, no specific local accumulation of FL4247 was noted in both studies.
The biodistribution was also assessed using one rat in which FL4247 was injected intravenously (I.V.) through the tail vein. To illustrate the capacity for image contrast, only 10% (one tenth or 1/10) of FL4247 was used with the normal rat (
Accumulation of morphologically-diverse collagen structures is a hallmark of atherosclerotic inflammation and unstable atherosclerotic plaques (Sukhova 1999; Penz 2005; Adiguzel 2009; Reininger 2010) as well as fibrotic diseases in general (Wynn 2004). Such de-structurization of aligned tissues is also mimicked by some animal models of vascular injury induced by FeCl3 application to blood vessels (Eckly 2011). Trp-rich hairpin peptides are shown here to respond to unpolymerized type-I collagen and to interfere with the formation of hydrogels by type-I collagen, which mimic the physicochemical environment of normal and inflamed tissues (Houdijk 1985; Eckly 2011; Torbet 2007). The application of ferric chloride on venous and arterial vessels was therefore the method used to produce the inflamed vasculature in the animal model. The carotid artery (arterial model) and the abdominal vena cava (venous model) were the chosen vessels for the imaging experiments with injured blood vessels undergong thrombosis.
The FeCl3 rat venous model of thrombosis was generated as described (Wang 2005) with some modifications. Briefly, rats were anesthetized with a 2.5% isoflurane/oxygen mixture and placed on a heat source (35-37° C.). The vena cava was then exposed via a midline incision and the region between the renal and iliolumbar veins was isolated. One minute after drug administration, a piece of filter paper (Gel Blot Paper, GB003, Whatman, Piscataway, N.J., USA; 7 mm diameter) saturated with 10% FeCl3 (EMD Chemicals Inc., Gibbstown, N.J., USA) was placed on the exposed surface of the vena cava and incubated for 3.5 minutes. During the application of FeCl3, the abdominal region was covered with aluminum foil. At the end of the incubation period, the filter paper was removed and the exposed viscera covered with a saline-soaked gauze.
In a first experiment, FL4247 was injected immediately after ferric chloride application with the imaging session started right after. Whole-body (rat) imaging data did not reveal any fluorescence at the site of vascular injury. However side-by-side ex vivo imaging of the injured abdominal vena cava and the thrombus produced by the vessel injury showed fluorescence retention by the injured vascular wall. In another experiment, a waiting time of 30 minutes was observed between the end of the ferric chloride application and the I.V. injection of FL4247 through the tail vein. In this experiment, whole-body imaging showed the presence of a strong fluorescence signal (
In order to clarify the specificity of FL4247 for tissue localization, two rats with venous thrombosis were imaged following I.V. injection through the tail vein of the unreacted dye purified from the preparation of the fluorescently-labelled compounds (e.g. FL4247). In these imaging experiments, there were also a 30-minute waiting time after introduction of FeCl3 to the exposed vena cava to ensure the development of substantial vascular injury and the deposition of a thrombus. In contrast to what was observed with FL4247 (
In summary, fluorescent intensities were retained by the injured blood vessel for rats with venous thrombosis (induced by application of 10% FeCl3 on the vena cava blood vessel), even at the one-hour time point when most injected FL4247 has cleared through the kidney to the bladder into the urine. Retention of FL4247 was substantial even with a five-fold reduction of the dose of the injected FL4247. In addition, a more elaborated vessel injury along with a blood clot (e.g. that evolved for 30 minutes after application of FeCl3) retained a higher level of FL4247 fluorescence as compared to FL4247 injection immediately before FeCl3 application. The observed fluorescence retention with FL4247 appears to be a result of the specific targeting of the CBLH peptide in FL4247, as the dye alone or a conjugate of the dye with a non-functional peptide GEWTYDDATKTFTVTEGGC based on the gb1 sequence (SEQ ID NO: 70) did not accumulate to significant levels in the injured vein.
Tissue localization of the representative compound FL4247 containing a collagen-binding hairpin peptide was further examined using a bleomycin-induced mouse model of pulmonary fibrosis. After injection through the tail vein, a high degree of FL4247 fluorescence was retained in the area of the lung for fibrotic mice. This is accompanied by fluorescence localization into generally inflamed tissues in the oral cavities of these mice, which are induced through either mechanical or contact damages. It can therefore be concluded that FL4247 has an affinity for inflamed/fibrosing tissues in vivo, very likely due to specific binding of the collagen-responsive hairpin moiety to newly-secreted collagen enriched in these tissue environments.
Specifically, pulmonary instillation of bleomysin was used to initiate pulmonary fibrosis in a mouse model using immune-compromised nude mice. The objective was to determine if the fluorescent FL4247 compound would localize in fibrotic tissues of the lung induced by bleomycin. Three mice with pulmonary fibrosis received FL4247 intravenously and one intraperitonealy at a dose level twice those used for biodistribution studies with mice (
All patents, patent applications and publications referred to herein and throughout the application are hereby incorporated by reference.
Ni F, Zhu Y, Scheraga H A. (1995) Thrombin-bound Structures of Designed Analogs of Human Fibrinopeptide A Determined by Quantitative Transferred NOE Spectroscopy: A New Structural Basis for Thrombin Specificity. J. Mol. Biol. (1995) 252, 656-671.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2013/000894 | 10/18/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61716093 | Oct 2012 | US |
