Synthetic peptides for use in vivo in thrombus detection

This invention relates to the development and use of peptides for thrombus detection both in human beings and animals, but primarily, of course, in the detection of human disease. The peptides described herein are also useful in targeting other sites <i>in vivo,</i> e.g. tumours, containing an RGD binding size.

In <nplcit id="ncit0001" npl-type="s"><text>1984 Pierschbacher and Ruoslahti (Nature, 309, 30-33</text></nplcit>), showed evidence that the cell attachment activity of fibronectin could be mimicked by small synthetic peptide fragments. The amino acid sequence responsible for this activity was shown to be Arg-Gly-Asp-Ser (RGDS) and it was demonstrated that synthetic peptides containing this sequence were able to inhibit attachment of NRK cells (cells from a neuroblastoma cell line) to fibronectin coated substrates. The inhibition obtained with RGDS containing peptides was shown to be dose-related, whilst peptides which did not contain the RGDS sequence failed to inhibit cell attachment. The serine residue of the tetrapeptide has shown to be non-essential, although only conservative substitutions may be made in order to retain biological activity.

The RGDS sequence has been shown to occur in fibrinogen, fibronectin and von Willebrand factor. Receptors for these proteins are expressed on the platelet membrane surface following platelet activation. Cross-linking of platelets via these cytoadhesive proteins accounts for the platelet-platelet interactions within a thrombus. It has also been demonstrated that RGDS containing synthetic peptides are capable of inhibiting platelet aggregation <i>in vitro,</i><nplcit id="ncit0002" npl-type="s"><text>Biochem. 1989, 28, 2909-2914</text></nplcit>. This would suggest a specific interaction with the GP IIb/IIIa (glycoprotein fibrinogen receptor) complex present on the platelet membrane surface, which contains the fibrinogen binding domains. Extension of the RGDS sequence, by one amino acid residue at the carboxy and amino terminal, results in a ten-fold reduction in its biological activity, although further extension is not associated with a further reduction in binding capacity. Substitution of the serine residue by phenylalanine results in an anti-aggregatory peptide which is 4 to 5 times more potent than RGDS. There has also been suggestion that the residue corresponding to serine in the RGDS sequence may impart a degree of recognition specificity for different RGDS receptors. This raises the possibility that both specificity and affinity could be modified by substitution around the RGD sequence. RGD binding sites are also known to occur on cell adhesion molecules (CAMs) and some tumours.

The present invention involves a novel approach to <i>in vivo</i> thrombus or tumour detection and which comprises the use of a radioactively labelled peptide comprising the amino acid sequence arginine-glycine-aspartic acid (RGD) in the manufacture of a composition for <i>in vivo</i> imaging or detection of a thrombus or tumour by binding <i>in vivo</i> to RGD binding sites on the thrombus or tumour. In thrombus detection, the composition is useful for intravenous injection into the patient (which term herein includes both humans and animals, unless the context requires otherwise) of a radioactively labelled peptide having therein an RGD (Arg-Gly-Asp)-containing sequence having a specific binding affinity for the platelet GP IIb/IIIa complex, and detecting the presence, if present, of the bound label on the thrombus. Present methods of thrombus detection using labelled antibodies require several hours due to the slow rate of diffusion of the antibody through the system; using labelled peptides in accordance with the present invention is expected to enable thrombus detection in a matter of minutes, thus greatly facilitating diagnosis and treatment, and at a very early stage.

Suitable radioactive labels for use in the construction of radioactively labelled peptides include: Tc^99m, I¹²³ and In¹¹¹, and will be attached to peptide in known manner, for example, via a cystine residue in the peptide. Other suitable techniques are described in <nplcit id="ncit0003" npl-type="s"><text>Science, 220, 613-615</text></nplcit>; <nplcit id="ncit0004" npl-type="s"><text>Int. J. Nucl. Med. Biol., 12, 3-8</text></nplcit>; <nplcit id="ncit0005" npl-type="s"><text>J. Nucl. Med., 27, 27, 685-693</text></nplcit> and <nplcit id="ncit0006" npl-type="s"><text>J. Nucl. Med., 26, 293-299</text></nplcit>.

Subject to the dictates of suitability for parenteral administration and utility, i.e. high affinity and specificity for the GP IIB/IIIA complex, the precise amino acid sequence of the peptide in terms of composition and length will not be particularly critical, although for practical reasons, e.g. economy and ease of synthesis, relatively short chain peptides will be preferred containing, for example, from 3 to 10 amino acid residues.

Suitable peptides containing an RGD sequence, preferably an RGDS or RGDF are available from a variety of different sources, or can be manufactured quite readily using conventional peptide synthesis procedures, and, in particular, using a conventional peptide synthesiser.

For <i>in vivo</i> thrombus detection, a parenterally administrable solution of the radioactively labelled peptide containing an RGD sequence and a parenterally administrable carrier may be employed.

Before proceeding further with the detailed description of this invention, and for the avoidance of doubt, the amino acid sequences referred to herein are identified by either their three letter abbreviations or single letter codes, as follows: <ul id="ul0001" list-style="none" compact="compact"><li>arginine = arg. or R</li><li>aspartic acid = asp. or D</li><li>glycine = gly. or G</li><li>serine = ser. or S</li><li>tyrosine = tyr. or Y</li><li>phenylalanine = phe. or F</li><li>cysteine = cys. or C</li></ul>

Reference is also made hereinafter to the accompanying figure, which is a radiograph taken of a rabbit following intravenous administration of a radioactively labelled peptide according to this invention, and showing the localisation of the peptide in an artificially induced thrombus in the left ear.

Referring to the invention in slightly more detail, studies have been conducted using four peptides (RGDSY, RGDFY, RGDSYC and RGDSCRGDSY) to evaluate their potential as thrombus imaging agents.

The effect of these peptides on ADP-induced platelet aggregation was determined and compared with peptide RGDS which is known to inhibit platelet aggregation. The results (table 1) demonstrate that all four peptides studied are capable of inhibiting platelet aggregation at high concentrations and are virtually equipotent with RGDS. This suggests that inclusion of amino acids into these peptide sequences, to permit radiolabelling, does not destroy their ability to bind platelets (a prerequisite for thrombus imaging applications).

The second study involved radiodination of RGDSY, RGDFY, RGDSYC and RGDSCRGDSY with subsequent analysis of their ability to bind activated platelets in whole blood. The results (Table 2) indicate that all four peptides can bind platelets in ADP stimulated blood and that higher incorporation can be achieved in clotted blood.

One study was performed using RGDSY, labelled with the radioisotope iodine-123, injected into a rabbit who had a preformed thrombus in the microvasculature of the ear. The imaging studies, shown in the accompanying figure demonstrates a rapid uptake onto this thrombus (within 2 minutes of injection), which persisted for the period of study (20 minutes).

These data demonstrate that the four peptides studied are capable of binding to platelets, can be radiolabelled with gamma-emitting isotopes and are incorporated into platelet aggregates in stimulated and clotted blood. This provides good potential for thrombus detection and diagnosis by these peptides <u style="single">in vivo</u> which has been confirmed, in an experimental animal model, using one of the peptides. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="28mm" /><colspec colnum="4" colname="col4" colwidth="28mm" /><colspec colnum="5" colname="col5" colwidth="30mm" /><colspec colnum="6" colname="col6" colwidth="29mm" /><thead><row><entry namest="col1" nameend="col6" align="center" valign="top">inhibition of ADP (1x10^-5M) - induced platelet aggregation by the peptides: RGDS, RGDSY, RGDFY, RGDSYC and RGDSCRGDSY</entry></row><row><entry namest="col1" nameend="col3" align="center" valign="top">(peptide)</entry><entry namest="col4" nameend="col6" align="center" valign="top">percentage inhibition</entry></row></thead><tbody><row><entry>mM</entry><entry>RGDS</entry><entry>RGDSY</entry><entry>RGDFY</entry><entry>RGDSYC</entry><entry>RGDSCRGDSY</entry></row><row rowsep="0"><entry>0.1</entry><entry align="center">40/37</entry><entry align="center">5/13</entry><entry align="center">32</entry><entry align="center">25</entry><entry align="center">17</entry></row><row rowsep="0"><entry>0.2</entry><entry align="center">70/65</entry><entry align="center">10/21</entry><entry align="center">55</entry><entry align="center">-</entry><entry align="center">57</entry></row><row><entry>0.4</entry><entry align="center">86/80</entry><entry align="center">43/68</entry><entry align="center">80</entry><entry align="center">-</entry><entry align="center">79</entry></row></tbody></tgroup></table></tables><tables id="tabl0002" num="0002"><table frame="none"><tgroup cols="5" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="30mm" /><colspec colnum="2" colname="col2" colwidth="34mm" /><colspec colnum="3" colname="col3" colwidth="34mm" /><colspec colnum="4" colname="col4" colwidth="35mm" /><colspec colnum="5" colname="col5" colwidth="35mm" /><thead><row><entry namest="col1" nameend="col5" align="left" valign="top"><u style="single">Table 2</u> Binding of the radiolabelled peptides: RGDSY, RGDFY, RGDSYC and RGDSCRGDSY to ADP stimulated and clotted blood.</entry></row><row><entry namest="col1" nameend="col3" align="center" valign="top">(peptide)</entry><entry namest="col4" nameend="col5" align="center" valign="top">(bound peptide) ng</entry></row><row><entry align="center" valign="top">ng</entry><entry rowsep="1" align="center" valign="top">RGDSY</entry><entry rowsep="1" align="center" valign="top">RGDFY</entry><entry rowsep="1" align="center" valign="top">RGDSYC</entry><entry rowsep="1" align="center" valign="top">RGDSCRGDSY</entry></row><row><entry namest="col1" nameend="col5" align="left" valign="top">ADP Stimulated</entry></row></thead><tbody><row><entry>1</entry><entry align="char" char="." charoff="43">0.05</entry><entry align="char" char="." charoff="43">0.01</entry><entry align="char" char="." charoff="43">0.03</entry><entry align="char" char="." charoff="43">0.01</entry></row><row><entry>10</entry><entry align="char" char="." charoff="43">0.64</entry><entry align="char" char="." charoff="43">1.00</entry><entry align="char" char="." charoff="43">0.94</entry><entry align="char" char="." charoff="43">0.85</entry></row><row><entry>100</entry><entry align="char" char="." charoff="43">9.80</entry><entry align="char" char="." charoff="43">4.46</entry><entry align="char" char="." charoff="43">9.85</entry><entry align="char" char="." charoff="43">9.07</entry></row></tbody></tgroup><tgroup cols="5" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="30mm" /><colspec colnum="2" colname="col2" colwidth="34mm" /><colspec colnum="3" colname="col3" colwidth="34mm" /><colspec colnum="4" colname="col4" colwidth="35mm" /><colspec colnum="5" colname="col5" colwidth="35mm" /><thead><row><entry namest="col1" nameend="col5" align="left" valign="top">clotted blood</entry></row></thead><tbody><row><entry>1</entry><entry align="char" char="." charoff="43">0.27</entry><entry align="char" char="." charoff="43">0.41</entry><entry align="char" char="." charoff="43">0.18</entry><entry align="char" char="." charoff="43">0.28</entry></row><row><entry>10</entry><entry align="char" char="." charoff="43">0.85</entry><entry align="char" char="." charoff="43">2.14</entry><entry align="char" char="." charoff="43">2.26</entry><entry align="char" char="." charoff="43">2.64</entry></row><row><entry>100</entry><entry align="char" char="." charoff="43">17.27</entry><entry align="char" char="." charoff="43">18.12</entry><entry align="char" char="." charoff="43">27.08</entry><entry align="char" char="." charoff="43">29.33</entry></row></tbody></tgroup></table></tables>

The above results demonstrate the applicability of the invention over a range of peptides of different sizes all containing an RGD sequence. The actual length of the peptides is not critical, but for practical purposes the chain lengths may range from 3 to 10 amino acids. preferably 4 to 10 and, as already indicated, either consisting of or comprising an RGDS or RGDF sequence. Many such peptides are already available as known commercial products. Where not so available they can be readily synthesised by known peptide synthesis and/or using known peptide synthesisers. Similarly said peptides can be radioactively labelled by known techniques, for example, by iodination with I¹²³ of a terminal tyrosine (Y) residue incorporated into the oligopeptide.

The detailed preparation of radioactively labelled peptides according to this invention is illustrated by the following example.

Example

Preparation of radioactively labelled (I

1

2

3

) peptides: RGDSY, RGDFY, RGDSYC and RGDSCRDSY

Iodogen tubes were prepared by dissolving Iodogen (1, 3, 4, 6-Tetrachloro3α, 6α-diphenylglycouril) in chloroform at a concentration of 1mg.ml^-1. Aliquots of 50µl(50µg lodogen) were dispensed into polypropylene cryo-tubes and the chloroform evaporated to dryness. These tubes were then stored desiccated at -20°C until required.

Prior to radiolabelling the peptides were dissolved in phosphate buffered saline (PBS) at a concentration of 50µg.ml^-1. RGDSYC and RGDSCRGDSY were first dissolved in a small volume of dimethyl sulphoxide (DMSO) such that the final concentration of DMSO in PBS was 1% v/v.

Iodogen tubes were equilibrated to room temperature before the addition of 200µl peptide solution and 1-10µl of ¹²³l (in aqueous solution). The reaction mixture was then left for 15 min. at room temperature with occasional shaking. Following the incubation period the reaction mixture was removed and passed through a Sephadex G10 column which had been equilibrated with PBS. The column, which separates radiolabelled peptide from free iodine was eluted with PBS and 2ml fractions collected. Radioactivity in the fractions was measured and the eluted peptides, represented by the first radioactive peak from the column, collected and stored at 4°C until required.

The utility of the radioactively labelled peptides in <u style="single">in vivo</u> thrombus detection is illustrated by the following experiment.

Experiment

Intravenous administration of radioactively labelled (I

123

) RGDSY to thrombitic rabbits.

A male New Zealand White rabbit (3kg) was sedated by intramuscular injection of Hypnorm (0.4ml.kg^-1) and then anaesthetised by intravenous injection of Midazolam (2mg.kg^-1),

Two permanent disc magnets were positioned externally in the region of the jugular vein and the rabbit was then injected with 0.2g carbonyl iron microspheres suspended in lml. of contrast media (Omnipaque) via an artery of the left ear. This procedure causes microthrombi in the capillary beds of the ear, whilst iron particles passing through the ear are trapped by the magnetic field and induce thrombus formation in the jugular vein. ¹²³I-RGDSY was injected intravenously into the contralateral ear 60 min after injection of iron. Dynamic imaging by gamma camera was performed using a 1 min frame rate for 20 min with the camera positioned anteriorly to include both ears, head and neck regions in the field of view.

Following intravenous administration of the labelled peptide, the rabbit was radiographed and the resulting radiograph is presented in the accompanying figure. As indicated by the radiograph, there was rapid uptake of the peptide by a thrombus in the jugular vein (arrow 1) and by multiple tiny thrombi in the left ear (arrow 2). The latter, in particular, demonstrates the possible utility of the invention in the detection of small thrombi <u style="single">in vivo</u> and the possibility of early diagnosis and treatment.