Abstract:
The invention relates to radiolabeled imaging of a mammalian body. The invention in particular provides for reagents labeled with technetium-99m for such imaging. The invention provides peptides which bind technetium-99m and which can be targeted to specific sites within a mammalian body.

Description:
This application is a continuation of application Ser. No. 08/264,176, filed Jun. 22, 1994, which was a divisional application Ser. No. 07/653,012, filed Feb. 8, 1991 both now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to radiodiagnostic reagents and, more particularly, to polypeptides useful for producing technetium (Tc-99m) labeled radiodiagnostic agents. The invention relates to Tc-99m labeled reagents, kits for making such reagents, and methods for using such reagents. 
     2. Description of the Prior Art 
     U.S. Pat. No. 4,861,869 (Nicolotti) describes coupling agents of the formula: ##STR1## wherein R 2  and R 3  are the same or different and each represents a radical selected from the group consisting of alkyls having from 1 to 6 carbon atoms, aryls having from 6 to 8 carbon atoms and aklaryls having 7 to 9 carbon atoms, any of which can be substituted with one or more hydroxyl, alkoxy, carboxy or sulfonate groups; n is either 1 or 2; and X is an activating group capable of forming an amide bond with an alpha or beta amino group of a biologically useful protein or polypeptide molecule. 
     U.S. Pat. No. 4,861,869 also describes compounds such as S-benzoylmercaptoacetylglyclglyclglycine. 
     The coupling agents are bound to large peptides such as antibodies or fragments thereof and complexed to Tc-99m. 
     U.S. Pat. Nos. 4,571,430, 4,575,556 and 4,434,151 (Byrne et al.) describe compounds of the formula: ##STR2## wherein R is hydrogen or lower alkyl, R 1  and R 2  are individually hydrogen or lower alkyl or taken together form oxo; R 3  is an amino protecting group where R 1  and R 2  taken together form oxo; R 4  is hydrogen or lower alkyl; R 5  is hydrogen or a thiol protecting group; and y and z are integers from 0 to 2; which are bifunctional chelating agents and as such can couple radionuclides to terminal amino-containing compounds capable of localizing in an organ or tissue which is desired to be imaged. 
     Bryson et al., Inorg. Chem. 27:2154-2161 (1988) and Inorg. Chem. 29:2948-2951 (1990), describes thiolate ligands for complexing with technetium of the formula: ##STR3## 
     European Patent Application No. 86100360.6, filed Jan. 13, 1986, describes dithio, diamino, or diamidocarboxylic acids or amine complexes useful for making technetium imaging agents. 
     Other references of interest include Khaw et al., J. Nucl. Med. 23:1011 (1982); Rhodes, B. A., Sem. Nucl. Med. 4:281 (1974); Davidson et al., Inorg. Chem. 20:1629 (1981); and Byrne and Tolman, J. Nucl. Med. 24:126 (1983). See particularly Fritzberg et al., J. Nucl. Med. 23:592 (1982); Fritzberg et al., ibid. 23:17 (1982), for descriptions of mercaptoacetyl derivatives of ethylene diamine carboxylic acid derivates. See also U.S. Pat. Nos. 4,434,151, 4,444,690 and 4,472,509. 
     European Patent Application 88104755.9 describes various S-protected mercaptoacetylglycylglycine chelating groups bound to large proteins such as antibodies. 
     European Patent Application 84109831.2 describes technetium complexes of compounds of the formula I and II: ##STR4## wherein R and R 6  are each selected from hydrogen, substituted or unsubstituted lower alkyl or --COR where R 9  is selected from hydroxy, substituted or unsubstituted lower alkoxy, substituted or unsubstituted amino, glycine ester, or an activated leaving group; R 1  is selected from hydrogen, or substituted or unsubstituted lower alkyl; R 2  and R 3  are each selected from hydrogen or a thiol protecting group; and R 4 , R 5 , R 7 , and R 8  are each selected from hydrogen or lower alkyl; and salts thereof. These complexes were used primarily as renal function monitoring agents. 
     Arginylglycylaspartate (Arg--Gly--Asp or RGD) and derivative peptides are known to bind to blood clots (see U.S. Pat. Nos. 4,792,525, 4,857,508 and 4,578,079) and RGD derivatives have been labeled with technetium as imaging agents, Journal of Nuclear Medicine 31, pp. 757, No. 209 (1990). 
     SUMMARY OF THE INVENTION 
     The invention encompasses polypeptides for labeling with technetium-99m and imaging target sites within a mammalian body comprising (a) a specific binding polypeptide region which specifically binds to the target site to be imaged, and (b) a technetium binding region of the formula Cp(aa)Cp wherein Cp is a protected cysteine and (aa) is an amino acid and wherein the technetium binding region is covalently bound to the specific binding polypeptide region. The invention includes technetium-99m complexes and methods for using the technetium-99m complexes to image target sites within a mammalian body. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The Cp(aa)Cp technetium binding group is covalently linked to the specific binding polypeptide preferably by one or more amino acids, most preferably glycine. Alternatively, the Cp(aa)Cp technetium binding group may be directly covalently linked to the specific binding polypeptide or other covalent linking groups can be used such as bifunctional amino/carboxy compounds which are not naturally-occurring amino acids. 
     Representative specific binding polypeptide sequences are: 
     Artherosclerotic Plaque Binding Peptides 
     YRALVDTLK (SEQ ID NO: 1) 
     RALVDTLK (SEQ ID NO: 2) 
     RALVDTLKFVTQAEGAK (SEQ ID NO: 3) 
     YAKFRETLEDTRDRMY (SEQ ID NO: 4) 
     AKFRETLEDTRDRMY (SEQ ID NO: 5) 
     YAALDLNAVANKIADFEL (SEQ ID NO: 6) 
     AALDLNAVANKIADFEL (SEQ ID NO: 7) 
     YRALVDTLKFVTEQAKGA (SEQ ID NO: 8) 
     RALVDTLKFVTEQAKGA (SEQ ID NO: 9) 
     YRALVDTEFKVKQEAGAK (SEQ ID NO: 10) 
     RALVDTEFKVKQEAGAK (SEQ ID NO: 11) 
     YRALVDTLKFVTQAEGAK (SEQ ID NO: 12) 
     Peptides Targeted to Infections and Atherosclerotic Plaque 
     VGVAPGVGVAPGVGVAPG (SEQ ID NO. 13) 
     VPGVGVPGVGVPGVGVPGVG (SEQ ID NO. 14) 
     formyl.Nleu.LF.Nleu.YK (SEQ ID NO. 15) 
     formyl MIFL (SEQ ID NO. 16) 
     formyl MLFK (SEQ ID NO. 17) 
     formyl MLFI (SEQ ID NO. 18) 
     formyl MFIL (SEQ ID NO. 19) 
     formyl MFLI (SEQ ID NO. 20) 
     formyl MLIF (SEQ ID NO. 21) 
     formyl MILF (SEQ ID NO. 22) 
     TKPR (SEQ ID NO. 23) 
     VGVAPG (SEQ ID NO. 24) 
     formyl MLF (SEQ ID NO. 25) 
     Thrombus 
     NDGDFEEIPEEYLQ (SEQ ID NO. 26) 
     NDGDFEEIPEEY(SO 3  Na)LQ (SEQ ID NO. 27) 
     GPRG (SEQ ID NO. 28) 
     Platelets 
     D-Phe.PRPGGGGNGDFEEIPEEYL (SEQ ID NO. 29) 
     RRRRRRRRRGDV (SEQ ID NO. 30) 
     PLYKKIIKKLLES (SEQ ID NO. 31) 
     RGD (SEQ ID NO. 32) 
     RGDS (SEQ ID NO. 33) 
     Infection and Atherosclerotic Plaque 
     YIGSR (SEQ ID NO. 34) 
     CH 2  CO.YIGSRC (SEQ ID NO. 35) 
     Alzheimers Disease (Amyloid Plaque) 
     EKPLQNFTLSFR (SEQ ID NO. 36) 
      Single letter abbreviations for amino acids can be found in G. Zubay, Biochemistry (2nd ed.), 1988, (MacMillan Publishing: New York), p.33.! 
     In the Cp(aa)Cp, the Cp is a protected cysteine where the S-protecting groups are the same or different and may be but not limited to: 
     --CH 2  -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl); 
     --CH--(aryl) 2 , (aryl is phenyl or alkyl or alkyloxy substituted phenyl); 
     --C--(aryl) 3 , (aryl is phenyl or alkyl or alkyloxy substituted phenyl); 
     --CH 2  -(4-methoxyphenyl); 
     --CH--(4-pyridyl)(phenyl) 2  ; 
     --C(CH 3 ) 3   
     --9-phenylfluorenyl; 
     --CH 2  NHCOR (R is unsubstituted or substituted alkyl or aryl); 
     --CH 2  --NHCOOR (R is unsubstituted or substituted alkyl or aryl); 
     --CONHR (R is unsubstituted or substituted alkyl or aryl); 
     --CH 2  --S--CH 2  -phenyl 
     When Cp--gly--Cp is combined with technetium, the following complex with the protecting groups removed is formed: ##STR5## 
     The preferred protecting group has the formula --CH 2  --NHCOR wherein R is a lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substituted with lower alkyl, hydroxyl, lower alkoxy, carboxy, or lower alkoxycarbonyl. 
     Compounds of the present invention can generally advantageously be prepared on an peptide synthesizer. Compounds of this invention are advantageous in that they are soluble and the sulfur is stabilized. 
     In forming the complex of radioactive technetium with the compounds of this invention, the technetium complex, a salt of technetium-99m pertechnetate, is reacted with the compound of this invention in the presence of a reducing agent such as stannous chloride, ferrous ion or sodium dithionite. These technetium labeled complexes can also be made by exchange of a prereduced technetium-99m complex. The complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a compound to be labeled and a sufficient amount of reducing agent to label the compound with technetium-99m. Alternatively, the complex may be formed by reacting the compound of this invention with a pre-formed labile complex of technetium and another compound. This process is known as ligand exchange, is well known to those skilled in the art, and the labile complex may be formed using such compounds as tartrate, citrate, gluconate or mannitol, for example. Among the technetium-99m pertechnetate salts are included the alkali metal salts such as the sodium salt or ammonium salts, or lower alkyl ammonium salts. The reaction of the compound of this invention with pertechnetate or preformed labile complex can be carried out in an aqueous medium at room temperature. The anionic complex which has a charge of -1 is formed in the aqueous medium in the form of a salt with a suitable cation such as sodium, ammonium cation, mono, di- or tri-lower alkyl amine cation, etc. Any conventional salt of the anionic complex with, a pharmaceutically acceptable cation can be used in accordance with this invention. 
     In carrying out the reaction of the compounds of this invention with pertechnetate or a labile complex to form the anionic complex, the thiol protecting group is cleaved. Therefore, this reaction not only introduces the radioactive metal into the compound but also cleaves the thiol protecting group. All of the aforementioned thiol protecting groups are cleaved by a reaction of salts of radioactive metals in accordance with this invention. 
     In forming the complex the radioactive material has a suitable amount of radioactivity. In forming the Tc-99m radioactive anionic complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 milliCuries (mCi) to 100 mCi per ml. 
     The complex can be used for visualizing organs such as the kidney for diagnosing disorders in these organs, tumors and blood clots can also be imaged. In accordance with this invention, the anionic complex either as a complex or as a salt with a pharmaceutically acceptable cation is administered in a single unit injectable dose. Any of the common carriers such as sterile saline solution, plasma, etc., can be utilized after the radiolabeling for preparing the injectable solution to diagnostically image various organs, clots, tumors and the like in accordance with this invention. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from about 0.01 ml to about 10 ml. After intravenous administration, imaging of the organ in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after injecting into patients. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos. Any conventional method of imaging for diagnostic purposes can be utilized in accordance with this invention. 
     The complexes may be administered intravenously in any conventional medium for intravenous injection such as an aqueous saline medium, or in blood plasma medium. Such medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Among the preferred mediums are normal saline and plasma. 
     The methods for making and labeling these compounds are more fully illustrated in the following examples. 
    
    
     EXAMPLE 1 
     Cys(Acm)GlyCys(Acm)GlyGlyArgGlyAspSer (SEQ ID NO. 37) 
     The title compound was prepared on a 0.25 millimole scale using an Applied Biosystems Model 431A peptide Synthesizer, N-terminus Fmoc protection and HMP resin (see Scheme). The product was cleaved from the resin using 95% trifluoroacetic acid at room temperature for 3 hours. Work-up and high performance liquid chromatography (HPLC) purification (using a Vydac 2.20 cm×25 cm, 10 um, C-18 column with a 20-minute gradient of 0.1% trifluoroacetic acid to 70% acetonitrile/0.1% trifluoroacetic acid at a flow rate of 25 ml/min) gave 50 mg of the title compound, 95% pure. (HPLC peak eluted at 5.5 min; Pos. ion FABMS Calc MM 952.97, Found 953). ##STR6## 
     EXAMPLE 2 
     Radiolabeling of Compound of Example 1 with Tc-99m 
     0.3 mg of the compound prepared as in Example 1 was dissolved in 0.3 ml of 0.05 M potassium phosphate buffer (pH 7.4) containing 0.5 mM EDTA. Tc-99m gluceptate was prepared by reconstituting a Glucoscan vial (E. I. DuPont de Nemours, Inc.) with 1.0 ml of Tc-99m sodium pertechnetate containing 26 mCi. After 15 minutes at room temperature, 75 ul of Tc-99m gluceptate was added to 0.3 mg of the compound prepared as in Example 1 and boiled for 45 minutes. 
     The extent of Tc-99m labeling of the peptide was determined by chomatography using Merck silica gel 60 F 250  aluminum-backed strips which were spotted with 10 ul of sample and chromatographed with acetonitrile:0.5 M sodium chloride solvent (15:85) approximately 2% of Tc-99m radioactivity remained at R f  0.0, confirming that no significant Tc-99m colloids or aggregates were generated. 
     The Tc-99m labeled peptide purity was determined by HPLC using a Brownlee Spheri-5 (5 um) resin, RP-18, 220×4.6 mm column and the following gradient: 0% A (CH 3  CN:H 2  O:TFA, 70:30:0.1) and 100% B (0.1% TFA in H 2  O) to 100% A+0% B over 10 minutes at 1.5 ml/min; and then held at the 100% A solvent for 5 minutes. This protocol yielded 100% of the radiometric species detected (by in-line NaI detector) as a single species (retention time=10.9 min). Tc-99m gluceptate and Tc-99m sodium pentechnetate elute between 1 and 4 minutes under identical conditions, confirming the identity of the Tc-99m labeled peptide isolated. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 37(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:TyrArgAlaLeuValAspThrLeuLys15(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:ArgAlaLeuValAspThrLeuLys15(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:ArgAlaLeuValAspThrLeuLysPheValThrGlnAlaGluGlyAla151015Lys(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:TyrAlaLysPheArgGluThrLeuGluAspThrArgAspArgMetTyr151015(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:AlaLysPheArgGluThrLeuGluAspThrArgAspArgMetTyr151015(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TyrAlaAlaLeuAspLeuAsnAlaValAlaAsnLysIleAlaAspPhe151015GluLeu(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:AlaAlaLeuAspLeuAsnAlaValAlaAsnLysIleAlaAspPheGlu151015Leu(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:TyrArgAlaLeuValAspThrLeuLysPheValThrGluGlnAlaLys151015GlyAla(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:ArgAlaLeuValAspThrLeuLysPheValThrGluGlnAlaLysGly151015Ala(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:TyrArgAlaLeuValAspThrGluPheLysValLysGlnGluAlaGly151015AlaLys(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:ArgAlaLeuValAspThrGluPheLysValLysGlnGluAlaGlyAla151015Lys(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:TyrArgAlaLeuValAspThrLeuLysPheValThrGlnAlaGluGly151015AlaLys(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:ValGlyValAlaProGlyValGlyValAlaProGlyValGlyValAla151015ProGly(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:ValProGlyValGlyValProGlyValGlyValProGlyValGlyVal151015ProGlyValGly20(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-Nle/note= &#34;Amino terminal formyl norleucine residue&#34;(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 4(D) OTHER INFORMATION: /label=Nleu/note= &#34;Norleucine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:XaaLeuPheXaaTyrLys15(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl-methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:MetIlePheLeu(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:MetLeuPheLys1(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:MetLeuPheIle1(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:MetPheIleLeu1(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:MetPheLeuIle1(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:MetLeuIlePhe1(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=formyl-M/note= &#34;Amino terminal formyl methionine residue&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:MetIleLeuPhe1(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:ThrLysProArg1(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:ValGlyValAlaProGly15(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:MetLeuPhe1(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:AsnAspGlyAspPheGluGluIleProGluGluTyrLeuGln1510(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 12(D) OTHER INFORMATION: /label=Tyrosine-SO3-Na/note= &#34;The tyrosine derivative at this positionhas been substituted at the phenolic hydroxyl withsodium sulfate&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:AsnAspGlyAspPheGluGluIleProGluGluTyrLeuGln1510(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:GlyProArgGly1(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label= D- Phe/note= &#34;The amino terminal phenylalanine residueis in the D stereochemical configuration&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:PheProArgProGlyGlyGlyGlyAsnGlyAspPheGluGluIlePro151015GluGluTyrLeu20(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:ArgArgArgArgArgArgArgArgArgGlyAspVal1510(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 13 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:ProLeuTyrLysLysIleIleLysLysLeuLeuGluSer1510(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:ArgGlyAsp1(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:ArgGlyAspSer1(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:TyrIleGlySerArg15(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 6(D) OTHER INFORMATION: /label=Cys-S-CH2-CO-/note= &#34;The side-chain sulfur atom of the carboxylterminal cysteine residue is carbomethoxylated andesterified to the amino group of the amino(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:TyrIleGlySerArgCys15(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:GluLysProLeuGlnAsnPheThrLeuSerPheArg1510(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /label=Cys-Acm/note= &#34;This cysteine residue is protected byesterification with acetic a...&#34;(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 3(D) OTHER INFORMATION: /label=Cys-Acm/note= &#34;This cysteine residue is protected byesterification with acetic a...&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:CysGlyCysGlyGlyArgGlyAspSer15__________________________________________________________________________