Patent application title: Retro-Inversion Peptides That Target GIT Transport Receptors and Related Methods
Inventors:
Daniel Joseph O'Mahony (Dublin, IE)
IPC8 Class: AA61K916FI
USPC Class:
424491
Class name: Particulate form (e.g., powders, granules, beads, microcapsules, and pellets) coated (e.g., microcapsules) containing proteins and derivatives
Publication date: 2009-09-24
Patent application number: 20090238886
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Patent application title: Retro-Inversion Peptides That Target GIT Transport Receptors and Related Methods
Inventors:
Daniel Joseph O'Mahony
Agents:
MYERS BIGEL SIBLEY & SAJOVEC
Assignees:
Origin: RALEIGH, NC US
IPC8 Class: AA61K916FI
USPC Class:
424491
Abstract:
Retro-inverted forms of GIT targeting agents that target specific receptor
sites in vivo and/or promote uptake of active agents and/or enhance
active site delivery across the GIT into the systemic circulation are
provided. These retro-inverted peptides and compositions containing these
retro-inverted peptides can be used to deliver an active agent, such as a
drug or a drug-containing nano- or microparticle for treatment of a
condition in a subject in need of the drug, in vivo. Additionally, the
invention provides antibodies which are capable of immunospecifically
binding the retro-inverted peptides.Claims:
1. A composition comprising a d-form retro-inverted peptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1
(ZElan144), SEQ ID NO:2 (ZElan145) and SEQ ID NO:3 (ZElan146), wherein
said peptide binds to a domain of a gastro-intestinal tract transport
receptor selected from the group consisting of amino acids 29-273 of
human intestinal oligopeptide transporter (HPT1), amino acids 391-571 of
human oligopeptide transporter (hPEPT1), amino acids 387-685 of human D2
clone (D2H), and amino acids 272-667 of human sucrase isomaltose (hSI),
wherein said peptide is no more than 50 amino acid residues, bound to an
material comprising an active agent, wherein said active agent treats a
mammalian disease or disorder,wherein said mammalian disease or disorder
is selected from the group consisting of hypertension, diabetes,
osteoporosis, hemophilia, anemia, cancer, migraine, and angina
pectoris,wherein the active agent is a drug selected from the group
consisting of a peptide, a hormone, an analgesic, an anti-migraine agent,
an anti-coagulant agent, a cardiovascular agent, an anti-emetic agent, a
narcotic agonist, a chelating agent, an anti-anginal agent, a
chemotherapeutic agent, a sedative, an anti-neoplasitc agent, a
prostaglandin, an antidiuretic agent, an anti-sense oligonucleotide, a
gene, a gene-correcting hybrid oligonculeotide, a ribozyme, an aptameric
oligonucleotide, a triple-helix forming oligoncuelotide, a signal
transduction pathway inhibitor, a tyrosine kinase inhibitor, a
DNA-modifying agent, a non-viral gene delivery system and a viral vector
gene system; and/orwherein the active agent is a drug selected from the
group consisting of insulin, calcitonin gene regulating protein, atrial
natriuretic protein, colony stimulating factor, betaseron, erythropoiten,
α-interferon, β-interferon, γ-interferon, somatropin,
somatotropin, somatostatin, somatomedins, luteinizing hormone-releasing
hormone, tissueplasminogen activator, growth hormone releasing hormone,
oxytocin, estrdiol, growth hormones, leuprolide acetate, factor VIII,
interleukins, fentanyl, sufentanil, butorphanol, buprenophrine,
levorphanol, morphine, hydromorphone, hydrocodone, oxymorphone,
methadone, lidocaine, bupivacaine, diclophenac, naproxen, paverin,
heparin, hiruden, scopolamine, ondasetron, domperidone, etoclopramide,
diltiazem, clonidine, nifedipine, verapamil, isocorbide-5-mononitrate,
benzodiazeines, phenothiozines, naltrexone, naloxone, deferoxamine,
desmopressin, vasopressin, nitroglycerin, 5-fluorouracil, bleomycin,
prostaglandins and vincristine.
2. The composition of claim 1, wherein the material is a particle containing the active agent.
3. The composition of claim 1, wherein the material is a slow-release device containing the active agent.
4. The composition of claim 1, wherein the peptide is covalently or non-covalently bound to the material.
5. A composition comprising a chimeric protein bound to ameterial comprising an active agent, in which the chimeric protein comprises a d-form peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 (ZElan144), SEQ ID NO:2 (ZElan145) and SEQ ID NO:3 (ZElan146) fused via a covalent bond to an amino acid sequence of a second protein, in which the active agent treats a mammalian disease or disorder, wherein said mammalian disease or disorder is selected from the group consisting of hypertension, diabetes, osteoporosis, hemophilia, anemia, cancer, migraine, and angina pectoris, wherein said peptide binds to a domain of a gastro-intestinal tract transport receptor selected from the group consisting of amino acids 29-273 of human intestinal oligopeptide transporter (HPT1), amino acids 391-571 of human oligopeptide transporter (hPEPT1), amino acids 387-685 of human D2 clone (D2H), and amino acids 272-667 of human sucrase isomaltose (hSI),wherein the active agent is a drug selected from the group consisting of a peptide, a hormone, an analgesic, an anti-migraine agent, an anti-coagulant agent, a cardiovascular agent, an anti-emetic agent, a narcotic agonist, a chelating agent, an anti-anginal agent, a chemotherapeutic agent, a sedative, an anti-neoplasitc agent, a prostaglandin, an antidiuretic agent, an anti-sense oligonuclaotide, a gene, a gene-correcting hybrid oligonculeotide, a ribozyme, an aptameric oligonucleotide, a triple-helix forming oligoncuelotide, a signal transduction pathway inhibitor, a tyrosine kinase inhibitor, a DNA-modifying agent, a non-viral gene delivery system and a viral vector gene system; and/orwherein the active agent is a drug selected from the group consisting of insulin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoiten, α-interferon, β-interferon, γ-interferon, somatropin, somatotropin, somatostatin, somatomedins, luteinizing hormone-releasing hormone, tissueplasminogen activator, growth hormone releasing hormone, oxytocin, estrdiol, growth hormones, leuprolide acetate, factor VIII, interleukins, fentanyl, sufentanil, butorphanol, buprenophrine, levorphanol, morphine, hydromorphone, hydrocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclophenac, naproxen, paverin, heparin, hiruden, scopolamine, ondasetron, domperidone, etoclopramide, diltiazem, clonidine, nifedipine, verapamil, isocorbide-5-mononitrate, benzodiazeines, phenothiozines, naltrexone, naloxone, deferoxamine, desmopressin, vasopressin, nitroglycerin, 5-fluorouracil, bleomycin, prostaglandins and vincristine.
6. A composition comprising a d-form retro-inverted peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 (ZElan144), SEQ ID NO:2 (ZElan145) and SEQ ID NO:3 (ZElan146), wherein said peptide binds to a domain of a gastro-intestinal tract transport receptor selected from the group consisting of amino acids 29-273 of human intestinal oligopeptide transporter (HPT1), amino acids 391-571 of human oligopeptide transporter (hPEPT1), amino acids 387-685 of human D2 clone (D2H), and amino acids 272-667 of human sucrase isomaltose (hSI), wherein said peptide is no more than 50 amino acid residues, non-covalently bound to a particle containing a drug, wherein the drug is selected from the group consisting of a peptide, a hormone, an analgesic, an anti-migraine agent, an anti-coagulant agent, a cardiovascular agent, an anti-emetic agent, a narcotic agonist, a chelating agent, an anti-anginal agent, a chemotherapeutic agent, a sedative, an anti-neoplasitc agent, a prostaglandin, an antidiuretic agent, an anti-sense oligonucleotide, a gene, a gene-correcting hybrid oligonculeotide, a ribozyme, an aptameric oligonucleotide, a triple-helix forming oligoncuelotide, a signal transduction pathway inhibitor, a tyrosine kinase inhibitor, a DNA-modifying agent, a non-viral gene delivery system and a viral vector gene system; and/orwherein the drug is selected from a group consisting of insulin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoiten, α-interferon, β-interferon, γ-interferon, somatropin, somatotropin, somatostatin, somatomedins, luteinizing hormone-releasing hormone, tissueplasminogen activator, growth hormone releasing hormone, oxytocin, estrdiol, growth hormones, leuprolide acetate, factor VIII, interleukins, fentanyl, sufentanil, butorphanol, buprenophrine, levorphanol, morphine, hydromorphone, hydrocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclophenac, naproxen, paverin, heparin, hiruden, scopolamine, ondasetron, domperidone, etoclopramide, diltiazem, clonidine, nifedipine, verapamil, isocorbide-5-mononitrate, benzodiazeines, phenothiozines, naltrexone, naloxone, deferoxamine, desmopressin, vasopressin, nitroglycerin, 5-fluorouracil, bleomycin, prostaglandins and vincristine.
7. A composition comprising a d-form retro-inverted peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 (ZElan144), SEQ ID NO:2 (ZElan145) and SEQ ID NO:3 (ZElan146), wherein said peptide binds to a domain of a gastro-intestinal tract transport receptor selected from the group consisting of amino acids 29-273 of human intestinal oligopeptide transporter (HPT1), amino acids 391-571 of human oligopeptide transporter (hPEPT1), amino acids 387-685 of human D2 clone (D2H), and amino acids 272-667 of human sucrase isomaltose (hSI), wherein said peptide is no more than 50 amino acid residues, covalently bound to a drug, wherein the drug is selected from a group consisting of a peptide, a hormone, an analgesic, an anti-migraine agent, an anti-coagulant agent, a cardiovascular agent, an anti-emetic agent, a narcotic agonist, a chelating agent, an anti-anginal agent, a chemotherapeutic agent, a sedative, an anti-neoplasitc agent, a prostaglandin, an antidiuretic agent, an anti-sense oligonucleotide, a gene, a gene-correcting hybrid oligonculeotide, a ribozyme, an aptameric oligonucleotide, a triple-helix forming oligoncuelotide, a signal transduction pathway inhibitor, a tyrosine kinase inhibitor, a DNA-modifying agent, a non-viral gene delivery system and a viral vector gene system; and/orwherein the drug is selected from the group consisting of insulin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoiten, α-interferon, β-interferon, γ-interferon, somatropin, somatotropin, somatostatin, somatomedins, luteinizing hormone-releasing hormone, tissueplasminogen activator, growth hormone releasing hormone, oxytocin, estrdiol, growth hormones, leuprolide acetate, factor VIII, interleukins, fentanyl, sufentanil, butorphanol, buprenophrine, levorphanol, morphine, hydromorphone, hydrocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclophenac, naproxen, paverin, heparin, hiruden, scopolamine, ondasetron, domperidone, etoclopramide, diltiazem, clonidine, nifedipine, verapamil, isocorbide-5-mononitrate, benzodiazeines, phenothiozines, naltrexone, naloxone, deferoxamine, desmopressin, vasopressin, nitroglycerin, 5-fluorouracil, bleomycin, prostaglandins and vincristine.
8. The composition of claim 1 wherein said peptide increases the transport of the active agent through human or animal gastro-intestinal tissue.
9. The composition of claim 1 which targets the active agent to a selected site or selected tissue in a human or animal.
10. A pharmaceutical composition comprising the composition of claim 1 in a pharmaceutically acceptable carrier suitable for use in humans in vivo.
11. A nanoparticle or microparticle formed from a composition comprising a d-form retro-inverted peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 (ZElan144), SEQ ID NO:2 (ZElan145) and SEQ ID NO:3 (ZElan146), wherein said peptide binds to a domain of a gastro-intestinal tract transport receptor selected from the group consisting of amino acids 29-273 of human intestinal oligopeptide transporter (HPT1), amino acids 391-571 of human oligopeptide transporter (hPEPT1), amino acids 387-685 of human D2 clone (D2H), and amino acids 272-667 of human sucrase isomaltose (hSI), wherein said peptide is no more than 50 amino acid residues, wherein the nanoparticle or microparticle is a drug-loaded nanoparticle or microparticle or a drug-encapsulating nanoparticle or microparticle, wherein the drug is selected from a group consisting of a peptide, a hormone, an analgesic, an anti-migraine agent, an anti-coagulant agent, a cardiovascular agent, an anti-emetic agent, a narcotic agonist, a chelating agent, an anti-anginal agent, a chemotherapeutic agent, a sedative, an anti-neoplasitc agent, a prostaglandin, an antidiuretic agent, an anti-sense oligonucleotide, a gene, a gene-correcting hybrid oligonculeotide, a ribozyme, an aptameric oligonucleotide, a triple-helix forming oligoncuelotide, a signal transduction pathway inhibitor, a tyrosine kinase inhibitor, a DNA-modifying agent, a non-viral gene delivery system and a viral vector gene system; and/orwherein the drug is selected from a group consisting of insulin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoiten, α-interferon, β-interferon, γ-interferon, somatropin, somatotropin, somatostatin, somatomedins, luteinizing hormone-releasing hormone, tissueplasminogen activator, growth hormone releasing hormone, oxytocin, estrdiol, growth hormones, leuprolide acetate, factor VIII, interleukins, fentanyl, sufentanil, butorphanol, buprenophrine, levorphanol, morphine, hydromorphone, hydrocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclophenac, naproxen, paverin, heparin, hiruden, scopolamine, ondasetron, domperidone, etoclopramide, diltiazem, clonidine, nifedipine, verapamil, isocorbide-5-mononitrate, benzodiazeines, phenothiozines, naltrexone, naloxone, deferoxamine, desmopressin, vasopressin, nitroglycerin, 5-fluorouracil, bleomycin, prostaglandins and vincristine.
12. The composition of claim 1 wherein the active agent is insulin or leuprolide.
13. The composition of claim 5 wherein the active agent is insulin or leuprolide.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation application of U.S. application Ser. No. 09/443,986, filed Nov. 19, 1999, allowed, which claims the benefit of U.S. Provisional Application Ser. No. 60/109,038, filed Nov. 19, 1998, which applications are incorporated by reference herein in their entireties.
FIELD OF THE INVENTION
[0002]The present invention relates generally to peptides that are capable of targeting or specifically binding gastro-intestinal tract (GIT) transport receptors. In particular, the invention relates to retro-inverted forms of peptide sequences and motifs, as well as derivatives thereof, which enhance drug delivery and transport through tissue, such as epithelial cells lining the luminal side of the GIT. Production of peptides and antibodies is also provided. The invention further relates to pharmaceutical compositions, formulations and related methods.
BACKGROUND OF THE INVENTION
[0003]Proteases cleave peptide bonds between adjacent L-amino acids, rendering these peptides susceptible to degradation in the GIT. Artificial proteins or peptides composed of D-amino acids are largely resistant to proteolytic degradation. However, when D-amino acids are substituted for all L-amino acids in a peptide/protein, the corresponding D-peptide/protein is a mirror image of the original peptide/protein and is likely to have modified or lost biological activity because of this change in conformation. Retro-inverted peptides are peptides having all D-amino acids but are synthesized in the reverse order of sequence compared to the original L-peptide/protein. The carboxy terminus of the original peptide/protein becomes the amino terminus (and vice versa) of the retro-inverted peptide/protein and the resulting side chain surface of the retro-inverted peptide/protein is similar to the original L-peptide/protein. The net result of combining D-enantiomers and reverse synthesis is that the positions of carbonyl and amine groups in each amide bond are exchanged while the position of side-chain groups is preserved (Brady, L. and Dodson, G., Nature, 368L:692-693 (1994); Jameson et al., Nature, 368; 744-746 (1994)). This alteration in the protein backbone is self compensating in that hydrogen-bond donors become hydrogen-bond acceptors (amide carbonyl groups) and vice-versa. When the position of the side-chains relative to the backbone are unchanged the modified surface of the retro-inverted peptide/protein is largely unaltered compared to the original L-peptide/protein.
[0004]Previously, as disclosed and claimed in WO 98/51325, which is hereby incorporated by reference in its entirety, we have identified random peptides and their fragments, motifs, derivatives, analogs or peptidomimetics thereof which are capable of specific binding to GIT transport receptors such as the D2H, hSI, HPT1 and hPEPT1 receptors (hereinafter "GIT targeting agents"). These GIT targeting agents are capable of facilitating transport of an active agent through a human or animal gastro-intestinal tissue and have use, for example, in facilitating transport of active agents from the lumenal side of the GIT into the systemic blood system and/or in targeting active agents to the GIT. Thus, for example, by binding (covalently or noncovalently) the GIT targeting agent to an orally administered active agent, the active agent can be targeted to specific receptor sites or transport pathways which are known to operate in the human gastrointestinal tract, thus facilitating its absorption into the systemic system. Preferably, the active agent is a drug or a drug-containing nano- or microparticle.
SUMMARY OF THE INVENTION
[0005]Surprisingly, we have found that retro-inverted forms of the GIT targeting agents target specific receptor sites in vivo and/or promote uptake of active agents and/or enhance active agent delivery across the GIT into the systemic circulation. By using retro-inversion D-peptide synthesis, we have discovered retro-inverted D-peptides of the GIT targeting agents that retain the same function of the GIT targeting agents but have enhanced stability to proteases in the human or animal GIT. These retro-inverted peptides and compositions containing these retro-inverted peptides can be used to deliver an active agent, such as a drug or a drug-containing nano- or microparticle for treatment of a condition in a subject in need of the drug, in vivo by any of the uses or methods disclosed in the above-referenced WO 98/51325. Additionally, the invention provides antibodies which are capable of immunospecifically binding the retro-inverted peptides of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006]FIG. 1 shows the systemic blood glucose levels following intestinal administration of control (PBS); ZElan 021 coated insulin-containing particles, ZElan 018 coated insulin-containing particles, ZElan091 coated insulin-containing particles, ZElan129 coated insulin-containing particles and ZElan 144 coated insulin-containing particles according to this invention (300 iu insulin loading); and
[0007]FIG. 2 shows the systemic insulin levels following intestinal administration of control ZElan 021 coated insulin-containing particles, ZElan 018 coated insulin-containing particles, ZElan091 coated insulin-containing particles, ZElan129 coated insulin-containing particles and ZElan144 coated insulin-containing particles according to this invention (300 iu insulin loading).
DETAILED DESCRIPTION OF THE INVENTION
[0008]The present invention relates to retro-inverted peptides (also referred to herein as "targeting retro-inverted peptides" or "targeting retro-inversion peptides") that target specific receptor sites in vivo and/or promote uptake of active agents and/or enhance active agent delivery across the GIT into the systemic, portal or hepatic circulation. In particular, these retro-inverted peptides specifically bind to one or more of the human gastro-intestinal tract receptors HPT1, HPEPT1, D2H or hSI or their equivalents in other mammals and have general utility in targeting active agents to selected sites and/or selected tissues in the body in which the receptors are expressed. These peptides are synthesized from D-amino acids and have a reverse sequence order of the GIT targeting agents disclosed and claimed in the above-referenced WO 98/51325. The present invention also relates to derivatives (including but not limited to fragments) of these retro-inverted peptides, which derivatives are functionally similar to the retro-inverted peptides (that is, capable of displaying one or more known functional activities of the retro-inverted peptides). These functional activities include but are not limited to the ability to bind or to compete with binding to the gastro-intestinal tract receptors HPT1, HPEPT1, D2H or hSI or the ability to be bound by an antibody directed against the retro-inverted peptide. Derivatives can be tested for the desired activity by procedures known in the art, including binding to a receptor domain or to Caco-2 cells, in vitro, or to intestinal tissue, in vitro or in vivo.
[0009]Derivatives can be made by altering the retro-inverted peptides sequences by substitutions, additions or deletions that provide for functionally equivalent activity. Derivatives include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the retro-inverted peptide including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequences resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
[0010]Included within the scope of the invention are retro-inverted peptides or derivatives which are modified. e.g., by glycosylation, acetylation, phosphorylation, amidation, derivitization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques. In a specific embodiment, the amino- and/or carboxy-termini are modified. Furthermore, is desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the retro-inverted peptides sequence. Non-classical amino acids include but are not limited to α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general.
[0011]The present invention also relates to therapeutic and diagnostic methods and compositions containing the targeting retro-inverted peptides.
[0012]The invention provides compositions comprising the targeting retro-inversion peptides of the invention bound to a material comprising an active agent. Such compositions have use in targeting the active agent to the GIT and/or in facilitating transfer through the lumen of the GIT into the systemic circulation in a human or animal subject. The retro-inverted D-peptides also have general utility in targeting active agents to selected sites/selected tissues in a human or animal subject in which the receptors or other related receptors are expressed. For instance, where the retro-inverted D-peptides bind to other receptors, such as related receptors, splice variants of the receptors or related receptors which exist as a superfamily, or where the peptides bind through non-specific interactions, such as non-specific ion-pairings, hydrogen bonding or hydrophobic pairings, they can be used to deliver drugs to tissues or cell types in mammals or humans that express these receptors. Additionally, when the active agent is an imaging agent, such compositions can be administered in vivo to image selected sites/selected tissues, such as the GIT (or particular transport receptors thereof). Other active agents include but are not limited to: any drug or antigen or any drug- or antigen-loaded or drug- or antigen-encapsulated nanoparticle, microparticle, liposome, or micellar formulation capable of eliciting a biological response in a human or animal. Examples of drug- or antigen-loaded or drug- or antigen-encapsulated formulations include those in which the active agent is encapsulated or loaded into nano- or microparticles, such as biodegradable nano- or microparticles, and which have the targeting retro-inversion peptide adsorbed, coated or covalently bound, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle. Additionally, the targeting retro-inverted peptide can form the nano- or microparticle itself or the targeting retro-inverted peptide can be covalently attached to the polymer or polymers used in the production of the biodegradable nano- or microparticles or drug-loaded or drug-encapsulated nano- or microparticles or the peptide can be directly conjugated to the active agent. Such conjugation to active agents include proteins in which the retro-inverted peptide is conjugated directly to the protein or peptide active agent of interest. Additionally, the retro-inverted peptides of this invention can be attached to the building blocks or subunits or polymer monomers used in the synthesis of the base polymers.
[0013]In a preferred embodiment, the invention provides for treatment of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic"). Such "Therapeutics" include but are not limited to: targeting retro-inversion peptide bound to an active agent of value in the treatment or prevention of a disease or disorder (preferably a mammalian, most preferably human, disease or disorder). The active agent is preferably a drug.
[0014]Any drug known in the art may be used, depending upon the disease or disorder to be treated or prevented, and the type of subject to which it is to be administered. As used herein, the term "drug" includes, without limitation, any pharmaceutically active agent. Representative drugs include, but are not limited to, peptides or proteins, hormones, analgesics, anti-migraine agents, anti-coagulant agents, anti-emetic agents, cardiovascular agents, anti-hypertensive agents, narcotic antagonists, chelating agents, anti-anginal agents, chemotherapy agents, sedatives, anti-neoplastics, prostaglandins, and antidiuretic agents. Typical drugs include peptides, proteins or hormones such as insulin, calcitonin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoietin (EPO), interferons such as α, β or γ interferon, somatropin, somatotropin, somatostatin, insulin-like growth factor (somatomedins), luteinizing hormone releasing hormone (LHRH), tissue plasminogen activator (TPA), growth hormone releasing hormone (GHRH), oxytocin, estradiol, growth hormones, leuprolide acetate, factor VIII, interleukins such as interleukin-2, and analogs thereof; analgesics such as fentanyl, sufentanil, butorphanol, buprenorphine, levorphanol, morphine, hydromorphone, hydrocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen, paverin, and analogs thereof; anti-migraine agents such as heparin, hirudin, and analogs thereof; anti-coagulant agents such as scopolamine, ondansetron, domperidone, etoclopramide, and analogs thereof; cardiovascular agents, anti-hypertensive agents and vasodilators such as diltiazem, clonidine, nifedipine, verapamil, isosorbide-5-mononitrate, organic nitrates, agents used in treatment of heart disorders and analogs thereof; sedatives such as benzodiazeines, phenothiozines and analogs thereof; narcotic antagonists such as naltrexone, naloxone and analogs thereof; chelating agents such as deferoxamine and analogs thereof; anti-diuretic agents such as desmopressin, vasopressin and analogs thereof; anti-angina agents such as nitroglycerine and analogs thereof; anti-neoplastics such as 5-fluorouracil, bleomycin and analogs thereof; prostaglandins and analogs thereof; and chemotherapy agents such as vincristine and analogs thereof. Representative drugs also include but are not limited to antisense oligonucleotides, genes, gene correcting hybrid oligonucleotides, ribozymes, aptameric oligonucleotides, triple-helix forming oligonucleotides, inhibitors of signal transduction pathways, tyrosine kinase inhibitors and DNA modifying agents. Drugs that can be used also include, without limitation, systems containing gene therapeutics, including non-viral systems for therapeutic gene delivery and viral vector systems for therapeutic genes which are modified with a retro-inversion peptide post virus purification.
[0015]In a preferred embodiment, a Therapeutic is therapeutically or prophylactically administered to a human patient.
[0016]Additional descriptions and sources of Therapeutics that can be used according to the invention are found in various Sections herein.
[0017]The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention. In a preferred aspect, the Therapeutic is substantially purified. The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably a human.
[0018]As will be clear, any disease or disorder of interest amenable to therapy or prophylaxis by providing a drug in vivo systemically or by targeting a drug (by linkage to a targeting retro-inversion peptide of the present invention) in vivo to the GIT or other selected sites, selected tissues or cell types which contain the receptor or other receptors, such as related receptors, splice variants of the receptors, related receptors which exist as a superfamily or to which the retro-inverted peptide interacts through non-specific interaction, such as non-specific ion-pairings or hydrogen bondings or hydrophobic pairings (using any route of administration) can be treated or prevented by administration of a Therapeutic of the invention. Such diseases may include but are not limited to hypertension, diabetes, osteoporosis, hemophilia, anemia, cancer, migraine, and angina pectoris, to name but a few.
[0019]Any route of administration known in the art may be used, including but not limited to oral, nasal, topical, intravenous, intraperitoneal, intradermal, mucosal, intrathecal, intramuscular, etc. Preferably, administration is oral; in such an embodiment the targeting retro-inverted peptide according to this invention acts advantageously to facilitate transport of the therapeutic active agent through the lumen of the GIT into the portal, hepatic or systemic circulation.
[0020]The present invention also provides therapeutic compositions or formulations. In a specific embodiment of the invention, a targeting retro-inversion peptide is associated with a therapeutically or prophylactically active agent, preferably a drug or drug-containing nano- or microparticle. More preferably, the active agent is a drug encapsulating or drug loaded nano- or microparticle, such as a biodegradable nano- or microparticle, in which the peptide is physically adsorbed or coated or covalently bonded, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle. Alternatively, the peptide can form the nano- or microparticle itself or can be directly conjugated to the active agent. Preferably the particles range in size from 10 nm and 500 μm, more preferably 50 to 800 nm, most preferably 200-600 nm.
[0021]Thus, in a specific embodiment, a targeting retro-inversion peptide is bound to a slow-release (controlled release) device containing a drug. In a specific embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard-et al., J. Neurosurg. 71:105 (1989)).
[0022]The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, sorbitol, trehelose and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
[0023]The active agent of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0024]The amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
[0025]According to this invention, a targeting retro-inverted peptide may also be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and a Fab expression library.
[0026]These antibodies can be used in methods relating to the localization and activity of the targeting retro-inversion peptide sequences of the invention, e.g., for imaging these peptides after in vivo administration (e.g., to monitor treatment efficacy), measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. For instance, antibodies or antibody fragments specific to a domain of a targeting retro-inversion peptide, such as a dansyl group or some other epitope introduced into the peptide, can be used to 1) identify the presence of the peptide on a nanoparticle or other substrate; 2) quantify the amount of peptide on the nanoparticle; 3) measure the level of the peptide in appropriate physiological samples; 4) perform immunohistology on tissue samples; 5) image the peptide after in vivo administration; 6) purify the peptide from a mixture using an immunoaffinity column, 7) bind or fix the peptide to the surface of nanoparticle or 8) when a tag is also added to either an active-agent containing particle or the active agent itself, track the fate of both the particle/active agent and the targeting retro-inversion peptide so as to determine if and/or where they become separated. Use 7 above envisions attaching the antibody (or fragment of the antibody) to the surface of drug-loaded nanoparticles or other substrates and then incubating this conjugate with the peptide. This procedure results in binding of the peptide in a certain fixed orientation, resulting in a particle that contains the peptide bound to the antibody in such a way that the peptide is fully active. Additionally, antibodies or antibody fragments specific to a domain of a targeting retro-inverted peptide 9) can be used in confocal microscopy imaging techniques or other imaging techniques in order to demonstrate or confirm or identify the location or localization of the peptide on the surface of a nano- or microparticle, 10) can be used in confocal microscopy imaging techniques or other imaging techniques in order to demonstrate or confirm or identify the location or localization of the peptide on the surface of a nanoparticle or microparticle which has also been loaded with a fluorescent agent, 11) in the case of nanoparticles or microparticles coated with the peptide which have been sliced into two halves by a microtone or other suitable techniques, the antibody can be used in suitable quantitative techniques such as confocal microscopy imaging techniques or other quantitative imaging techniques in order to identify or quantitate the relative distribution of the peptide between the surface of the nanoparticle or microparticle and the sub-surface interior matrix of the nanoparticles or microparticles, 12) can be used in confocal microscopy imaging techniques or other imaging techniques in order to demonstrate or confirm or identify the location of a peptide on the surface of a nanoparticle or microparticle which has been loaded with a fluorescent agent such as TRME or fluorascene, 13) can be used to identify which epitope or domain of the peptide is responsible for identification by the antibody; peptide derivatives such as cyclic forms or derivatives containing intra-chain disulphide bonds or other intra-chain bonds can also be used in mapping studies in order to identify which domain or epitope of the peptide is responsible for recognition by the antibody; 14) in the case of peptide derivatives in which the epitope or domain responsible for binding to a target receptor is flanked by di-sulphide bond or other intra-chain bonds and in which this domain is also responsible for binding to the antibody, the antibody can be used to determine if that epitope or domain is exposed or available for binding to the antibody when the peptide or derivative is coated onto the surface of a nanoparticle, microparticle or other substance, 15) can be used where the epitope or domain on the peptide which binds to the target receptors in the human gastro-intestinal tract or the target receptors on model epithelial cells such as Caco-2 cells or polarised Caco-2 cells and where this epitope or domain on the peptide is also responsible for binding by the antibody, the antibody can be used in competition studies to compete for the binding of the peptide to its target receptor sites and 16) where the epitope or domain on the peptide which binds to the target receptors in the human gastro-intestinal or the target receptors on model epithelial cells such as Caco-2 cells or polarised Caco-2 cells and where this epitope or domain on the peptide is also responsible for binding by the antibody, the antibody can be used in competition studies in which nanoparticles or microparticles are coated with the peptide and are used in cell binding studies and/or in receptor binding studies.
[0027]Abtides (or Antigen binding peptides) specific to a domain of targeting retro-inverted peptide, such as a dansyl group or some other epitope introduced into the peptide, can be used for the same purposes identified above for antibodies.
[0028]The retro-inverted peptides of this invention may be prepared by methods that are known in the art. For example, in brief, solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids. The protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. The coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Pat. No. 4,244,946. Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178 and in U.S. Pat. Nos. 4,305,872 and 4,316,891. In a preferred aspect, an automated peptide synthesizer is employed.
[0029]By way of example but not limitation, peptides can be synthesized on an Applied Biosystems Inc. ("ABI") model 431A automated peptide synthesizer using the "Fastmoc" synthesis protocol supplied by ABI, which uses 2-(1H-Benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate ("HBTU") (R. Knorr et al., 1989, Tet. Lett., 30:1927) as coupling agent. Syntheses can be carried out on 0.25 mmol of commercially available 4-(2',4'-dimethoxyphenyl-(9-fluorenyl-ethoxycarbonyl)-aminomethyl)-phenox- y polystyrene resin ("Rink resin" from Advanced ChemTech) (H. Rink, 1987, Tet. Lett. 28:3787). Fmoc amino acids (1 mmol) are coupled according to the Fastmoc protocol. The following side chain protected Fmoc amino acid derivatives are used: FmocArg(Pmc)OH; FmocAsn(Mbh)OH; FmocAsp(tBu)OH; FmocCys(Acm)OH; FmocGlu(tBu)OH; FmocGln(Mbh)OH; FmocHis(Tr)OH; FmocLys(Boc)OH; FmocSer(tBu)OH; FmocThr(tBu)OH; FmocTyr(tBu)OH. [Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; tBu, tert-butyl; Fmoc, 9-fluorenylmethoxycarbonyl; Mbh, 4,4'-dimethoxybenzhydryl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Tr, trityl].
[0030]Synthesis is carried out using N-methylpyrrolidone (NMP) as solvent, with HBTU dissolved in N,N-dimethylformamide (DMF). Deprotection of the Fmoc group is effected using approximately 20% piperidine in NMP. At the end of each synthesis the amount of peptide present is assayed by ultraviolet spectroscopy. A sample of dry peptide resin (about 3-10 mg) is weighed, then 20% piperidine in DMA (10 ml) is added. After 30 min sonication, the UV (ultraviolet) absorbance of the dibenzofulvene-piperidine adduct (formed by cleavage of the N-terminal Fmoc group) is recorded at 301 nm. Peptide substitution (in mmol g-1) can be calculated according to the equation:
substitution = A × v 7800 × w × 1000 ##EQU00001##
where A is the absorbance at 301 nm, v is the volume of 20% piperidine in DMA (in ml), 7800 is the extinction coefficient (in mol-1 dm3 cm-1) of the dibenzofulvene-piperidine adduct, and w is the weight of the peptide-resin sample (in mg).
[0031]Finally, the N-terminal Fmoc group is cleaved using 20% piperidine in DMA, then acetylated using acetic anhydride and pyridine in DMA. The peptide resin is thoroughly washed with DMA, CH2Cl2 and finally diethyl ether.
[0032]By way of example but not limitation, cleavage and deprotection can be carried out as follows: The air-dried peptide resin is treated with ethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole (PhSMe) for approximately 20 min. prior to addition of 95% aqueous trifluoracetic acid (TFA). A total volume of approximately 50 ml of these reagents are used per gram of peptide-resin. The following ratio is used: TFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5). The mixture is stirred for 3 h at room temperature under an atmosphere of N2. The mixture is filtered and the resin washed with TFA (2×3 ml). The combined filtrate is evaporated in vacuo, and anhydrous diethyl ether added to the yellow/orange residue. The resulting white precipitate is isolated by filtration. See King et al., 1990, Int. J. Peptide Protein Res. 36:255-266 regarding various cleavage methods.
[0033]Purification of the synthesized peptides can be carried out by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography, high performance liquid chromatography (HPLC)), centrifugation, differential solubility, or by any other standard technique.
[0034]The peptides of the present invention may be linked to other molecules (e.g., a detectable label, a molecule facilitating adsorption to a solid substratum, or a toxin, according to various embodiments of the invention) by methods that are well known in the art. Such methods include the use of homobifunctional and heterobifunctional cross-linking molecules.
[0035]The homobifunctional molecules have at least two reactive functional groups, which are the same. The reactive functional groups on a homobifunctional molecule include, for example, aldehyde groups and active ester groups. Homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and subaraldehyde. The use of glutaraldehyde as a cross-linking agent was disclosed by Poznansky et al., 1984, Science 223:1304-1306.
[0036]Homobifunctional molecules having at least two active ester units include esters of dicarboxylic acids and N-hydroxysuccinimide. Some examples of such N-succinimidyl esters include disuccinimidyl suberate and dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonic acid and bis-sulfonate salts such as their sodium and potassium salts. These homobifunctional reagents are available from Pierce, Rockford, Ill.
[0037]The heterobifunctional molecules have at least two different reactive groups. Some examples of heterobifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson et al., 1978, Biochem J. 173:723-737), sodium S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and 4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene. N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examples of heterobifunctional reagents comprising reactive groups having a double bond that reacts with a thiol group include succinimidyl 4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidyl m-maleimidobenzoate.
[0038]Other heterobifunctional molecules include succinimidyl 3-(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl 4-(N-maleimidomethyl-cyclohexane)-1-carboxylate, maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate salt of succinimidyl m-maleimidobenzoate is preferred. Many of the above-mentioned heterobifunctional reagents and their sulfonate salts are available from Pierce.
[0039]Additional information regarding how to make and use these as well as other polyfunctional reagents may be obtained from the following publications or others available in the art: Carlsson et al., 1978, Biochem. J. 173:723-737; Cumber et al., 1985, Methods in Enzymology 112:207-224; Jue et al., 1978, Biochem 17:5399-5405; Sun et al., 1974, Biochem. 13:2334-2340; Blattler et al., 1985, Biochem. 24:1517-152; Liu et al., 1979, Biochem. 18:690-697; Youle and Neville, 1980, Proc. Natl. Acad. Sci. USA 77:5483-5486; Lerner et al., 1981, Proc. Natl. Acad. Sci. USA 78:3403-3407; Jung and Moroi, 1983, Biochem. Biophys. Acta 761:162; Caulfield et al., 1984, Biochem. 81:7772-7776; Staros, 1982, Biochem. 21:3950-3955; Yoshitake et al., 1979, Eur. J. Biochem. 101:395-399; Yoshitake et al., 1982, J. Biochem. 92:1413-1424; Pilch and Czech, 1979, J. Biol. Chem. 254:3375-3381; Novick et al., 1987, J. Biol. Chem. 262:8483-8487; Lomant and Fairbanks, 1976, J. Mol. Biol. 104:243-261; Hamada and Tsuruo, 1987, Anal. Biochem. 160:483-488; Hashida et al., 1984, J. Applied Biochem. 6:56-63.
[0040]Additionally, methods of cross-linking are reviewed by Means and Feeney, 1990, Bioconjugate Chem. 1:2-12.
Synthesis and Characterization of Targeting Retro-Inverted Peptides
[0041]Similar to that described in the above-referenced WO 98/51325, synthetic dansylated peptides were manufactured at Genosys Biotechnologies, UK and Anaspec Inc., USA. Characterisation profiles included appearance, solubility, HPLC and mass spectrometry (minimum purity>95%). Table 1 shows the primary sequences for both the retro-inverted peptide synthesized by reference to particular GIT targeting agents and the original GIT targeting agents themselves.
TABLE-US-00001 TABLE 1 Name Description Sequence ZElan144 PAX2 15 mer fragment - D K(dns)-rtrlrrnhsshkant form retroinversion ZElan145 P31 16 mer fragment - D K(dns)-gphrrgrpnsrsskrt form retroinversion ZElan146 HAX42 14 mer fragment - K(dns)-gtsngngccnydgp D form retroinversion ZElan129 PAX2 15 mer fragment K(dns)-TNAKHSSHNRRLRTR ZElan031 P31 16 mer fragment K(dns)- TRKSSRSNPRGRRHPG ZElan091 HAX42 14 mer fragment K(dns)-PGDYNCCGNGNSTG
Analysis of Binding of Dansylated Peptides to Caco-2 Cell Membrane Fractions by ELISA
[0042]Similar to the methods described in the above-referenced WO 98/51325, Caco-2 cell membrane (P100) and cytosolic (S100) fractions were prepared using a modification of the method described in Kinsella, B. T., O'Mahony, D. J. and G. A. FitzGerald, 1994, J. Biol. Chem. 269(47): 29914-29919. Confluent Caco-2 cell monolayers (grown in 75 cm2 flasks for up to 1 week at 37° C. and 5% CO2) were washed twice in Dulbecco's PBS (DPBS) and the cells were harvested by centrifugation at 1000 rpm after treatment with 10 mM EDTA-DPBS. The cells were washed 3 times in DPBS and the final cell pellet was resuspended in 3 volumes of ice cold HED buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride (PMSF)). The cells were allowed to swell for 5 min on ice prior to homogenization for 30 sec. The homogenates were centrifuged at 40,000 rpm for 45 min at 4° C. The supernatant (S100) was removed and the pellet (P100) was resuspended in HEDG buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 100 mM NaCl, 10% glycerol, 1 mM PMSF). Protein concentrations were determined using the Bradford assay (Bradford, M. M., 1976, Anal. Biochem. 72: 248-254).
[0043]Binding of peptides to membrane (P100) was assessed by detection of the dansyl moiety incorporated in the peptide. Costar ninety six well ELISA plates were coated with P100 fractions (100 μg/ml in 0.05 M NaHCO3 (pH 9.6); 100 μl/well) overnight at 4° C. The plates were blocked with 2% Marvel-DPBS for 1 h at room temperature and washed 3 times in 1% DPBS-Tween. Peptides (200 μg/ml in 2% Marvel-DPBS) were serially diluted on the plates and incubated for 1 h at room temperature. The plates were washed 5 times and the dansylated peptides were detected using i) mouse anti-dansyl antiserum (Cytogen DB3-226.3; 1:1340 dilution in 2% Marvel-DPBS) or ii) rabbit anti-dansyl antiserum (La Jolla Diagnostics LAJD-119; 1:1000 dilutions) for 1 h at room temperature. The plates were washed 3 times prior to incubation with i) goat anti-mouse IgGλ:HRP antibody (Southern Biotechnology 1060-05; 1:10,000 dilution in 2% Marvel-DPBS) or ii) anti-rabbit IgG HRP (Sigma A-0545, 1:8,000) for 1 h at room temperature. After 3 washes, reactions were visualized using K Blue Substrate and Red Stop Solution (Neogen Co. 300176 & 301475, respectively) at 650 nm.
[0044]ZElan021, full length HAX42 [K(dns)-SDHALGTNLRSDNAKEPG DYNCCGNGNSTGRKVFNRRRPSAIPT] was given the arbitrary value of 1.00 for binding to P100 at a given peptide concentration determined from the signal-to-noise ratio data. Table 2 shows the results of P100 assays with the HAX42 related peptides ZElan021, ZElan091 and ZElan146. Assay number 1 was at 20 μg/ml; 2 and 3 were at 50 μg/ml; and 4 through 8 were at 25 μg/ml. The results for the retro-inverted form, ZElan 146 show reasonable binding compared to the HAX42 fragment ZElan091 and that the activity of the GIT targeting agent was not eliminated when converted to its retro-inverted form.
TABLE-US-00002 TABLE 2 P100 assay number Peptide 1 2 3 4 5 6 7 8 ZElan021 1.00 1.00 1.00 1.00 1.00 1.00 1.14 0.94 ZElan091 2.02 1.37 1.20 0.85 ZElan146 0.36 0.72
[0045]KD values, or the concentration of the peptide required to reach half maximal binding to Caco-2 P100 fractions, are given in Table 3 for ZElan021, full length HAX42, [K(dns)-SDHALGTNLRSDNAKEPGDYNCCGNGNSTGR KVFNRRRPSAIPT], HAX42 fragment ZElan091, and the retro-inverted form of this fragment, ZElan146 and well as for ZElan018, full length PAX2, [K(dns)-STPPSREAYSRPYSVDS DSDTNAKHSSHNRRLRTRSRPNG], PAX2 fragment ZElan129, and the retro-inverted form of this fragment, ZElan144.
TABLE-US-00003 TABLE 3 KD Name Sequence (μmol) ZElan018 K(dns)-STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPNG >50.0 ZElan129 K(dns)-TNAKHSSHNRRLRTR 29.6 ZElan144 K(dns)-rtrlrrnhsshkant 28.8 ZElan021 K(dns)-SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPSAIPT 6.7 ZElan091 K(dns)-PGDYNCCGNGNSTG 0.75 ZElan146 K(dns)-gtsngngccnydgp 21.65
Manufacture and Analysis of Peptide-Coated Insulin Loaded PLGA Particles
[0046]Insulin-loaded PLGA particles are coated with retro-inverted peptides according to this invention or GIT targeting agents by the coacervation processes described in the above-referenced WO 98/51325. In particular, solid particles containing an active agent are formed from a polymer and have a particle size of between about 10 nm and 500 μm, most preferably 50 to 800 nm. In addition the particles contain targeting retro-inverted peptides which are incorporated into the particles using a number of methods as outlined below and described in the above-referenced WO 98/51325.
[0047]The organic phase (B) polymer of the general method given below may be soluble, permeable, impermeable, biodegradable or gastroretentive. The polymer may consist of a mixture of polymer or copolymers and may be a natural or synthetic polymer. Representative biodegradable polymers include without limitation polyglycolides; polylactides; poly(lactide-co-glycolides), including DL, L and D forms; copolyoxalates; polycaprolactone; polyesteramides; polyorthoesters; polyanhydrides; polyalkylcyanoacrylates; polyhydroxybutyrates; polyurethanes; albumin; casein; citosan derivatives; gelatin; acacia; celluloses; polysaccharides; alginic acid; polypeptides; and the like, copolymers thereof, mixtures thereof and stereoisomers thereof. Representative synthetic polymers include alkyl celluloses; hydroxalkyl celluloses; cellulose ethers; cellulose esters; nitrocelluloses; polymers of acrylic and methacrylic acids and esters thereof; dextrans; polyamides; polycarbonates; polyalkylenes; polyalkylene glycols; polyalkylene oxides; polyalkylene terephthalates; polyvinyl alcohols; polyvinyl ethers; polyvinyl esters; polyvinyl halides; poyvinylpyrrolidone; polysiloxanes and polyurethanes and co-polymers thereof.
[0048]Typically, particles are formed using the following general method:
[0049]An aqueous solution (A) of a polymer, surface active agent, surface stabilising or modifying agent or salt, or surfactant preferably a polyvinyl alcohol (PVA) or derivative with a % hydrolysis 50-100% and a molecular weight range 500-500,000, most preferably 80-100% hydrolysis and 10,000-150,000 molecular weight, is introduced into a vessel. The mixture (A) is stirred under low shear conditions at 10-2000 rpm, preferably 100-600 rpm. The pH and/or ionic strength of this solution may be modified using salts, buffers or other modifying agents. The viscosity of this solution may be modified using polymers, salts, or other viscosity enhancing or modifying agents.
[0050]A polymer, preferably poly(lacide-co-glycolide), polylactide, polyglycolide or a combination thereof or in any enantiomeric form or a covalent conjugate of the these polymers with a targeting ligand is dissolved in water miscible organic solvents to form organic phase (B). Most preferably, a combination of acetone and ethanol is used in a range of ratios from 0:100 acetone: ethanol to 100:0 acetone: ethanol depending upon the polymer used.
[0051]Additional polymer(s), peptide(s) sugars, salts, natural/biological polymers or other agents may also be added to the organic phase (B) to modify the physical and chemical properties of the resultant particle product.
[0052]A drug or bioactive substance may be introduced into either the aqueous phase (A) or the organic phase (B). A targeting retro-inversion peptide or GIT targeting agent may also be introduced into either the aqueous phase (A) or the organic phase (B) at this point.
[0053]The organic phase (B) is added into the stirred aqueous phase (A) at a continuous rate. The solvent is evaporated, preferably by a rise in temperature over ambient and/or the use of a vacuum pump. The particles are now present as a suspension (C). A targeting retro-inversion peptide or GIT targeting agent may be introduced into the stirred suspension at this point.
[0054]A secondary layer of polymer(s), peptide(s) sugars, salts, natural/biological polymers or other agents may be deposited on to the pre-formed particulate core by any suitable method at this stage.
[0055]The particles (D) are then separated from the suspension (C) using standard colloidal separation techniques, preferably by centrifugation at high `g` force, filtration, cross-flow filtration, gel permeation chromatography, affinity chromatography or charge separation techniques. The supernatant is discarded and the particles (D) re-suspended in a washing solution (E) preferably water, salt solution, buffer or organic solvent(s). The particles (D) are separated from the washing liquid in a similar manner as previously described and re-washed, commonly twice. A targeting ligand may be dissolved in washing solution (E) at the initial, intermediate and/or at the final washing stage and may be used to wash the particles (D).
[0056]The particles may then be dried. Particles may then be further processed for example, tabletted, encapsulated or spray dried.
[0057]The release profile of the particles formed above may be varied from immediate to controlled or delayed release dependent upon the formulation used and/or desired.
[0058]Drug loading may be in the range 0-90% w/w. Targeting retro-inversion peptide or GIT targeting agent loading may be in the range 0-90% w/w.
[0059]Insulin-loaded PLGA (RG504H) nanoparticles were manufactured as given above for the following targeting ligands: full length HAX42 (ZElan021), full length PAX2 (ZElan018), HAX42 fragment ZElan091, PAX2 fragment ZElan129, HAX42 fragment retro-inverted peptide ZElan146 and PAX2 fragment retro-inverted peptide ZElan144. Bovine insulin potency (HPLC) and peptide loading (dansyl fluorescence) were assessed prior to analysis of insulin delivery in Wistar rats using the open loop model. Table 4 shows the insulin potency and targeting peptide loading of the PLGA particles.
TABLE-US-00004 TABLE 4 Peptide Loading Peptide Insulin Potency (mg/g) (μg/mg) ZELAN018 47.0 1.68 ZELAN 129 59.0 0.85 ZELAN 144 58.2 0.68 ZELAN021 49.7 2.63 ZELAN 091 57.1 1.87 ZELAN 146 53.8 1.77
Animal Studies
[0060]In vivo assessment of oral insulin bioavailability of various targeting retro-inversion peptides was undertaken using open-loop studies study in which the test solution containing nanoparticles described above (Table 4) was injected directly into the ileum in Wistar rats similar to the protocols described in the above-referenced WO 98/51325. In short, Wistar rats (300-350 g) were fasted for 4 hours and anaesthetized by intramuscular administration 15 to 20 minutes prior to administration of the test solution with a solution of ketamine [0.525 ml of ketamine (100 mg/ml) and 0.875 ml of acepromazine maleate-BP ACP (2 mg/ml)]. The rats were then injected with a test solution (injection volume: 1.5 ml PBS) intra-duodenally at 2-3 cm below the pyloris. Insulin (fast-acting bovine; 28.1 iu/mg) was incorporated in the particles as described above for a total of 300 iu insulin (approximately 210 mg particles). Blood glucose values for the rats were measured using a Glucometer® (Bayer; 0.1 to 33.3 m/mol/L); plasma insulin values were measured using a Phadeseph RIA Kit® (Upjohn Pharmacia; 3 to 240 μU/ml-assayed in duplicate). Systemic and portal blood was sampled.
[0061]Study groups included animals receiving test solutions containing particles coated with the following peptides shown in Table 4. Control groups included: 1) PBS control (1.5 ml) Open-Loop; 2) Insulin solution (1 iu/0.2 ml) subcutaneous; 3) Insulin particles, no peptide.
[0062]Table 5 shows the insulin bioavailability for the insulin-loaded nanoparticles described above (surface modified with targeting retro-inversion peptide or GIT targeting agent) expressed as a % bioavailability of the administered oral dose compared to the reference insulin sub-cutaneous dose. FIGS. 1 and 2 show the (1) systemic blood glucose and (2) insulin levels following intestinal administration of control (PBS); ZElan 021 coated insulin-containing particles, ZElan 018 coated insulin-containing particles, ZElan091 coated insulin-containing particles, ZElan129 coated insulin-containing particles and ZElan 144 coated insulin-containing particles according to this invention (300 iu insulin loading).
TABLE-US-00005 TABLE 5 % Insulin Targeting Ligand Bio-availability HAX42 ZELAN021 12.6 PAX2 ZELAN018 13.04 HAX42 14 MER ZELAN 091 11.4 HAX42 14 MER ZELAN 146 (retro-inversion) 2.1 PAX2 15 MER ZELAN 144 (retro-inversion) 10.8 PAX2 15 MER ZELAN129 14.3
[0063]The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Sequence CWU
1
85115PRTArtificial SequencePAX2 15 mer fragment-D form retroinversion 1Arg
Thr Arg Leu Arg Arg Asn His Ser Ser His Lys Ala Asn Thr1 5
10 15216PRTArtificial SequenceP31 16
mer fragment- D form retroinversion 2Gly Pro His Arg Arg Gly Arg Pro Asn
Ser Arg Ser Ser Lys Arg Thr1 5 10
15314PRTArtificial SequenceHAX42 14 mer fragment-D form
retroinversion 3Gly Thr Ser Asn Gly Asn Gly Cys Cys Asn Tyr Asp Gly Pro1
5 10415PRTArtificial SequencePAX2 15 mer
fragment 4Thr Asn Ala Lys His Ser Ser His Asn Arg Arg Leu Arg Thr Arg1
5 10 15516PRTArtificial
SequenceP31 16 mer fragment 5Thr Arg Lys Ser Ser Arg Ser Asn Pro Arg Gly
Arg Arg His Pro Gly1 5 10
15614PRTArtificial SequenceHAX42 14 mer fragment 6Pro Gly Asp Tyr Asn
Cys Cys Gly Asn Gly Asn Ser Thr Gly1 5
10740PRTArtificial SequencePAX2 full length 7Ser Thr Pro Pro Ser Arg Glu
Ala Tyr Ser Arg Pro Tyr Ser Val Asp1 5 10
15Ser Asp Ser Asp Thr Asn Ala Lys His Ser Ser His Asn
Arg Arg Leu 20 25 30Arg Thr
Arg Ser Arg Pro Asn Gly 35 40845PRTArtificial
SequenceHAX42 full length, N-terminal Lysine is dansylated 8Lys Ser
Asp His Ala Leu Gly Thr Asn Leu Arg Ser Asp Asn Ala Lys1 5
10 15Glu Pro Gly Asp Tyr Asn Cys Cys
Gly Asn Gly Asn Ser Thr Gly Arg 20 25
30Lys Val Phe Asn Arg Arg Arg Pro Ser Ala Ile Pro Thr 35
40 45916PRTArtificial SequenceZElan 144;
PAX2 15 mer fragment-D form retroinversion with additional L-lysine
in position 1 9Lys Arg Thr Arg Leu Arg Arg Asn His Ser Ser His Lys Ala
Asn Thr1 5 10
151017PRTArtificial SequenceZElan 145; P31 16 mer fragment- D form
retroinversion with additional L-lysine in position 1 10Lys Gly Pro His
Arg Arg Gly Arg Pro Asn Ser Arg Ser Ser Lys Arg1 5
10 15Thr1115PRTArtificial SequenceZElan 146;
HAX42 14 mer fragment-D form retroinversion with additional
L-Lysine in position 1 11Lys Gly Thr Ser Asn Gly Asn Gly Cys Cys Asn Tyr
Asp Gly Pro1 5 10
151216PRTArtificial SequenceZElan 129; PAX2 15 mer fragment with
additional L-Lysine in position 1 12Lys Thr Asn Ala Lys His Ser Ser His
Asn Arg Arg Leu Arg Thr Arg1 5 10
151317PRTArtificial SequenceZElan 031; P31 16 mer fragment with
additional L-Lysine in position 1 13Lys Thr Arg Lys Ser Ser Arg Ser
Asn Pro Arg Gly Arg Arg His Pro1 5 10
15Gly1415PRTArtificial SequenceZElan 091; HAX42 14 mer
fragment with additional L-lysine in position 1 14Lys Pro Gly Asp
Tyr Asn Cys Cys Gly Asn Gly Asn Ser Thr Gly1 5
10 151541PRTArtificial SequencePAX2 full length,
N-terminal Lysine is dansylated 15Lys Ser Thr Pro Pro Ser Arg Glu
Ala Tyr Ser Arg Pro Tyr Ser Val1 5 10
15Asp Ser Asp Ser Asp Thr Asn Ala Lys His Ser Ser His Asn
Arg Arg 20 25 30Leu Arg Thr
Arg Ser Arg Pro Asn Gly 35 401644PRTArtificial
SequenceS15 44 mer fragment L-form 16Arg Ser Gly Ala Tyr Glu Ser Pro Asp
Gly Arg Gly Gly Arg Ser Tyr1 5 10
15Val Gly Gly Gly Gly Gly Cys Gly Asn Ile Gly Arg Lys His Asn
Leu 20 25 30Trp Gly Leu Arg
Thr Ala Ser Pro Ala Cys Trp Asp 35
401744PRTArtificial SequenceS21 44 mer fragment L-form 17Ser Pro Arg Ser
Phe Trp Pro Val Val Ser Arg His Glu Ser Phe Gly1 5
10 15Ile Ser Asn Tyr Leu Gly Cys Gly Tyr Arg
Thr Cys Ile Ser Gly Thr 20 25
30Met Thr Lys Ser Ser Pro Ile Tyr Pro Arg His Ser 35
401844PRTArtificial SequenceS22 44 mer fragment L-form 18Ser Ser Ser
Ser Asp Trp Gly Gly Val Pro Gly Lys Val Val Arg Glu1 5
10 15Arg Phe Lys Gly Arg Gly Cys Gly Ile
Ser Ile Thr Ser Val Leu Thr 20 25
30Gly Lys Pro Asn Pro Cys Pro Glu Pro Lys Ala Ala 35
401944PRTArtificial SequenceSni10 44 mer fragment L-form 19Arg
Val Gly Gln Cys Thr Asp Ser Asp Val Arg Arg Pro Trp Ala Arg1
5 10 15Ser Cys Ala His Gln Gly Cys
Gly Ala Gly Thr Arg Asn Ser His Gly 20 25
30Cys Ile Thr Arg Pro Leu Arg Gln Ala Ser Ala His 35
402039PRTArtificial SequenceSni28 39 mer fragment L-form
20Ser His Ser Gly Gly Met Asn Arg Ala Tyr Gly Asp Val Phe Arg Glu1
5 10 15Leu Arg Asp Arg Trp Asn
Ala Thr Ser His His Thr Arg Pro Thr Pro 20 25
30Gln Leu Pro Arg Gly Pro Asn 352141PRTArtificial
SequenceSni34 41 mer fragment L-form 21Ser Pro Cys Gly Gly Ser Trp Gly
Arg Phe Met Gln Gly Gly Leu Phe1 5 10
15Gly Gly Arg Thr Asp Gly Cys Gly Ala His Arg Asn Arg Thr
Ser Ala 20 25 30Ser Leu Glu
Pro Pro Ser Ser Asp Tyr 35 402239PRTArtificial
SequenceSni38 39 mer fragment L-form 22Arg Gly Ala Ala Asp Gln Arg Arg
Gly Trp Ser Glu Asn Leu Gly Leu1 5 10
15Pro Arg Val Gly Trp Asp Ala Ile Ala His Asn Ser Tyr Thr
Phe Thr 20 25 30Ser Arg Arg
Pro Arg Pro Pro 352344PRTArtificial SequenceSni45 44 mer fragment
L-form 23Ser Gly Gly Glu Val Ser Ser Trp Gly Arg Val Asn Asp Leu Cys Ala1
5 10 15Arg Val Ser Trp
Thr Gly Cys Gly Thr Ala Arg Ser Ala Arg Thr Asp 20
25 30Asn Lys Gly Phe Leu Pro Lys His Ser Ser Leu
Arg 35 402444PRTArtificial SequenceSniAX2 44 mer
fragment L-form 24Ser Asp Ser Asp Gly Asp His Tyr Gly Leu Arg Gly Gly Val
Arg Cys1 5 10 15Ser Leu
Arg Asp Arg Gly Cys Gly Leu Ala Leu Ser Thr Val His Ala 20
25 30Gly Pro Pro Ser Phe Tyr Pro Lys Leu
Ser Ser Pro 35 402539PRTArtificial SequenceSniAX4
39 mer fragment L-form 25Arg Ser Leu Gly Asn Tyr Gly Val Thr Gly Thr Val
Asp Val Thr Val1 5 10
15Leu Pro Met Pro Gly His Ala Asn His Leu Gly Val Ser Ser Ala Ser
20 25 30Ser Ser Asp Pro Pro Arg Arg
352638PRTArtificial SequenceSniAX6 38 mer fragment L-form 26Arg Thr
Thr Thr Ala Lys Gly Cys Leu Leu Gly Ser Phe Gly Val Leu1 5
10 15Ser Gly Cys Ser Phe Thr Pro Thr
Ser Pro Pro Pro His Leu Gly Tyr 20 25
30Pro Pro His Ser Val Asn 352739PRTArtificial
SequenceSniAX8 39 mer fragment L-form 27Ser Pro Lys Leu Ser Ser Val Gly
Val Met Thr Lys Val Thr Glu Leu1 5 10
15Pro Thr Glu Gly Pro Asn Ala Ile Ser Ile Pro Ile Ser Ala
Thr Leu 20 25 30Gly Pro Arg
Asn Pro Leu Arg 352839PRTArtificial SequenceDAB3 39 mer fragment
L-form 28Arg Trp Cys Gly Ala Glu Leu Cys Asn Ser Val Thr Lys Lys Phe Arg1
5 10 15Pro Gly Trp Arg
Asp His Ala Asn Pro Ser Thr His His Arg Thr Pro 20
25 30Pro Pro Ser Gln Ser Ser Pro
352944PRTArtificial SequenceDAB7 44 mer fragment L-form 29Arg Trp Cys Gly
Ala Asp Asp Pro Cys Gly Ala Ser Arg Trp Arg Gly1 5
10 15Gly Asn Ser Leu Phe Gly Cys Gly Leu Arg
Cys Ser Ala Ala Gln Ser 20 25
30Thr Pro Ser Gly Arg Ile His Ser Thr Ser Thr Ser 35
403039PRTArtificial SequenceDAB10 39 mer fragment L-form 30Ser Lys
Ser Gly Glu Gly Gly Asp Ser Ser Arg Gly Glu Thr Gly Trp1 5
10 15Ala Arg Val Arg Ser His Ala Met
Thr Ala Gly Arg Phe Arg Trp Tyr 20 25
30Asn Gln Leu Pro Ser Asp Arg 353138PRTArtificial
SequenceDAB18 38 mer fragment L-form 31Arg Ser Ser Ala Asn Asn Cys Glu
Trp Lys Ser Asp Trp Met Arg Arg1 5 10
15Ala Cys Ile Ala Arg Tyr Ala Asn Ser Ser Gly Pro Ala Arg
Ala Val 20 25 30Asp Thr Lys
Ala Ala Pro 353244PRTArtificial SequenceDAB24 44 mer fragment
L-form 32Ser Lys Trp Ser Trp Ser Ser Arg Trp Gly Ser Pro Gln Asp Lys Val1
5 10 15Glu Lys Thr Arg
Ala Gly Cys Gly Gly Ser Pro Ser Ser Thr Asn Cys 20
25 30His Pro Tyr Thr Phe Ala Pro Pro Pro Gln Ala
Gly 35 403344PRTArtificial SequenceDAB30 44 mer
fragment L-form 33Ser Gly Phe Trp Glu Phe Ser Arg Gly Leu Trp Asp Gly Glu
Asn Arg1 5 10 15Lys Ser
Val Arg Ser Gly Cys Gly Phe Arg Gly Ser Ser Ala Gln Gly 20
25 30Pro Cys Pro Val Thr Pro Ala Thr Ile
Asp Lys His 35 403444PRTArtificial SequenceDAX15
44 mer fragment L-form 34Ser Glu Ser Gly Arg Cys Arg Ser Val Ser Arg Trp
Met Thr Thr Trp1 5 10
15Gln Thr Gln Lys Gly Gly Cys Gly Ser Asn Val Ser Arg Gly Ser Pro
20 25 30Leu Asp Pro Ser His Gln Thr
Gly His Ala Thr Thr 35 403539PRTArtificial
SequenceDAX23 39 mer fragment L-form 35Arg Glu Trp Arg Phe Ala Gly Pro
Pro Leu Asp Leu Trp Ala Gly Pro1 5 10
15Ser Leu Pro Ser Phe Asn Ala Ser Ser His Pro Arg Ala Leu
Arg Thr 20 25 30Tyr Trp Ser
Gln Arg Pro Arg 353644PRTArtificial SequenceDAX24 44 mer fragment
L-form 36Arg Met Glu Asp Ile Lys Asn Ser Gly Trp Arg Asp Ser Cys Arg Trp1
5 10 15Gly Asp Leu Arg
Pro Gly Cys Gly Ser Arg Gln Trp Tyr Pro Ser Asn 20
25 30Met Arg Ser Ser Arg Asp Tyr Pro Ala Gly Gly
His 35 403736PRTArtificial SequenceDAX27 36 mer
fragment L-form 37Ser His Pro Trp Tyr Arg His Trp Asn His Gly Asp Phe Ser
Gly Ser1 5 10 15Gly Gln
Ser Arg His Thr Pro Pro Glu Ser Pro His Pro Gly Arg Pro 20
25 30Asn Ala Thr Ile
353844PRTArtificial SequenceDCX8 44 mer fragment L-form 38Arg Tyr Lys His
Asp Ile Gly Cys Asp Ala Gly Val Asp Lys Lys Ser1 5
10 15Ser Ser Val Arg Gly Gly Cys Gly Ala His
Ser Ser Pro Pro Arg Ala 20 25
30Gly Arg Gly Pro Arg Gly Thr Met Val Ser Arg Leu 35
403944PRTArtificial SequenceDCX11 44 mer fragment L-form 39Ser Gln
Gly Ser Lys Gln Cys Met Gln Tyr Arg Thr Gly Arg Leu Thr1 5
10 15Val Gly Ser Glu Tyr Gly Cys Gly
Met Asn Pro Ala Arg His Ala Thr 20 25
30Pro Ala Tyr Pro Ala Arg Leu Leu Pro Arg Tyr Arg 35
404044PRTArtificial SequenceDCX26 44 mer fragment L-form
40Ser Gly Arg Thr Thr Ser Glu Ile Ser Gly Leu Trp Gly Trp Gly Asp1
5 10 15Asp Arg Ser Gly Tyr Gly
Trp Gly Asn Thr Leu Arg Pro Asn Tyr Ile 20 25
30Pro Tyr Arg Gln Ala Thr Asn Arg His Arg Tyr Thr
35 404139PRTArtificial SequenceDCX33 39 mer fragment
L-form 41Arg Trp Asn Trp Thr Val Leu Pro Ala Thr Gly Gly His Tyr Trp Thr1
5 10 15Arg Ser Thr Asp
Tyr His Ala Ile Asn Asn His Arg Pro Ser Ile Pro 20
25 30His Gln His Pro Thr Pro Ile
354244PRTArtificial SequenceDCX36 44 mer fragment L-form 42Ser Trp Ser
Ser Trp Asn Trp Ser Ser Lys Thr Thr Arg Leu Gly Asp1 5
10 15Arg Ala Thr Arg Glu Gly Cys Gly Pro
Ser Gln Ser Asp Gly Cys Pro 20 25
30Tyr Asn Gly Arg Leu Thr Thr Val Lys Pro Arg Thr 35
404337PRTArtificial SequenceDCX39 37 mer fragment L-form 43Ser
Gly Ser Leu Asn Ala Trp Gln Pro Arg Ser Trp Val Gly Gly Ala1
5 10 15Phe Arg Ser His Ala Asn Asn
Asn Leu Asn Pro Lys Pro Thr Met Val 20 25
30Thr Arg His Pro Thr 354444PRTArtificial
SequenceDCX42 44 mer fragment L-form 44Arg Tyr Ser Gly Leu Ser Pro Arg
Asp Asn Gly Pro Ala Cys Ser Gln1 5 10
15Glu Ala Thr Leu Glu Gly Cys Gly Ala Gln Arg Leu Met Ser
Thr Arg 20 25 30Arg Lys Gly
Arg Asn Ser Arg Pro Gly Trp Thr Leu 35
404539PRTArtificial SequenceDCX45 39 mer fragment L-form 45Ser Val Gly
Asn Asp Lys Thr Ser Arg Pro Val Ser Phe Tyr Gly Arg1 5
10 15Val Ser Asp Leu Trp Asn Ala Ser Leu
Met Pro Lys Arg Thr Pro Ser 20 25
30Ser Lys Arg His Asp Asp Gly 354638PRTArtificial
SequencePAX9 38 mer fragment L-form 46Arg Trp Pro Ser Val Gly Tyr Lys Gly
Asn Gly Ser Asp Thr Ile Asp1 5 10
15Val His Ser Asn Asp Ala Ser Thr Lys Arg Ser Leu Ile Tyr Asn
His 20 25 30Arg Arg Pro Leu
Phe Pro 354739PRTArtificial SequencePAX14 39 mer fragment L-form
47Arg Thr Phe Glu Asn Asp Gly Leu Gly Val Gly Arg Ser Ile Gln Lys1
5 10 15Lys Ser Asp Arg Trp Tyr
Ala Ser His Asn Ile Arg Ser His Phe Ala 20 25
30Ser Met Ser Pro Ala Gly Lys 354844PRTArtificial
SequencePAX15 44 mer fragment L-form 48Ser Tyr Cys Arg Val Lys Gly Gly
Gly Glu Gly Gly His Thr Asp Ser1 5 10
15Asn Leu Ala Arg Ser Gly Cys Gly Lys Val Ala Arg Thr Ser
Arg Leu 20 25 30Gln His Ile
Asn Pro Arg Ala Thr Pro Pro Ser Arg 35
404939PRTArtificial SequencePAX16 39 mer fragment L-form 49Ser Trp Thr
Arg Trp Gly Lys His Thr His Gly Gly Phe Val Asn Lys1 5
10 15Ser Pro Pro Gly Lys Asn Ala Thr Ser
Pro Tyr Thr Asp Ala Gln Leu 20 25
30Pro Ser Asp Gln Gly Pro Pro 355044PRTArtificial
SequencePAX17 44 mer fragment L-form 50Ser Gln Val Asp Ser Phe Arg Asn
Ser Phe Arg Trp Tyr Glu Pro Ser1 5 10
15Arg Ala Leu Cys His Gly Cys Gly Lys Arg Asp Thr Ser Thr
Thr Arg 20 25 30Ile His Asn
Ser Pro Ser Asp Ser Tyr Pro Thr Arg 35
405139PRTArtificial SequencePAX18 39 mer fragment L-form 51Ser Phe Leu
Arg Phe Gln Ser Pro Arg Phe Glu Asp Tyr Ser Arg Thr1 5
10 15Ile Ser Arg Leu Arg Asn Ala Thr Asn
Pro Ser Asn Val Ser Asp Ala 20 25
30His Asn Asn Arg Ala Leu Ala 355239PRTArtificial
SequencePAX35 39 mer fragment L-form 52Arg Ser Ile Thr Asp Gly Gly Leu
Asn Glu Val Asp Leu Ser Ser Val1 5 10
15Ser Asn Val Leu Glu Asn Ala Asn Ser His Arg Ala Tyr Arg
Lys His 20 25 30Arg Pro Thr
Leu Lys Arg Pro 355344PRTArtificial SequencePAX38 44 mer fragment
L-form 53Ser Ser Lys Val Ser Ser Pro Arg Asp Pro Thr Val Pro Arg Lys Gly1
5 10 15Gly Asn Val Asp
Tyr Gly Cys Gly His Arg Ser Ser Ala Arg Met Pro 20
25 30Thr Ser Ala Leu Ser Ser Ile Thr Lys Cys Tyr
Thr 35 405444PRTArtificial SequencePAX40 44 mer
fragment L-form 54Arg Ala Ser Thr Gln Gly Gly Arg Gly Val Ala Pro Glu Phe
Gly Ala1 5 10 15Ser Val
Leu Gly Arg Gly Cys Gly Ser Ala Thr Tyr Tyr Thr Asn Ser 20
25 30Thr Ser Cys Lys Asp Ala Met Gly His
Asn Tyr Ser 35 405539PRTArtificial SequencePAX43
39 mer fragment L-form 55Arg Trp Cys Glu Lys His Lys Phe Thr Ala Ala Arg
Cys Ser Ala Gly1 5 10
15Ala Gly Phe Glu Arg Asp Ala Ser Arg Pro Pro Gln Pro Ala His Arg
20 25 30Asp Asn Thr Asn Arg Asn Ala
355639PRTArtificial SequencePAX45 39 mer fragment L-form 56Ser Phe
Gln Val Tyr Pro Asp His Gly Leu Glu Arg His Ala Leu Asp1 5
10 15Gly Thr Gly Pro Leu Tyr Ala Met
Pro Gly Arg Trp Leu Arg Ala Arg 20 25
30Pro Gln Asn Arg Asp Arg Gln 355738PRTArtificial
SequencePAX46 38 mer fragment L-form 57Ser Arg Cys Thr Asp Asn Glu Gln
Cys Pro Asp Thr Gly Thr Arg Ser1 5 10
15Arg Ser Val Ser Asn Ala Arg Tyr Phe Ser Ser Arg Leu Leu
Lys Thr 20 25 30His Ala Pro
His Arg Pro 355839PRTArtificial SequenceP31 39 mer fragment L-form
58Ser Ala Arg Asp Ser Gly Pro Ala Glu Asp Gly Ser Arg Ala Val Arg1
5 10 15Leu Asn Gly Val Glu Asn
Ala Asn Thr Arg Lys Ser Ser Arg Ser Asn 20 25
30Pro Arg Gly Arg Arg His Pro 355944PRTArtificial
SequenceP90 44 mer fragment L-form 59Ser Ser Ala Asp Ala Glu Lys Cys Ala
Gly Ser Leu Leu Trp Trp Gly1 5 10
15Arg Gln Asn Asn Ser Gly Cys Gly Ser Pro Thr Lys Lys His Leu
Lys 20 25 30His Arg Asn Arg
Ser Gln Thr Ser Ser Ser Ser His 35
406039PRTArtificial Sequence5PAX3 39 mer fragment L-form 60Arg Pro Lys
Asn Val Ala Asp Ala Tyr Ser Ser Gln Asp Gly Ala Ala1 5
10 15Ala Glu Glu Thr Ser His Ala Ser Asn
Ala Ala Arg Lys Ser Pro Lys 20 25
30His Lys Pro Leu Arg Arg Pro 356139PRTArtificial
Sequence5PAX5 39 mer fragment L-form 61Arg Gly Ser Thr Gly Thr Ala Gly
Gly Glu Arg Ser Gly Val Leu Asn1 5 10
15Leu His Thr Arg Asp Asn Ala Ser Gly Ser Gly Phe Lys Pro
Trp Tyr 20 25 30Pro Ser Asn
Arg Gly His Lys 356239PRTArtificial Sequence5PAX7 39 mer fragment
L-form 62Arg Trp Gly Trp Glu Arg Ser Pro Ser Asp Tyr Asp Ser Asp Met Asp1
5 10 15Leu Gly Ala Arg
Arg Tyr Ala Thr Arg Thr His Arg Ala Pro Pro Arg 20
25 30Val Leu Lys Ala Pro Leu Pro
356344PRTArtificial Sequence5PAX12 44 mer fragment L-form 63Arg Gly Trp
Lys Cys Glu Gly Ser Gln Ala Ala Tyr Gly Asp Lys Asp1 5
10 15Ile Gly Arg Ser Arg Gly Cys Gly Ser
Ile Thr Lys Asn Asn Thr Asn 20 25
30His Ala His Pro Ser His Gly Ala Val Ala Lys Ile 35
406439PRTArtificial SequenceHAX9 39 mer fragment L-form 64Ser Arg
Glu Glu Ala Asn Trp Asp Gly Tyr Lys Arg Glu Met Ser His1 5
10 15Arg Ser Arg Phe Trp Asp Ala Thr
His Leu Ser Arg Pro Arg Arg Pro 20 25
30Ala Asn Ser Gly Asp Pro Asn 356544PRTArtificial
SequenceHAX35 44 mer fragment L-form 65Glu Trp Tyr Ser Trp Lys Arg Ser
Ser Lys Ser Thr Gly Leu Gly Asp1 5 10
15Thr Ala Thr Arg Glu Gly Cys Gly Pro Ser Gln Ser Asp Gly
Cys Pro 20 25 30Tyr Asn Gly
Arg Leu Thr Thr Val Lys Pro Arg Lys 35
406644PRTArtificial SequenceHAX40 44 mer fragment L-form 66Arg Glu Phe
Ala Glu Arg Arg Leu Trp Gly Cys Asp Asp Leu Ser Trp1 5
10 15Arg Leu Asp Ala Glu Gly Cys Gly Pro
Thr Pro Ser Asn Arg Ala Val 20 25
30Lys His Arg Lys Pro Arg Pro Arg Ser Pro Ala Leu 35
406744PRTArtificial SequenceHAX42 44 mer fragment L-form 67Ser
Asp His Ala Leu Gly Thr Asn Leu Arg Ser Asp Asn Ala Lys Glu1
5 10 15Pro Gly Asp Tyr Asn Cys Cys
Gly Asn Gly Asn Ser Thr Gly Arg Lys 20 25
30Val Phe Asn Arg Arg Arg Pro Ser Ala Ile Pro Thr 35
406844PRTArtificial SequenceHCA3 44 mer fragment L-form
68Arg His Ile Ser Glu Tyr Ser Phe Ala Asn Ser His Leu Met Gly Gly1
5 10 15Glu Ser Lys Arg Lys Gly
Cys Gly Ile Asn Gly Ser Phe Ser Pro Thr 20 25
30Cys Pro Arg Ser Pro Thr Pro Ala Phe Arg Arg Thr
35 406938PRTArtificial SequenceH40 38 mer fragment
L-form 69Ser Arg Glu Ser Gly Met Trp Gly Ser Trp Trp Arg Gly His Arg Leu1
5 10 15Asn Ser Thr Gly
Gly Asn Ala Asn Met Asn Ala Ser Leu Pro Pro Asp 20
25 30Pro Pro Val Ser Thr Pro
357039PRTArtificial SequencePAX2 39 mer fragment L-form 70Ser Thr Pro Pro
Ser Arg Glu Ala Tyr Ser Arg Pro Tyr Ser Val Asp1 5
10 15Ser Asp Ser Asp Thr Asn Ala Lys His Ser
Ser His Asn Arg Arg Leu 20 25
30Arg Thr Arg Ser Arg Pro Asn 35711827PRTArtificial SequencehSI
receptor 71Met Ala Arg Lys Lys Phe Ser Gly Leu Glu Ile Ser Leu Ile Val
Leu1 5 10 15Phe Val Ile
Val Thr Ile Ile Ala Ile Ala Leu Ile Val Val Leu Ala 20
25 30Thr Lys Thr Pro Ala Val Asp Glu Ile Ser
Asp Ser Thr Ser Thr Pro 35 40
45Ala Thr Thr Arg Val Thr Thr Asn Pro Ser Asp Ser Gly Lys Cys Pro 50
55 60Asn Val Leu Asn Asp Pro Val Asn Val
Arg Ile Asn Cys Ile Pro Glu65 70 75
80Gln Phe Pro Thr Glu Gly Ile Cys Ala Gln Arg Gly Cys Cys
Trp Arg 85 90 95Pro Trp
Asn Asp Ser Leu Ile Pro Trp Cys Phe Phe Val Asp Asn His 100
105 110Gly Tyr Asn Val Gln Asp Met Thr Thr
Thr Ser Ile Gly Val Glu Ala 115 120
125Lys Leu Asn Arg Ile Pro Ser Pro Thr Leu Phe Gly Asn Asp Ile Asn
130 135 140Ser Val Leu Phe Thr Thr Gln
Asn Gln Thr Pro Asn Arg Phe Arg Phe145 150
155 160Lys Ile Thr Asp Pro Asn Asn Arg Arg Tyr Glu Val
Pro His Gln Tyr 165 170
175Val Lys Glu Phe Thr Gly Pro Thr Val Ser Asp Thr Leu Tyr Asp Val
180 185 190Lys Val Ala Gln Asn Pro
Phe Ser Ile Gln Val Ile Arg Lys Ser Asn 195 200
205Gly Lys Thr Leu Phe Asp Thr Ser Ile Gly Pro Leu Val Tyr
Ser Asp 210 215 220Gln Tyr Leu Gln Ile
Ser Ala Arg Leu Pro Ser Asp Tyr Ile Tyr Gly225 230
235 240Ile Gly Glu Gln Val His Lys Arg Phe Arg
His Asp Leu Ser Trp Lys 245 250
255Thr Trp Pro Ile Phe Thr Arg Asp Gln Leu Pro Gly Asp Asn Asn Asn
260 265 270Asn Leu Tyr Gly His
Gln Thr Phe Phe Met Cys Ile Glu Asp Thr Ser 275
280 285Gly Lys Ser Phe Gly Val Phe Leu Met Asn Ser Asn
Ala Met Glu Ile 290 295 300Phe Ile Gln
Pro Thr Pro Ile Val Thr Tyr Arg Val Thr Gly Gly Ile305
310 315 320Leu Asp Phe Tyr Ile Leu Leu
Gly Asp Thr Pro Glu Gln Val Val Gln 325
330 335Gln Tyr Gln Gln Leu Val Gly Leu Pro Ala Met Pro
Ala Tyr Trp Asn 340 345 350Leu
Gly Phe Gln Leu Ser Arg Trp Asn Tyr Lys Ser Leu Asp Val Val 355
360 365Lys Glu Val Val Arg Arg Asn Arg Glu
Ala Gly Ile Pro Phe Asp Thr 370 375
380Gln Val Thr Asp Ile Asp Tyr Met Glu Asp Lys Lys Asp Phe Thr Tyr385
390 395 400Asp Gln Val Ala
Phe Asn Gly Leu Pro Gln Phe Val Gln Asp Leu His 405
410 415Asp His Gly Gln Lys Tyr Val Ile Ile Leu
Asp Pro Ala Ile Ser Ile 420 425
430Gly Arg Arg Ala Asn Gly Thr Thr Tyr Ala Thr Tyr Glu Arg Gly Asn
435 440 445Thr Gln His Val Trp Ile Asn
Glu Ser Asp Gly Ser Thr Pro Ile Ile 450 455
460Gly Glu Val Trp Pro Gly Leu Thr Val Tyr Pro Asp Phe Thr Asn
Pro465 470 475 480Asn Cys
Ile Asp Trp Trp Ala Asn Glu Cys Ser Ile Phe His Gln Glu
485 490 495Val Gln Tyr Asp Gly Leu Trp
Ile Asp Met Asn Glu Val Ser Ser Phe 500 505
510Ile Gln Gly Ser Thr Lys Gly Cys Asn Val Asn Lys Leu Asn
Tyr Pro 515 520 525Pro Phe Thr Pro
Asp Ile Leu Asp Lys Leu Met Tyr Ser Lys Thr Ile 530
535 540Cys Met Asp Ala Val Gln Asn Trp Gly Lys Gln Tyr
Asp Val His Ser545 550 555
560Leu Tyr Gly Tyr Ser Met Ala Ile Ala Thr Glu Gln Ala Val Gln Lys
565 570 575Val Phe Pro Asn Lys
Arg Ser Phe Ile Leu Thr Arg Ser Thr Phe Ala 580
585 590Gly Ser Gly Arg His Ala Ala His Trp Leu Gly Asp
Asn Thr Ala Ser 595 600 605Trp Glu
Gln Met Glu Trp Ser Ile Thr Gly Met Leu Glu Phe Ser Leu 610
615 620Phe Gly Ile Pro Leu Val Gly Ala Asp Ile Cys
Gly Phe Val Ala Glu625 630 635
640Thr Thr Glu Glu Leu Cys Arg Arg Trp Met Gln Leu Gly Ala Phe Tyr
645 650 655Pro Phe Ser Arg
Asn His Asn Ser Asp Gly Tyr Glu His Gln Asp Pro 660
665 670Ala Phe Phe Gly Gln Asn Ser Leu Leu Val Lys
Ser Ser Arg Gln Tyr 675 680 685Leu
Thr Ile Arg Tyr Thr Leu Leu Pro Phe Leu Tyr Thr Leu Phe Tyr 690
695 700Lys Ala His Val Phe Gly Glu Thr Val Ala
Arg Pro Val Leu His Glu705 710 715
720Phe Tyr Glu Asp Thr Asn Ser Trp Ile Glu Asp Thr Glu Phe Leu
Trp 725 730 735Gly Pro Ala
Leu Leu Ile Thr Pro Val Leu Lys Gln Gly Ala Asp Thr 740
745 750Val Ser Ala Tyr Ile Pro Asp Ala Ile Trp
Tyr Asp Tyr Glu Ser Gly 755 760
765Ala Lys Arg Pro Trp Arg Lys Gln Arg Val Asp Met Tyr Leu Pro Ala 770
775 780Asp Lys Ile Gly Leu His Leu Arg
Gly Gly Tyr Ile Ile Pro Ile Gln785 790
795 800Glu Pro Asp Val Thr Thr Thr Ala Ser Arg Lys Asn
Pro Leu Gly Leu 805 810
815Ile Val Ala Leu Gly Glu Asn Asn Thr Ala Lys Gly Asp Phe Phe Trp
820 825 830Asp Asp Gly Glu Thr Lys
Asp Thr Ile Gln Asn Gly Asn Tyr Ile Leu 835 840
845Tyr Thr Phe Ser Val Ser Asn Asn Thr Leu Asp Ile Val Cys
Thr His 850 855 860Ser Ser Tyr Gln Glu
Gly Thr Thr Leu Ala Phe Gln Thr Val Lys Ile865 870
875 880Leu Gly Leu Thr Asp Ser Val Thr Glu Val
Arg Val Ala Glu Asn Asn 885 890
895Gln Pro Met Asn Ala His Ser Asn Phe Thr Tyr Asp Ala Ser Asn Gln
900 905 910Val Leu Leu Ile Ala
Asp Leu Lys Leu Asn Leu Gly Arg Asn Phe Ser 915
920 925Val Gln Trp Asn Gln Ile Phe Ser Glu Asn Glu Arg
Phe Asn Cys Tyr 930 935 940Pro Asp Ala
Asp Leu Ala Thr Glu Gln Lys Cys Thr Gln Arg Gly Cys945
950 955 960Val Trp Arg Thr Gly Ser Ser
Leu Ser Lys Ala Pro Glu Cys Tyr Phe 965
970 975Pro Arg Gln Asp Asn Ser Tyr Ser Val Asn Ser Ala
Arg Tyr Ser Ser 980 985 990Met
Gly Ile Thr Ala Asp Leu Gln Leu Asn Thr Ala Asn Ala Arg Ile 995
1000 1005Lys Leu Pro Ser Asp Pro Ile Ser
Thr Leu Arg Val Glu Val Lys 1010 1015
1020Tyr His Lys Asn Asp Met Leu Gln Phe Lys Ile Tyr Asp Pro Gln
1025 1030 1035Lys Lys Arg Tyr Glu Val
Pro Val Pro Leu Asn Ile Pro Thr Thr 1040 1045
1050Pro Ile Ser Thr Tyr Glu Asp Arg Leu Tyr Asp Val Glu Ile
Lys 1055 1060 1065Glu Asn Pro Phe Gly
Ile Gln Ile Arg Arg Arg Ser Ser Gly Arg 1070 1075
1080Val Ile Trp Asp Ser Trp Leu Pro Gly Phe Ala Phe Asn
Asp Gln 1085 1090 1095Phe Ile Gln Ile
Ser Thr Arg Leu Pro Ser Glu Tyr Ile Tyr Gly 1100
1105 1110Phe Gly Glu Val Glu His Thr Ala Phe Lys Arg
Asp Leu Asn Trp 1115 1120 1125Asn Thr
Trp Gly Met Phe Thr Arg Asp Gln Pro Pro Gly Tyr Lys 1130
1135 1140Leu Asn Ser Tyr Gly Phe His Pro Tyr Tyr
Met Ala Leu Glu Glu 1145 1150 1155Glu
Gly Asn Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met 1160
1165 1170Asp Val Thr Phe Gln Pro Thr Pro Ala
Leu Thr Tyr Arg Thr Val 1175 1180
1185Gly Gly Ile Leu Asp Phe Tyr Met Phe Leu Gly Pro Thr Pro Gln
1190 1195 1200Val Ala Thr Lys Gln Tyr
His Glu Val Ile Gly His Pro Val Met 1205 1210
1215Pro Ala Tyr Trp Ala Leu Gly Phe Gln Leu Cys Arg Tyr Gly
Tyr 1220 1225 1230Ala Asn Thr Ser Glu
Val Arg Glu Leu Tyr Asp Ala Met Val Ala 1235 1240
1245Ala Asn Ile Pro Tyr Asp Val Gln Tyr Thr Asp Ile Asp
Tyr Met 1250 1255 1260Glu Arg Gln Leu
Asp Phe Thr Ile Gly Glu Ala Phe Gln Asp Leu 1265
1270 1275Pro Gln Phe Val Asp Lys Ile Arg Gly Glu Gly
Met Arg Tyr Ile 1280 1285 1290Ile Ile
Leu Asp Pro Ala Ile Ser Gly Asn Glu Thr Lys Thr Tyr 1295
1300 1305Pro Ala Phe Glu Arg Gly Gln Gln Asn Asp
Val Phe Val Lys Trp 1310 1315 1320Pro
Asn Thr Asn Asp Ile Cys Trp Ala Lys Val Trp Pro Asp Leu 1325
1330 1335Pro Asn Ile Thr Ile Asp Lys Thr Leu
Thr Glu Asp Glu Ala Val 1340 1345
1350Asn Ala Ser Arg Ala His Val Ala Phe Pro Asp Phe Phe Arg Thr
1355 1360 1365Ser Thr Ala Glu Trp Trp
Ala Arg Glu Ile Val Asp Phe Tyr Asn 1370 1375
1380Glu Lys Met Lys Phe Asp Gly Leu Trp Ile Asp Met Asn Glu
Pro 1385 1390 1395Ser Ser Phe Val Asn
Gly Thr Thr Thr Asn Gln Cys Arg Asn Asp 1400 1405
1410Glu Leu Asn Tyr Pro Pro Tyr Phe Pro Glu Leu Thr Lys
Arg Thr 1415 1420 1425Asp Gly Leu His
Phe Arg Thr Ile Cys Met Glu Ala Glu Gln Ile 1430
1435 1440Leu Ser Asp Gly Thr Ser Val Leu His Tyr Asp
Val His Asn Leu 1445 1450 1455Tyr Gly
Trp Ser Gln Met Lys Pro Thr His Asp Ala Leu Gln Lys 1460
1465 1470Thr Thr Gly Lys Arg Gly Ile Val Ile Ser
Arg Ser Thr Tyr Pro 1475 1480 1485Thr
Ser Gly Arg Trp Gly Gly His Trp Leu Gly Asp Asn Tyr Ala 1490
1495 1500Arg Trp Asp Asn Met Asp Lys Ser Ile
Ile Gly Met Met Glu Phe 1505 1510
1515Ser Leu Phe Gly Ile Ser Tyr Thr Gly Ala Asp Ile Cys Gly Phe
1520 1525 1530Phe Asn Asn Ser Glu Tyr
His Leu Cys Thr Arg Trp Met Gln Leu 1535 1540
1545Gly Ala Phe Tyr Pro Tyr Ser Arg Asn His Asn Ile Ala Asn
Thr 1550 1555 1560Arg Arg Gln Asp Pro
Ala Ser Trp Asn Glu Thr Phe Ala Glu Met 1565 1570
1575Ser Arg Asn Ile Leu Asn Ile Arg Tyr Thr Leu Leu Pro
Tyr Phe 1580 1585 1590Tyr Thr Gln Met
His Glu Ile His Ala Asn Gly Gly Thr Val Ile 1595
1600 1605Arg Pro Leu Leu His Glu Phe Phe Asp Glu Lys
Pro Thr Trp Asp 1610 1615 1620Ile Phe
Lys Gln Phe Leu Trp Gly Pro Ala Phe Met Val Thr Pro 1625
1630 1635Val Leu Glu Pro Tyr Val Gln Thr Val Asn
Ala Tyr Val Pro Asn 1640 1645 1650Ala
Arg Trp Phe Asp Tyr His Thr Gly Lys Asp Ile Gly Val Arg 1655
1660 1665Gly Gln Phe Gln Thr Phe Asn Ala Ser
Tyr Asp Thr Ile Asn Leu 1670 1675
1680His Val Arg Gly Gly His Ile Leu Pro Cys Gln Glu Pro Ala Gln
1685 1690 1695Asn Thr Phe Tyr Ser Arg
Gln Lys His Met Lys Leu Ile Val Ala 1700 1705
1710Ala Asp Asp Asn Gln Met Ala Gln Gly Ser Leu Phe Trp Asp
Asp 1715 1720 1725Gly Glu Ser Ile Asp
Thr Tyr Glu Arg Asp Leu Tyr Leu Ser Val 1730 1735
1740Gln Phe Asn Leu Asn Gln Thr Thr Leu Thr Ser Thr Ile
Leu Lys 1745 1750 1755Arg Gly Tyr Ile
Asn Lys Ser Glu Thr Arg Leu Gly Ser Leu His 1760
1765 1770Val Trp Gly Lys Gly Thr Thr Pro Val Asn Ala
Val Thr Leu Thr 1775 1780 1785Tyr Asn
Gly Asn Lys Asn Ser Leu Pro Phe Asn Glu Asp Thr Thr 1790
1795 1800Asn Met Ile Leu Arg Ile Asp Leu Thr Thr
His Asn Val Thr Leu 1805 1810 1815Glu
Glu Pro Ile Glu Ile Asn Trp Ser 1820
182572685PRTArtificial SequenceD2H receptor 72Met Ala Glu Asp Lys Ser Lys
Arg Asp Ser Ile Glu Met Ser Met Lys1 5 10
15Gly Cys Gln Thr Asn Asn Gly Phe Val His Asn Glu Asp
Ile Leu Glu 20 25 30Gln Thr
Pro Asp Pro Gly Ser Ser Thr Asp Asn Leu Lys His Ser Thr 35
40 45Arg Gly Ile Leu Gly Ser Gln Glu Pro Asp
Phe Lys Gly Val Gln Pro 50 55 60Tyr
Ala Gly Met Pro Lys Glu Val Leu Phe Gln Phe Ser Gly Gln Ala65
70 75 80Arg Tyr Arg Ile Pro Arg
Glu Ile Leu Phe Trp Leu Thr Val Ala Ser 85
90 95Val Leu Val Leu Ile Ala Ala Thr Ile Ala Ile Ile
Ala Leu Ser Pro 100 105 110Lys
Cys Leu Asp Trp Trp Gln Glu Gly Pro Met Tyr Gln Ile Tyr Pro 115
120 125Arg Ser Phe Lys Asp Ser Asn Lys Asp
Gly Asn Gly Asp Leu Lys Gly 130 135
140Ile Gln Asp Lys Leu Asp Tyr Ile Thr Ala Leu Asn Ile Lys Thr Val145
150 155 160Trp Ile Thr Ser
Phe Tyr Lys Ser Ser Leu Lys Asp Phe Arg Tyr Gly 165
170 175Val Glu Asp Phe Arg Glu Val Asp Pro Ile
Phe Gly Thr Met Glu Asp 180 185
190Phe Glu Asn Leu Val Ala Ala Ile His Asp Lys Gly Leu Lys Leu Ile
195 200 205Ile Asp Phe Ile Pro Asn His
Thr Ser Asp Lys His Ile Trp Phe Gln 210 215
220Leu Ser Arg Thr Arg Thr Gly Lys Tyr Thr Asp Tyr Tyr Ile Trp
His225 230 235 240Asp Cys
Thr His Glu Asn Gly Lys Thr Ile Pro Pro Asn Asn Trp Leu
245 250 255Ser Val Tyr Gly Asn Ser Ser
Trp His Phe Asp Glu Val Arg Asn Gln 260 265
270Cys Tyr Phe His Gln Phe Met Lys Glu Gln Pro Asp Leu Asn
Phe Arg 275 280 285Asn Pro Asp Val
Gln Glu Glu Ile Lys Glu Ile Leu Arg Phe Trp Leu 290
295 300Thr Lys Gly Val Asp Gly Phe Ser Leu Asp Ala Val
Lys Phe Leu Leu305 310 315
320Glu Ala Lys His Leu Arg Asp Glu Ile Gln Val Asn Lys Thr Gln Ile
325 330 335Pro Asp Thr Val Thr
Gln Tyr Ser Glu Leu Tyr His Asp Phe Thr Thr 340
345 350Thr Gln Val Gly Met His Asp Ile Val Arg Ser Phe
Arg Gln Thr Met 355 360 365Asp Gln
Tyr Ser Thr Glu Pro Gly Arg Tyr Arg Phe Met Gly Thr Glu 370
375 380Ala Tyr Ala Glu Ser Ile Asp Arg Thr Val Met
Tyr Tyr Gly Leu Pro385 390 395
400Phe Ile Gln Glu Ala Asp Phe Pro Phe Asn Asn Tyr Leu Ser Met Leu
405 410 415Asp Thr Val Ser
Gly Asn Ser Val Tyr Glu Val Ile Thr Ser Trp Met 420
425 430Glu Asn Met Pro Glu Gly Lys Trp Pro Asn Trp
Met Ile Gly Gly Pro 435 440 445Asp
Ser Ser Arg Leu Thr Ser Arg Leu Gly Asn Gln Tyr Val Asn Val 450
455 460Met Asn Met Leu Leu Phe Thr Leu Pro Gly
Thr Pro Ile Thr Tyr Tyr465 470 475
480Gly Glu Glu Ile Gly Met Gly Asn Ile Val Ala Ala Asn Leu Asn
Glu 485 490 495Ser Tyr Asp
Ile Asn Thr Leu Arg Ser Lys Ser Pro Met Gln Trp Asp 500
505 510Asn Ser Ser Asn Ala Gly Phe Ser Glu Ala
Ser Asn Thr Trp Leu Pro 515 520
525Thr Asn Ser Asp Tyr His Thr Val Asn Val Asp Val Gln Lys Thr Gln 530
535 540Pro Arg Ser Ala Leu Lys Leu Tyr
Gln Asp Leu Ser Leu Leu His Ala545 550
555 560Asn Glu Leu Leu Leu Asn Arg Gly Trp Phe Cys His
Leu Arg Asn Asp 565 570
575Ser His Tyr Val Val Tyr Thr Arg Glu Leu Asp Gly Ile Asp Arg Ile
580 585 590Phe Ile Val Val Leu Asn
Phe Gly Glu Ser Thr Leu Leu Asn Leu His 595 600
605Asn Met Ile Ser Gly Leu Pro Ala Lys Ile Arg Ile Arg Leu
Ser Thr 610 615 620Asn Ser Ala Asp Lys
Gly Ser Lys Val Asp Thr Ser Gly Ile Phe Leu625 630
635 640Asp Lys Gly Glu Gly Leu Ile Phe Glu His
Asn Thr Lys Asn Leu Leu 645 650
655His Arg Gln Thr Ala Phe Arg Asp Arg Cys Phe Val Ser Asn Arg Ala
660 665 670Cys Tyr Ser Ser Val
Leu Asn Ile Leu Tyr Thr Ser Cys 675 680
6857311PRTArtificial Sequencebinding 11 mer fragment L-form 73Xaa
Thr Xaa Xaa Ser Xaa Xaa Xaa Asn Xaa Arg1 5
10748PRTArtificial Sequencebinding 8 mer fragment L-form 74Asp Xaa Asp
Xaa Arg Arg Xaa Xaa1 57510PRTArtificial Sequencebinding 10
mer fragment L-form 75Val Arg Ser Gly Cys Gly Xaa Xaa Ser Ser1
5 107611PRTArtificial Sequencebinding 11 mer
fragment L-form 76Asn Thr Arg Lys Ser Ser Arg Ser Asn Pro Arg1
5 107711PRTArtificial Sequencebinding 11 mer
fragment L-form 77Ser Thr Lys Arg Ser Leu Ile Tyr Asn His Arg1
5 107810PRTArtificial Sequencebinding 10 mer
fragment L-form 78Ser Thr Gly Arg Lys Val Phe Asn Arg Arg1
5 107911PRTArtificial Sequencebinding 11 mer fragment
L-form 79Thr Asn Ala Lys His Ser Ser His Asn Arg Arg1 5
10808PRTArtificial Sequencebinding 8 mer fragment L-form
80Asp Ser Asp Val Arg Arg Pro Trp1 5818PRTArtificial
Sequencebinding 8 mer fragment L-form 81Ala Ala Asp Gln Arg Arg Gly Trp1
5828PRTArtificial Sequencebinding 8 mer fragment L-form
82Asp Gly Arg Gly Gly Arg Ser Tyr1 5834PRTArtificial
Sequencebinding 4 mer fragment L-form 83Arg Val Arg Ser18412PRTArtificial
Sequencebinding 12 mer fragment L-form 84Ser Val Arg Ser Gly Cys Gly Phe
Arg Gly Ser Ser1 5 108511PRTArtificial
Sequencebinding 11 mer fragment L-form 85Ser Val Arg Gly Gly Cys Gly Ala
His Ser Ser1 5 10
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