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WO1993020834A1 - Procedes et compositions d'administration orale de substances therapeutiques - Google Patents

Procedes et compositions d'administration orale de substances therapeutiques Download PDF

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Publication number
WO1993020834A1
WO1993020834A1 PCT/US1993/002874 US9302874W WO9320834A1 WO 1993020834 A1 WO1993020834 A1 WO 1993020834A1 US 9302874 W US9302874 W US 9302874W WO 9320834 A1 WO9320834 A1 WO 9320834A1
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Prior art keywords
carrier
receptor
egf
therapeutic agent
chimeric molecule
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PCT/US1993/002874
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English (en)
Inventor
Patricia A. Gonnella
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the delivery of therapeutic agents in animals, including humans.
  • the common routes of therapeutic agent administration are enteral (oral ingestion) and parenteral (intravenous, subcutaneous, and intramuscular) routes of administration.
  • the intravenous route is advantageous for emergency use when very rapid increases in blood levels of the therapeutic agent are necessary. Further, the intravenous route allows for easy dosage adjustments and is useful for administration of large volumes of a drug when diluted.
  • intravenous drug administration suffers from numerous limitations. One problem is the risk of adverse effects resulting from the rapid accumulation of high concentration of the therapeutic agent in plasma and/or tissues. Also, the intravenous route requires repeated injections which may cause discomfort to the subject. Further, the repeated injections may be complicated by local infections at the site of needle insertion. Other routes of parenteral administration are painful for subjects, especially if frequent administration is required.
  • Subcutaneous injection is sometimes used for delivery of therapeutic agents that are not irritating.
  • this mode of administration is not suitable for delivering large volumes nor is it suitable for administering irritating substances which may cause pain or necrosis at the site of injection.
  • the intramuscular route cannot be used during anticoagulant medication and may interfere with the interpretation of certain diagnostic tests.
  • it is sometimes suitable for administering therapeutic agents in moderate volumes, oily substances, and some irritating substances.
  • Oral administration of drugs is generally more convenient, economical, and acceptable. However, oral administration is limited where the therapeutic agent is not efficiently absorbed by the gastrointestinal tract Absorption by the gastrointestinal tract may be inefficient for poorly soluble, slowly absorbed, or unstable therapeutic preparations. Many important therapeutic agents which need to be administered frequently are not effectively absorbed when administered orally and hence must be delivered by injection methods.
  • the efficacy of orally administered therapeutic agents depends, in a large part, on the agent being absorbed from the gastrointestinal tract into the circulation.
  • Some investigators have attempted to circumvent the above-noted problems through intranasal administration of a therapeutic agent to a subject through the use of a fusidic acid derivative adjuvant
  • Others have attempted to effect therapeutic agent penetration across skin through use of penetration enhancers such as chelating agents, bile salts, surfactants, acylcholines and acylcarnitines.
  • penetration enhancers such as chelating agents, bile salts, surfactants, acylcholines and acylcarnitines.
  • Penetration enhancers such as Azone, oleic acid, decy nethyl sulfoxide and propylene glycol have recently been shown to promote the penetration of the anticancer drug 5-fluorouraciI in hairless rat skin.
  • Chimeric peptides were developed consisting of a transportable peptide (such as insulin, transferrin, insulin-like growth factor I, insulin-like growth --actor II, basic albumin and prolactin) conjugated to hydrophilic peptide neuropharmaceutical agents which alone are not generally transported across the blood-brain barrier.
  • a transportable peptide such as insulin, transferrin, insulin-like growth factor I, insulin-like growth --actor II, basic albumin and prolactin
  • Absorption enhancing agents for the gastrointestinal system can be found in: Fix, J., Controlled Release :151-156 (1987); Sugibayashi et al,
  • Delivery of a therapeutic agent to a subject is achieved by administering to the subject a chimeric molecule wherein the chimeric molecule comprises a therapeutic agent to be delivered conjugated to a suitable carrier wherein the carrier is capable of transport across epithelial cells via transcytosis. Delivery of therapeutic agents across epithelial cells in the liver, kidney, and gastrointestinal tract can occur by the chimeric molecules of the present invention.
  • a therapeutic agent can be delivered to a subject's circulatory system by transcytosis of the carrier across epithelial cells of the gastrointestinal tract.
  • the present invention obviates the need for injection of therapeutic agents unable to cross the gastrointestinal barrier.
  • Methods and compositions are provided for delivery of therapeutic agents to a subject
  • the methods involve conjugating a therapeutic agent to be delivered to a suitable carrier wherein the carrier is capable of effectuating delivery via transcytosis.
  • the resultant conjugate is a chimeric molecule which acts as a vehicle for delivery of the therapeutic agent
  • the therapeutic agent can be dehvered to a subject's circulatory system when the chimeric molecules of the present invention are administered to the subject orally.
  • the chimeric molecule is absorbed into the circulation from the gastrointestinal tract by transcytosis.
  • subject is intended both human and non-human animal subjects who are administered the chimeric molecules of the present invention. Specifically intended are- mammalian subjects. More specifically intended are human subjects.
  • therapeutic agent is intended drugs and/or medicinal peptides useful for treating a medical or veterinary disorder, preventing a medical or veterinary disorder, or regulating the physiology of a human being or animal.
  • Drags for which the method of administration of the invention is particularly important are peptides.
  • Suitable peptides include, but are not limited to, insulin, proinsulin, glucagon, parathyroid hormone and antagonists of it, calcitonin, vasopressin, renin, prolactin, growth hormone, thyroid stimulating hormone, corticotropin, follicle stimulating hormone, luteinizing hormone, chorionic gonadotropin, atrial peptides (a natriuretic factor), interferon, tissue plasminogen activator, gamma globulin, factor VIII, and analogs and/or chemical modifications of these peptides.
  • the invention can also be used to administer hormone releasing hormones, e.g., growth hormones releasing hormone, corticotropin releasing factor, luteinizing hormone releasing hormone, growth hormone release inhibiting hormone (somatostatin) and thyrotropin releasing hormone.
  • hormone releasing hormones e.g., growth hormones releasing hormone, corticotropin releasing factor, luteinizing hormone releasing hormone, growth hormone release inhibiting hormone (somatostatin) and thyrotropin releasing hormone.
  • suitable drags include the physiologically active enzymes: transferases, hydrolases, isomerases, proteases, ligases, and oxidoreductases such as esterases, phosphatases, glycosidases and peptidases; enzyme inhibitors such as leupeptin, chymostatin and pepstatin; and growth factors such as tumor angiogenesis factor, epidermal growth factor, nerve growth factor and insulin-like growth factors.
  • Suitable drags are those normally absorbed only to a limited extent across the gastrointestinal mucosa after oral administration; e.g., antihistamines (e.g. diphenylhydramine and chlorpheniramine), and drugs affecting the cardiovascular (e.g., antihypertensives), renal, hepatic and immune systems (including vaccines).
  • antihistamines e.g. diphenylhydramine and chlorpheniramine
  • drugs affecting the cardiovascular e.g., antihypertensives
  • renal, hepatic and immune systems including vaccines
  • sympathomimetic drugs such as the catecholamines (e.g., epinephrine) and non-catecholamines (e.g., phenylephrine and pseudoephedrine) may be administered according to the method of the present invention.
  • Drugs such as anti-infective agents may also be administered according to the method of the present invention.
  • antibiotics such as the aminoglycosides (e.g., streptomycin, gentamicin, kanamycin, etc.) are normally not adequately absorbed after oral administration, and may therefore be advantageously administered by the method of the invention.
  • Drags may also be administered according to the invention, e.g., the many drags currently used to treat arthritis such as narcotic pain relievers.
  • Anti-inflammatory agents e.g., indomethacin, dexamethasone and triamcinolone
  • anti-tumor agents e.g., 5-fluorouracil and methotrexate
  • tranquilizers such as diazepam
  • Suitable drugs are the water insoluble, fat-soluble hydrophobic drugs, e.g., steroids, such as progesterone, estrogens (including contraceptives such as ethinyl estradiol) and androgens and their analogs, and the fat-soluble vitamins, e.g., vitamins A, D, E and K, and their analogs.
  • steroids such as progesterone
  • estrogens including contraceptives such as ethinyl estradiol
  • androgens and their analogs include the fat-soluble vitamins, e.g., vitamins A, D, E and K, and their analogs.
  • growth factors having receptors in epithelial cells of the gastrointestinal tract are used as carriers to transport therapeutic agents from the gastrointestinal tract into the circulation of a subject.
  • the resultant conjugate is a chimeric molecule which acts as a vehicle allowing oral delivery of a therapeutic agent into the subject's circulatory system.
  • growth factor fragments or analogs can also be used as carriers for delivery of therapeutic agents to the subject All that is required is that the growth factor fragment or analog can be conjugated to a therapeutic agent, binds the growth factor receptor and is capable of transepithelial transport via transcytosis.
  • carrier macromolecules which, when conjugated to a therapeutic agent, are capable of effecting delivery of the therapeutic agent to a subject via transcytosis.
  • carrier is intended growth factors, or fragments or analogs thereof, which bind a growth factor receptor and, when conjugated to a therapeutic agent, are capable of effectuating delivery of the therapeutic agent via transcytosis.
  • Suitable carriers include, but are not limited to: epidermal growth factor (EGF), transforming growth factor a (TGF- ⁇ ), and fragments or analogs of these growth factors wherein the fragments or analogs are capable of binding the growth factor receptors and are capable of effectuating oral delivery of a therapeutic agent to the circulation.
  • EGF epidermal growth factor
  • EPA 0 326 046 Proc. Natl Aca ⁇ ScL USA 81:1351 (1984); EPA 0 326 046; UK Patent Application GB 2 172 890 A; EPA 335 400, the disclosures of which are hereby incorporated by reference.
  • EGF EGF are known. EGF has three disulfide bonds which define three looped regions from residues 1-20, 14-31, and 32-53. EGF is a potent stimulator of cellular proliferation and inhibitor of gastric acid secretion.
  • Epidermal growth factor receptors have been ⁇ found in cells of the gastrointestinal tract, in hepatocytes and in kidney cells. In the gastrointestinal tract, these epidermal growth factor receptors have been shown to mediate transepithelial transport of epidermal growth factor into the circulation. This pathway is called receptor-mediated transcytosis. Transepithelial transport of epidermal growth factor can also occur by transcytosis which is not receptor-mediated.
  • the growth factor can be absorbed from the gastrointestinal tract by either receptor mediated transcytosis or by non-specific association. See Gonnella, et al, J. Clin. Invest 8022-32 (1987); Gonnella, et al, Advanced Drug Delivery Review 1:235-248 (1987); Thompson, J., Am. J. Physiol 25 G429-G435 (1988); Thornberg et al, Am. J. Physiol 2J5:G68-G71 (1987); Weaver et al, Gastroenterology 98:828-837 (1990); and Pothier et al, FEBS. Lett.
  • liver and kidney cells also are known to have epidermal growth factor receptors. See, for example, Dunn et al, J. Cell Biol 102:24-36 (1986) and Brandli et al, J. Biol Chem. 266 (75j:8560-8666 (1991), the disclosures of which are hereby incorporated by reference.
  • Substantial amounts of EGF are cleared from the circulation by hepatocytes via receptor mediated endocytosis and are subsequently degraded within lysosomes. See, Dunn et al, J. Cell Biol 102:24-36 (1986). Further, the EGF receptor is known to mediate uptake of EGF in kidney cells.
  • the present invention also provides a means for delivering therapeutic agents to the liver and the kidney. Drag transport in the intestine, liver and kidney is similar because in each case transport occurs across a barrier of epithelial cells. If the target for delivery is the liver or kidney, administration of the chimeric molecule may be parenteral rather than enteral.
  • Komoriya et al. provided biologically active, synthetic fragments of EGF and localized a major receptor-binding region of EGF. See, Komoriya et al, Proc. Natl Acad. Sci. USA 81:1351-1355 (1984). Synthetic peptide fragments of EGF were shown by Komoriya et al to compete with intact EGF in binding to the EGF receptors. Further, the synthetic fragments of the epidermal growth factor were shown to induce a series of cellular responses like those to EGF. Thus, EGF fragments or analogs which bind to the EGF receptor can be used as carriers in the present invention.
  • TGF- ⁇ Mature transforming growth factor ⁇
  • EGF- ⁇ is a 50-amino add containing polypeptide sharing about 32% sequence homology with EGF. Like EGF, TGF- ⁇ induces a potent mitogenic response in cells.
  • the amino add and nucleic add sequences for TGF- ⁇ are known. See, for example, Deiynck et al, Cell 55:287-297 (1984), the disclosure of which is hereby incorporated by reference. TGF- ⁇ competes with EGF for binding to the EGF receptor and exhibits all the apparent biological activities of EGF. Tarn et al, Proc. West Pharmacol Soc.
  • TGF- ⁇ synthesized several fragments of TGF- ⁇ to identify the active portion of this growth factor. These fragments, like intact TGF- ⁇ , bind EGF receptors which mediate all apparent biological activities of EGF. See, also, Tarn et al, InL J. Pept Protein. Res. 38(3):204-211 (1991) and Nestor et al, Biochem. and Biophys. Res. Comm. 129(1 )2' 26-232 (1985), the disclosures of which are hereby incorporated by reference.
  • intact TGF- ⁇ and TGF- ⁇ fragments and analogs which bind the EGF receptor can be used as carriers in the present invention.
  • chimeric molecules are formed by conjugating a therapeutic agent to a suitable carrier wherein the carrier is capable of transporting the conjugate across epithehal cells via transcytosis.
  • chimeric molecule is intended a conjugate molecule comprising a therapeutic agent conjugated to a suitable carrier wherein the carrier is capable of transporting, the conjugate across epithehal cells via transcytosis.
  • chimeric molecule is intended a conjugate molecule comprising a therapeutic agent conjugated to a growth factor or a growth factor fragment or analog wherein the growth factor or growth factor fragment or analog is capable of effectuating delivery of the therapeutic agent to a subject via transcytosis.
  • chimeric molecule is intended a conjugate molecule wherein a therapeutic agent is conjugated to EGF or fragments or analogs thereof or TGF- ⁇ or fragments or analogs thereof capable of effectuating deliveiy via transcytosis.
  • compositions of this invention can be employed in mixture with conventional exdpients, i.e., pharmaceutically acceptable organic or inorganic substances suitable for enteral or parenteral application which do not deleteriousry react with the chimeric molecules.
  • suitable pharmaceutically acceptable substances include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic add, viscous paraffin, perfume oil, f tty add monoglycerides and diglycerides, petroethral fatty add esters, hydroxymethylcellulose, pofyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents.
  • lubricants e.g., lubricants, preservatives, stabilizers, wetting agents, emulsi ⁇ ers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriousfy react with the chimeric molecules.
  • solutions preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories.
  • Ampules are convenient unit dosages.
  • enteral application particularly suitable are tablets, dragees or capsules having talc and/or a carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch.
  • a syrup, elixir or the like can be used wherein a sweetened vehicle is employed.
  • Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
  • the chimeric molecules of the .present invention are made by conjugating the therapeutic agent to be dehvered to a suitable carrier.
  • the conjugation needs to be carried out in a manner that will not prevent binding of the growth factor to the receptor.
  • EGF and TGF- ⁇ are necessary and suffident for binding to the epidermal growth factor receptor. Further, the conjugation of the carrier to the therapeutic agent must not prevent transcytosis of the resultant chimeric molecule.
  • the receptor binding regions of the EGF and TGF- ⁇ are known in the art See, for example, Nestor et al, Biochem. and Biophys. Res. Comm. 129(1)226-232 (1985); Tam et al, Proc West. Pharmacol Soc. 29:471-474 (1986); Tam et al, Inl J. PepL Protein Res. 38(3)204-211 (1991); and Komoriya et al, Proc. Nail Acad. ScL USA 81:1351-1355 (1984).
  • conjugation may be carried out using bifunctional reagents which are capable of reacting with each of the peptides (i.e., the medicinal peptide and carrier peptide) and forming a bridge between the two.
  • bifunctional reagents capable of reacting with each of the peptides (i.e., the medicinal peptide and carrier peptide) and forming a bridge between the two.
  • One preferred method of conjugation involves peptide thiolation wherein the two peptides are treated with reagents such asN-Succinimidyl 3-(2-pyridyldithio)propionate(SPDP) to form adisulfide bridge between the two peptides to form the chimeric molecule.
  • SPDP N-Succinimidyl 3-(2-pyridyldithio)propionate
  • conjugation agents may be used, so long as they provide linkage of the two peptides (i.e., the carrier peptide and the medicinal peptide) together without denaturing them. Preferably, the linkage can easily be broken once the chimeric molecule has entered the circulation.
  • Suitable examples of conjugation reagents include: glutaraldehyde and cystamine and EDAC. Conjugation of peptides using glutaraldehyde is described in Poznansky et al, Science 225:1304-1306 (1984), the disclosure of which is herein incorporated by reference. Conjugation of peptides using cystamine and EDAC is described in Ito et al, Mol Cell Endocrinol
  • conjugate chimeric molecules of the present invention can be in the form of a fusion peptide made by recombinant DNA techniques.
  • EGF EGF
  • examples of producing EGF by genetic engineering techniques are provided in EPA 0335400, EPA 0326046, and GB 2172 890.
  • An example of producing TGF- ⁇ by genetic engineering techniques is provided in Derynck et al, Cell 38281-291 (1984).
  • fusion peptides suitable as chimeric molecules for delivery of therapeutic agents to a subject.
  • the use of recombinant DNA techniques requires knowledge of the nucleic add sequence of the medicinal peptide to be dehvered.
  • the nucleic add fragment corresponding to the medicinal peptide is linked to a nucleic add fragment corresponding to a carrier to form a recombinant molecule.
  • the recombinant molecule is then operably linked to an expression vector and introduced into a host cell to enable expression of a fusion peptide useful as a chimeric molecule in the present invention. See Molecular Cloning A Laboratory Manual, Sambrook et al, eds. Cold Spring Harbor Laboratory, 2nd. Ed., Cold Spring Harbor, NY (1989).
  • mature EGF is a 53-amino add single chain polypeptide. The amino add and nucleic acid sequences of EGF are known.
  • each of the 53 amino acid residues of EGF can be used as a site for conjugating the therapeutic agent.
  • an EGF fragment encompassing amino acid residues 14-31 of mature EGF can be used as a carrier in the present invention.
  • one or more of these residues (14-31) can be used as a site for conjugating the therapeutic agent
  • mature TGF ⁇ is a 50-amino add polypeptide.
  • the amino add and nucleic add sequences of TGF ⁇ are known. Potentially, one or more of each of the 50 amino add residues of TGF ⁇ can be used as a site for conjugating the therapeutic agent.
  • a TGF ⁇ fragment encompassing amino acid residues 34-43 of mature TGF ⁇ can be used as a carrier in the present invention.
  • one or more of these residues can be used as a site for conjugating the therapeutic agent
  • a therapeutic agent has been conjugated to EGF or TGF ⁇ at one or more of the amino add residues specified above
  • the suitability of the resultant conjugate for purposes of the present invention can be tested following the protocols set forth in the Experimental section of the specification.
  • the chimeric molecules of the present invention can be administered to subjects at a dosage range of from about 15 mg to about 75 mg. However, the dosage range may differ depending on the subject and the therapeutic agent to be delivered.
  • Optimal dosages can be determined by one of ordinary skill in the art using conventional techniques. As a general rale, the dosage levels should correspond to the accepted and estabhshed dosages for the particular therapeutic agent to be dehvered.
  • TGF- ⁇ or EGF receptor-binding fragments alone and -in conjugation with a foreign peptide bind to preparations of brash border membrane vesicles.
  • Microvillous membrane fractions are prepared from adult rat and human small intestine by the calcium precipitation method (Schmitz et al, BBA 525:98-112 (1973), Kessler et al, BBA 506:136-154
  • Intestinal segments are removed from anesthetized rats or are obtained after a gastric bypass procedure (human tissue) and washed immediately with cold saline. Tissue processing is performed at 4°C according to the following method. Scrapings are homogenized in a conical binding tube in 500 mM mannitol, 10 mM hepes buffer pH 7.5. After a 1:6 dilution and filtration through fine nylon mesh (40 ⁇ m pore size), CaCl 2 is added to a final concentration of 10 mM. After 10 minutes and occasional mixing by inversion the homogenate is centrifuged at 2500 x g for 15 minutes.
  • a receptor-binding carrier peptide is derived from the A, B or C disulfide loop of TGF- ⁇ .
  • Synthetic peptides representing residues 8-50 (ABC loop), 8-21 (A loop), 16-32 (B loop), and 34-43 (C loop) are prepared by a stepwise solid phase method using a differential add lability protection scheme of N ⁇ -tertbutoxycarbonyl and side chain benzyl protecting groups.
  • synthetic peptides which are the third disulfide loop of TGF ⁇ are prepared by the stepwise solid phase method.
  • a receptor-binding carrier peptide from F-GF is derived from the three looped regions defined by disulfide bonds.
  • Synthetic peptides representing residues 1-53 are prepared by the stepwise solid phase method.
  • synthetic peptides which are a cychc fragment of EGF, containing residues 14-31 are prepared by the stepwise solid phase method.
  • TGF- ⁇ or EGF carrier peptide fragments alone and in conjugation with a foreign peptide are labeled with ⁇ 1 using iodobeads
  • Specifidry of binding is assessed by electron microscopic autoradiography.
  • the purpose of the following example is to show that the EGF or TGF ⁇ fragments (described above) alone and in conjugation with a foreign peptide are transported across the epithelium and are detected in the plasma and target tissues.
  • Iodinated EGF or TGF ⁇ receptor-binding fragments alone and in combination with a foreign peptide are injected into iigated loops in vivo of proximal or distal Tat intestine for time intervals of 30 minutes to 2 hours.
  • Ligated loops are prepared by making an incision along the midline of the abdomen and exposing a segment of intestine. Ligatures are passed through the mesentery without disturbing the mesenteric circulation. The distal hgature, placed 3 cm proximal to the cecum is tightened. A 27 gauge needle is passed through the proximal hgature, the ligature is tightened around the needle and the protein-label solution is injected.
  • the proximal hgature is tightened further to provide a leak free compartment
  • the ligated loop is exdsed intact
  • the loop lumen is rinsed with PBS and subsequently rinsed with fixative consisting of freshly depolymerized formaldehyde, 2.5% glutaraldehyde and 4 mM CaCl 2 in 0.1 M sodium cacodylate buffer, pH 7.4 Tissues are then immersed in a drop of fixative, chopped at 1 mm and processed for autoradiography.
  • tissue slices are rinsed in 0.1 M cacodylate buffer and postfixed in 1% osmium in cacodylate buffer.
  • tissues are stained en bloc in 1% uranyl acetate, dehydrated in graded ethanols and embedded in Epon-Araldite.
  • Epon-Araldite For light microscopic autoradiography, 1 ⁇ m sections are stained with iron hematoxylin, coated with Ilford K5 emulsion (diluted 1:1 with distilled water) exposed for 1-4 weeks and developed for 4 minutes in Kodak D-19 at 18°C.
  • EM electron microscopic
  • Grids are carbon coated, placed on a glass slide and coated with a thin film of Ilford L4 emulsion (diluted 1:4 with distilled water) by the loop method (Caro, L.G. and R.P. Vantubergen, /. Cell Biol 15:113-118 (1962)).
  • the emulsion coat consists of a monolayer of silver halide crystals as confirmed by electron microscopy of undeveloped grids.
  • the distribution of silver grains in subcellular compartments and at the basolateral surface are quantitated according to the following procedure. For each time interval 15 well-oriented cells sectioned along a central longitudinal axis are selected and all grains over these cells are counted. Compartments are demarcated into microvilh, apical vesicular compartments, multivesicular bodies (lysosomes), nucleus, and basal vesicular compartments. Grains are assigned to lateral or basal membranes if they span the membrane or lay within 900 nm, the estimated half-distance (HD) for Ilford ⁇ A emulsion exposed by 125 I (Salpeter et al, J.
  • HD estimated half-distance
  • Relative surface densities of cell compartments are calculated from the same section by outlining compartment boundaries overlaying a standard grid and scoring allocations of grid intersects (Weibel et al, In: Principles and techniques of electron microscopy, M. A. Hyat, editor, Van
  • samples of serum are precipitated with 20% trichloroacetic add (TCA) washed twice in acetone and solubilized in sample buffer containing SDS and mercaptoethanol. Aliquots, containing
  • Reversed-phase HPLC is performed on a C18 Bondapack column (Waters Instruments, Inc., Rochester, MN) equilibrated in 0.1% trifluoroacetic add (TFA) with 21% acetonitrile. Samples are diluted 1:1 with starting solvent, applied to a 7.8 x 30 cm column and the column is washed 5 minutes in the same solvent Material is eluted with a 20 minute acetonitrile gradient (21-63% in 0.1% TFA). Fractions are collected and radioactivity in each is measured in a gamma scintillation counter (75% efficiency, Packard Instruments Co., Downers Grove, JL).
  • Tissues are homogenized in a total volume of 9 ml ice-cold distilled water using a Waring blender in a prechilled microjar.
  • a Waring blender in a prechilled microjar.
  • One milliliter of cold 0.5 N HC1 is added and tubes are stoppered and hand mixed by inversion. Samples are kept on ice 30-60 minutes. After extraction and additional mixing, 0.1 or 1.0 ml aliquots of the homogenates are removed and counted in the gamma counter to determine total radioactivity. Samples are then transferred to 5/8 x 3 inch tubes and centrifuged at 100,000 x g for 40 minutes.
  • Supernatant are decanted and aliquots are counted to determine extraction efficiencies. Approximately 90% of the homogenate radioactivity is extracted by this method. After rehomoginization of the pellet, greater than 95% of the radioactivity is extracted.. Extracts are concentrated as required either by lyophilization or a Speed-Vac concentrator (Savant) and the residue is dissolved in 0.05 M phosphate buffer pH 7.4 and applied to a column. Plasma is obtained from the supernatant after centrifugation (9,600 x g) of heparin-treated blood and analyzed directly. The pellet of blood cells is treated the same as other tissue samples.
  • Intact peptides are detected based on their appearance in the excluded void volume following Sephadex chromatography.
  • the proportion of radioactivity in the void volume is calculated by summing the total cpm in this region and is expressed as a percent of the total radioactivity applied to the column. This percentage, along with the total radioactivities accumulated in specific tissues is used to calculate the total tissue recoveries of intact peptide. Results are expressed as a percentage of fed radioactivity.
  • Fractions containing intact or modified peptide are then further characterized by SDS gel autoradiography and reversed phase PHLC under dissociating conditions as detailed above.
  • B. The purpose of the following example is to determine the effect of the EGF-peptide or TFG ⁇ -peptide conjugate on the mitogenic activity of the epithelium.
  • Animals are fed peptides (80 ng to 1 ⁇ g) and simultaneously injected intraperitoneally with H-thymidine (1.5 ⁇ Ci/gm body weight) for 2, 6, 12, 23 and 41 hours. Segments of proximal and distal intestine are rinsed with fixative in situ removed and immediately immersed in fixative. Tissues are chopped at 0.55 mm, dehydrated in graded alcohols and embedded in Epon-Araldite. Light microscopic autoradiography is then performed according to the following method. Sections (l ⁇ ) are mounted on glass shdes and stained with iron-hematoxylin. Autoradiographs are prepared by coating shdes with Ilford K5 photographic emulsion. The shdes are kept refrigerated during exposure for 1 to 6 weeks, then developed.
  • total epithehal cells and labeled epithehal cells are counted to determine the labelling index in 50 crypt regions in well-oriented sections. Cells are considered labeled if three or more exposed silver grains overlay the nucleus. Different sections from the same specimen are separated by at least ten serial sections to avoid scoring labeled cells more than once.
  • Migration rate of enterocytes is calculated by measuring the distance between the crypt base and the foremost labeled cell in 20 well- oriented crypt-villus units.

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Abstract

Procédés et compositions pour l'administration de substances thérapeutiques à un sujet. Plus particulièrement, l'invention concerne l'administration orale d'une molécule chimèrique possédant une substance thérapeutique conjuguée à une molécule vecteur appropriée, ladite molécule vecteur étant capable de procéder à l'administration de la molécule chimère par transport transépithélial via la transcytose. La présente invention peut éviter de devoir recourir à l'injection de substances thérapeutiques incapables de franchir la barrière gastrointestinale.
PCT/US1993/002874 1992-04-10 1993-03-29 Procedes et compositions d'administration orale de substances therapeutiques Ceased WO1993020834A1 (fr)

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US07/866,054 1992-04-10

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010850A1 (fr) * 1995-09-21 1997-03-27 Andaris Limited Vehicules et stimulateurs de transcytose pour l'administration de medicaments
WO1997029779A3 (fr) * 1996-02-16 1997-10-30 Univ Minnesota Conjugues de egf-genisteine pour le traitement du cancer
WO1999004813A1 (fr) * 1997-07-24 1999-02-04 Brigham & Women's Hospital, Inc. Transport trans-epithelial d'agents therapeutiques specifique de recepteur
US5872223A (en) * 1994-08-19 1999-02-16 Regents Of The University Of Minnesota Immunoconjugates comprising tyrosine kinase inhibitors
EP0805628A4 (fr) * 1995-01-17 1999-03-24 Brigham & Womens Hospital Transport transepithelial specifique de recepteurs d'immunogenes
US6086875A (en) * 1995-01-17 2000-07-11 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of immunogens
US6485726B1 (en) 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6495526B2 (en) * 1996-01-23 2002-12-17 Gpc Biotech, Inc. Inhibitors of cell-cycle progression and uses related thereto
EP1165597A4 (fr) * 1999-03-08 2003-06-11 Univ Case Western Reserve Molecules bifonctionnelles pour administration d'agents therapeutiques
US6916789B2 (en) 2001-07-02 2005-07-12 Merrion Research I Limited Peyer's patch and/or M-cell targeting ligands
EP1670428A4 (fr) * 2003-10-10 2008-03-12 Univ Southern California Proteines hybrides g-csf-transferrine

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US5137877A (en) * 1990-05-14 1992-08-11 Bristol-Myers Squibb Bifunctional linking compounds, conjugates and methods for their production

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US5137877A (en) * 1990-05-14 1992-08-11 Bristol-Myers Squibb Bifunctional linking compounds, conjugates and methods for their production
US5137877B1 (en) * 1990-05-14 1996-01-30 Bristol Myers Squibb Co Bifunctional linking compounds conjugates and methods for their production

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PROC. NATL. ACAD. SCI., Volume 81, issued March 1984, A. KOMORIYA et al., "Biologically Active Synthetic Fragments of Epidermal Growth Factor: Localization of a Major Receptor-Binding Region", pages 1351-1355, Vol. 223. *
SCIENCE, Vol. 223, issued 09 March 1984, HANS MARQUARDT, "Rat Transforming Growth Factor Type I: Structure and Relation to Epidermal Growth Factor", pages 1079-1082. *
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5872223A (en) * 1994-08-19 1999-02-16 Regents Of The University Of Minnesota Immunoconjugates comprising tyrosine kinase inhibitors
US5911995A (en) * 1994-08-19 1999-06-15 Regents Of The University Of Minnesota EGF-genistein conjugates for the treatment of cancer
US7060274B2 (en) 1995-01-17 2006-06-13 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
EP1323346A3 (fr) * 1995-01-17 2003-11-26 Brigham And Women's Hospital, Inc. Transport épithélial spécifique de récepteurs d'immunogènes
EP0805628A4 (fr) * 1995-01-17 1999-03-24 Brigham & Womens Hospital Transport transepithelial specifique de recepteurs d'immunogenes
US7547436B2 (en) 1995-01-17 2009-06-16 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6030613A (en) * 1995-01-17 2000-02-29 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6086875A (en) * 1995-01-17 2000-07-11 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of immunogens
EP1658772A3 (fr) * 1995-01-17 2007-01-17 The Brigham And Women's Hospital, Inc. Transport épithélial spécifique de récepteurs d'immunogènes
US6485726B1 (en) 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US7067129B2 (en) 1995-01-17 2006-06-27 The Brigham And Woman's Hospital, Inc. Receptor specific transepithelial transport in therapeutics
WO1997010850A1 (fr) * 1995-09-21 1997-03-27 Andaris Limited Vehicules et stimulateurs de transcytose pour l'administration de medicaments
US6204054B1 (en) 1995-09-21 2001-03-20 Andaris Limited Transcytosis vehicles and enchancers for drug delivery
US6495526B2 (en) * 1996-01-23 2002-12-17 Gpc Biotech, Inc. Inhibitors of cell-cycle progression and uses related thereto
WO1997029779A3 (fr) * 1996-02-16 1997-10-30 Univ Minnesota Conjugues de egf-genisteine pour le traitement du cancer
EP1616574A1 (fr) 1997-07-24 2006-01-18 The Brigham And Women's Hospital, Inc. Transport trans-épithélial d'agents thérapeutiques spécifique de récepteur
WO1999004813A1 (fr) * 1997-07-24 1999-02-04 Brigham & Women's Hospital, Inc. Transport trans-epithelial d'agents therapeutiques specifique de recepteur
EP1165597A4 (fr) * 1999-03-08 2003-06-11 Univ Case Western Reserve Molecules bifonctionnelles pour administration d'agents therapeutiques
US6916789B2 (en) 2001-07-02 2005-07-12 Merrion Research I Limited Peyer's patch and/or M-cell targeting ligands
EP1670428A4 (fr) * 2003-10-10 2008-03-12 Univ Southern California Proteines hybrides g-csf-transferrine
US8188032B2 (en) 2003-10-10 2012-05-29 National Institutes Of Health (Nih) G-CSF transferrin fusion proteins

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