WO2004022657A2 - Verstärkung der resorption von substanzen über die haut und schleimhaut - Google Patents
Verstärkung der resorption von substanzen über die haut und schleimhaut Download PDFInfo
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- WO2004022657A2 WO2004022657A2 PCT/EP2003/009788 EP0309788W WO2004022657A2 WO 2004022657 A2 WO2004022657 A2 WO 2004022657A2 EP 0309788 W EP0309788 W EP 0309788W WO 2004022657 A2 WO2004022657 A2 WO 2004022657A2
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Definitions
- the invention relates to the enhancement of the absorption of substances via the skin and mucous membrane.
- the invention further relates to substances with an increased ability to be absorbed by the skin and mucous membrane, and to pharmaceutical compositions comprising such substances.
- Ner administration of biologically and therapeutically active substances by parenteral administration is often considered the most suitable type of ner administration if a quick and strong systemic effect is to be achieved and the active substance is not or only slightly from Body is resorbed or inactivated in the gastrointestinal tract or by liver metabolism.
- injection by Ner has a number of disadvantages.
- the use of sterile syringes and needles or other mechanical devices is required and pain, irritation and infection can occur, especially in the case of repeated injections.
- injections should only be given by trained people.
- Certain medicinal products can be administered to a patient transdermally (percutaneously, via the - uninjured - skin) or transmucosal (via the mucous membrane).
- This administration essentially involves applying the drug to the surface of the skin and / or the mucosa and penetrating the skin or mucosa through the drug into the patient's bloodstream.
- Cutaneous or mucosal administration is interesting in that it can produce a local as well as a systemic effect of a drug. Furthermore, this type of administration can be an alternative to parenteral administration be interesting if a quick onset of an effect of the administered drug is required.
- Non-invasive application also saves physician and patient the inconvenience and risk associated with injections and infusions, and can also be done by untrained people, i.e. also independently by the patient.
- This type of drug application is therefore associated with higher patient compliance than invasive techniques. This is especially true for topical (local) or enteral administration, i.e. H. administration by the oral or rectal route.
- topical administration of systemically active substances also has a significant advantage over the cases in which the substance is poorly absorbed orally, gastric intolerance occurs or the substance is metabolized immediately after absorption in the liver.
- another advantage is that topical administration can achieve a systemic effect at a lower dose than that required for oral administration.
- the skin and mucous membrane is a physical and physiological barrier that has to be exceeded when administering drugs that are intended to reach internal tissues.
- Oral drugs must also be resistant to the low pH and digestive enzymes in the gastrointestinal tract.
- a transdermal or transmucosal administration is therefore suitable only for those drugs that are good "absorbed by the skin or mucosa.
- the rate of absorption and the rate of absorption ie the ratio of the amount absorbed to the amount applied, and ultimately the achievable blood plasma level, ie the bioavailability of an active ingredient, depend, among other factors, on sufficient solubility in water, other chemical properties of the substance and the physiological conditions on the application. or absorption site.
- Many active pharmaceutical ingredients are extremely large and practically impermeable to the skin and mucous membrane.
- There are also many active pharmaceutical ingredients due to their poor water solubility to water insolubility, difficult to reabsorb through mucous membranes, which speaks against their application via these mucous membranes, for example by enteral (oral and rectal), nasal, buccal, vaginal or urethral routes.
- Absorption enhancers have been added to drugs to enhance their absorption through the skin or mucous membrane. These compounds increase the rate of permeation of the drug through the skin or mucous membrane.
- absorption enhancers examples include alcohols and glycols (US Pat. No. 5,296,222), urea derivatives, hyaluronic acids, N, N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), terpenes (DE-A-10053383), bile acid salts (JP-A-59- 130820), chelators (Cassidy and Tidball, J. Cell. Biol. 32, 685, 1967), surfactants (JP-A-4-247034, George et al, J. I fect. Dis. 136, 822, 1977), Salts of fatty acids (U.S.
- Patents 4,476,116 and 6,333,046) synthetic hydrophilic and hydrophobic compounds, biodegradable polymeric compounds and glycyrrhizic acid salts (JP-A-2-042027; US-A-6,333,046).
- absorption enhancers Various mechanisms for the action of absorption enhancers have been proposed. These mechanisms of action include at least for protein and peptide drugs (1) a reduction in the viscosity and / or elasticity of the mucous membranes, (2) an easier transcellular transport by increasing the fluidity of the bilayer of membranes and (3) an increase in the thermodynamic activity of drugs ( Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems 8, 91, 1991).
- a reduction in the viscosity and / or elasticity of the mucous membranes (2) an easier transcellular transport by increasing the fluidity of the bilayer of membranes and (3) an increase in the thermodynamic activity of drugs ( Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems 8, 91, 1991).
- absorption-enhancing product there is currently hardly any absorption-enhancing product on the market. The reasons for this include the low effectiveness and certainty regarding irritation and damage to the mucous membranes, the unpleasant taste and smell, etc.
- the absorption-enhancing effect largely depends on its concentration and it is believed that it is almost ineffective at a concentration of less than 50%. It also has adverse effects on the eyes and also has side effects on the skin.
- the absorption-enhancing effect of urea derivatives, hyaluronic acids, N, N-dimethylfoamamide and surfactants is low compared to dimethyl sulfoxide.
- absorption enhancers increase the absorption of all drugs.
- the absorption enhancer must therefore be tailored to the respective drug.
- absorption enhancers frequently irritate the mucosa or are unsuitable because of an unpleasant smell or taste, often lead to pain and liquorimation after a single administration or lead to irritation and inflammation of the mucosa after several applications.
- transferosomes are known from the prior art (DE 41 07 152, DE 41 07 153 and DE 44 47 287). They are used for the non-invasive administration of suitable active ingredients through the skin. Transferosomes are characterized by an improved penetration ability compared to other liposomes described for topical use. Transferosomes are usually much larger than conventional micelle-like carrier formulations and are therefore subject to different diffusion laws. The increased penetration ability is achieved through their special composition, which makes them sufficiently elastic (hyperflexible) to be able to overcome the constrictions in the barrier, for example in the skin.
- the object of the invention is to improve the absorption capacity per se of substances which are difficult to absorb via the skin and mucous membrane in order to provide a better absorption rate for these substances.
- This is intended to enable non-invasive use of substances which are normally not or only poorly absorbed by the skin or mucous membranes, without having to put up with a great deal of technical outlay and a high consumption of active ingredients.
- the object of the invention is achieved in that an agent which increases the absorption of a substance through the skin or mucosa is coupled with the substance and thus a higher bioavailability for the substance is provided.
- the combination of a substance and an absorption-enhancing agent according to the invention surprisingly enables an improvement in the absorption rate and / or permeation of substances through the skin and mucous membranes which were previously considered poor or non-absorbable.
- the strengthening effect (enhancer effect) of agents on the absorption of substances over or through the skin or mucous membranes makes application forms of therapeutic, diagnostic or cosmetic substances via the skin and mucosa such as the nasal mucosa, eye mucosa, tracheal / bronchial / lung mucosa, the mucous membrane of the rectum, the mucous membrane of the genital tract, the oral mucosa, the gastrointestinal mucosa, the vaginal mucous membrane, or the urinary mucosa to date, or also the urinary mucosa poorly or non-absorbable substances accessible.
- therapeutic, diagnostic or cosmetic substances via the skin and mucosa such as the nasal mucosa, eye mucosa, tracheal / bronchial / lung mucosa, the mucous membrane of the rectum, the mucous membrane of the genital tract, the oral mucosa, the gastrointestinal mucosa, the vaginal mucous membrane, or
- the absorption-enhancing agent increases the bioavailability of the substance. Despite the poor original absorption and the associated low bioavailability, a satisfactory absorption with all therapeutic consequences can thus be achieved and the dosage of the substance can also be reduced compared to the conventional dosage, or an improved effect can be achieved if the dosage remains the same.
- the invention thus relates in one aspect to a method for producing a percutaneous or transmucosal preparation, comprising the coupling of a substance with at least one agent which enhances the absorption of the substance through the skin or mucosa.
- the invention relates to a method for enhancing the bioavailability of a substance when applied to the skin or mucosa, comprising coupling the substance with at least one agent which increases the absorption of the substance by skin or mucosa.
- the invention also relates to a method for enhancing the ability of a substance to be absorbed by the skin or mucosa when it is applied, comprising coupling the substance to at least one agent which enhances the absorption of the substance by the skin or mucosa.
- the invention further relates to a method for enhancing the permeability (penetration ability) of a substance for skin or mucosa, comprising coupling the substance with at least one agent which enhances the absorption of the substance through skin or mucosa.
- the invention relates to the substances obtainable by the methods according to the invention with increased bioavailability, increased ability to be absorbed by skin or mucosa, and / or increased permeability (penetration ability) and pharmaceutical compositions comprising one or more of these substances.
- the invention relates to the use of the substances obtainable by the processes according to the invention with increased bioavailability, increased ability to be absorbed by skin or mucosa, and / or increased permeability (penetration ability) and their pharmaceutical compositions for application to the skin or mucosa and for Treatment (including prophylaxis and cosmetic treatment) and / or diagnosis of diseases which are usually treated, prevented or diagnosed with these substances without the change according to the invention.
- Fe he relates to methods of treatment (including prophylaxis and cosmetic treatment) and / or diagnosis of a disease in a patient, comprising the administration of a pharmaceutical composition comprising the substances obtainable by the method according to the invention with enhanced bioavailability, enhanced ability, of skin or Mucosa to be absorbed and / or increased permeability (penetration ability) to the patient, so that the concentration (local or systemic, preferably systemic) of the substance with increased bioavailability, increased ability to be absorbed by skin or mucosa, and / or increased permeability is sufficient to treat, prevent and / or diagnose the disease.
- a pharmaceutical composition comprising the substances obtainable by the method according to the invention with enhanced bioavailability, enhanced ability, of skin or Mucosa to be absorbed and / or increased permeability (penetration ability) to the patient, so that the concentration (local or systemic, preferably systemic) of the substance with increased bioavailability, increased ability to be absorbed by skin or mucosa, and
- the invention relates to a method for elucidating a mucosal, dermatological and / or systemic effect of a substance, in particular a pharmaceutical active substance, which is administered via the skin or mucous membrane of a pharmaceutical composition which enhances the substances obtainable by the method according to the invention Bioavailability, increased ability to be absorbed by skin or mucosa, and / or increased Contains permeability (penetration ability) in a mucosal, dermal and / or systemically effective amount to a patient.
- permeability penetration ability
- the absorption-enhancing agent can be covalently or non-covalently linked (coupled) to a substance.
- a bond is preferably a covalent bond.
- the linker is e.g. enzymatically or chemically, in particular by in vivo processes, so that the substance can be separated from the absorption-enhancing agent.
- the linker contains a cleavable ester or carbamate functionality or a peptide which can be recognized by a proteinase such as a proteinase which occurs in serum.
- the substance is separated from the absorption-enhancing agent after absorption through the skin or mucosa.
- the absorption enhancing agent is coupled to the substance multiple times, i.e. at least 2, preferably 2 to 10, more preferably 2 to 5, even more preferably 2 to 3, in particular 2 absorption-enhancing agents, which may be the same or different, are coupled to the substance (covalently and / or non-covalently).
- These multiply coupled absorption-enhancing agents can be connected to the substance as tandem constructs separately or in series, if appropriate separated by a linker. This preferably results in greater bioavailability, ability to be absorbed by the skin or mucosa, and / or increased permeability (penetration ability) than with a simple coupling of the absorption-enhancing agent.
- the absorption-enhancing agent is a polypeptide or protein.
- the polypeptide or protein preferably comprises a sequence derived from a virus and in particular a sequence derived from a surface protein of a virus or a derivative or a part thereof.
- the term "virus” encompasses DNA and RNA viruses, in particular adenoviruses, adeno-associated viruses, Vaccinia viruses, baculoviruses, hepatitis C viruses, hepatitis A viruses, influenza viruses, herpes viruses and Hepadna viruses.
- the peptide or protein comprises a sequence or a derivative derived from a hepatitis virus, Hepadna-Viras or HIN, in particular a hepatitis B nixus.
- the peptide or protein comprises an antenna-derived, an HIV-derived, or a VP22-derived herpesvirus sequence.
- virus includes those viruses which are present in humans, non-human primates or other animals, in particular mammals (such as cow, horse, pig, sheep, goat, dog and cat), birds (such as, for example, chicken) or Rodents (like mouse and rat) occur.
- mammals such as cow, horse, pig, sheep, goat, dog and cat
- birds such as, for example, chicken
- Rodents like mouse and rat
- polypeptide or protein which acts as an absorption-enhancing agent comprises a sequence which falls under the general formula below:
- XI, X6, X7, X9, XI 0 and XI 2 are variable
- X2 and X5 are hydrophobic amino acid residues
- X3, X4, X8 and XI 1 are hydrophilic amino acid residues.
- X7 is preferably a hydrophilic amino acid residue.
- the polypeptide or protein that acts as an absorption-enhancing agent comprises this sequence, wherein 1 or 2 amino acid residues, in particular 1 amino acid residue from XI to X12, deviate from this hydropathy distribution.
- Amino acid side chains with charged groups, hydrogen-bonding groups or dipoles can be classified as hydrophilic. In contrast, you can neutral organic amino acid side chains with a hydrocarbon character, which have no significant dipoles and do not have the ability to form hydrogen bonds, are classified as hydrophobic.
- the hydrophobic amino acids include alanine, valine, leucine, isoleucine, tryptophan, phenylalanine and methionine.
- the hydrophilic amino acids include glycine, serine, tyrosine, threonine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine and proline.
- a variable amino acid residue can be any of the amino acids listed above.
- XI is preferably proline, histidine, leucine or threonine, more preferably proline or threonine, especially proline.
- X2 is preferably alanine, valine, leucine, or isoleucine, more preferably leucine or isoleucine, especially leucine.
- X3 is preferably serine, asparagine, aspartic acid or glutamine, especially serine.
- X4 is preferably serine, glutamine, histidine or proline, more preferably serine, histidine or proline, in particular Serine.
- X5 is preferably alanine, valine, leucine or isoleucine, more preferably isoleucine or valine, especially isoleucine.
- X6 is preferably phenylalanine, serine, alanine, leucine, methionine or valine, more preferably phenylalanine or valine, especially phenylalanine.
- X7 is preferably serine, alanine, glycine, aspartic acid or proline, more preferably serine, aspartic acid or proline, especially serine.
- X8 is preferably arginine, histidine or threonine, more preferably arginine or histide, especially arginine.
- X9 is preferably isoleucine, threonine, methionine or valine, more preferably isoleucine or valine, especially isoleucine.
- XI 0 is preferably glycine, isoleucine, glutamine, aspartic acid or serine, more preferably glycine or serine, especially glycine.
- XI 1 is preferably aspartic acid, proline, threonine or serine, more preferably aspartic acid or threonine, especially aspartic acid.
- XI 2 is preferably proline, lysine, methionine, valine, isoleucine or threonine, in particular proline.
- polypeptide or protein that acts as a resorption enhancer comprises an amino acid sequence that falls under the general formula below:
- XI is a variable amino acid, preferably proline, histidine, leucine or threonine, more preferably proline or histidine, in particular proline,
- X2 is a hydrophobic amino acid, preferably alanine, valine, leucine or isoleucine, more preferably leucine or isoleucine, in particular leucine
- X6 is a variable amino acid, preferably phenylalanine, serine alanine, leucine, methionine or valine, more preferably phenylalanine or serine, in particular phenylalanine
- X7 is a variable amino acid, preferably serine, alanine, glycine, aspartic acid or proline, more preferably serine or alanine, in particular serine
- X9 is a variable amino acid, preferably isoleucine, threonine, methionine or valine, more preferably isoleucine or threonine, in particular isoleucine
- the polypeptide or protein which acts as an absorption-enhancing agent comprises an amino acid sequence which falls under the general formula below:
- X3 is a hydrophilic amino acid, preferably serine, asparagine, aspartic acid or
- X9 is a variable amino acid, preferably isoleucine, threonine, methionine or nalin, in particular nalin or isoleucine,
- XI 0 is a variable amino acid, preferably glycine, isoleucine, glutamine, aspartic acid or serine, in particular aspartic acid or glutamine,
- XI 1 is a hydrophilic amino acid, preferably aspartic acid, proline, or threonine
- X12 is a variable amino acid, preferably proline, lysine, methionine, nalin, isoleucine or threonine, in particular valine or methionine.
- polypeptide or protein which acts as an absorption-enhancing agent comprises an amino acid sequence which falls under the general formula below:
- XI 2 is a variable amino acid, preferably proline, lysine, methionine, valine, isoleucine or threonine, in particular isoleucine or threonine.
- the polypeptide or protein which acts as a absorption-enhancing agent comprises one of the amino acid sequences listed below or an amino acid sequence derived therefrom:
- polypeptide or protein that acts as an absorption enhancer comprises the amino acid sequence:
- polypeptides or proteins described according to the invention which act as absorption-enhancing agents, can also be derivatives thereof, in particular amino acid insertion variants, amino acid deletion variants and / or amino acid substitution variants.
- Amino acids are preferably replaced by others with similar properties such as hydrophobicity, hydrophilicity, electronegativity, volume of the side chain and the like (conservative substitution).
- Conservative substitutions involve, for example, the replacement of one amino acid by another, with both amino acids listed in the same group below:
- 1 to 6, preferably 1 to 4, more preferably 1 to 3, in particular 1 to 2, amino acids can be replaced in the polypeptides or proteins described according to the invention, which function as absorption-enhancing agents.
- polypeptides or proteins described according to the invention can also include non-naturally occurring amino acids such as D-amino acids, non-classical amino acids or chemical amino acid analogs.
- Non-classical amino acids and chemical amino acid analogs include, but are not limited to, ⁇ -aminobutyric acid, aminobutyric acids, aminohexanoic acids, aminopropionic acids, ⁇ -alanine, ⁇ -carboxyglutamic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine t-butylguanine, phenylglycine, cyclohexylalanine, P-alanine, fluoroamino acids, phenylalanine, the ring of which is methylated, and the like.
- Each amino acid residue can be replaced by a non-classical amino acid or a chemical amino acid analog.
- the polypeptide or protein which acts as a absorption-enhancing agent comprises an amino acid sequence or a sequence derived therefrom which has a hydropathy profile which corresponds to one or more of the amino acid sequences listed below:
- hydroopathy profile which corresponds to an amino acid sequence means that amino acid residues occur at corresponding positions of two or more amino acid sequences, each of which can be assigned hydrophilic, hydrophobic or variable amino acid residues.
- the polypeptide or protein which acts as an absorption-enhancing agent comprises an ajuino acid sequence which, with regard to at least 10, more preferably at least 11, in particular 12 amino acid residues, corresponds to the hydropathy profile of these amino acid sequences (1) to (9) either individually or in a summary of corresponds to two or more amino acid sequences.
- the absorption-enhancing polypeptide or protein can be attached to the N-, C-tenriinus, on a side chain and / or in internal as an insertion (internal) of the substance to be coupled.
- Peptides or proteins which contain the absorption-enhancing agent at the N- and / or C-terminus can be produced recombinantly in that a nucleic acid coding for the absorption-enhancing polypeptide or protein with the
- Nucleic acid which codes for the peptide or protein to be coupled is fused and the fused sequence is expressed, for example, in a cell. Furthermore, if the substance, which is a peptide or protein, is to contain the absorption-enhancing agent internally, the nucleic acid coding for the absorption-enhancing agent can be inserted into the nucleic acid coding for the substance.
- the invention also relates to such peptide / protein constructs and nucleic acids and derivatives coding therefor.
- peptide / protein constructs and nucleic acids or derivatives thereof are preferably recombinant constructs and not peptides / proteins or nucleic acids which contain the polypeptides or proteins described according to the invention which function as an absorption-enhancing agent or nucleic acids coding therefor, the term "naturally" being a peptide , Protein or a nucleic acid that occurs in nature, eg in an animal or a plant without human interference.
- a coupling of a peptide / protein substance with a polypeptide or protein, which acts as an absorption-enhancing agent, via the side chain (s) of the peptide / protein substance can, for example, via acidic amino acids and their amides such as aspartic acid, asparagine, glutamic acid and glutamine or basic Amino acids such as lysine and arginine are made directly or via a linker.
- any substance, inorganic or organic in nature can be coupled with an agent which enhances the absorption of the substance through the skin or mucosa.
- the substance can be absorbed, poorly absorbed or not absorbed.
- the substance is preferably an active pharmaceutical ingredient whose transdermal or transmucosal absorption can be improved.
- the pharmaceutical active ingredient can be of animal or vegetable origin, and is preferably a pure substance of animal or vegetable origin, or can be of synthetic origin.
- At least 2, preferably 2 to 4, more preferably 2 to 3, in particular 2 substances, which may be the same or different, are coupled to one another and this conjugate is preferably at least 1, preferably 1 to 5, more preferably 1 to 3, more preferably 1 or 2, in particular 1 identical or different absorption-enhancing means coupled.
- the substances and / or the absorption-enhancing agent (s) coupled via linker are coupled to one another and this conjugate.
- the substance coupled to the absorption enhancing agent may have its native (i.e. naturally occurring and active) structure or a modified structure.
- the term “modified structure” is understood to mean any non-native structure of the substance.
- a modified structure comprises, for example, a modified polypeptide or protein in which one or more modifications, in particular post-translational modifications, are missing and / or are additionally present compared to the native polypeptide or protein.
- Modifications, particularly post-translational modifications include, but are not limited to, glycosylation rings, oxidation of cysteine side chains, isomerizations of disulfide bridges and peptidyl-prolyl bonds, hydroxylations, carboxylations, acylations and the like.
- the substance, which is coupled to the absorption-enhancing agent can have an activity before or after transdermal or transmucosal absorption which corresponds to or is lower or higher than that of the native substance.
- the activity of the substance before or after transdermal or transmucosal absorption is less than 100%, less than 80%, less than 60% or less than 50% of the activity of the native substance.
- the substance has no activity, i.e. it is inactive compared to the native substance.
- the substance can be used in particular for immunization.
- a pharmaceutical active substance can comprise any biologically active substance which is selected from the group: analgesics, amino acids, anorectics, antibiotics, antiallergics, antiarrhythmics, anticholinergics, antidepressants, antidiabetics, antidots, antiemetics, antiepileptics, anti-infectious agents, antigens, antihistamines and Histamines, antihypertensives, anticoagulants, anticonvulsants, antibodies, antifungals, antineoplastics, antiphlogistics, antipsorics, antipyretics, Antiseptics, antitumor agents, antitussives (asthma drugs) and other respiratory medicines, antivirals and anti-cancer drugs, anti-wormers, anxiolytics, ophthalmic drugs (including anti-glaucoma drugs), beta-blockers, imaging agents, blood factors, bronchodilators, chaperones, chemokines, cholesterol drugs,
- Analgesics include, but are not limited to, fentanyl, Mo ⁇ hin, tramadol, hydrocodeine, methadone, lidocaine, diclofenac, paverin and the like.
- Antiarrhythmic drugs include substances that affect the process of excitation of the heart, preferably to treat irregular heartbeats.
- An example of a class of antiarrhythmics are beta blockers such as propanolol, alprenolol, timolol, nadoxolol and the like.
- Antibiotics, anti-infectious agents, antifungals and antivirals include, but are not limited to, tetracyclines, tetracycline-like antibiotics, erythromycin, 2-thiopyridine-N-oxide, halogen compounds (preferably iodine-containing compounds such as iodine-polyvinylpyrrolidone complex), ⁇ -lactam compounds such as penicilin - Compounds (e.g. penicilin G or V), cephalosporins, sulfonamide compounds, aminoglycoside compounds (such as streptomycin), amphothericin B, 5-iodo-2-deoxyuridine, gramicidin, nystatin and the like.
- halogen compounds preferably iodine-containing compounds such as iodine-polyvinylpyrrolidone complex
- ⁇ -lactam compounds such as penicilin - Compounds (e.g. penicilin G or
- Antidiuretics and diuretics include, but are not limited to, desmopressin, vasopressin, furosemide, and the like.
- antiemetics include pipamazine, chloromazine, dimenhydrinate, meclozin, metoclopramide, and the like.
- Antihistamines include compounds that inhibit the effects of histamine.
- Non-limiting examples include 3- (2-aminoethyl) pyrazole, cimetidine, cyproheptadine hydrochloride and the like.
- Antihypertensives, angina drugs and vasodilators include, but are not limited to, compounds such as clonidine, ⁇ -methyldopa, nitroglycerin, polynitrates of polyalcohols (e.g. erythritol tetranitrate and mannitol hexanitrate), papaverine, dipyridamole, nifedipine, diltiazem and the like.
- Anti-inflammatory drugs include, but are not limited to, steroidal and non-steroidal anti-inflammatory drugs. Examples include cortisone, hydrocortisone, betamethasone, dexamethasone, prednisolone, ibuprofen, aspirin, salicylic acid, flumethasone, fluprednisolone, aminopyrine, antipyrine, fluprofen and derivatives thereof.
- Antitussives include, but are not limited to, compounds such as cromoglycate and its derivatives, beclomethasone, budesonide, salbutamol, mometasone, terbutaline and the like. Contraceptives relate to compounds that prevent ovulation or implantation of the fertilized egg in the placenta in female patients or sperm maturation in male patients.
- Nonlimiting examples of mer are ethinyl estradiol, medroxyprogesterone acetate and anti-progestins (such as RU 486).
- Anti-migraine drugs include, but are not limited to, heparin, hirudin, and the like.
- muscle relaxants include, but are not limited to, cyclobenzapyrin hydrochloride, diazepam, alcuronium, vecuro ium, succinyldicholine, and the like.
- Narcotics and local anesthetics include, but are not limited to, benzocaine, procaine, propoxycaine, dibucain, lidocaine, naloxone, naltrexone, and derivatives thereof.
- Peptidomimetics and inverse peptides include peptide-like compounds that act as peptides but do not have the typical peptide structure.
- a non-limiting example of this is a peptide analog, which, in contrast to its native peptide, is composed only of D-amino acids.
- Potency enhancers include, but are not limited to, such pharmaceutical agents that increase a patient's libido and / or lead to prolonged sexual performance.
- Examples of potency-increasing agents are those that increase NO synthesis in the patient (e.g. sildenafine).
- Steroid hormones are those hormones that are derived from cholesterol.
- Steroid hormones include, but are not limited to, progestogens (such as progesterone), corticoids that include glucocorticoids (such as cortisone and cortisol) and mineral corticoids (such as aldosterone), sex hormones such as androgens (such as testosterone), and estrogens (such as estrone and oestradiol), and derivatives thereof (such as Dexamethasone, betamethasone, prednisolone, beclomethasone, mometasone and the like).
- the active ingredient can also be a nucleic acid or "antisense" nucleic acid or a derivative thereof.
- Antisense molecules or “antisense” nucleic acids can be used for regulation, in particular the reduction of the expression of a nucleic acid.
- the term “antisense molecule” or “antisense nucleic acid” relates to an oligonucleotide which is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide or modified oligodeoxyribonucleotide and which under physiological conditions hybridizes to DNA comprising a specific gene or mRNA whereby the transcription of this gene and / or the translation of this mRNA is inhibited.
- an “antisense molecule” also comprises a construct which contains a nucleic acid or a part thereof in a reverse orientation with respect to its natural promoter.
- An antisense transcript of a nucleic acid or a portion thereof can enter a duplex molecule with the naturally occurring mRNA that specifies the enzyme, thus preventing accumulation or translation of the mRNA into the active enzyme.
- an oligonucleotide is a "modified" oligonucleotide.
- the oligonucleotide can be modified in many different ways, for example to increase its stability or therapeutic effectiveness, without impairing its ability to bind to its target.
- the term “modified oligonucleotide” means an oligonucleotide in which (i) at least two of its nucleotides are linked to one another by a synthetic intemucleoside bond (ie an intemucleoside bond which is not a phosphodiester bond) and or (ii) a chemical group is covalently linked to the oligonucleotide that does not normally occur with nucleic acids.
- Preferred synthetic intemucleoside bonds are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
- modified oligonucleotide also includes oligonucleotides with a covalently modified base and / or sugar and oligonucleotides that are not in nature contain occurring nucleotides and / or nucleotide analogs.
- Modified oligonucleotides include, for example, oligonucleotides with sugar residues that are covalently bound to low molecular weight organic groups that are not a hydroxyl group at the 3 'position and not a phosphate group at the 5' position.
- Modified oligonucleotides can include, for example, a 2'-O-allylated ribose residue or another sugar instead of ribose such as arabinose.
- Modified oligonucleotides can also contain modified bases and / or base analogs such as 7-deazaadenosine, 7-deazaguanosine, isoguanosine, 2-thiopyrimidine, isocytidine, universal base and the like.
- the active ingredient can also be a gene, a gene correcting oligonucleotide, an aptameric oligonucleotide, triple helix nucleotide or a ribozyme.
- the active ingredient can also be a polypeptide or protein or a derivative thereof. Furthermore, it can be a conjugate of several peptides or proteins that have been chemically or genetically coupled to one another.
- the peptides or proteins used according to the invention can be derived from a natural source or can be recombinantly or chemically synthesized substances.
- the polypeptides and proteins used according to the invention are preferably isolated.
- isolated protein or isolated polypeptide” mean that the protein or polypeptide is separated from its natural environment.
- An isolated protein or polypeptide can be in a substantially purified state.
- substantially purified means that the protein or polypeptide is essentially free of other substances with which it is present in nature or in vivo.
- polypeptides or proteins which can be used according to the invention include, without limitation, antibiotics, hematopoietics, anti-infectious agents, anti-dementia agents, anti-viral agents, anti-tumor agents, anti-pyretic agents, analgesics, anti-inflammatory agents, anti-allergic agents, anti-depressant agents, anti-psoriatic agents, psychotropic agents, cardiotonic agents, vasodilator drugs, anti-antioxidants , Antihypertensives, antidiabetics, anticoagulants, cholesterol-lowering agents, therapeutic agents for osteoporosis, Hormones, vaccines and the like, as well as those polypeptides and proteins that have been described above as pharmaceutical active ingredients.
- Particularly preferred peptides or proteins include cytokines, peptide hormones, growth factors, factors of the cardiovascular system, factors of the central and peripheral nervous system, factors of the gastrointestinal system, factors of the immune system, enzymes and vaccines.
- Lymphokines monokines, hematopoietic factors and the like are particularly preferred.
- Lymphokines include interferons (e.g. ⁇ -, ⁇ - and ⁇ -interferon and their subtypes including IFN- ⁇ -2a, IFN- ⁇ -2b and IFN- ⁇ -n3), interleukins (e.g. interleukin 1-17) and the like.
- interferons e.g. ⁇ -, ⁇ - and ⁇ -interferon and their subtypes including IFN- ⁇ -2a, IFN- ⁇ -2b and IFN- ⁇ -n3
- interleukins e.g. interleukin 1-17
- Interferon is a term that generally encompasses a group of glycoproteins and proteins from vertebrates that are known to have various biological activities such as antiviral, antiproliferative and immunomodulating activities.
- the term “interferon” relates to native as well as recombinant proteins, and also proteins that are expressed in eukaryotic cells, in particular mammalian cells, and also prokaryotic cells.
- the term “interferon” includes both IFN-ß-la and IFN-ß-lb with respect to IFN- ⁇ .
- Interferons are secretory proteins that can be divided into two different subtypes.
- Type I interferons include in particular the members of the interferon- ⁇ multigen family (there are approximately 14-20 different IFN- ⁇ molecules), IFN- ⁇ (also called trophoblast-IFN), as well as IFN-ß and IFN- ⁇ ,
- the type I IFN genes are located as “clusters” on the short arm of chromosome 9. While IFN- ⁇ and IFN- ⁇ are preferably formed by cells of the hematopoietic system, IFN-ß is formed by non-hematopoietic cells, especially fibroblasts. IFN-ß is a glycoprotein (N-glycosylation), while most human IFN- ⁇ subspecies have no N-glycosylation. IFN- ⁇ and IFN-ß form dimers in the active form.
- IFN- ⁇ belongs to the type II interferons. IFN- ⁇ is a glycoprotein that is also present as a dimer in the active form. IFN- ⁇ is especially formed in CD4 + T helper cells and in almost all CD8 + cells. Despite a great functional similarity, there is no significant structural similarity between Type I and Type II interferons.
- Interferons are important pharmaceuticals for the therapy of e.g. viral diseases, tumor diseases and immune defects.
- Systemic application is usually intravenous, subcutaneous or intramuscular.
- local forms of application e.g. intratumoral injection and topical gel.
- IFN- ⁇ the partial acid instability of the molecule also limits oral use.
- cytokines include, but are not limited to, colony-stimulating factor 4, heparin-binding neutrotrophic factor (HBNF), midkin (MD) and thymopoeitin.
- HBNF heparin-binding neutrotrophic factor
- MD midkin
- thymopoeitin thymopoeitin
- monokines include interleukin-1, tumor necrosis factors (e.g. TNF- ⁇ and -ß), leukocyte-inhibiting factor (LIF) and the like.
- tumor necrosis factors e.g. TNF- ⁇ and -ß
- LIF leukocyte-inhibiting factor
- hematopoietic factors include, for example, erythropoietin, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF).
- Anticoagulants include blood clotting modifiers that circulate in the blood and control blood clotting.
- Non-limiting examples of this are factor I, II, III, V, VI, VII, Vi ⁇ , EX, X, XI and XII, ⁇ l-antitrypsin, ⁇ 2-macroglobulin, antithrombin III, heparin cofactor ⁇ , kallikrein, plasmin, plasminogen, Prokallikrein, Protein C, Protein S, thrombomodulin and the like.
- Peptide hormones include, for example, insulin, glucagon, growth hormone, luteinizing hormone releasing hormone (LH-RH), adrenocorticotrophin (ACTH), amylin, oxitozin, luteinizing hormone (LH), calcitonin, protein that controls the calcitonin gene, calcitonin N-terminal flanking peptide, somatotropin, somatostatin, somatomedin, tissue plasminogen activator (TPA), leuprolide acetate and the like.
- growth factors include, for example, nerve growth factor (NGF), epidermis growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), hematocyte growth hormone growth hormone (hormone hormone) ), human growth hormone (hGH) and the like.
- NGF nerve growth factor
- EGF epidermis growth factor
- FGF fibroblast growth factor
- IGF insulin-like growth factor
- TGF transforming growth factor
- PDGF platelet-derived growth factor
- hGH human growth hormone
- Factors of the cardiovascular system are, for example, factors that regulate blood pressure, arterosclerosis and the like, such as endotheline, endofelin inhibitors, endothelin antagonists, vasopressin (ADH), renin, angiotensin, atrial natriuretic factor (ANP) and the like.
- Hormones derived from peptides include, but are not limited to, activin, cholecystokinin (CCK), ciliary neurotrophic factor (CNTF), cortotropin-releasing factor (CRF or CRH), follicle-stimulating hormone (FSH), gastrin-inhibiting peptide ( GIP), gastrin releasing peptide, ghrelin, gonadotropin releasing factor (GnRF or GNRH), growth hormone releasing factor (GRF, GRH), human chorionic gonadotropin (hCH), inhibin A, inhibin B, leptin, lipotropm (LPH), ⁇ -Melanocyte stimulating hormone, ß-Melanocyte stimulating hormone, ⁇ - Melanocyte stimulating hormone, melatonin, motilin, pancreatic polypeptide, Parathyroid hormones (PTH), prolactin of the placenta, prolactin (PRL), prolactin release inhibiting factor (PIF),
- Central or peripheral nervous system factors include, for example, opioid peptides (e.g. enkephalins, endo ⁇ hins, Kvto ⁇ hine), neutrotrophic factor (NTF), tyroid hormone releasing hormone (TRH), neurotensin and the like.
- opioid peptides e.g. enkephalins, endo ⁇ hins, Kvto ⁇ hine
- NTF neutrotrophic factor
- TRH tyroid hormone releasing hormone
- Endophine or pharmacologically active derivatives thereof include, in a non-limiting manner, dermoophine, dynophine, ⁇ -endophine, ⁇ -endophine, ⁇ -endophine, ⁇ -endophine [Leu5] enkephalin, [Met5] £ nkephalin, substance P and the like.
- Factors of the gastrointestinal system are, for example, secretin and gastrin.
- Immune system factors are, for example, factors that control inflammation and neoplasms and factors that attack infectious microorganisms, such as antibodies, chemotactic peptides or bradykinins.
- an antibody can be a monoclonal antibody.
- the antibody is a chimeric or humanized antibody, a fragment of a natural antibody or a synthetic antibody, which can be produced by combinatorial techniques.
- Antibodies can also be coupled to specific diagnostic substances for the display of cells and tissues. They can also be coupled to therapeutically useful substances. Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, lopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate, and radiodiagnostics, including positron emitters such as fluorine-18 and carbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131 and indium-111, nuclear magnetic resonance nuclides such as fluorine and gadolinium.
- therapeutically useful substance means any therapeutically usable molecule, including anti-cancer agents, compounds provided with radioactive iodine, technetium or other radioisotopes, toxins, cytostatic or cytolytic drugs, etc.
- Anti-cancer agents include, for example, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, Chlorambucil, cisplatin, cyclophosphamide, cyclosporin, cytarabine, dacarbazine, dactinomycin, daunorabine, doxorubicin, taxol, etoposide, fluorouracil, interferon- ⁇ , lomustine, mercaptopurine, methotrexate, mitotan, procarbazine-HCl, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sulfin, vincristin sul
- Toxins can proteins like Pokeweed- antiviral protein, cholera toxin, pertu ssistoxin, ricin, gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin.
- Toxin residues can also be high-energy emitting radionulide such as cobalt-60.
- the substance is a dermatological agent.
- Dermatological agents include cosmetics such as sunscreens, the inner tissues of the skin (especially the tissues below the stratum corneum) from external factors such as UV rays in the UV-A and UV-B range (preferably radiation in the range from 280 to 400 nm) protect (e.g. p-aminobenzoic acid, p-dimethylaminobenzoic acid and its alkyl esters), skin lightening agents (e.g. hydroquinone), vitamins (e.g.
- dermatological agents include agents for itching and erythema (e.g. hydrocortisone), for acne (e.g. erythromycin or tetracycline), for He ⁇ es simplex (e.g. 5-iodo-2-deoxyuridine), for psoriasis or skin cancer (e.g. fluorouracil).
- erythema e.g. hydrocortisone
- acne e.g. erythromycin or tetracycline
- He ⁇ es simplex e.g. 5-iodo-2-deoxyuridine
- psoriasis or skin cancer e.g. fluorouracil
- the agent which enhances the absorption of a substance through the skin or mucosa is, if appropriate, with a particle biodegradable nanoparticles, optionally biodegradable microparticles, optionally biodegradable nanospheres, optionally biodegradable microspheres, a capsule, emulsion, micelle, a liposome, a non-viral vector system or a viral vector system, coupled or loaded with it.
- the particle is preferably a particle derived from a Viras (virus-like particle), which preferably binds non-specifically or specifically to cells and can introduce a nucleic acid into them.
- the particle contains a substance as described above, in particular a nucleic acid or a peptide or protein, which is to be absorbed by the skin or mucosa.
- a substance as described above in particular a nucleic acid or a peptide or protein, which is to be absorbed by the skin or mucosa.
- the particles preferably comprise: (a) a protein envelope, which preferably comprises a viral protein, a resorption enhancing agent, preferably a peptide or protein, and optionally a heterologous cell-specific binding site as a fusion molecule, and (b) a nucleic acid present in the protein envelope, the sequences for a virus-specific packaging signal and a structural gene.
- virus encompasses DNA and RNA viruses, in particular adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, hepatitis C viruses, hepatitis A viruses, influenza viruses and Hepadna viruses.
- adenoviruses in particular adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, hepatitis C viruses, hepatitis A viruses, influenza viruses and Hepadna viruses.
- HBV HBV
- WHV woodchuck hepatitis virus
- GSHV ground squirrel hepatitis virus
- RBSFIV red-bellied squirrel hepatitis viras
- DHV Pekin duck hepatitis viras
- HHN hereon hepatitis virus "heron hepatitis virus ")
- the expression “structural gene” encompasses any gene which codes for a polypeptide or protein, such as
- the agent that enhances the resorption of a substance through the skin or mucosa can be adsorbed, non-covalently or covalently coupled, either directly or via a linker, to the particle, to the particle (s) used for particle synthesis ) Polymer (s) or monomer (s) or bound to other constituents of the particle.
- the particle is loaded with a therapeutic, prophylactic or diagnostic substance, the agent which increases the absorption of a substance through the skin or mucosa being bound to or loaded with the particle.
- a particle according to the invention can be produced by customary methods.
- Substances, in particular peptides or proteins, which are coupled according to the invention to a absorption-enhancing agent, in particular polypeptide or protein, can be used as immunogens to induce the production of antibodies which preferably bind the immunogen in an immunospecific manner.
- the invention also relates to a method for producing antibodies, comprising inducing antibody production by administering substances, in particular peptides or proteins, which are coupled with resorption-enhancing agents according to the invention, to a living being, in particular a human or an animal, and one Isolation of these antibodies.
- Figure 1 is a bar graph showing the amount of IFN- ⁇ detected in serum 4h and 8h after oral administration of IFN- ⁇ -la-TLM (TLM-1 and TLM-2).
- Nl negative control 1 (untreated animals);
- N2 negative control 2 (feeding rank of PreSlPreS2);
- N3 negative control 3 '(feeding rank of commercial recombinant IFN-ß-la).
- Figure 2 is a bar graph showing the amount of IFN- ⁇ detected in serum 4h and 8h after dermal administration of IFN- ⁇ -la-TLM (TLM).
- Nl negative control 1 (untreated animals);
- N2 Negative control 2 (dermal administration of commercially available recombinant IFN-ß-la).
- the term "reso ⁇ tion” is understood to mean the absorption of substances from the body surface.
- the resection comprises in particular a resection through the skin (i.e. transdermally, percutaneously) or via the mucosa (mucous membrane) (i.e. transmucosal), preferably in the blood, lymphatic system and / or lower skin layers, from where it can be distributed throughout the organism.
- the reso ⁇ tion can take place via the passive mechanism of diffusion but also via active transport mechanisms.
- a substance that is coupled to a resorption enhancing agent preferably enters the outermost layer of the skin (stratum corneum) in the event of resorption through the skin or mucosa of a patient.
- the substance coupled with the absorption-enhancing agent reaches the layers below.
- the substance coupled with the absorption-enhancing agent is released into the bloodstream.
- reinforcement refers to an increase, increase or improvement over a previous state.
- enhancement of reso ⁇ tion refers to an increase in reso ⁇ tion, i.e. a larger amount of a substance is absorbed in a certain period of time, in particular by increasing the speed at which a substance penetrates a body barrier such as the skin and mucous membrane.
- bioavailability characterizes the speed and the extent to which the therapeutically effective portion of a drug is released and reabsorbed from the respective drug forms or is available at the site of action. It can be determined by measuring the drug concentration in the body fluids and the acute pharmacological effect.
- permeability refers to the property, such as skin and mucosa to have a substance pass •.
- permeability and penetration refer to the ability of a substance to cross such a barrier.
- transdermal or transmucosal preparation refers to a substance, in particular a pharmaceutical active ingredient, which was originally not or only poorly absorbed by the skin or mucosa, but was modified so that it is absorbed by the skin or mucosa and therefore for administration to the skin or mucosa is suitable.
- Mucosa or “mucous membrane” according to the invention can be any mucous membrane of a mammal, including humans.
- mucous membranes examples include the mucous membrane of the gastrointestinal tract (eg intestinal mucosa, gastric mucosa), eye mucosa, nasal mucosa, tracheal / bronchial / lung mucosa, mucous membrane of the oral cavity, rectum, genital tract, vagina, ureter and the like.
- the mucous membrane is preferably a mucous membrane of the nose, mouth or gastrointestinal tract.
- Transdermal administration or “transmucosal administration” means delivery through the skin or mucosa.
- agents which increase the absorption of a substance are those substances or preparations which promote the transport of other substances through barriers and constrictions, in particular permeation obstacles, and preferably their bioavailability, ability, to be absorbed and / or to increase permeability (penetration ability).
- the obstacles to permeation include, in particular, human and animal skin layers, in particular Der is (in particular stratum comeum) and mucosa.
- the agent that enhances the absorption of a substance through the skin or mucosa is free of toxic side effects.
- Non-covalent bonds include, but are not limited to, ionic interactions, hydrogen bonds, van der Waals interactions (hydrophobic interactions), and bonds that result from the inclusion of one compound in another (eg, in crown ethers and cage compounds) ).
- Covalent coupling of, for example, peptides and proteins can be carried out using coupling agents such as N, N'-dicyclohexylcarbodiimide (DCC) or N, N'-diisopropylcarbodiimide (DIPCDI) or by recombinant techniques in a manner known per se.
- DCC N, N'-dicyclohexylcarbodiimide
- DIPCDI N, N'-diisopropylcarbodiimide
- a nucleic acid is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
- nucleic acids include genomic DNA, cDNA, mRNA, rRNA, tRNA, recombinantly produced and chemically synthesized molecules.
- a nucleic acid can be present as a single-stranded or double-stranded and linear or covalently circular molecule.
- “derivative” of a nucleic acid means that single or multiple nucleotide substitution, deletion and / or addition are present in the nucleic acid. Furthermore, the term “derivative” also includes chemical derivatization of a nucleic acid on a base, a sugar or phosphate of a nucleotide. The term “derivative” also encompasses nucleic acids which contain non-naturally occurring nucleotides and nucleotide analogs.
- isolated nucleic acid means that the nucleic acid (i) has been amplified in vitro, for example by polymerase chain reaction (PCR), (ii) has been produced recombinantly by cloning, (iii) has been purified, for example by cleavage and gel electrophoretic separation, or (iv) synthesized, for example by chemical synthesis.
- An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant DNA techniques.
- RNA or of RNA and protein are used according to the invention in its most general meaning and encompasses the production of RNA or of RNA and protein. It also includes partial expression of nucleic acids. Furthermore, the expression can be transient or stable.
- sequence derived from an amino acid sequence relates to derivatives of the former sequence.
- “Derivatives” of a protein or polypeptide or an amino acid sequence in the sense of this invention include amino acid insertion variants, amino acid deletion variants and / or amino acid substitution variants.
- Amino acid insertion variants include amino- and / or carboxy-terminal fusions, as well as insertions of single or several amino acids in a certain amino acid sequence.
- amino acid sequence variants with an insertion one or more amino acid residues are introduced into a predetermined position in an amino acid sequence, although a random insertion with suitable screening of the resulting product is also possible.
- Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence.
- Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place.
- the modifications are preferably at positions in the amino acid sequence that are not conserved between homologous proteins or polypeptides.
- Amino acids are preferably replaced by others with similar properties, such as 1 hydrophobicity, hydrophilicity, electronegativity, volume of the side chain and the like (conservative substitution). For example, conservative substitutions involve the replacement of one amino acid by another, with both amino acids listed in the same group below:
- amino acid variants described above can easily be made using known peptide synthesis techniques such as e.g. through “Solid Phase Synthesis” (Merrifield,
- “derivatives” of proteins or polypeptides also include single or multiple substitutions, deletions and / or additions of any molecules which are associated with the enzyme, such as carbohydrates, lipids and / or proteins or polypeptides.
- derivatives of proteins or polypeptides include those modified analogs which are characterized by glycosylation, acetylation, phosphoryl, or amidation, palmitoylation, myristylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective / blocking groups, proteolytic cleavage or binding to one Antibodies or to another cellular ligand arise.
- Derivatives of proteins or polypeptides can also be prepared by other processes, such as, for example, by chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 2 , acetylation ring, formylation ring, oxidation, reduction or by metabolic synthesis in the presence of tunicamycin.
- a part or fragment of a polypeptide or protein has a functional property of the polypeptide or protein from which it is derived. Such functional properties include the interaction with other molecules such as antibodies, polypeptides or proteins, the selective binding of nucleic acids and an enzymatic activity.
- a part or fragment of a peptide or protein preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids from the peptide or protein.
- the terms “active pharmaceutical ingredient,“ pharmaceutically active substance ”or“ pharmaceutically active ” refer to any agent that can be used in therapy (including prophylaxis) or diagnosis.
- the agent is in particular any therapeutic or prophylactic agent used in treatment (including prevention, relief or cure) of a disease, ailments or an injury to a patient and has the desired biological or pharmacological effect.
- a pharmaceutical active substance can be a "dermally active dermatological active substance or a" systemically active dermatological active substance.
- the term "dermally active dermatological agent as used herein refers to those chemical and biochemical substances which, when applied to a patient's skin, produce a beneficial topical effect, which are cosmetic or therapeutic in nature (e.g. a mitigation of a skin disease)
- the term "systemic dermatological agent as used herein refers to those chemical and biochemical substances which, when applied to a patient's skin, enter the bloodstream and have a therapeutic effect.
- the ; Terms "dermatologically active dermatological active substance and" systemically active dermatological active substance should not be mutually exclusive, since a number of pharmaceutical active substances are both dermally and systemically active.
- a pharmaceutical active substance can also be a "mucosal active mucosal active substance or a" systemic active mucosal active substance, the terms “mucosal active mucosal active substance and” systemic active mucosal active substance being a dermatological active substance or "systemic dermatological active substance” as defined above Active substance have corresponding meaning.
- the active pharmaceutical ingredient is preferably formulated in neutral or salt form.
- Pharmaceutically acceptable salts include, but are not limited to, those that form with free amino or carboxyl groups.
- Suitable acids for the preparation of acid addition salts are inorganic acids, such as HC1, HBr, H SO 4 , HNO, H PO 4 and the like, and organic acids, such as acetic acid, propionic acid, oxalic acid, Maleic acid, malonic acid, succinic acid, malic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
- Basic compounds that can form salts with the carboxyl groups include, but are not limited to, NaOH, KOH, NH, Ca (OH) 2 , iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.
- the pharmaceutical active ingredient can also be a pharmaceutical precursor which can be activated before, during or after penetration of the active ingredient through the skin or mucosa.
- drug precursor refers to an agent that is inactive but is convertible to an active form via an enzymatic, chemical, or physical activation.
- compositions can be prepared in a manner known per se and usually contain suitable pharmaceutically acceptable auxiliaries and carriers.
- pharmaceutically acceptable refers to a substance which causes no or only a slight significant irritation or toxicity in the patient being treated and which does not cancel or interact with the biological activity and properties of the active ingredient.
- carrier relates to one or more compatible solid or liquid fillers, diluents, adjuvants, excipients or capsule substances which are suitable for administration to a human being.
- carrier relates to an organic or inorganic component, natural or synthetic in nature, in which the active ingredient is combined to facilitate application
- the ingredients of the pharmaceutical composition of the invention are usually such that there will be no interaction that would significantly affect the desired pharmaceutical efficacy.
- the carriers are sterile liquids such as water or oils, including those derived from petroleum, animals or plants or of synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, sunflower oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be used as aqueous carriers.
- auxiliaries and carriers are acrylic and methacrylic derivatives, alginic acid, sorbic acid derivatives such as ⁇ -octadecyl- ⁇ -hydroxypoly (oxyethylene) -5-sorbic acid,
- Amino acids and their derivatives especially amine compounds such as choline, lecithin and phosphatidylcholine, gum arabic, flavorings, ascorbic acid, carbonates such as sodium, potassium, magnesium and calcium carbonate and hydrogen carbonate, hydrogen phosphates and phosphates of sodium, potassium, calcium and magnesium, Carmellose sodium, dimeticone, colors, flavors, buffer substances, preservatives, thickeners, plasticizers, gelatin, glucose syrups, malt, highly disperse silicon dioxide, hydromellose, benzoates, especially sodium and potassium benzoate, macrogol, skimmed milk powder, magnesium oxide, fatty acids and stearic acids and their derivatives and salts and their derivatives and salts Stearates, especially magnesium and potassium stearate, fatty acid esters and mono- and diglycerides of fatty acids, natural and artificial waxes such as beeswax, yellow wax and montanglycol wax, chlorides, especially sodium chloride, polyvidone, polyethylene glycols, poly
- the pharmaceutical compositions can preferably also additionally contain wetting agents, emulsifiers and / or pH-buffering agents.
- the pharmaceutical compositions can contain an additional absorption enhancer. These additional resorption enhancers can, if desired, replace an equimolar amount of the vehicle in the composition.
- additional absorption enhancers include, but are not limited to, eucalyptol, N, N-diethyl-m-toluamide, polyoxyalkylene alcohols (such as propylene glycol and polyethylene glycol), N-methyl-2-pyrrolidone, isopropyl myristate, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), Dimefhylacetamide (DMA), urea, diethanolamine, triethanolamine and the like (see e.g. Percutaneous Penetration Enhancers, ed. Smith et al. (CRC Press, 1995)).
- the amount of additional absorption enhancer in the composition may depend on the desired effects to be achieved.
- protease inhibitor can be incorporated into the composition according to the invention in order to avoid degradation of a peptide or protein active ingredient and thereby to increase the bioavailability.
- protease inhibitors include, but are not limited to, aprotinin, leupepsin, pepstatin, ⁇ 2-macroglobulin and trypsin inhibitor. These inhibitors can be used alone or in combination.
- compositions according to the invention can be provided with one or more coatings.
- the solid oral dosage forms are preferably provided with an enteric coating or are in the form of an enteric, hardened soft gelatin capsule.
- Dosage forms can include materials that release the pharmaceutically active substance in a specific portion of the gastrointestinal tract, thereby enhancing site-directed delivery.
- compositions described herein can also be administered as a sustained release formulation (ie, a formulation that causes slow release of the drug after administration).
- sustained release formulations are known.
- the pharmaceutical compositions of the invention may be formulated for administration by any transdermal or transmucosal route, including, for example, topical, oral, enteral, intracranial, sublingual, nasal, buccal, vaginal, ocular or urethral.
- Enteral, more preferably oral dosage forms are particularly preferred, in particular enteric formulations and retarded formulations of oral forms.
- rectal dosage forms such as suppositories, vaginal dosage forms such as suppositories, and nasally applicable preparations such as nasal sprays are also possible.
- the pharmaceutical composition is incorporated into the matrix of a plaster in order to release the substance, in particular the pharmaceutical active substance, which is coupled to the absorption-enhancing agent to the skin over a longer period of time.
- the pharmaceutical formulations are, for example, in the form of tablets, suppositories, troches, dragees, drops, solutions, suspensions, emulsions (preferably oil-in-water or water-in-oil emulsions), ointments, gels, pastes, films, juices , Syrups, nasal sprays, vaginal suppositories or tablets, capsules, granules, pellets, microtablets, powders, rectal suppositories, rectal capsules, aerosols, shampoos or sprays.
- Hard or soft gelatin capsules are particularly preferred, optionally with an enteric coating, hardened soft gelatin capsules are very particularly preferred.
- the pharmaceutical composition can be an indirect dosage form such as an oral formulation for administration to the gastric or intestinal mucosa.
- the composition can also be administered directly to a mucous membrane.
- the pharmaceutical compositions are preferably topically or orally administrable drugs.
- the term “patient” means human, not human primacy or another animal, in particular mammal such as cow, horse, pig, sheep, goat, dog, cat, birds such as chicken or rodent such as mouse and rat. In a particularly preferred embodiment, the patient is a human.
- compositions according to the invention are preferably sterile and are administered in effective amounts.
- An "effective amount” refers to the amount that, alone or together with other doses, achieves a desired reaction or physiological effect.
- the desired response is to inhibit the course of the disease. This includes slowing the progression of the disease and, in particular, stopping the progression of the disease.
- the desired response in treating a disease or condition may also be to delay the onset or prevent the onset of the disease or condition.
- the effective amount can be selected according to the activity of the specific active pharmaceutical ingredient and its therapeutically effective dose. However, it is preferred to incorporate a slightly larger amount than the desired dose since the bioavailability of any active substance can never be 100%, i.e. the dose administered is not completely absorbed.
- physiologically active peptides or proteins are broken down by digestive juices in the gastrointestinal tract or hydrolyzed by enzymes in the gastrointestinal tract.
- An effective amount of a pharmaceutical composition is also determined by factors such as the patient's condition to be treated, the severity of the disease, the patient's individual parameters, including age, physiological condition, height and weight, duration of treatment, type of accompanying therapy ( if available), the specific route of administration, the desired administration period and similar factors. In the event that a patient's response to an initial dose is inadequate, higher doses (or effectively higher doses obtained by another, more localized route of administration) can be used.
- higher doses can be achieved by increasing the amount of absorption-enhancing agent, the concentration of the substance (in particular the pharmaceutical active ingredient) and / or the amount of additional absorption-enhancing agent in the formulation, and increasing the area to which the formulation is applied or a combination of them.
- PQe8 expression vectors were produced which code for IFN-ß in fusion with the sequence PLSSIFSRIGDP (TLM) at the 5 ⁇ and 3 ⁇ end.
- TLM sequence PLSSIFSRIGDP
- cDNAs were amplified by means of PCR which code for IFN-ß-specific fusion proteins which comprise the TLM sequence at the N- or C-terminal end.
- the forward primers had a BamHI-specific interface at their 5 "end and a HindII-specific interface at the 3 ⁇ end.
- a cDNA was amplified which contains the sequence coding for the TLM at the 5 "end.
- a sequence was amplified which contains the TLM-specific sequence at the 3" end .
- the IFN-ß-specific cDNA was amplified without 5 ⁇ - or 3 "-specific extensions by combining the primers C / B.
- the respective PCR products were purified by means of "PCR purification spin columns" in accordance with the manufacturer's instructions (Quiagen), BamHI / HindIII cleaved and cleaned again. The fragments restricted in this way were ligated into the BamHI / HindIII cleaved and dephosphorylated bacterial expression vector pQe8 (Quiagen).
- the vector pQe8 contains the sequence coding for an amino-terminal hexa-His tag, so that all IFN-ß-specific proteins were formed as hexa-His fusion proteins.
- the ligation approach was used to transform competent bacteria (DH5 ⁇ ).
- the amp resistance encoded on the plasmid pQe8 allowed selection on media containing amp.
- Plasmid DNA was isolated from the clones growing under these conditions and analyzed by means of BamHI / HindIII restriction. Positive clones were then characterized by sequencing.
- IFN-ß-specific fusion proteins The formation of IFN-ß-specific fusion proteins was induced as follows:
- the elution was carried out weakly bound proteins by means of a buffer containing 50 mM NaH 2 PO 4/300 mM NaCl / 20 mM imidazole, pH 8.0. Elution of the specifically bound hexa-His-tagged IFN-beta fusion proteins was effected by a linear gradient between buffer containing 50 mM NaH 2 PO 4/300 mM NaCl / 20 mM imidazole, pH 8.0 and a buffer of 50 mM NaH PO 4 / 300mM NaCl / 250mM imidazole, pH 8.0. The detection of eluted proteins was carried out by simultaneous detection of the absorption at 215, 260 and 280 nm. The eluate was collected in fractions of 1 ml.
- the isolation was carried out using an AEKTA Explorer or AEKTA Purifier system.
- a "reversed phase" chromatography was carried out in individual cases using an RP18 column.
- the eluate of the Ni-NTA column was diluted 1: 5 with the running buffer of the RP column (0.1% TFA in H 2 O) and loaded onto the column. Elution was carried out using a linear gradient between 0.1 / TFA in H 2 0 and 80% acetonitrile / H 2 O.
- the purity of the proteins isolated in this way was analyzed by SDS-PAGE according to Laemmli.
- the gels were stained using Coomassie or subjected to silver staining (according to Heukeshoven / Dernick).
- the identity of the detected protein bands with IFN-ß (IFN-ß-lb) was verified by Western blotting.
- the proteins were transferred to a PVDF membrane by means of electroblottings using the semi-dry method (Kyshe / Andersen).
- An IFN-ß-specific sheep serum was used to label the transferred IFN-ß-specific protein.
- the detection was carried out fluorographically using a peroxidase-conjugated secondary antibody using the ECL system (Amersham).
- the human hepatoma cell line huH7 was incubated for 30 min in the presence of 0.5 ⁇ M IFN-ß-lb-specific proteins in medium. To remove surface-bound IFNs, the cells were washed for 5 seconds with Na 2 CO 3 / NaHCO 3 buffer, pH 9.5 and then in PBS after removing the medium. After scraping, the cells were gently disrupted using a Potter homogenizer. After the undigested cells and the cell nuclei had been separated by centrifugation at 13 kUpm in an Eppendorf centrifuge for 30 seconds, the lysate was subjected to a differential centrifugation.
- the cytosol and the microsomal fraction could be isolated by ultra-centrifugation at 100,000 rpm (430,000 g) for 18 min.
- the cell fractions isolated in this way were subjected to SDS-PAGE and then analyzed by means of Western blotting using the IFN- ⁇ -specific serum.
- the Western blotting analysis of the subcellular fractionation showed that only TLM-IFN-ß-lb, but not wt-IFN is detectable in the cytosol.
- the detection of extracellularly added TLM-IFN-ß-lb in the cytosol confirms the cell permeability and underlines that the uptake was not via an endosome-associated pathway.
- the human hepatoma cell line huH7 and COS cells (hamsters) were incubated in the medium for 30 min in the presence of 0.5 ⁇ M IFN- ⁇ -lb-specific proteins.
- To remove surface-bound IFNs were the cells after removal of the medium '5 sec with Na 2 CO 3 / NaHCO 3 buffer, pH 9.5 and then washed in PBS. The washed cells were fixed for 10 min in ice-cold ethanol / DAPI (for staining the cell nucleus). After fixation, rehydration was carried out in PBST for 30 min. Non-specific binding sites were blocked using 10% BSA. HuNN-ß-specific sheep serum was used to label the IFN-ß. The detection was carried out by a Cy3-coupled secondary antibody. A Leica fluorescence microscope was used for evaluation.
- TLM-IFN-ß-lb can be detected well in both the huH7 and COS cells. It can be detected in almost all cells. TLM-IFN-ß-lb is homogeneously distributed over the cell; no specific accumulation in individual subcellular compartments can be observed.
- mice were kept without food overnight. The following morning the animals were given a weighed feedress soaked in IFN-ß-lb-specific protein solution. By weighing the pellet after the end of the feeding trial, the amount of the orally ingested IFN could be determined. The animals were sacrificed using CO 2 and the blood was removed as EDTA blood using cardiac puncture. After the cellular components had been separated off, the serum was analyzed using Western blotting or a huIFN-ß-specific ELISA.
- the Elisa values were standardized to the absorbed IFN-ß-lb amount (amount of feed) and related to the c / o value.
- the c / o value was set to 1.
- Example 5 Production and use of IFN- ⁇ -TLM with the help of an eukaryotic IFN- ⁇ -TLM-specific expression vector
- the cDNA coding for IFN-ß specific fusion proteins was amplified.
- This cDNA codes for a complete IFN- ⁇ -specific fusion protein which comprises the cell permeability-mediating TLM coding sequence in the open reading frame at the C-terminal.
- the primers were designed in such a way that the amplificate had a BamHI-specific interface at its 5 "end and 3" end.
- the PCR product was purified by means of "PCR purification spin columns" according to the manufacturer's instructions (Quiagen), BamHI cleaved and cleaned again.
- the fragments restricted in this way were ligated into the BamHI-cleaved and dephosphorylated eukaryotic expression vector pCDNA.3.1 (Invitrogen).
- the ligation approach was used to transform competent bacteria (DH5 ⁇ ).
- the amp resistance coded on the plasmid allowed selection on media containing amp.
- Plasmid DNA was isolated from the clones propagated under these conditions and initially analyzed by means of BamHI restriction. Positive clones were then characterized by sequencing and checked for their orientation.
- IFN-ß-specific fusion proteins in which IFN-ß was glycosylated as in the native protein were formed as described below:
- the fractions identified as IFN-ß positive by West blot analysis using a huIFN-ß-specific antiseram were pooled and further purified on a MonoQ ion exchange column. Elution was carried out by a linear gradient of 20 to 1000 mM NaCl, buffered in 40 mM Tris with a pH of 7.5 and 2% ethanol. As determined by Westem blot analysis, silver-stained SDS gels and analytical HPLC, IFN-ß-la-TLM was isolated in this way in a purity of over 90%.
- HepG2.2.15 cells a stable HBV-producing cell line
- IFN-ß-la-TLM IFN-ß-la-TLM
- IFN-ß can bind to cells that are not infected with a Viras and thereby induces the formation of, inter alia, the 2 ', 5' ⁇ oligoadenylate synthetase, which leads to a breakdown of viral RNA (cf. e.g. Takane et al, Jpn. J. Pharmacol. 90, 304-312, 2002).
- mice were kept without food for 18 hours. At the beginning of the trial, the animals received a weighed piece of toast (about 3.5 to 4.5 g), the 10 4 U IF ⁇ -ß-la-TLM
- Example 5 contained (TLM). Animals that none were used as negative controls
- Fig. 1 shows that after oral administration of IFN-ß-la-TLM for 4h or 8h, the amount of IFN-ß in the serum was significantly increased, the amount after 4h was about twice as high as the amount after 8h. In contrast, no significant increase in IFN-ß was found in serum in any negative control. Consequently, the results show that the coupling of TLM to IFN-ß significantly increased the absorption of IFN-ß via the mucosa.
- mice were carefully sheared so as not to injure the skin and kept for 4 or 8 hours with an outwardly impermeable gauze bandage (2 ⁇ 6 cm, 2-ply), which in 10 4 U IFN-ß-la-TLM Example 5 had been soaked (TLM).
- Animals that were not subjected to treatment (Nl) or animals that were exposed to commercially available recombinant IFN-ß-la (N2) were used as controls. The animals were sacrificed using CO 2 and the blood was removed as EDTA blood using cardiac puncture. After separating cellular components, the serum was analyzed using a commercial huIFN-ß-specific ELISA. Different amounts of commercially available recombinant IFN-ß-la (krIFN-ß-la) were measured for a calibration curve. Tables 4 and 5 below show the measured values obtained (mean from two measurements):
- Table 5 Measured values, mean values and calculated amounts after dermal administration of IFN-ß-la-TLM (TLM) or for the controls (Nl and N2)
- FIG. 2 shows that after dermal administration of LFN-ß-la-TLM for 4 h and 8 h, an increased amount of IFN-ß was present in the serum, whereas no significant amount of IFN-ß could be detected in the serum in the controls , The results thus show that the coupling of TLM to IFN-ß increases the absorption of IFN-ß through the skin. It was surprisingly found that the amount of IFN- ⁇ detectable in the serum increased by a factor of 2 during the test period. Such a depot effect, as is typical for subcutaneous administration, is thus also achieved by the method according to the invention without the need for an invasive application.
- Example 8 Detection of the functionality of the orally ingested IFN-ß-la-TLM
- HBV-producing cell line HepG2.2.15 The functionality was examined as described above using the HBV-producing cell line HepG2.2.15. The cells were laid out in 24-well plates. After 24 h, the medium was changed and replaced by medium which was diluted 1: 1 with the mouse sera which are identified by a star in Example 6, Table 2 (IFN- ⁇ sera). Untreated cells (Nl) and mouse seram from untreated animals (N2) served as controls. This procedure was repeated after 24 h and after a further 24 h the amount of virus in the supernatant was quantified using taqman PCR (Stoeckl et al., 2003). Table 6 shows the values obtained (HBV genome / ml) as the mean of a duplicate determination.
- B6 mice (9 animals) were kept without food for 18 hours. At the start of the experiment, the animals were given a weighed piece of toast bread (about 3.5 to 4.5 g), which had been soaked with 1 ml of a 200 ⁇ M PreSlPreS2 solution.
- the PreSlPreS2 protein contains the HBV-TLM endogenously at its C-terminus. Animals remained untreated as negative controls (5 animals). The animals were fed for 8 hours. The animals were sacrificed using CO 2 and the blood was removed as EDTA blood using cardiac puncture. After separating cellular components, the serum was analyzed by Western blot analysis using a PreSlPreS2-specific serum. The Western blots showed that under these conditions 9 of 9 animals showed PreSlPreS2 protein in the serum, but not in the controls.
- the animals were kept as described above and over a Feeded with PreSlPreS2 protein every 14 weeks. A total of 6 weeks after the first feeding, the animals were sacrificed as described above and the serum was obtained. Blot strips were produced on which one lane was loaded with cytochrome c (200 ng), one lane with PreSlPreS2 protein (20 ng) and one lane with the heavy IgG chain (marker). These strip blots were incubated with the sera obtained. The detection of the bound antibodies was carried out using a peroxidase-coupled anti-mouse IgG-specific secondary antibody.
- B6 mice (9 animals) were carefully sheared so as not to injure the skin and kept for 8 hours with an externally impermeable gauze bandage (2 ⁇ 6 cm, 2-ply) which had been soaked in 1 ml of a 200 ⁇ M PreSlPreS2 solution was. Untreated animals served as controls (4 animals). The animals were sacrificed using CO 2 and the blood was cardiac punctured as EDTA blood. After separating cellular components, the serum was analyzed by Western blot analysis using a PreSlPreS2-specific serum.
- the Western blots showed that under these conditions 8 of 9 animals showed the PreSlPreS2 protein in the serum, but not in the controls. 4 shows a typical example of a Western blot of this test series (lane 1: positive control; lanes 2 to 5: sera from untreated animals; lanes 6 to 9: sera from animals to which PreSlPreS2 was dermally administered).
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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AU2003273817A AU2003273817A1 (en) | 2002-09-04 | 2003-09-03 | Increasing of the resorption of substances via skin and mucous membranes |
CA002497696A CA2497696A1 (en) | 2002-09-04 | 2003-09-03 | Increasing the absorption of substances via the skin and mucous membranes |
JP2004533464A JP2005537328A (ja) | 2002-09-04 | 2003-09-03 | 皮膚および粘膜を介した物質の吸収の増大 |
US10/526,846 US20070172516A1 (en) | 2002-09-04 | 2003-09-03 | Increasing of the resorption of substances via skin and mucous membranes |
EP03757780A EP1534315A2 (de) | 2002-09-04 | 2003-09-03 | Verstarkung der resorption von substanzen über die haut und schleimhaut |
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DE10240894.7 | 2002-09-04 | ||
DE10240894A DE10240894A1 (de) | 2002-09-04 | 2002-09-04 | Verstärkung der Resorption von Subtanzen über die Haut und Schleimhaut |
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WO2004022657A2 true WO2004022657A2 (de) | 2004-03-18 |
WO2004022657A8 WO2004022657A8 (de) | 2004-04-22 |
WO2004022657A3 WO2004022657A3 (de) | 2004-09-02 |
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US (1) | US20070172516A1 (de) |
EP (1) | EP1534315A2 (de) |
JP (1) | JP2005537328A (de) |
AU (1) | AU2003273817A1 (de) |
CA (1) | CA2497696A1 (de) |
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WO2024168055A1 (en) * | 2023-02-08 | 2024-08-15 | Navigen, Inc. | Tnf-alpha binding agents and methods of using the same |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4545985A (en) * | 1984-01-26 | 1985-10-08 | The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services | Pseudomonas exotoxin conjugate immunotoxins |
EP0335476A2 (de) * | 1984-02-08 | 1989-10-04 | Cetus Corporation | Rekombinante Verfahren für die Herstellung von Ricin-A, Ricin-B, Ricin oder Diphterietoxin-A oder AB'-Fragment, Wirte und Vektoren dafür und Konjugate, die Ricintoxin A-Kette oder Diphterietoxin enthalten |
US5725852A (en) * | 1992-04-17 | 1998-03-10 | Takeda Chemical Industries, Ld. | Transmucosal therapeutic composition |
US5766620A (en) * | 1995-10-23 | 1998-06-16 | Theratech, Inc. | Buccal delivery of glucagon-like insulinotropic peptides |
WO1999005302A1 (en) * | 1997-07-24 | 1999-02-04 | The Perkin-Elmer Corporation | Conjugates of transporter peptides and nucleic acid analogs, and their use |
DE19850718C1 (de) * | 1998-11-03 | 2000-05-18 | Hildt Eberhardt | Zellpermeabilität-vermittelndes Polypeptid |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
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DE19904800C1 (de) * | 1999-02-05 | 2001-02-08 | Eberhard Hildt | Partikel zur Gentherapie |
US6887462B2 (en) * | 2001-04-09 | 2005-05-03 | Chiron Corporation | HSA-free formulations of interferon-beta |
US6835810B2 (en) * | 2002-05-13 | 2004-12-28 | Geneshuttle Biopharma, Inc. | Fusion protein for use as vector |
-
2002
- 2002-09-04 DE DE10240894A patent/DE10240894A1/de not_active Ceased
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2003
- 2003-09-03 CA CA002497696A patent/CA2497696A1/en not_active Abandoned
- 2003-09-03 US US10/526,846 patent/US20070172516A1/en not_active Abandoned
- 2003-09-03 JP JP2004533464A patent/JP2005537328A/ja active Pending
- 2003-09-03 EP EP03757780A patent/EP1534315A2/de not_active Withdrawn
- 2003-09-03 AU AU2003273817A patent/AU2003273817A1/en not_active Abandoned
- 2003-09-03 WO PCT/EP2003/009788 patent/WO2004022657A2/de active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4545985A (en) * | 1984-01-26 | 1985-10-08 | The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services | Pseudomonas exotoxin conjugate immunotoxins |
EP0335476A2 (de) * | 1984-02-08 | 1989-10-04 | Cetus Corporation | Rekombinante Verfahren für die Herstellung von Ricin-A, Ricin-B, Ricin oder Diphterietoxin-A oder AB'-Fragment, Wirte und Vektoren dafür und Konjugate, die Ricintoxin A-Kette oder Diphterietoxin enthalten |
US5725852A (en) * | 1992-04-17 | 1998-03-10 | Takeda Chemical Industries, Ld. | Transmucosal therapeutic composition |
US5766620A (en) * | 1995-10-23 | 1998-06-16 | Theratech, Inc. | Buccal delivery of glucagon-like insulinotropic peptides |
WO1999005302A1 (en) * | 1997-07-24 | 1999-02-04 | The Perkin-Elmer Corporation | Conjugates of transporter peptides and nucleic acid analogs, and their use |
DE19850718C1 (de) * | 1998-11-03 | 2000-05-18 | Hildt Eberhardt | Zellpermeabilität-vermittelndes Polypeptid |
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DE10240894A1 (de) | 2004-03-11 |
WO2004022657A8 (de) | 2004-04-22 |
AU2003273817A8 (en) | 2004-03-29 |
WO2004022657A3 (de) | 2004-09-02 |
CA2497696A1 (en) | 2004-03-18 |
AU2003273817A1 (en) | 2004-03-29 |
JP2005537328A (ja) | 2005-12-08 |
EP1534315A2 (de) | 2005-06-01 |
US20070172516A1 (en) | 2007-07-26 |
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