Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to pharmaceutical preparations and medicaments for intraarticular administration, containing pepstatin and their preparation and in particular their use in the treatment and / or prophylaxis of osteoarthritis, traumatic cartilage injuries, arthritis, pain, allodynia or hyperalgesia, particularly preferably in osteoarthritis.
• Osteoarthritis is the most prevalent joint disease in the world, and radiological evidence of osteoarthritis is found in the majority of over-65s. Despite this vital importance to the health system, the causes of osteoarthritis remain unclear and effective
• arthrosis refers to a "joint wear and tear" that exceeds the age-usual level
• Ostearthritis secondary arthritis or associated with congestion caused by overburdening (secondary inflammatory reaction) (activated arthritis).
• the Anglo-American literature distinguishes between osteoarthrosis (O. osteoarthritis [OA]), in which the destruction of the articular surfaces are likely to be mainly due to stress, and the arthritis (arthritis, rheumatoid arthritis [RA]), in which the joint degeneration by an inflammatory component in the foreground.
• osteoarthrosis O. osteoarthritis [OA]
• RA rheumatoid arthritis
• the Arthrosis alcaptonurica is an increased deposition of
• osteoarthritis which expresses an apparent absence of a causative disorder (HI Roach and S. Tilley, Bone and Osteoarthritis F. Bronner and MC Farach-Carson (Editors), Springer, Germany , Volume 4, 2007).
• Antibiotics of the type of gyrase inhibitors fluoroquinolones, such as ciprofloxacin, levofloxacin. These drugs cause complexation of magnesium ions in poorly vascularized tissues (hyaline articular cartilage, tendon tissue), resulting in irreversible damage to the connective tissue. These damages are more pronounced in children and adolescents usually in the growth phase.
• Joint internal structure favor osteoarthritis.
• mechanical overloads, (micro) traumas, de-stabilization of joints caused by loss of safety mechanisms, as well as genetic factors are risk factors for
• Nitric oxide temporarily increased. The same could be done by high
• the treatment of osteoarthritis has two goals. On the one hand the freedom from pain under normal stress and on the other hand, the prevention of mechanical restrictions or changes in a joint. In the long term, these goals can not be achieved by pain treatment as a purely symptomatic therapeutic approach, as this can not stop the progression of the disease. If the latter is to be achieved, cartilage destruction must be stopped. Since the articular cartilage can not regenerate in the adult patient, the elimination of pathogenetic factors such as joint dysplasias or malpositions that lead to increased punctual pressure of the
• the extracellular matrix which consists primarily of collagens, proteoglycans and water.
• the enzymes involved in extracellular matrix degradation are the metalloproteases, aggrecanases, and cathepsin enzymes. But other enzymes can in principle
• Neutrophil elastase, tryptase and chymase Neutrophil elastase, tryptase and chymase.
• Cathepsins belong to the papain superfamily of lysosomal proteases. Cathepsins are involved in the normal proteolysis and turnover of target proteins and tissues as well as in the initiation of
• cathepsin activity is not fully understood.
• one induced cathepsin expression prevents B-cells deprived of serum from apoptosis and that treatment of the cells with antisense oligonucleotides of cathepsin B induces apoptosis (Shibata et al. (1998) Biochem. Biophys. Res. Commun. 251: 199-20; Isahara et al. (1999) Neuroscience, 91: 233-249).
• Cathepsins are synthesized as inactive zymogens on ribosomes and transferred into the lysosomal system. After proteolytic cleavage of the N-terminal propeptide, the cathepsin concentration in the acid medium of the lysosomes increases up to 1 mM and the cathepsins are released from the lysosomes into the extracellular medium.
• the cathepsins one distinguishes the cysteine cathepsins B, C, H, F, K, L, O, S, V and W, the aspartyl cathepsins D and E and the serine cathepsin G.
• Cathepsin K inhibitors for the treatment of arthritis and cathepsin S inhibitors for the treatment of arthritis, neuropathic pain and psoriasis.
• the aspartyl proteases also include HIV
• Cathepsin D is normally involved in the degradation of intracellular or phagocytosed proteins and thus plays an important role in protein metabolism (Heiseth, et al., Proc. Natl. Acad., USA 81, 3302-3306, 1984) in protein catabolism (Kay , et al., Intracellular Protein Catabolism (Eds., Katunuma, et al., 155-162, 1989) and US Pat
• Elevated Cathepsin D levels are associated with a number of diseases. Increased cathepsin D levels correlate with poor prognosis in breast cancer and with increased cell invasion and increased risk of metastases, as well as shorter relapse-free ones
• Cathepsin D and other proteases such as collagenase, produced in close proximity to a growing tumor, could thereby degrade the extracellular matrix in the tumor environment, thereby mediating the detachment of tumor cells and invasion of new tissues via the lymphatic and circulatory systems (Liotta LA , Scientific American Feb: 54, 1992; Liotta LA and Stetler-Stevenson WG, Cancer Biol. 1: 99, 1990; Liaudet E., Cell Growth Differ. 6: 1045-1052, 1995; Ross JS, Am. J. Clin Pathol, 104: 36-41, 1995, Dickinson AJ, J. Urol., 154: 237-241, 1995).
• Cathepsin D is also associated with degenerative changes in the brain, such as Alzheimer's disease. So is
• Catepsin D is associated with the cleavage of the amyloid ⁇ -protein precursor or a mutant precursor, which expresses the expression of the amyloid protein in Acid., 87: 3861, 1990; Ladror, US et al., J. Biol. Chem. 269: 18422, 1994, Evin G., Biochemistry 34 : 14185-14192, 1995).
• the amyloid ⁇ -protein which results from the proteolysis of the amyloid ⁇ -protein precursor, leads to the formation of plaques in the brain and appears to be responsible for the development of Alzheimer's disease.
• Cathepsin D probably plays an essential role in the development of arthrosis at various levels.
• elevated mRNA levels of cathepsin D are measured (Clements DN et al., Arthritis Res. Ther. 2006; 8 (6): R158; Ritchlin C. et al , Scand J. Immunol., 40: 292-298, 1994).
• Devauchelle V. et al. shows different levels of expression of cathepsin D in osteoarthritis in human patients
• cathepsin D The lysosomal endopeptidase cathepsin D is the most abundant proteinase in the chondrocytes (Ruiz-Romero C. et al., Proteomics, 2005, 5 (12): 3048-59). The proteolytic activity of cathepsin D has also been demonstrated in cultured synovium from osteoarthritis patients (Bo G.P. et al., Clin. Rheumatol., 2009, 28 (2): 191-9) and also in
• Arthrosis also has a direct correlation between low pH in the joint tissue and the severity and progression of the disease
• pepstatin a peptide originally isolated from a Streptomyces culture.
• Pepstatin is effective against pepsin, cathepsin and renin.
• Aspartyl proteases or cathepsin D are frequently described as target proteins for drugs for the treatment of neurodegenerative diseases, cognitive disorders, dementia, Alzheimer's disease, cancer, malaria, HIV infection and diseases of the cardiovascular and vascular systems and inhibitors of aspartyl proteases or cathepsin D are used to treat them Diseases disclosed, for example, in WO 2009013293, EP 1987834, EP 1872780, EP 1867329, EP 1745778, EP 1745777, EP 1745776, WO 1999002153, WO 1999055687, US 6150416, WO
• cathepsin D inhibitors and the two model compounds pepstatin and ritonavir effectively inhibit cathepsin D activity, they have a rather low selectivity over others
• RAS renin-angiotensin system
• Synovial fluid from patients with rheumatoid arthritis (see page 731, right column, penultimate paragraph).
• Tables 1 and 2 Powell et al. Disclosed that most of the modified and unmodified peptides of 10 to 25 amino acids tested had a half-life of less than one hour in the media tested, human plasma (HS), synovial fluid (SF), fetal calf serum (FCS) or mouse liver homogenate (MLH) (see page 735, right column, last
• Intraarticular administration of peptidic compounds also occurs in synovial fluid due to the expected low half-life. but in particular due to the expected low residence time in the joint capsule (diffusion through the synovial membrane and degradation) and due to the expected by the diffusion into the plasma
• the object of the present invention was therefore to find new drugs and pharmaceutical preparations which are used for the prevention and
• Treatment of osteoarthritis can be used and are sufficiently stable in local or intraarticular application in synovial fluid and diffuse only slightly through the synovial membrane into the plasma and thus have a long residence time in the joint capsule, so that after injection, the drug concentration over as long as possible
• Period remains in the therapeutically effective range.
• pepstatin is surprisingly suitable for pharmaceuticals and pharmaceutical preparations which are applied locally or intra-articularly for the prevention and treatment of osteoarthritis and are maintained at high levels over a long period of time, so that the
• Medicaments and pharmaceutical preparations according to the invention need only be administered intra-articularly at most weekly, preferably at intervals of one to several months.
• Pepstatin inhibits cathepsin D highly effectively and minor side effects are to be expected in the case of intraarticular administration for the treatment of osteoarthritis, since pepstatin only achieves low systemic levels (plasma levels) when administered intra-articularly due to its long residence time in the joint capsule and slow release from the synovium.
• the high residence time of pepstatin in the joint capsule is surprising and therapeutically valuable, since starting from the
• the invention therefore relates to a pharmaceutical preparation for intraarticular administration containing pepstatin ((3S, 4S) -3-hydroxy-4- [(S) -2 - ((3S, 4S) -3-hydroxy-6-methyl-4 - ⁇ (S) -3-methyl-2 - [(S) -3-methyl-2- (3-methyl-butyrylamino) -butyrylamino] -butyrylamino ⁇ -heptanoylamino) -propionylamino] -6-methyl-heptanoic acid) and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including mixtures thereof in all ratios.
• pepstatin ((3S, 4S) -3-hydroxy-4- [(S) -2 - ((3S, 4S) -3-hydroxy-6-methyl-4 - ⁇ (S) -3-methyl-2 - [(S) -3-methyl-2- (3-methyl-
• the invention also relates to such an invention
• composition containing further carrier and / or
• compositions according to the invention containing at least one further active pharmaceutical ingredient.
• the invention also relates to pharmaceutical preparations for intraarticular administration, containing peptidic cathepsin D inhibitors which have similar properties as pepstatin, namely a cathepsin D inhibition in the nanomolar range, a high stability in
• Synovial fluid and a high residence time in the joint capsule Synovial fluid and a high residence time in the joint capsule.
• Pepstatin contains several chiral centers, so that the use of the optically active forms (stereoisomers), the enantiomers, racemates, Diastereomers and hydrates and solvates of pepstatin is the subject of the invention.
• prodrug compounds By pharmaceutically or physiologically harmless derivatives is meant e.g. Salts of pepstatin, as well as so-called prodrug compounds.
• prodrug compounds By prodrug compounds is meant by e.g. Alkyl or acyl groups (see also the following amino and hydroxy protecting groups), sugars or oligopeptides modified pepstatin derivatives which are rapidly cleaved or released in the organism to the effective pepstatin molecules. These include biodegradable polymer derivatives of pepstatin, as described e.g. in Int. J. Pharm. 115 (1995), 61-67.
• Pepstatin can be used in its final non-salt form.
• the present invention also encompasses the use of pepstatin in the form of its pharmaceutically acceptable salts which can be derived from various organic and inorganic bases by art-known procedures.
• pharmaceutically acceptable salts which can be derived from various organic and inorganic bases by art-known procedures.
• pepstatin contains a carboxylic acid group
• one of its suitable salts can be formed by reacting pepstatin with a suitable base to the corresponding base addition salt.
• bases include, for example, alkali metal hydroxides, including potassium hydroxide, sodium hydroxide and lithium hydroxide; Alkaline earth metal hydroxides such as
• Barium hydroxide and calcium hydroxide Barium hydroxide and calcium hydroxide; Alkali metal alcoholates, e.g.
• the basic salts of pepstatin include aluminum,
• Salts of pepstatin derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, e.g. Arginine, betaine, caffeine,
• Preferred metals are sodium, potassium, magnesium and calcium.
• Preferred organic amines are N, N'-dibenzylethylenediamine,
• the base addition salts of pepstatin are prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt in a conventional manner.
• the free acid can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.
• the free acid forms differ in some sense from their corresponding salt forms with respect to certain physical ones Properties such as solubility in polar solvents; However, in the context of the invention, the salts otherwise correspond to their respective free acid forms.
• pharmaceutically acceptable salt in the present context is to be understood as an active substance which contains pepstatin in the form of one of its salts, in particular if this salt form corresponds to the active ingredient in the art
• acceptable salt form of the active ingredient may provide this drug with a desired pharmacokinetic property that it has not previously possessed, and may even positively affect the pharmacodynamics of that drug in terms of its therapeutic efficacy in the body.
• Solvent molecules Pepstatin understood, which form due to their mutual attraction. Solvates are, for example, hydrates, such as monohydrates or dihydrates or alkoxides, i.
• pepstatin is well tolerated and has valuable pharmacological properties, as it selectively inhibits aspartyl proteases and in particular cathepsin D.
• Another object of the invention is therefore the use of pepstatin for the manufacture of a medicament for intra-articular administration for the treatment and / or prophylaxis of diseases caused by cathepsin D and / or by cathepsin D-mediated signal transduction mediated and / or propagated.
• the invention thus also relates in particular to a medicament for intraarticular administration containing pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including mixtures thereof in all ratios for use in the treatment and / or prophylaxis of physiological and / or or or pathophysiological conditions.
• Physiological and / or pathophysiological conditions are physiological and / or pathophysiological conditions that are medically relevant, such as illnesses and / or pathophysiological conditions.
• Another object of the invention is a medicament for
• intraarticular administration containing pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including mixtures thereof in all proportions for use in the treatment and / or prophylaxis of physiological and / or pathophysiological conditions selected from the group consisting of osteoarthritis , traumatic cartilage injuries and arthritis, especially rheumatoid arthritis. Particularly preferred is the
• Pain is a complex sensory perception, which has the character of a warning and guidance signal as an acute event, but when chronic pain has lost it and in this case (as a chronic pain syndrome) today seen as an independent disease and should be treated.
• hyperalgesia in medicine one
• Stimuli that can trigger pain include pressure, heat, cold or inflammation.
• Hyperalgesia is a form of hyperesthesia, the generic term for excessive sensitivity to a stimulus. Allodynia in medicine is a sensation of pain that is triggered by stimuli that usually do not cause pain.
• Another object of the invention is a medicament for intra-articular application containing pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including mixtures thereof in all proportions for use in the treatment and / or prophylaxis of physiological and / or pathophysiological conditions selected from the group consisting of pain, allodynia and hyperalgesia.
• a particularly preferred object of the invention is a
• Medicament for intra-articular application containing pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates,
• Prodrugs and stereoisomers including mixtures thereof in all proportions for use in the treatment and / or prophylaxis of physiological and / or pathophysiological conditions selected from the group consisting of osteoarthritis, traumatic cartilage injuries, arthritis, pain, allodynia and hyperalgesia, more preferably for use in the treatment and / or prophylaxis of osteoarthritis.
• Medicament for intraarticular administration for the treatment and / or prophylaxis of physiological and / or above
• a preferred subject of the invention is thus the use of a pharmaceutical preparation according to the invention for intra-articular
• physiological and / or pathophysiological conditions selected from the group consisting of arthrosis, traumatic cartilage injuries, arthritis, pain, allodynia or hyperalgesia.
• a particularly preferred subject of the invention is the
• a preferred subject of the invention is thus also the use of pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including mixtures thereof in all ratios for intraartlkulMC application in the treatment and / or prophylaxis of physiological and / or
• pathophysiological conditions selected from the group consisting of osteoarthritis, traumatic cartilage injuries, arthritis, pain, allodynia and hyperalgesia.
• a particularly preferred subject of the invention is the
• the invention also relates to the use of peptidic
• Pepstatin namely a cathepsin D inhibition in the nanomolar range, high stability in synovial fluid and a high residence time in the joint capsule, for intra-articular administration in the treatment and / or prophylaxis of physiological and / or pathophysiological conditions selected from the group consisting of osteoarthritis, traumatic
• Cartilage injuries, arthritis, pain, allodynia and hyperalgesia particularly preferred in the treatment and / or prophylaxis of osteoarthritis.
• Pepstatin exhibits beneficial biological activity which is readily detectable in enzyme assays and animal studies as described in the Examples. In such enzyme-based assays, pepstatin exhibits and effects an inhibiting effect, usually documented by IC 50 values in a suitable range, preferably in the micromolar range, and most preferably in the nanomolar range.
• Pepstatin can be administered to humans or animals, particularly mammals, such as monkeys, horses, dogs, cats, rats or mice, and used in the therapeutic treatment of the human or animal body as well as in the control of the diseases listed above. It can also be used as a diagnostic or as a reagent.
• Pepstatin can be used for the preparation of pharmaceutical preparations for intraarticular administration, in particular by non-chemical means. Here it is together with at least one solid, liquid and / or semi-liquid carrier and / or excipient and optionally in combination with one or more others
• Active ingredients into a suitable dosage form.
• Another object of the invention are therefore in particular also pharmaceutical preparations for intraarticular application, containing pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including their
• the invention also relates in particular to a method for
• pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers including mixtures thereof in all proportions together with a solid, liquid or semi-liquid carrier and / or excipient and optionally with another Brings drug ingredient in a suitable dosage form.
• the pharmaceutical preparations according to the invention can be used as medicaments in human or veterinary medicine.
• the patient or host may belong to any mammalian species, e.g. a primate species, especially humans; Rodents, including mice, rats and hamsters; Rabbits; Horses, cattle, dogs, cats, etc. Animal models are of interest for experimental studies, providing a model for the treatment of human disease.
• Suitable carrier substances are organic or inorganic substances which are suitable for intraarticular administration and with which do not react according to the invention. Which excipients are suitable for the desired drug formulation is familiar to the person skilled in the art on the basis of his specialist knowledge.
• solvents such as water, physiological saline or alcohols such as ethanol, propanol or glycerol
• sugar solutions such as glucose or mannitol or a mixture of said solvents and other drug carriers can stabilizers and / or wetting agents, emulsifiers, salts to
• Solubilizers can be used. If desired, can be used.
• Preparations or medicaments according to the invention contain one or more further active ingredients, for example one or more vitamins or active substances which are effective in the prophylaxis and / or treatment of the above-mentioned medical indications.
• Medicaments contain one or more further active ingredients and / or one or more potentiators (adjuvants).
• pharmaceutical formulation and “pharmaceutical
• pharmaceutically acceptable refers to drugs, precipitation reagents, carriers, excipients, stabilizers, solvents, and other agents which inhibit the administration of the resulting pharmaceutical preparations without undesired
• pepstatin preferably has the advantage that a direct use is possible and no further before use in pharmaceutical formulations
• a particularly preferred subject of the invention are also provided.
• compositions for intraarticular administration containing pepstatin in precipitated non-crystalline, precipitated crystalline or in dissolved or suspended form, and optionally excipients and / or adjuvants and / or other pharmaceutical active ingredients.
• pepstatin makes it possible to prepare highly concentrated formulations without unfavorable undesirable aggregations of pepstatin. So can Pepstatin with aqueous
• Solvents or in aqueous media ready to use solutions with high content of active ingredients.
• Pepstatin and / or its physiologically acceptable salts and solvates can also be lyophilized and the resulting lyophilisates can be used, for example, for the preparation of injection preparations for intraarticular administration.
• Aqueous preparations for intra-articular application can be prepared by dissolving pepstatin in an aqueous solution or
• a solution or suspension with a defined concentration of pepstatin with defined volumes of stock solutions containing the said further auxiliaries in a defined concentration, and optionally diluted with water to the precalculated concentration.
• the adjuvants may be added in solid form.
• the resulting aqueous Solution or suspension can then be mixed with the respectively required amounts of stock solutions and / or water.
• pepstatin can also be dissolved or suspended directly in a solution containing all other auxiliaries.
• pepstatin-containing solutions or suspensions having a pH of 4 to 10, preferably having a pH of 5 to 9, and an osmolality of 250 to 350 mOsmol / kg can be prepared.
• the pharmaceutical preparation can thus be administered largely painless intra-articularly directly.
• the preparation for intra-articular administration may also contain infusion solutions, such as e.g.
• Glucose solution isotonic saline or Ringer's solution, which may also contain other active ingredients, are added, so that larger amounts of active ingredient can be applied.
• Pepstatin is physiologically well tolerated, easy to prepare, precisely metered and preferably over the duration of storage and transport and in multiple freezing and thawing processes in terms of content
• Refrigerator temperature (2-8 ° C) and at room temperature (23-27 ° C) and 60% relative humidity (r.F.) are preferably stored stable over a period of at least three months to two years.
• pepstatin can be stably stored by drying and, if necessary, by solution or suspension in a ready-to-use
• drying methods include, but are not limited to, nitrogen gas drying, vacuum oven drying, lyophilization, organic solvent washing and subsequent air drying.
• Fluid bed drying fluidized bed drying, spray drying,
• the term "effective amount” means the amount of a drug or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal or human, such as is sought or sought by a researcher or physician.
• terapéuticaally effective amount means an amount that, as compared to a corresponding subject that has not received that amount, results in: improved
• therapeutically effective amount also includes the amounts effective to increase normal physiological function.
• Pepstatin and / or its physiologically acceptable salts and solvates is generally used analogously to known, commercially available preparations or preparations.
• the dosage depends on the age, sex, weight and health status and constitution of the patient, as well as the severity of his illness and other individual factors.
• intra-articular administration are preferably administered intra-articularly weekly to annually, more preferably 4-day to half-yearly, most preferably monthly to quarterly.
• Another object of the invention is therefore the inventive use of a pharmaceutical preparation according to the invention, wherein the pharmaceutical preparation according to the invention is administered intra-articularly as follows:
• the invention thus also relates to the use of pepstatin and / or one of its physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers, including mixtures thereof in all ratios for intraarticular administration in the treatment and / or prophylaxis of physiological and / or pathophysiological conditions selected from the group consisting of osteoarthritis,
• Hyperalgesia particularly preferred in the treatment and / or prophylaxis of osteoarthritis, wherein pepstatin and / or one of its physiological
• the individual dosage and administration intervals for a patient will also depend on a large number of individual factors, such as the potency of the particular compound used, age, body weight, general health, sex, diet, time of administration and route of administration
• bioavailability One measure of the uptake of a drug into an organism is its bioavailability. Is the drug in the form of a Injection solution supplied intra-articularly to the organism, its absolute bioavailability, ie the proportion of the drug that passes unchanged into the joint space, is 100%. Pharmacokinetic data, ie bioavailability, can be obtained analogously to the method of J. Shaffer et al. (J. Pharm. Sciences, 88 (1999), 313-318).
• such drugs can be with one of the im
• Drugs can be adapted for administration by intra-articular route.
• Such medicaments may be prepared by any method known in the pharmaceutical art, for example by administering the active ingredient with the carrier (s) or excipient (s).
• the intra-articular application has the advantage that the compound according to the invention is applied directly into the vicinity of the articular cartilage in the synovial fluid and from there can also diffuse into the cartilaginous tissue.
• Pharmaceutical compositions according to the invention can thus also be injected directly into the joint space and thus directly on the
• Compound of the invention is also suitable for the production of intraarticularly administered medicaments with controlled, uniform and / or delayed release of active ingredient (slow-release, sustained-release, controlled release).
• pepstatin is also suitable for the production of depot formulations which are advantageous for the patient, since a
• Medicaments adapted for intra-articular administration include aqueous and non-aqueous sterile injection solutions
• Formulation isotonic with the synovial fluid of the treated Recipient is included; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickeners.
• the formulations may be presented in single or multi-dose containers, such as sealed vials and vials, and stored in freeze-dried (lyophilized) condition so that only the addition of the sterile carrier liquid, eg water for injection, is required immediately before use. the recipe
• Injection solutions and suspensions prepared can be prepared from sterile powders, granules and tablets.
• Pepstatin can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
• liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamella
• phospholipids e.g. Cholesterol, stearylamine or phosphatidylcholines.
• Pepstatin may also be targeted with soluble polymers
• Drug carriers are coupled.
• Such polymers can be any polymers.
• Pepstatin to a class of biodegradable polymers suitable for the controlled release of a drug, e.g. Polylactic acid, polyepsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates, polylactic acid, polymers such as conjugates between dextran and methacrylates, polyphosphoesters, various polysaccharides and
• a drug e.g. Polylactic acid, polyepsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates, polylactic acid, polymers such as conjugates between dextran and methacrylates, polyphosphoesters, various polysaccharides and
• Block copolymers of hydrogels to be coupled are Block copolymers of hydrogels to be coupled. It is understood that the pharmaceutical compositions according to the invention, in addition to the above-mentioned particularly mentioned constituents, may contain other agents customary in the art with respect to the respective type of pharmaceutical formulation.
• compositions of the present invention may be used to provide additive or synergistic effects in certain known therapies, and / or may be used to treat them
• compositions according to the invention may contain, in addition to pepstatin, further active pharmaceutical ingredients, e.g. to
• Pepstatin was synthesized and characterized. However, the preparation and characterization of pepstatin is also feasible in other ways for the skilled person.
• Pepstatin is also characterized by a high selectivity for cathepsin D over renin (IC 50 > 10000nM), good cartilage penetration and no measurable toxicity or genotoxicity.
• IC 50 > 10000nM IC 50 > 10000nM
• MCA (7-methoxycoumarin-4-yl) acetyl
• the reference substance was pepstatin A (Sigma-Aldrich).
• the substrate used was MCA-GKPILFFRLK (Dnp) dR-NH 2 (Enzo Life Sciences, Lörrach).
• Cathepsin D (Sigma-Aldrich) isolated from the human liver was used as enzyme in a final concentration of 1.4 nM.
• the assay was performed in 100 mM sodium acetate buffer, 1.25% (v / v) DMSO, 0.25% (w / v) chaps, pH 5.5.
• To each 4 pl Cathepsin D solution 2 ⁇ each substance solution were added with serially diluted substance concentration and incubated for 10 min at room temperature. The reaction was started by adding 2 ⁇ l of substrate solution (final concentration 5 ⁇ ).
• GAGs glycosaminoglycans
• Dimethylmethylene blue hydrochloride makes use of the decrease in absorption at 633 nm. Since it is also possible to work at very low GAG concentrations, no dye / GAG complex precipitates even after a long incubation of DMMB with GAG, as happens with other measuring methods sometimes after only a short time. To determine the concentration, a calibration line with chondroitin sulfate is carried. Based on the GAG values, an IC 50 value can be calculated, ie one
• DMEM DMEM without FBS
• the pH is adjusted by addition of MES and monitored at the pH meter, adding 1% Pen / Strep and 30 pg / ml
• the substances are used in the desired concentration and cultured for 3 days.
• the compounds to be tested are tested in a first experiment in a concentration of 1 ⁇ and 1% DMSO.
• Substances with an> 50% effect on GAG release (equivalent to ⁇ 50% of the control in the assay explorer) will be tested in a next experiment at 100 nM and 1% DMSO. Substances that are among these
• Pepstatin A As a positive control Pepstatin A is used with a concentration of 0.01 ⁇ .
• the assay window is defined by the control (pH 5.5), defined as 0% effect, and the control pH 5.5 + 0.01 ⁇ pepstatin A, defined as 100% effect.
• the cell culture supernatants After 3 days of incubation, the cell culture supernatants are collected and stored at -20 ° C or measured directly. The amount of released GAG is measured photometrically.
• the cell culture supernatants (200 ⁇ ) are either measured directly or stored at -20 ° C. To ensure accurate determination of concentration (pg / ml GAG in supernatant) of GAG, the readings must be in the linear range of the standard curve. To ensure this, various dilutions are routinely presented (1/5, 1/10, 1/20, 1/40). The dilutions are made with medium and submitted to automated (Hamilton) in a 384 multiwell plate (15 ⁇ ). Also automated (or with multichannel pipette) add 60 ⁇ DMMB solution. A fast color reaction is performed which is then measured at 633 nm with a plate reader (e.g., Envision).
• a plate reader e.g., Envision
• the data are provided by the MTP reader as csv or xls files and stored as raw data based on this format (xls) or used to calculate the percentage effect of the respective connection. 5.) Quality controls
• Pepstatin control (pH 5.5 + 0.01 ⁇ pepstatin A). This substance non-specifically blocks the activity of most proteases and thus determines the maximum possible effect of a compound. 6.
• Pepstatin shows an IC 5 o value in the nanomolar range in the GAG assay (see Table 1).
• a carrageenan solution (CAR, 1%, 50 ⁇ ) was injected intra-articularly on one side into a rat knee joint.
• the non-injected side was used for control purposes.
• Six animals per group were used.
• the swelling was determined by means of a micrometer screw (medial-lateral on the knee joint) and the thermal hyperalgesia was determined by means of a directed infrared light source according to the Hargreaves method (Hargreaves et al., 1988) on the underside of the foot. Since the place of inflammation (knee joint) from the place of
• Measurement (lower paw) is called secondary thermal hyperalgesia, the mechanisms of which are important for finding effective analgesics.
• Test animal (as an expression of reaching the pain threshold) or when reaching a given maximum temperature by automatic
• the infrared light source Turn off the infrared light source finished. As long as the animal is sitting still, light reflected from the paw is registered. Pulling the paw off this reflection, whereupon the infrared light source is switched off and the time from switching on and off is registered.
• the device is calibrated so that the infrared light source in 0s raises the skin temperature to approximately 45 degrees Celsius (Hargreaves et al., 1988). For testing, a device manufactured by Ugo Basile for this purpose is used.
• cathepsin D inhibitors were made intraarticularly 30 minutes before the CAR.
• triamcinolone TAC
• the solvent vehicle
• Hyperalgesia is expressed as the difference in withdrawal times between the inflamed and non-inflamed paws.
• pepstatin was able to dose-dependently reduce the extent of thermal hyperalgesia.
• pepstatin exerts an anti-hyperalgesic effect. This can be postulated because pepstatin has no effect on the inflammatory swelling and thus on the trigger of hyperalgesia. It can therefore be assumed that pepstatin has a pain-reducing effect in humans.
• Example 5 Stability of pepstatin in bovine synovial fluid
• bovine explants for the diffusion chamber or other assays
• cattle hooves metalacarpal joints
• bovine knee are used.
• the synovial fluid can be made from both
• synovial fluid Carefully remove synovial fluid from the joint with a 10 ml syringe and cannula and place in prepared 2 ml Eppendorf tubes.
• the Eppendorf vessels are labeled depending on the animal (cattle pass is available). It is important to ensure that no blood enters the joint space during the preparation of the joints. If this is the case, the synovial fluid turns reddish and must therefore be discarded.
• the synovial fluid is generally highly viscous and clear to yellowish in color. The collection along with a macroscopic analysis of the synovial fluid is documented.
• bovine synovial fluids a pool of 4 different bovine synovial fluids is mixed. For this purpose approx. 1 ml per SF are used. The mixture is placed directly in a 5 ml glass jar to minimize any absorption effects. The SFs are mixed thoroughly but gently. There should be no air bubbles or foam. This is done using a lowest-level vortex device. The compounds to be tested are in one
• the samples can be frozen at -20 ° C.
• the negative control used is SF without substance.
• SF with 1 ⁇ substance is used. This corresponds to the 0h value and thus 100% stability.
• the samples are stored in "low-binding" Eppendorf tubes at -20 ° C. The samples are then quantitatively measured
• Pepstatin stays in for a period of at least two weeks
• the frozen joints were briefly thawed, minced with the bone shears as best as possible and then added 4 times the volume of 80% ethanol.
• the mixture was then homogenized with the Ultraturrax, the extract was shaken at RT for 20 min and then stored at -20 ° C. for at least 30 min. It was then centrifuged for 5 min at 13,000 rpm, a 10 ⁇ aliquot of the supernatant 1: 5000 with internal
• Diluted standard solution transferred to a PCR plate and analyzed.
• 20 ⁇ plasma was mixed with 20 ⁇ internal standard solution, added 100 ⁇ methanol and shaken for 5 min. Thereafter, the extracts were stored for at least 30 min at -20 ° C and then centrifuged for 5 min at 13000 rpm. 80 ⁇ of the supernatant was transferred to a PCR plate and analyzed.
• Pepstatin a so-called flip-flop kinetics, i. the terminal
• Plasma half-life is determined not by the elimination, but by the release of pepstatin from the suspension and diffusion through the synovial membrane. Pepstatin was detected in plasma at very low concentrations up to 28 days after administration (see Figure 2).
• Example 7 Pharmacokinetic data after intravenous (iv) and oral (po) administration
• pepstatin The pharmacokinetic parameters of pepstatin were determined in Wistar rats (BW about 250 g) after administration of the test substance in a cocktail of up to 4 substances.
• the test substances were dissolved in DMSO / PEG200 / water (2/60/38 v / v) to a final concentration of 0.8 mg / mL and a dose of 0.2 mg / kg i.v. and 0.5 mg / kg orally.
• Blood samples (200 ⁇ ) were taken under light isoflurane anesthesia through the sublingual vein at the following times after administration: iv: 0.1, 0.5, 1, 2, 4, 6 and 24 h; po: 0.25, 0.5, 1, 2, 4, 6 and 24 h.
• the blood samples were taken in centrifuge tubes containing Li-heparin
• pepstatin After intravenous administration, pepstatin shows a very high clearance (> 100% hepatic blood flow), an average volume of distribution and consequently a very short plasma half-life (about 0.14 h). After oral administration, all plasma concentrations were below the detection limit (see Figure 3).
• Example 8 In vivo Effectiveness of Pepstatin in Osteoarthritis - ACLT tMx Model As experimental animals, rats were chosen. After shaving and disinfection, the area of operation is opened by means of a medial skin incision about 1 cm long. The knee capsule is prepared and the medial
• Patellar band shown. After opening the joint capsule, severing the medial parapatellar ligaments and shifting the patella laterally, the anterior cruciate ligament becomes curved, blunt-ended
• anterior and posterior meniscus fixing ligaments are prepared and severed and the meniscus removed tMx
• the patella is repositioned and the medial patellar ligament is fixed again by means of a continuous suture while closing the capsule.
• the muscles are also sutured.
• the skin is then closed by means of single stitches.
• the duration of the procedure is about 10 minutes.
• the animals are anesthetized with 1, 5-2 vol% isoflurane. Before injection, buprenorphine is injected subcutaneously. The
• Injection area is gently shaved and disinfected.
• the knee joint is placed in a slightly flexed position and the substance or vehicle to be tested is injected into the joint.
• 1mg pepstatin was used in 30 ⁇ .
• As a negative control only one vehicle was injected into an experimental group.
• As a positive control the unoperated hind leg of the rat was used in each case. Processing in Histology
• the sampled tissue samples are fixed for at least 72 hours in paraformaledhyd (4%) and then under running for 24 hours Rinsed tap water. The samples are then decalcified by Osteosoft over a period of 4 weeks. This was followed by infiltration of the tissue with paraffin and the preparation of histological sections with a thickness of 7 ⁇ m. For assessment, the sections were stained with Safranin O Fast Green.
• the rating system is based on the work of V.B. Kraus et al (Osteoarthritis & Cartilage, 18, S3, 2010).
• Pepstatin also shows significant in vivo efficacy in osteoarthritis (see Figure 4)
• Example 9 Microdialysis
• the free drug levels at the site of action are crucial.
• the distribution space in the synovial fluid in this regard is of great interest.
• the synovial fluid is a complex matrix for the analytical determination of peptides and, on the other hand, only the free, i. the non-protein portion of the drug is relevant to the effect.
• the microdialysis procedure was used. The obtained eluate from the microdialysis allows an analysis of free drug levels from the complex matrix of the synovial fluid.
• Reason is that a
• Microdialysis membrane (microdialysis membrane) is used, which can pass only molecules up to a certain size. This "pore size" determines in addition to the flow rate of the eluate and the available free concentration of the drug in the synovial fluid as the driving force for the mass transfer, the concentration of the drug in the eluate.
• pore size determines in addition to the flow rate of the eluate and the available free concentration of the drug in the synovial fluid as the driving force for the mass transfer, the concentration of the drug in the eluate.
• Triamcinolone was used here in the already dissolved crystal suspension, whereas the pepstatin crystals were added directly and not dissolved to the synovial fluid. 3 mg of drug per ml of synovial fluid were used in a glass jar, which roughly corresponds to the concentration ratios after an intra-articular injection into the knee joint. Surprisingly, and in contrast to triamcinolone, very high levels of unbound pepstatin were measured. Even the more or less constant levels speak for a fast dissolution rate with high stability of pepstatin in

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