EP1984016A1 - Utilisation du facteur semblable à l'insuline 3 dans le traitement de l'ostéoporose et des troubles osseux - Google Patents

Utilisation du facteur semblable à l'insuline 3 dans le traitement de l'ostéoporose et des troubles osseux

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Publication number
EP1984016A1
EP1984016A1 EP06708356A EP06708356A EP1984016A1 EP 1984016 A1 EP1984016 A1 EP 1984016A1 EP 06708356 A EP06708356 A EP 06708356A EP 06708356 A EP06708356 A EP 06708356A EP 1984016 A1 EP1984016 A1 EP 1984016A1
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EP
European Patent Office
Prior art keywords
insl3
bone
insulin
peptide
osteoporosis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06708356A
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German (de)
English (en)
Inventor
Carlo Foresta
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Associazione Foresta Per La Ricerca Nella Riproduzione Umana
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Associazione Foresta Per La Ricerca Nella Riproduzione Umana
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Publication of EP1984016A1 publication Critical patent/EP1984016A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention concerns a method for the treatment of mammals having or being at substantial risk of developing osteoporosis.
  • the invention is also directed to the fields of bone loss, growth and degeneration, and to the use of compounds and compositions thereof for use as pharmaceuticals. Background
  • Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. It is a significant cause of mortality and morbidity in the elderly people, both of individual and community perspective. Osteoporosis affects an estimated 75 million people in Europe, the United States and Japan. [003] There are a number of causes of osteoporosis. Hormone deficiencies (estrogen in women and androgen in men) are the leading cause. It is well known that women are at greater risk of osteoporosis than men. Women experience a sharp acceleration of bone loss during the five years following menopause.
  • HRT hormone replacement therapy
  • bisphosphonates bisphosphonates
  • calcitonin bisphosphonates
  • Other adjuncts to these therapies may be recommended, including adequate calcium intake, vitamin D and weight bearing exercise.
  • Other drugs may be used in conjunction with these therapies, including tamoxifen (commonly used as an adjunct in the treatment of breast cancer) , thiazide diuretics (used in the treatment of hypertension) and sodium fluoride which is presently undergoing evaluation by the Food and Drug Administration in order to be approved for the treatment of osteoporosis.
  • Estrogen is known to reduce fractures, and is an example of an anti-resorptive agent.
  • Black, et al . (EP 0605193Al) report that estrogen, particularly when taken orally, lowers plasma levels of low density lipoproteins (LDL' s) and raises those of the beneficial high density lipoproteins (HDL f s).
  • LDL' s low density lipoproteins
  • HDL f s beneficial high density lipoproteins
  • estrogen failed to restore bone back to young adult levels in the established osteoporotic skeleton.
  • long-term estrogen therapy has been implicated in a variety of disorders, including an increase in the risk of uterine cancer, endometrial cancer and possibly breast cancer, causing many women to avoid this treatment.
  • the significant undesirable effects associated with estrogen replacement therapy support the need to develop alternative therapies for osteoporosis that have the desirable effect on serum LDL but do not cause undesirable effects.
  • Bisphosphonates are non-hormonal treatments for osteoporosis which work by "switching off” the cells that break down bone, allowing the bone building cells to work more efficiently.
  • Calcitriol an active form of vitamin D given to post-menopausal women who have osteoporosis in the spine. Calcitriol improves the absorption of calcium from the gut, as calcium cannot be absorbed without vitamin D.
  • Calcitonin is a hormone made by the thyroid gland which prevents the cells that break down bone from working properly, improving the action of bone building cells.
  • Calcitonin is presently the only other FDA approved treatment. The drug acts by slowing the rate of bone loss and relieves bone pain.
  • drawbacks with calcitonin are that it must be injected daily, it can cause nausea and it is very expensive compared with estrogen replacement therapy.
  • Salcatonin (Calsynar) is licensed for the treatment of post-menopausal osteoporosis.
  • Testosterone is a treatment for men who are deficient in the male sex hormone, but it can also increase bone density in men with osteoporosis who have normal testosterone levels. It is available as injections or implants.
  • Anabolic steroids can increase bone and muscle mass and may be helpful in the very elderly who are frail and also in people with spinal fractures. Injections are carefully monitored due to side effects.
  • SERMs Selective Estrogen Receptor Modulators
  • SERMs Selective Estrogen Receptor Modulators
  • SERMs Selective Estrogen Receptor Modulators
  • One form, raloxifene is licensed for the prevention and treatment of osteoporosis in postmenopausal women.
  • United States Patent 6,300,309 describes a method of treatment of osteoporosis in higher mammals exhibiting decreased cortical bone mineral density and preventing osteoporosis due to cortical bone mineral density reduction in such mammals.
  • the treatment is based on Insulin-like Growth Factor I as an active agent.
  • Pharmaceutical compositions useful in the treatment of osteoporosis are also described.
  • In United States Patent 6,548,482 a substantially pure complex of insulin growth factor II E polypeptide and insulin growth factor BP2 polypeptide is described. Methods for treating an osteoporosis patient and targeting a compound to the skeletal extracellular matrix of a patient are also described.
  • a process of treating or alleviating the symptoms of pathological conditions where bone density is decreased is described in United States Patent 6,846,907.
  • the process comprises inhibiting, in a mammalian patient suffering from such a condition, the formation in vivo of a tertiary complex of IL-Il, its cell surface membrane receptor and the cell surface glycoprotein gpl30.
  • examples of such substances are recombinant soluble IL-Il receptor mutants modified, as compared with native IL-Il receptor, at their gpl30 binding site, and peptides which can interact with IL- 11.
  • the process of the invention not only inhibits bone resorption and hence bone loss, but also increases the process of bone formation to increase bone density.
  • Active agent drug
  • drug drug
  • pharmaceutically active agent are used interchangeably herein to refer to a chemical material or compound that induces a desired effect.
  • the primary active agents herein is insulin-like factor 3, although combination therapy wherein a insulin-like factor 3 is administered with one or more additional active agents is also within the scope of the present invention. Included are derivatives and analogs of those compounds or classes of compounds not specifically mentioned but which also induce the desired effect.
  • Carriers or “vehicles” as used herein refer to carrier materials suitable for administration of the active agent or drug. Carriers and vehicles useful herein include any such materials known in the art which is nontoxic and does not interact with other components of the composition in a deleterious manner.
  • Effective or “therapeutically effective” amount/ dose of a drug or pharmacologically active agent is meant a nontoxic but sufficient amount/ dose of the drug or agent to provide the desired effect, i.e., an amount sufficient to slow, stop, or reverse the bone density reduction rate in a patient exhibiting bone density reduction.
  • “Pharmacologically active” (or simply “active") as in a “pharmacologically active” derivative or metabolite, refers to a derivative or metabolite having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • pharmaceutically acceptable refers to a derivative (e.g., a salt) of an active agent, it is to be understood that the compound is pharmacologically active as well, i.e., therapeutically effective for the treatment of osteoporosis.
  • Parenteral administration used herein may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • the formulation for parenteral administration may be in an aqueous or non-aqueous sterile injectable solutions. Such solutions may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • the formulation for parenteral administration may be presented as an aqueous or nonaqueous sterile suspension which may include suspending agents and thickening agents .
  • Oral administration is used herein to mean administration of a active agent, drug or pharmacologically active agent" in a pharmaceutical vehicle convenient for that administrative route.
  • the medicament may be presented as tablets, capsules, ingestible liquid or a powder preparation.
  • Such formulations can include pharmaceutically acceptable carriers known to those skilled in the art.
  • Formulations suitable for oral administration further include lozenges, pastilles, aerosols and mouthwashes.
  • Transmucosal drug delivery is meant administration of a drug to the mucosal surface of an individual so that the drug passes through the mucosal tissue and into the individual's blood stream.
  • Transmucosal drug delivery may be “buccal” or “transbuccal, "referring to delivery of a drug by passage through an individual's buccal mucosa and into the bloodstream.
  • transmucosal drug delivery is “sublingual” drug delivery, which refers to delivery of a drug by passage of a drug through an individual's sublingual mucosa and into the bloodstream.
  • Treating” and “treatment” as used herein refer to a reduction in severity and/or frequency of symptoms, elimination of symptoms and/ or underlying cause, prevention of the occurrence of symptoms and/ or their underlying cause and improvement or remediation of damage .
  • the present invention is based on the surprising discovery that following clinical observations more than 50% of the subjects with a mutation of INSL3 gene or INSL3 receptor (LGR8) gene showed osteoporosis.
  • 2/3 of the subjects with INSL3 mutation and 7/14 subjects with LGR8 mutation presented said pathology. All these subjects were young (less than 35 years old) and did not have other osteoporosis causes, particularly all had normal levels of testosterone.
  • the idea was that the system INSL3- LGR8 played a possible role at the bone level, and its alteration could cause osteoporosis.
  • an in vitro study has been carried out involving different steps.
  • the present invention is directed to synthesized or recombinant compositions derived from the deduced amino acid sequences for Insulin-like factor 3 for the treatment of diseases and disorders which may be treated with insulin-like factor 3.
  • the composition comprises the full-length amino acid sequence for chain B of INSL3. In another embodiment of the present invention, the composition comprises the full-length amino acid sequence for INSL3. In yet another embodiment of the present invention, the composition comprises a INSL3 protein derivative wherein the protein is shortened at either or both its 3 f and 5 f ends of either or both the A- or B-chains . Specifically, the A chain may be as short as fifteen amino acids in length and the B chain may be as short as thirteen amino acids in length.
  • Non limitative examples of preferred conditions to be treated are those arising from reduction of bone density and loss of bone mass. Specifically osteoporosis and osteppenia.
  • the therapeutic and/or prophylactic treatment of the invention is effected by the chain B.
  • the medicament is, preferably, administered intravenous or intramuscular.
  • the amount of insulin-like 3 peptide ranges from dosage from 1 ⁇ g/kg body weight to 1 mg/kg body weight every 1-7 days for a period of 1-6 months.
  • the insulin-like 3 peptide is contained in a pharmaceutical composition.
  • the insulin-like 3 peptide is administered to a human subject.
  • Figure 1 depicts the structure of Insulin-like factor 3.
  • Leydig cell-specific insulin-like peptide (Ley IL)
  • Leydig cell-specific insulin-like peptide (Ley IL)
  • LEF relaxin-like factor
  • INSL3 insulin-like Factor 3
  • the insulin-like hormone superfamily comprises insulin, relaxin and insulin-like growth factors I and II.
  • the members of this family are characterized by a signal peptide, a B-chain, a connecting C-peptide, and an A-chain.
  • the INSL3 protein has been characterized also as insulin-like, rather than either IGF-like or relaxin-like, based upon the protein's C-peptide chain length. More specifically, the C-peptide length of the INSL3 protein is 49 amino acids, as compared to the 35 amino acid length of proinsulin C-peptide, the twelve amino acid length of the, known proIGF C-peptides and the over one-hundred amino acid C-peptide length of prorelaxin. [047] Finally, INSL3 has been designated insulin-like based on the observation that the protein is expressed exclusively in prenatal and postnatal testicular Leydig cells. Burkhardt, et al . , supra.
  • INSL3 is expressed in various reproductive tissues and is also regarded a marker of differentiation in human testicular Leydig cells. On the basis of the protein's similarities to insulin and the source of such protein, it was reported that the Ley I-L protein is implicated in testicular function. Id., (Adham, et al . , 1993, J. Bio. Chem. 268 (35) : 26668-6672) .
  • the INSL3 is a circulating peptide hormone. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation- dependent manner.
  • INSL3 expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population.
  • the ruminant ovary has a very high level of INSL3 expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent (Ivell R, Bathgate RA. Biol Reprod 2002 Sep; 67 (3) : 699-705) .
  • EP083187lBl describes the relaxin like factor and its use in the treatment of cardiovascular disease, neurodegenerative or neurologic disease, sinus bradycardia, depression, hair loss and diseases related to uncontrolled or abnormal collagen or fibronectin formation.
  • INSL3 shares primary and secondary structural homology with relaxin, insulin and the other members of the insulin-related family of hormones. INSL3 is structurally closer to insulin than relaxin.
  • the structures of INSL3 is formed by the cleavage of the pro-hormone peptide into three chains (A, B and C) , removal of the C chain and the formation of three disulfide bridges between six invariant cysteine residues found on the A and B chains, to produce an active protein.
  • the deduced primary structure of INSL3 is set forth at FIG. 1.
  • the amino acid sequence SEQ. I. D. N.
  • DisulfideBridges are located at : AChain : Cys lo -Cys 15
  • INSL3 may be produced using techniques previously disclosed as useful in producing relaxin and insulin. For example, the cDNA for INSL3 disclosed in Burkhardt, et al., 1994, Genomics 20:13-19 and Adham, et al . , 1994, J. Biol. Chem.
  • 268:26668-26672 may be used to recombinantly produce INSL3 according to processes previously described as useful in recombinantly manufacturing relaxin (e.g., U.S. Pat. Nos . 4,758,516, 4,871,670, 4,835,251, 5,464,756). Similarly, such sequence information may be used to synthesize INSL3 according to the methods of Bullesbach and Schwabe, 1991, J. Biol. Chem. 266:10754-10761.
  • Derivatives and analogs of INSL3 also may be synthesized according to the methods of Bullesbach and Schwabe, supra. Alternatively, such derivatives and analogs may be produced recombinantly using, for example, site directed mutagenesis techniques as set forth in Tsurushita, et al . , 1988, Gene 62:135-139. [056] The scope of this invention is not limited by the production techniques herein described. According to the invention, production of INSL3 and its derivatives and analogs may be carried out by any techniques or methods .
  • plasmids comprising INSL3 wild-type have been created and pancreatic HIT-T 15 cells (ATTC CRL-1777) have been transfected with said plasmids to produce a large amount of INSL3 peptide.
  • plasmids comprising LGR8 wild-type have been created and T-293 cells, derived from human embryonic kidney (HEK) fibroblast (ATCC CRL-1573) , have been transfected with said plasmids .
  • T-293 cells so transfected and M-63 cells have been stimulated with increasing concentration of synthetic INSL3. Both cases produced an increase of cAMP depending to the INSL3 concentrations .
  • Osteoporosis is a bone disease in which the amount of bone is decreased and the structural integrity of trabecular bone is impaired. Cortical bone becomes more porous and thinner. This makes the bone weaker and more likely to fracture.
  • Osteopenia which is a loss of bone mass or the presence of less than normal amount of bone, can arise from a decrease in muscle activity, which may occur as the result of a bone fracture, bed rest, fracture immobilization, joint reconstruction, arthritis, and the like. Osteopenia, if not treated, may result in osteoporosis .
  • Osteoporosis may be based on the bone density. Standardized bone density measurements of the total hip, "normal” bone is greater than 833 mg/cm 2 . “Osteopenia” is between 833 and 648mg/cm 2 . Osteoporosis is lower than 648mg/cm 2 , and "Severe (established) osteoporosis” is when there has been a fragility fracture. [064] Osteoporosis literally means “porous bones”. The bones in the skeleton are made of a thick outer shelf and a strong inner mesh filled with collagen (protein) , calcium salts and other minerals.
  • Osteoporosis occurs when the holes between bone become bigger, making it fragile and liable to break easily. Osteoporosis usually affects the whole skeleton but it most commonly causes breaks (fractures) to bone in the wrist, spine and hip. Old bone is broken down by cells called osteoclasts and replaced by bone building cells called osteoblasts. This process of renewal is termed bone turnover. [065] Osteoporosis encompasses a broad range of clinical syndromes having varying etiologies . In postmenopausal women, for example, two distinct types of osteoporsis have been identified.
  • Type I osteoporosis occurs mainly in the early postmenopausal period from about age 50-65. It is characterized by excessive resorption, primarily in trabecular bone. Vertebral fractures are common. If given prior to significant bone loss, treatment which decreases or prevents bone resorption (such as with estrogen or calcitonin) is considered effective therapy.
  • Type II osteoporosis (a.k.a. senile osteoporosis) occurs essentially in all aging women and, to a lesser extent, in men. It is characterized by proportionate loss of cortical bone as well as trabecular bone. Here decreased bone formation plays a major role, if not a more important role than increased bone resorption. Fractures of the hip are characteristic of this type of osteoporosis.
  • INSL3 peptide is administered to an individual exhibiting bone degenerative disease. More particularly, INSL3 is administered to an individual suffering from osteoporosis or osteopenia. Specifically, INSL3 is administered to an individual exhibiting bone density reduction, loss of bone mass and/or the presence of less than normal amount of bone leading to impairment of structural integrity of the trabecular bone or porous and thin cortical bone. INSL3 peptide may be administered by way of a pharmaceutically acceptable carrier and/or comprised in a pharmaceutical composition .
  • INSL3 may be administered in conjunction with a another active agent.
  • the INSL3 and the second active agent can be administered simultaneously or sequentially with either the INSL3 or the second active agent being administered first.
  • Both the inhibitor and the second active agent can be administered by the same modality (and even in the same formulation) or they can be administered in different formulations and/or by different modalities.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans .
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al . , 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi) .
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the relaxin-like activity and effects .
  • Administration of INSL3, with other active agents can be via any of the accepted modes of administration for agents that serve similar utilities, preferably by systemic administration.
  • the INSL3 will be administered at a dosage that is able to slow, stop, or reverse the bone density reduction rate in a patient exhibiting bone density reduction.
  • a daily dose is from about 1 ⁇ g/kg to 1 mg/kg of body weight per day, preferably about 1 ⁇ g/kg to 100 ⁇ g/kg, and most preferably about 1 ⁇ g/kg to 10 ⁇ g/kg. Dosage levels depend on whether INSL3 is administered alone or in combination with a second active agent.
  • the dosage range would be about 70 ⁇ g to 70 mg per day, preferably about 70 ⁇ g to 7 mg per day, and most preferably about 70 ⁇ g to 700 ⁇ g per day.
  • the amount of INSL3 administered will, of course, be dependent on the subject and the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician.
  • INSL3 for treatment of the above conditions, any pharmaceutically acceptable mode of administration can be used.
  • INSL3 can be administered either alone or in combination with other pharmaceutically acceptable excipients, including solid, semi-solid, liquid or aerosol dosage forms, such as, for example, tablets, capsules, powders, liquids, gels, suspensions, suppositories, aerosols or the like.
  • INSL3 can also be administered in sustained or controlled release dosage forms (e.g., employing a slow release bioerodable delivery system) , including depot injections, osmotic pumps (such as the Alzet implant made by Alza) , pills, transdermal (including electrotransport) patches, and the like, for prolonged administration at a predetermined rate, preferably in unit dosage forms suitable for single administration of precise dosages.
  • the compositions will typically include a conventional pharmaceutical carrier or excipient and INSL3.
  • these compositions may include other active agents, carriers, adjuvants, etc.
  • a sustained/controlled release INSL3 formulation was a selectively permeable outer barrier with a drug dispensing opening, and an inner INSL3-containing portion designed to deliver dosage of RLF progressively diminished as a predetermined rate (e.g. containing about 30 mg of INSL3 in a matrix for delivery of initially about 500 ⁇ g per day diminishing as a rate of 10 ⁇ g per day) .
  • a sustained/controlled release INSL3 formulation has a selectively permeable outer barrier with a drug dispensing opening, a first inner relaxin-containing portion designed for steady state release of relaxin at a therapeutically effective daily dosage (e.g.
  • the pharmaceutically acceptable composition will contain about 0.1% to 90%, preferably about 0.5% to 50%, by weight of INSL3, either alone or with suitable pharmaceutical excipients, carriers, etc.
  • compositions of this invention are administered to a patient suffering from a bone degenerative disease, in an amount sufficient to cure or at least partially modify the issue of the condition and its complications
  • the INSL3 derivatives and analogs thereof are typically combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition.
  • Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound (s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent (s).
  • physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes .
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions .
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides .
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be the VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol .
  • the VPD co-solvent system (VPD: 5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.
  • This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a cosolvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. [093] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs .
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid or gel phase carriers or excipients .
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Formulations of INSL3 may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients .
  • the particles of the formulation may advantageously have diameters of less than 50 microns, preferably less than 10 microns. See, e.g., U.S. Pat. No. 5,364,838, which discloses a method of administration for insulin that can be adapted for the administration of INSL3 in the present invention.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores .
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses .
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas .
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas .
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the active agent (s) and on the particular physio-chemical characteristics of the active agent (s).
  • the excipients are preferably sterile and generally free of undesirable matter.
  • compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, etc. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the patient .
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration.
  • Parenteral administration is generally characterized by injection, either subcutaneously, intradermally, intramuscularly or intravenously, preferably subcutaneously.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, and the like.
  • the percentage of INSL3 contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages . Preferably the composition will comprise 0.2-2% of the INSL3 in solution.
  • the liposomes will be targeted to and taken up selectively by the tissue.
  • the human osteoblast carcinoma cell line MG-63 (ATCC, Manassas, VA) were propagated in D-MEM/F-12 (Invitrogen, Milan, Italy) supplemented with 10% FBS (Celbio, Milan, Italy) , 1 rtiM Glutamine (Invitrogen, Milan, Italy) , and an antibiotic mixture of Penicillin
  • HIT-T15 cell line ATCC, Manassas, VA
  • RPMI 1640 medium Invitrogen, Milan, Italy
  • HIT-T15 and 293T/17 cells were seeded into 25cm 2 cell culture flasks at high density in order to achieve 60-80% confluency.
  • RNA from human osteoblast cell line was isolated with TRIzol reagent (Invitrogen, Milan, Italy) . The amount of RNA isolated was determined by spectrometry at 260 and 280 nm.
  • Total RNA ( ⁇ 50 ng) obtained from the osteoblast cell line was used for first strand cDNA synthesis employing the Sensiscript Reverse Transcriptase kit (Qiagen, Milan, Italy) according to the manufacture's instructions: IX Buffer RT, 0.5 rtiM each dNTP, 10 ⁇ M random examer primers (Invitrogen, Milan, Italy) , 10 U RNase inhibitor, l ⁇ l Sensiscript Reverse Transcriptase in a final volume of 20 ⁇ l . Products of RT reactions (cDNAs) were tested by PCR employing specific oligonucleotide primers for the housekeeping gene ⁇ -actin.
  • Expression of LGR8/INSL3 Polymerase Chain Reaction (PCR)
  • PCRs were performed on cDNA obtained by osteoblast cell line, Human Multiple Tissue cDNA (MTC) panel I and II (BD Clonetech, Palo Alto, CA) and Prostate, Testis and Fetal Lung Marathon-Ready cDNA (BD Clonetech) .
  • MTC Human Multiple Tissue cDNA
  • BD Clonetech Palo Alto, CA
  • Prostate Testis and Fetal Lung Marathon-Ready cDNA
  • PCR reactions were carried out in a final volume of 25 ⁇ l containing 5 ⁇ l of cDNA, IX Taq Gold Buffer, 1.5 rtiM MgCl 2 , 100 ⁇ M each dNTP, 100 ng each primer and 1 U Taq Gold DNA-polymerase (Roche, Mannheim, Germany) .
  • the PCR cycles consisted of an initial denaturation for 10 min at 95 0 C, annealing for 45 sec, elongation for 1 min at 72 0 C all followed by 40 cycles of denaturation at 95 0 C for 45 sec, annealing for 45 sec and elongation at 72 0 C for 1 min and a final extension cycle at 72 0 C for 10 min.
  • the annealing temperature was optimized for each fragment and reactions were performed in a PTC-100 Thermal cycler MJ Research, Inc.
  • LGR8/INSL3 expression of LGR8/INSL3 in mammalian cells
  • LGR8 cDNA expression construct hLGR8-FLAG in pCR3.1
  • INSL3 cDNA expression construct hINSL3 in pcDNA3.1 myc/His B
  • Plasmids were purified with QIAfilter Plasmid Midi kit (Qiagen, Milan, Italy) .
  • Cellular lysate cell lines were obtained by a physical procedure (freezing in liquid nitrogen and defrost in water at 37 0 C) . Lysates were denatured with SDS and 2-beta-mercaptoethanol, and fractionated using SDS-PAGE. After blotting onto Hybond ECL Nitrocellulose Membrane (Amersham Biosciences, Uppsala, Sweden) and blocking with a 0.1% milk solution (Bio-Rad, Milan, Italy), blots were incubated at 4 0 C with the anti-LGR8 antibody diluted 1:1500 overnight.
  • Recombinant wild- type INSL3 peptide were obtained by transfecting pancreatic HIT cells grown in a T-25 flask with 5 ⁇ g of the expression construct encoding corresponding peptide using Fugene 6 (Roche, Indianapolis, IN) . Medium from cells transfected with pCR3.1 vector was used as a control .
  • To measure INSL3 hormone production in the culture media of HIT-T15 and osteoblast line we used the novel INSL3/Insulin-like 3 (RLF) (Human) EIA kit (Phoenix Pharmaceutical, Belmont, CA) .
  • RLF novel INSL3/Insulin-like 3
  • DEXA analysis showed normal values for the 20 controls, whereas 2 of 3 patients with INSL3 mutations and 7/14 patients with LGR8 mutations showed BMD values at or below -2.5 S. D. (osteoporosis) .
  • RT-PCR analysis showed the presence of LGR8 and INSL3 mRNA in the osteoblast MG-63 cell line, confirmed by sequencing of the PCR products. Different human tissues showed expression of INSL3 and/or LGR8, as previously demonstrated in other reports.
  • Western blot analysis carried out on total protein extracts from MG-63, 293T/17 cell lines and human commercial protein extracts with the anti-LGR8 identified the 86 kDa band corresponding to the molecular weight of the receptor. We didn't find LGR8 expression in vector cell line (negative control) .
  • Immunohistochemical analysis for LGR8 on MG-63 cell line showed a clear the transmembrane localization of the receptor.
  • HIT pancreatic cell line

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Abstract

La présente invention concerne une méthode de traitement de mammifères souffrant d'ostéoporose ou présentant un risque substantiel de développer cette maladie. La présente invention concerne également les domaines de la perte, de la croissance et de la dégénérescence osseuses, ainsi que l'emploi de composés et de compositions incluant lesdits composés en tant que produits pharmaceutiques.
EP06708356A 2006-02-17 2006-02-17 Utilisation du facteur semblable à l'insuline 3 dans le traitement de l'ostéoporose et des troubles osseux Withdrawn EP1984016A1 (fr)

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US20130072995A1 (en) * 2011-07-11 2013-03-21 Terrance Ransbury Catheter system for acute neuromodulation
IT201900000367A1 (it) * 2019-01-10 2020-07-10 Altergon Sa Composizioni comprendenti un peptide in grado di stimolare la via del segnale gprc6a-dipendente

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