ITFI20120146A1 - NEW THERAPY FOR ALCAPTONURIA TREATMENT - Google Patents
NEW THERAPY FOR ALCAPTONURIA TREATMENT Download PDFInfo
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- ITFI20120146A1 ITFI20120146A1 IT000146A ITFI20120146A ITFI20120146A1 IT FI20120146 A1 ITFI20120146 A1 IT FI20120146A1 IT 000146 A IT000146 A IT 000146A IT FI20120146 A ITFI20120146 A IT FI20120146A IT FI20120146 A1 ITFI20120146 A1 IT FI20120146A1
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- Italy
- Prior art keywords
- methotrexate
- pharmaceutically acceptable
- treatment
- pharmaceutical compositions
- alkaptonuria
- Prior art date
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- 238000011282 treatment Methods 0.000 title claims description 20
- 206010001689 alkaptonuria Diseases 0.000 title claims description 19
- 238000002560 therapeutic procedure Methods 0.000 title description 3
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 38
- 229960000485 methotrexate Drugs 0.000 claims description 38
- 150000003839 salts Chemical class 0.000 claims description 16
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 201000010394 Ochronosis Diseases 0.000 claims description 9
- 208000029347 ochronosis disease Diseases 0.000 claims description 9
- 239000003085 diluting agent Substances 0.000 claims description 4
- 230000005764 inhibitory process Effects 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 4
- DASQOOZCTWOQPA-GXKRWWSZSA-L methotrexate disodium Chemical compound [Na+].[Na+].C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 DASQOOZCTWOQPA-GXKRWWSZSA-L 0.000 claims description 3
- 230000003442 weekly effect Effects 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 229940102223 injectable solution Drugs 0.000 claims 1
- IGMNYECMUMZDDF-UHFFFAOYSA-N homogentisic acid Chemical compound OC(=O)CC1=CC(O)=CC=C1O IGMNYECMUMZDDF-UHFFFAOYSA-N 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 9
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- OUBCNLGXQFSTLU-UHFFFAOYSA-N nitisinone Chemical compound [O-][N+](=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C1C(=O)CCCC1=O OUBCNLGXQFSTLU-UHFFFAOYSA-N 0.000 description 6
- 229960001721 nitisinone Drugs 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 5
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- 230000007774 longterm Effects 0.000 description 5
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- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 208000037340 Rare genetic disease Diseases 0.000 description 2
- 102000054727 Serum Amyloid A Human genes 0.000 description 2
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- 238000010874 in vitro model Methods 0.000 description 2
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- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- AOFUBOWZWQFQJU-SNOJBQEQSA-N (2r,3s,4s,5r)-2,5-bis(hydroxymethyl)oxolane-2,3,4-triol;(2s,3r,4s,5s,6r)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O.OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O AOFUBOWZWQFQJU-SNOJBQEQSA-N 0.000 description 1
- KKADPXVIOXHVKN-UHFFFAOYSA-N 4-hydroxyphenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=C(O)C=C1 KKADPXVIOXHVKN-UHFFFAOYSA-N 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 208000036487 Arthropathies Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BQENDLAVTKRQMS-SBBGFIFASA-L Carbenoxolone sodium Chemical compound [Na+].[Na+].C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C([O-])=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](OC(=O)CCC([O-])=O)C1(C)C BQENDLAVTKRQMS-SBBGFIFASA-L 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical class OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
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- 150000002148 esters Chemical class 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 229910052700 potassium Inorganic materials 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Obesity (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
TITOLO TITLE
NUOVA TERAPIA PER IL TRATTAMENTO DELL’ALCAPTONURIA NEW THERAPY FOR THE TREATMENT OF ALCAPTONURIA
DESCRIZIONE DESCRIPTION
Campo dell’Invenzione Field of Invention
La presente invenzione riguarda in generale il campo farmaceutico, e più precisamente si riferisce all’uso di un composto noto nel trattamento di una malattia genetica rara, denominata “alcaptonuria†. The present invention generally relates to the pharmaceutical field, and more precisely it refers to the use of a known compound in the treatment of a rare genetic disease, called â € œalcaptonuriaâ €.
Stato dell’Arte State of the art
L’alcaptonuria à ̈ una malattia genetica rara, di natura ereditaria, correlata al metabolismo della tirosina. Questa malattia à ̈ infatti dovuta alla mancanza di un enzima nel fegato, l’omogentisato 1,2-deossigenasi, che à ̈ responsabile in condizioni normali della trasformazione dell’acido omogentisinico, un intermedio nel metabolismo della tirosina, mentre il difetto dell’enzima causa appunto l’accumulo tossico dell’acido nel sangue. Alkaptonuria is a rare genetic disease, inherited in nature, related to the metabolism of tyrosine. This disease is in fact due to the lack of an enzyme in the liver, the homogentisate 1,2-deoxygenase, which is responsible under normal conditions for the transformation of homogentisinic acid, an intermediate in the metabolism of tyrosine, while the defect of The enzyme causes the toxic accumulation of acid in the blood.
L’acido omogentisinico accumulato viene escreto nelle urine in grandi quantità e, ossidandosi facilmente, conferisce loro un caratteristico colore nero, che rappresenta spesso il primo e più sicuro segno della malattia. L’acido omogentisinico in eccesso va inoltre ad accumularsi nel tessuto connettivo in forma di un pigmento scuro, creando danni in particolare alle giunture, al sistema cardiovascolare, ai reni, alla pelle e alle ghiandole, e dando luogo ad una situazione patologica nota come “ocronosi†che nelle cartilagini può degenerare in artropatie dette appunto “ocronotiche†. The accumulated homogentisinic acid is excreted in the urine in large quantities and, oxidizing easily, gives them a characteristic black color, which is often the first and surest sign of the disease. The excess homogentisinic acid also accumulates in the connective tissue in the form of a dark pigment, creating damage in particular to the joints, the cardiovascular system, the kidneys, the skin and the glands, and giving rise to a pathological situation known as â € œochronosisâ € which in cartilages can degenerate into arthropathies called â € œochronoticâ €.
Fino ad oggi non à ̈ stato ancora trovato un trattamento efficace dell’alcaptonuria. I regimi dietetici poveri di tirosina hanno dato scarsi risultati, anche per le difficoltà dei pazienti a mantenere un regime dietetico restrittivo per l’intera durata della propria vita. Ci sono inoltre studi che suggerivano un effetto protettivo da parte dell’acido ascorbico, ma questi stessi studi hanno poi mostrato risultati contraddittori. Più recentemente, à ̈ stato studiato anche il trattamento con nitisinone, un potente inibitore dell’enzima che catalizza la conversione dell’idrossifenilpiruvato ad acido omogentisinico; il nitisinone ha dimostrato di abbassare notevolmente l’escrezione urinaria di acido omogentisinico, ma non vi sono dati clinici che suggeriscano un’efficacia del trattamento a lungo termine; al contrario, uno studio clinico a lungo termine sull’uso di nitisinone con 40 pazienti à ̈ stato completato nel 2009, con risultati deludenti (Introne W.J. et al. Mol. Gen. Metabol. 2011, 103(4):307-314). Inoltre non si ha alcuna evidenza che il nitisinone prevenga o curi l’ocronosi conseguente all’alcaptonuria. An effective treatment for alkaptonuria has not yet been found. Dietary diets low in tyrosine have given poor results, also due to the difficulty of patients to maintain a restrictive diet for the entire duration of their life. There are also studies that suggested a protective effect of ascorbic acid, but these same studies have also shown contradictory results. More recently, treatment with nitisinone, a powerful inhibitor of the enzyme that catalyzes the conversion of hydroxyphenylpyruvate to homogentisinic acid, has also been studied; nitisinone has been shown to significantly lower urinary excretion of homogentisinic acid, but there are no clinical data to suggest long-term efficacy of the treatment; on the contrary, a long-term clinical study on the use of nitisinone with 40 patients was completed in 2009, with disappointing results (Introne W.J. et al. Mol. Gen. Metabol. 2011, 103 (4): 307-314 ). Furthermore, there is no evidence that nitisinone prevents or cures ochronosis resulting from alkaptonuria.
L’esigenza di un trattamento farmacologico efficace dell’alcaptonuria e delle sue complicanze, in particolare di un trattamento efficace anche a lungo termine, à ̈ pertanto tuttora sentita. The need for effective pharmacological treatment of alkaptonuria and its complications, in particular for effective treatment even in the long term, is therefore still felt.
Sommario dell'invenzione Summary of the invention
Ora le Richiedenti hanno trovato che il metotrexato, un composto noto ed utilizzato in campo farmaceutico nel trattamento di alcune neoplasie e patologie autoimmuni, può essere usato anche nel trattamento dell’alcaptonuria e delle sue complicanze, risultando efficace anche a lungo termine. In particolare, il metotrexato à ̈ risultato capace di inibire l’ocronosi. Now the Applicants have found that methotrexate, a compound known and used in the pharmaceutical field in the treatment of some neoplasms and autoimmune pathologies, can also be used in the treatment of alkaptonuria and its complications, resulting also effective in the long term. In particular, methotrexate was found to be capable of inhibiting ochronosis.
Rappresenta pertanto oggetto dell'invenzione l’uso di quantità di metotrexato o suoi sali farmaceuticamente accettabili efficaci nel trattamento dell’alcaptonuria. Therefore, the subject of the invention is the use of quantities of methotrexate or its pharmaceutically acceptable salts effective in the treatment of alkaptonuria.
Composizioni farmaceutiche comprendenti una quantità di metotrexato o suoi sali farmaceuticamente accettabili efficace per l’uso nel trattamento dell’alcaptonuria, in combinazione con eccipienti e/o diluenti farmaceuticamente accettabili, rappresentano ulteriore oggetto dell'invenzione. Pharmaceutical compositions comprising an amount of methotrexate or its pharmaceutically acceptable salts effective for use in the treatment of alkaptonuria, in combination with pharmaceutically acceptable excipients and / or diluents, represent a further object of the invention.
Altre importanti caratteristiche dell'invenzione sono riportate nella seguente descrizione dettagliata. Other important features of the invention are reported in the following detailed description.
Descrizione dettagliata dell'invenzione Detailed description of the invention
Il metotrexato chimicamente à ̈ un derivato dell’acido glutammico, il cui nome completo à ̈ acido N-[4-[[2,4-diammino-6-pteridinil)metil]metilammino]benzoil]-L-glutammico; può essere derivatizzato in vario modo, attraverso le sue funzioni amminiche e acido carbossiliche, in particolare può essere salificato per ottenere sali farmaceuticamente accettabili. Chemically methotrexate is a derivative of glutamic acid, whose full name is N- [4 - [[2,4-diamino-6-pteridinyl) methyl] methylamino] benzoyl] -L-glutamic acid; it can be derivatized in various ways, through its amino and carboxylic acid functions, in particular it can be salified to obtain pharmaceutically acceptable salts.
Secondo l’invenzione l’uso di metotrexato o suoi sali farmaceuticamente accettabili, somministrato in quantità efficace a soggetti umani, à ̈ in grado di inibire l’ocronosi e risulta dunque utile nel trattamento dell’alcaptonuria e di qualsiasi altra malattia o disfunzione, a cui sia correlata l’insorgenza di condizioni ocronotiche delle cartilagini. Tale uso secondo l’invenzione si à ̈ rivelato inoltre efficace nell’inibire<3>SIB – BI1081F According to the invention, the use of methotrexate or its pharmaceutically acceptable salts, administered in effective quantities to human subjects, is able to inhibit ochronosis and is therefore useful in the treatment of alkaptonuria and any other disease or dysfunction, to which the onset of ochronotic conditions of the cartilages is correlated. This use according to the invention has also proved effective in inhibiting <3> SIB â € “BI1081F
l’ocronosi, e dunque nel trattamento delle suddette malattie, sia nel breve sia nel lungo termine. ochronosis, and therefore in the treatment of the aforementioned diseases, both in the short and long term.
Il metotrexato per l’attuazione dell’invenzione può essere anche in forma di sali, ottenuti ad esempio salificando la funzione acida con opportune basi quali quelle 5 contenenti metalli alcalini e alcalino terrosi (sodio, potassio, magnesio e calcio) oppure ammine organiche opportune. Eventualmente, può essere salificato anche con aminoacidi. Particolarmente preferito secondo l’invenzione, à ̈ l’uso di metotrexato sale sodico, in cui le due funzioni acido carbossilico del metotrexato sono salificate con sodio. The methotrexate for the implementation of the invention can also be in the form of salts, obtained for example by salifying the acid function with suitable bases such as those 5 containing alkaline and alkaline earth metals (sodium, potassium, magnesium and calcium) or organic amines appropriate. Eventually, it can also be salified with amino acids. Particularly preferred according to the invention is the use of methotrexate sodium salt, in which the two carboxylic acid functions of methotrexate are salified with sodium.
10 Il metotrexato ed i suoi sali possono essere usati, da soli o in combinazione di due o p iù compost i , in composizioni farmaceutiche con eccipienti e/o diluenti farmaceuticamente accettabili, ed eventuali ulteriori principi attivi. Il metotrexato può essere presente nelle composizioni come tale o in forma di esteri o sali farmaceuticamente accettabili, solubili o disperdibili in acqua o olio, dove con il termine 15 “sale farmaceuticamente accettabile†si intende un sale preparato con acidi o basi, organici o inorganici, non tossici, farmaceuticamente accettabili. 10 Methotrexate and its salts can be used, alone or in combination of two or more compost ies, in pharmaceutical compositions with pharmaceutically acceptable excipients and / or diluents, and possible further active ingredients. Methotrexate can be present in the compositions as such or in the form of pharmaceutically acceptable esters or salts, soluble or dispersible in water or oil, where the term 15 `` pharmaceutically acceptable salt '' means a salt prepared with acids or bases, organic or inorganic, non-toxic, pharmaceutically acceptable.
Le presenti composizioni farmaceutiche possono essere formulate in varie forme farmaceutiche, per diverse vie di somministrazione, ad esempio come composizioni orali o soluzioni iniettabili, preferibilmente soluzioni iniettabili, in qualsiasi forma 20 farmaceutica adatta a questi tipi di somministrazione, ad esempio in compresse, capsule, pillole, polveri, granuli, soluzioni e sospensioni. Soluzioni iniettabili per la somministrazione intramuscolare sono particolarmente preferite secondo l’invenzione. The present pharmaceutical compositions can be formulated in various pharmaceutical forms, for different routes of administration, for example as oral compositions or injectable solutions, preferably injectable solutions, in any pharmaceutical form 20 suitable for these types of administration, for example in tablets, capsules, pills, powders, granules, solutions and suspensions. Injectable solutions for intramuscular administration are particularly preferred according to the invention.
Il metotrexato o suoi sali possono essere somministrati secondo l’invenzione in combinazione con diluenti ed eccipienti farmaceuticamente accettabili, scelti 25 opportunamente in base alla forma di somministrazione usata secondo la pratica farmaceutica convenzionale. The methotrexate or its salts can be administered according to the invention in combination with pharmaceutically acceptable diluents and excipients, suitably selected 25 on the basis of the administration form used according to conventional pharmaceutical practice.
Nell'ambito della presente invenzione per “quantità efficace†si intende una quantità efficace di un prodotto che à ̈ sufficiente ad ottenere la risposta terapeutica desiderata senza effetti collaterali, come ad esempio tossicità , irritazioni o risposte 30 allergiche, con un rapporto rischio / beneficio ragionevole e adeguato alle circostanze. In the context of the present invention, `` effective quantity '' means an effective quantity of a product that is sufficient to obtain the desired therapeutic response without side effects, such as toxicity, irritation or allergic responses, with a risk / reasonable benefit appropriate to the circumstances.
La specifica “quantità efficace†può pertanto variare di volta in volta a seconda delle particolari condizioni da trattare, dalla durata del trattamento, dall’eventuale cosomministrazione di altri farmaci, e delle condizioni del soggetto da trattare. La quantità efficace di metotrexato secondo l’invenzione può essere compresa tra 7 e 25 mg a settimana, e preferibilmente à ̈ 15 mg/settimana, vantaggiosamente in un'unica somministrazione settimanale. The specific â € œeffective quantityâ € can therefore vary from time to time depending on the particular conditions to be treated, the duration of the treatment, the possible co-administration of other drugs, and the conditions of the subject to be treated. The effective quantity of methotrexate according to the invention can be between 7 and 25 mg per week, and preferably 15 mg / week, advantageously in a single weekly administration.
Con l’espressione “capace di inibire†l’ocronosi, come usata nella presente invenzione, si intende capace di prevenire o ridurre la progressione della condizione patologica in questione e/o le sue complicazioni nel paziente trattato; mentre con il termine “trattamento†dell’alcaptonuria si intende l’inibizione, la regressione o quantomeno l’arresto o il rallentamento nella progressione della malattia in questione. With the expression â € œcapable of inhibitingâ € ™ ochronosis, as used in the present invention, it is meant capable of preventing or reducing the progression of the pathological condition in question and / or its complications in the treated patient; while the term â € œtreatmentâ € of alkaptonuria means inhibition, regression or at least the arrest or slowdown in the progression of the disease in question.
Parte Sperimentale Experimental Part
Condrociti e colorazione con rosso Congo Chondrocytes and Congo red staining
Per lo studio à ̈ stato adottato un modello in vitro di condrociti umani, messo a punto dagli stessi inventori e descritto dettagliatamente in Tinti et al., Rheumatology 201150:271-277. Condrociti sono stati seminati su una piastra da 24 pozzetti in misura di 4x10<4>cellule per pozzetto, e cresciuti fino a quasi confluenza, pre-incubati per 24 ore in assenza ed presenza di 10<-9>M di metotrexato disciolto in DMEM (Dulbecco’s Modified Eagle’s Medium) contenente il 10% di siero fetale bovino, come mezzo di coltura. Questo mezzo di coltura à ̈ stato poi rimosso e sostituito con un mezzo di coltura contenente una concentrazione 10<-9>M di metotrexato e 0,33 mM di acido omogentisinico. Dopo 8 giorni le cellule sono state fissate con il 70% di etanolo per 15 minuti ed osservate al microscopio dopo averle colorate con rosso Congo, seguendo un protocollo modificato per consentire una perfetta distinzione tra collagene ed amiloide. La birifrangenza del rosso Congo à ̈ stata quantificata nelle immagini polarizzate usando il software Photoshop CSII (Adobe System, Mountain View, CA, USA). Tre campi 20x sono stati selezionati come quelli che riflettono meglio la birifrangenza complessiva del rosso Congo delle cellule contenute sull’intero vetrino. L’area totale nera à ̈ stata selezionata automaticamente. Successivamente, la selezione à ̈ stata invertita per mettere in evidenza le aree di birifrangenza. Il numero di pixels di birifrangenza à ̈ stato estrapolato e normalizzato con il numero totale di cellule in ciascun campo. Infine, sulla base del rapporto pixels birifrangenti/cellule, à ̈ stata calcolata la percentuale di inibizione della birifrangenza da parte del metotrexato nelle cellule trattate con acido omogentisinico. An in vitro model of human chondrocytes was adopted for the study, developed by the same inventors and described in detail in Tinti et al., Rheumatology 201150: 271-277. Chondrocytes were seeded on a 24-well plate at the rate of 4x10 <4> cells per well, and grown to near confluence, pre-incubated for 24 hours in the absence and presence of 10 <-9> M of methotrexate dissolved in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum as a culture medium. This culture medium was then removed and replaced with a culture medium containing a 10 <-9> M concentration of methotrexate and 0.33 mM of homogentisinic acid. After 8 days the cells were fixed with 70% ethanol for 15 minutes and observed under the microscope after having stained them with Congo red, following a modified protocol to allow a perfect distinction between collagen and amyloid. Congo red birefringence was quantified in polarized images using Photoshop CSII software (Adobe System, Mountain View, CA, USA). Three 20x fields were selected as best reflecting the overall Congo red birefringence of the cells contained on the entire slide. The total black area has been automatically selected. Subsequently, the selection was reversed to bring out the areas of birefringence. The number of birefringence pixels was extrapolated and normalized with the total number of cells in each field. Finally, on the basis of the birefringent pixels / cells ratio, the percentage of inhibition of birefringence by methotrexate in cells treated with homogentisinic acid was calculated.
I risultati della colorazione birifrangente ottenuta con rosso Congo nel modello cellulare condrocitico di alcaptonuria da noi messo a punto, mostrano come il metotrexato sia in grado di inibire la formazione di amiloide indotta da acido omogentisinico. La birifrangenza del rosso Congo à ̈ osservabile in condrociti umani trattati con acido omogentisinico 0,33 mM. Il pretrattamento con metotrexato 10<-9>M ha inibito la produzione cellulare di amiloide. L’inibizione à ̈ stata significativa, con una riduzione della produzione cellulare di amiloide valutata pari al 97,2%. The results of the birefringent staining obtained with Congo red in the chondrocytic cell model of alkaptonuria we developed show how methotrexate is able to inhibit the formation of amyloid induced by homogentisinic acid. Congo red birefringence is observed in human chondrocytes treated with 0.33 mM homogentisinic acid. Pretreatment with 10 <-9> M methotrexate inhibited cellular production of amyloid. The inhibition was significant, with an estimated 97.2% reduction in amyloid cell production.
Saggio delle citochine Cytokine assay
Il rilascio di citochine pro-infiammatorie da condrociti trattati con acido omogentisinico o con acido omogentisinico e metotrexato, Ã ̈ stato misurato con Bioplex (Bio-Rad, Milano, Italia). Per il confronto dei dati rilevati, sono stati utilizzati tests ANOVA, nei quali si sono considerate come significative differenze di P inferiori a 0,05. The release of pro-inflammatory cytokines from chondrocytes treated with homogentisinic acid or with homogentisinic acid and methotrexate, was measured with Bioplex (Bio-Rad, Milan, Italy). For the comparison of the data collected, ANOVA tests were used, in which significant differences in P lower than 0.05 were considered as significant.
Si à ̈ osservato come le cellule umane condrocitiche trattate con acido omogentisinico producano massicce dosi di citochine pro-infiammatorie e come il trattamento con metotrexato sia in grado di ridurre fortemente i livelli di tali citochine pro-infiammatorie sino a riportarli nella maggior parte dei casi a livelli di normalità . It has been observed that human chondrocytic cells treated with homogentisinic acid produce massive doses of pro-inflammatory cytokines and how the treatment with methotrexate is able to strongly reduce the levels of these pro-inflammatory cytokines until they return in most cases to levels of normality.
Risultati Results
Dagli esperimenti condotti à ̈ emerso come l’acido omogentisinico inducesse la produzione di amiloide ed il rilascio di siero amiloide A, e come il metotrexato fosse in grado di inibire l’amiloide in vitro e di ridurre i livelli di citochine pro-infiammatorie tipiche di un quadro di flogosi cronica. From the experiments carried out it emerged that homogentisinic acid induced the production of amyloid and the release of serum amyloid A, and how methotrexate was able to inhibit amyloid in vitro and reduce the levels of pro-inflammatory cytokines typical of a chronic inflammation picture.
Per correlare direttamente la produzione ed il rilascio di siero amiloide A da condrociti di donatori ammalati di alcaptonuria e l’accumulo di acido omogentisinico tipico dell’ocronosi alcaptonurica, abbiamo adottato il modello in vitro descritto sopra. Condrociti umani primari coltivati in presenza di un eccesso di acido omogentisinico entro l’intervallo di acido omogentisinico circolante in pazienti affetti da alcaptonuria, rilasciavano livelli più alti di siero amiloide A rispetto al controllo (dati non mostrati). I depositi di amiloide sono stati rivelati con la colorazione al rosso Congo in condrociti trattati con acido omogentisinico, provando così il coinvolgimento dell’acido omogentisinico nella formazione di amiloide. Questo modello ha inoltre consentito una analisi semiquantitativa della produzione di amiloide causata dall’aggiunta di acido omogentisinico e la sua riduzione in misura del 97,2% a seguito del trattamento con metotrexato. Bassi dosaggi di metotrexato, quali quelli proposti nell’ambito della presente invenzione, sono generalmente ben tollerati, consentendo comunque di bloccare o addirittura revertire la progressione della malattia. To directly correlate the production and release of amyloid serum A from chondrocytes of donors suffering from alkaptonuria and the accumulation of homogentisinic acid typical of alkaptonuric ochronosis, we adopted the in vitro model described above. Primary human chondrocytes cultured in the presence of an excess of homogentisinic acid within the range of circulating homogentisinic acid in patients with alkaptonuria, released higher levels of serum amyloid A than the control (data not shown). The amyloid deposits were revealed with Congo red staining in chondrocytes treated with homogentisinic acid, thus proving the involvement of homogentisinic acid in the formation of amyloid. This model also allowed a semi-quantitative analysis of amyloid production caused by the addition of homogentisinic acid and its reduction by 97.2% following treatment with methotrexate. Low dosages of methotrexate, such as those proposed in the context of the present invention, are generally well tolerated, allowing in any case to block or even reverse the progression of the disease.
Il trattamento con metotrexato o suoi sali si à ̈ perciò rivelato come l’unico attualmente prospettabile nella terapia dell’alcaptonuria, e più in generale negli stati patologici a cui à ̈ legato uno stato di ocronosi delle cartilagini; e inoltre rappresenta comunque il trattamento da utilizzare per quei pazienti sintomatici per i quali i test clinici con nitisinone sopra menzionato ha recentemente fallito, ovvero per tutti quei pazienti che non tollerano il nitisinone. Treatment with methotrexate or its salts has therefore proved to be the only one currently feasible in the therapy of alkaptonuria, and more generally in pathological states to which a state of ochronosis of the cartilages is linked; it also represents the treatment to be used for those symptomatic patients for whom the clinical tests with nitisinone mentioned above have recently failed, that is, for all those patients who do not tolerate nitisinone.
Claims (13)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT000146A ITFI20120146A1 (en) | 2012-07-12 | 2012-07-12 | NEW THERAPY FOR ALCAPTONURIA TREATMENT |
| PCT/IB2013/055519 WO2014009860A1 (en) | 2012-07-12 | 2013-07-05 | Novel therapy in the treatment of alkaptonuria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT000146A ITFI20120146A1 (en) | 2012-07-12 | 2012-07-12 | NEW THERAPY FOR ALCAPTONURIA TREATMENT |
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| Publication Number | Publication Date |
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| ITFI20120146A1 true ITFI20120146A1 (en) | 2014-01-13 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IT000146A ITFI20120146A1 (en) | 2012-07-12 | 2012-07-12 | NEW THERAPY FOR ALCAPTONURIA TREATMENT |
Country Status (2)
| Country | Link |
|---|---|
| IT (1) | ITFI20120146A1 (en) |
| WO (1) | WO2014009860A1 (en) |
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- 2013-07-05 WO PCT/IB2013/055519 patent/WO2014009860A1/en active Application Filing
Non-Patent Citations (3)
| Title |
|---|
| LAURA TINTI ET AL: "Evaluation of antioxiodant drugs for the treatment of ochronotic alkaptonuria in an in vitro human cell model", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 225, no. 1, 1 October 2010 (2010-10-01), pages 84 - 91, XP055047533, ISSN: 0021-9541, DOI: 10.1002/jcp.22199 * |
| LIA MILLUCCI ET AL: "Alkaptonuria is a novel human secondary amyloidogenic disease", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - MOLECULAR BASIS OF DISEASE, vol. 1822, no. 11, 1 November 2012 (2012-11-01), pages 1682 - 1691, XP055047517, ISSN: 0925-4439, DOI: 10.1016/j.bbadis.2012.07.011 * |
| MOLLY YANCOVITZ ET AL: "Alkaptonuria", DERMATOLOGY ONLINE JOURNAL, 1 November 2010 (2010-11-01), XP055047695, Retrieved from the Internet <URL:http://dermatology.cdlib.org/1611/articles/6_2010-04-20/yancovitz.html> [retrieved on 20121213] * |
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| WO2014009860A1 (en) | 2014-01-16 |
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