FORMULATIONS COMPRISING SOLVENT / DETERGENT-TREATED PLASMA (PLASMA S / D) AND USES THEREOF

专利摘要:
The present invention relates to pharmaceutical formulations comprising solvent / detergent (S / D) treated plasma and a glycosaminoglycan such as hyaluronic acid, and their use for treating diseases, in particular musculoskeletal diseases.
公开号:BE1021755B1
申请号:E2013/0645
申请日:2013-09-26
公开日:2016-01-15
发明作者:Enrico Bastianelli；Valentina Albarani
申请人:Bone Therapeutics S.A.；Enrico Bastianelli S.P.R.L.；
IPC主号:

专利说明:

Formulations comprising solvent / detergent treated plasma (S / D plasma) and uses thereof
Field The invention relates to pharmaceutical formulations and their use for treating diseases such as musculoskeletal diseases, and more particularly bone or joint diseases.
Context
Musculoskeletal diseases or disorders can affect bones, muscles, joints, cartilage, tendons, ligaments, and other connective tissues that support and fix tissues and organs together. These diseases may develop over time or may result, for example, from excessive use of the musculoskeletal system or trauma. Musculoskeletal diseases can be difficult to diagnose and / or treat because of the close relationship of the musculoskeletal system with other internal systems.
A potential and promising approach for the treatment of musculoskeletal diseases and in particular bone diseases and joint diseases is the transplantation of mesenchymal stem cells (MSCs) capable of undergoing osteogenic or chondrogenic differentiation or cells that are engaged in a lineage. osteoblastic or chondroblastic.
MSCs have previously been used to treat bone disorders (Gangji et al, Expert Opin Biol Ther, 2005, Vol 5, 437-42). However, although such relatively undifferentiated stem cells can be transplanted, they are not involved in the osteoblastic or chondroblastic lineage and thus a considerable proportion of transplanted stem cells thus can not ultimately contribute to the formation of the desired tissue. In addition, the amount of such stem cells obtainable from any possible source tissues is frequently unsatisfactory. The local administration of pharmaceutical active substances, and in particular the localized intra- or peri-bone or intra- or peri-articular administration thereof, is of great interest in musculoskeletal diseases, since it contributes to increase the local concentration and effectiveness of the components and avoid potential systemic side effects. For example, intra-articular injection of high molecular weight hyaluronic acid (HA) is effective in restoring the mechanical integrity of joints affected by osteoarthritis. However, HA of high molecular weight has the disadvantage of not producing a completely satisfactory scaffold due to gelation. In another example, bone marrow implantation in the osteonecrotic zone has beneficial effects in patients with non-traumatic aseptic osteonecrosis of the femoral head (Gangji et al., 2005, supra).
Therefore, there is a continuing need for new and / or improved pharmaceutical formulations configured for localized administration, for example for intraosseous, peri-bone, intra-articular or peri-articular administration, or for intra-tendinous administration, peri-tendinous, intra-ligamentary or peri-ligamentous. Particularly useful formulations can exhibit or achieve a gel consistency after administration, such that the pharmaceutical active ingredients included in the formulations are retained in the gel in situ and are gradually released from the gel, i.e. prolonged or slow release. In addition, when the formulations contain therapeutic cells, the gel consistency can produce a cell-supporting environment and can ensure the proximity of cells and potential beneficial substances, such as growth factors that stimulate survival, proliferation ( propagation) and / or cell differentiation.
Fresh frozen plasma (FFP) is prepared in global blood banks as a by-product of erythrocyte concentrate preparation. In order to improve the safety of FFP, a solvent / detergent (S / D) treatment of plasma was developed in the 1980s, and led to a product manufactured in 1992. S / D treatment and plasma S / D treated are described, inter alia, in Horowitz et al. 1992; US 4,764,369; EP 0 322 786; and US 2002/018777. The S / D treatment of plasma breaks the membranes of the lipid-enveloped viruses, cells, and most protozoa, leaving labile coagulation factors intact. The treatment has variable efficacy against bacteria and is substantially ineffective against non-lipid enveloped viruses. Compared with FFP, S / D-treated plasma has an extremely high safety with respect to transfusion-associated acute lung injury (TRALI), a significantly lower probability of causing allergic reactions, and a low lot variation at lot in terms of plasma levels of clotting factors. JP2011229508 (abstract) relates to a cell composition comprising backbone myoblasts incorporated into plasma-comprising gel. WO 2009/016451 relates to the use of a substance derived from homologous or autologous blood alone or in combination with hyaluronic acid to treat joint disease, joint pain or skin. Park et al. 2009 relates to the combination of fibrin and hyaluronic acid as a cell delivery vehicle for rabbit chondrocytes with applications in cartilage repair. summary
The present inventors have discovered pharmaceutical formulations that solve one or more of the above-mentioned problems in the art.
One aspect of the invention relates to a pharmaceutical formulation comprising solvent / detergent treated plasma (S / D plasma) and a glycosaminoglycan.
As indicated in the Examples section, formulations applying the principles of the present invention have the advantageous property of in vitro gelling in contact with the body fluids of subjects, such as human subjects, in particular serum or synovial fluids, e.g. of patients with osteoarthritis of the knee or bone diseases. It is disclosed that such formulations applying the principles of the present invention gel in vitro in contact with body fluids such as whole blood, and thus have the property of in situ gelation (in situ coagulation) when brought into contact with each other. with body fluids such as whole blood. For example, the present formulation may advantageously constitute a natural environment of autologous growth factors of whole blood. The formulations exhibit particularly satisfactory gel-forming behavior in situ (i.e., when administered at a given site or site (e.g., bone, joint, tendon, or ligament) of the body of a subject), producing advantageously viscous formulations. For example, once injected into joint defects, the present formulations allow gelation in situ and restore the physiological and rheological states of an arthritic joint such as an arthritic knee joint. In addition, this viscous quality can provide localized and / or sustained release of formulation components, namely S / D plasma, which may include beneficial biological substances such as endogenous growth factors, and / or glycosaminoglycan. The viscous quality may also make it possible to incorporate additional active pharmaceutical substance (s) (such as, without limitation, a cell composition, a pharmaceutical active compound, a protein, a peptide, and / or a small organic molecule) in the formulations, thereby obtaining localized and / or sustained release of such component (s). The inventors furthermore contemplate that the viscous quality may protect the formulation components and / or the additional active pharmaceutical substance (s) included therein against the physical environment, such as to improve the overall stability (e.g., stability against enzymatic degradation) of the components and ingredients in vivo. The inventors further postulate that the viscous quality can make it possible to improve articular function by prolonged lubricating action on the joint, so as to better restore the mechanical integrity of the joint. The inventors furthermore envisage that the formulations may make it possible to improve bone healing by osteoinductive action.
The pharmaceutical formulation may typically comprise one or more pharmaceutically acceptable excipients (e.g., solvents, carriers, diluents, etc.), particularly excipients compatible with the intended mode of administration of the formulation, such as, in particular , parenteral administration and, preferably, intraosseous, peri-bone, intra-articular, or peri-articular administration of the formulation, or for intraspinous, peritendinous, intra-ligamentary or peri-articular administration. ligament of the formulation.
The pharmaceutical formulation may be produced by a method comprising combining (e.g., mixing or inclusion in a kit of components) S / D plasma and glycosaminoglycan; such methods are presently included. Therefore, the glycosaminoglycan may be conveniently designated exogenous, or exogenously added to the S / D plasma, or additionally added to the S / D plasma.
The pharmaceutical formulation may be configured for separate, simultaneous or sequential administration in any order of the S / D plasma and the glycosaminoglycan and, when included, the active pharmaceutical substance (s) (s). ) additional. Accordingly, the pharmaceutical formulation may be a mixture of all its individual components, or may be a combination, such as a kit of components, comprising the individual constituents separately or comprising a mixture (s) of two or more, but not all, among the individual constituents. Therefore, the formulation may consist of a component kit comprising S / D plasma and a glycosaminoglycan.
The pharmaceutical formulation can be provided in a medical device. Such a medical device advantageously allows parenteral administration, such as intraosseous administration, peri-bone, intra-articular, or peri-articular, or intra-tendinous, peritendinous, intra-ligament or peri-tendinous administration. -ligamental, from the formulation to a subject requiring this one.
Preferably, the S / D plasma may be human S / D plasma, so that pharmaceutical formulations comprising human S / D plasma are particularly suitable for administration to human subjects.
In some embodiments, the glycosaminoglycan may be selected from the group consisting of hyaluronic acid and derivatives thereof, a proteoglycan and derivatives thereof, a chondroitin sulfate, a keratan sulfate, a chitosan and derivatives thereof, chitin and derivatives thereof. The formulation may include one or more glycosaminoglycans. The . formulation may therefore comprise a glycosaminoglycan or a mixture of glycosaminoglycans selected from the group consisting of hyaluronic acid and derivatives thereof, a proteoglycan and derivatives thereof, a chondroitin sulfate, a keratan sulfate, a chitosan and derivatives thereof, chitin and derivatives thereof.
Without limitation, the pharmaceutical formulation may comprise the glycosaminoglycan at a concentration in the range of about 0.10 mg / ml to about 200 mg / ml, preferably about 1.0 mg / ml to about 100 mg / ml, more preferably from about 2.0 mg / ml to about 50 mg / ml, for example, about 10 mg / ml, about 20 mg / ml, about 30 mg / ml or about 40 mg / ml.
Typically, intra- or peri-bone injection or intra- or peri-articular injection may have a volume of between about 2 ml and about 4 ml.
When the glycosaminoglycan has therapeutic benefit on its own, it can be included in a therapeutically effective amount, such as the exemplary amounts mentioned in this paragraph.
In particularly preferred embodiments, the glycosaminoglycan may be hyaluronic acid or a derivative thereof.
Without limitation, suitable derivatives may be hyaluronic acid salts, such as, preferably, sodium hyaluronate. Hyaluronic acid or a derivative thereof may have a low molecular weight (<900 kDa) or high (> 900 kDa). It may be particularly preferred hyaluronic acid or derivatives thereof having a high molecular weight (> 900 kDa). For example, the hyaluronic acid or derivative thereof may have a molecular weight in the range of about 1 x 106Da to about 6x106Da or greater, for example in the range of about 1 x 106 Da to about 4 x 106 Da, for example in the range of about 1.3 x 106 Da to about 3 x 106 Da.
Without limitation, the pharmaceutical formulation may include hyaluronic acid or a derivative thereof at a concentration in the range of about 0.10 mg / ml to about 200 mg / ml, preferably about 1.0 mg / ml. ml to about 100 mg / ml, more preferably from about 2.0 mg / ml to about 50 mg / ml.
In some embodiments, the pharmaceutical formulation may comprise one or more other components in addition to S / D plasma and glycosaminoglycan. In other embodiments, S / D plasma and glycosaminoglycan may be the only components of the formulation; therefore, in such embodiments, the pharmaceutical formulation may consist of or consist essentially of S / D plasma and glycosaminoglycan. In other additional embodiments, the pharmaceutical formulation may comprise one or more other components in addition to S / D plasma and glycosaminoglycan, but such additional components are not pharmaceutical active ingredients.
In some embodiments, the pharmaceutical formulation may advantageously further comprise one or more pharmaceutical active ingredients. The applicability of the present invention is not limited to any pharmaceutical active substance or class of pharmaceutical active substances. The pharmaceutical active substance can be pharmacologically active itself, or it can be converted into a pharmacologically active species by a chemical or enzymatic process in the body, i.e. the pharmaceutical active substance can be a prodrug. The present pharmaceutical formulations may be particularly useful for weakly stable pharmaceutical active ingredients. Illustrative, non-limiting examples of weakly stable pharmaceutical active ingredients include peptides and proteins such as growth factors, peptidomimetic active substances, antibodies and vaccines, a small interfering RNA (siRNA), DNA, hormones etc.
The term "pharmaceutical active substance" also covers all pharmacologically active salts, esters, N-oxides or prodrugs of the subject compound or substance.
In addition, a combination of two or more pharmaceutical active substances or combinations of doses can be included as a pharmaceutical component. In this case, the release of each active substance may be identical or different, such as, for example in the case of a combination of two active substances in which the first is presented in an immediate release form and the second in a form controlled release. Similarly, a combination of immediate release and controlled release form can also be obtained for the same active substance to produce a rapid and prolonged effect.
In some embodiments, the pharmaceutical formulation may further comprise serum. The addition of serum to the present formulation may allow some degree of improved gelation of the formulation.
For example, the serum may be allogeneic or autologous to the subject receiving the formulation. Preferably, the serum may be human serum, so that pharmaceutical formulations further comprising human serum are particularly suitable for administration to human subjects. For example, the formulation may contain serum and S / D plasma so that the value calculated by (volume of serum in formulation) / (serum volume in formulation + plasma volume S / D in formulation) is from about 0.01 to about 0.40, preferably from about 0.05 to about 0.15, more preferably from about 0.10.
In some embodiments, the pharmaceutical formulation may further comprise whole blood or a fractionated whole blood component. The addition of whole blood or said fractionated component, preferably whole blood, to the present formulation may at least partially allow the gelling of the formulation to be improved.
The present formulations comprising whole blood or said fractionated component thereof advantageously comprise growth factors derived from platelets useful in regenerative medicine, particularly for stimulating the repair of bone, cartilage, tendon or ligament defects. or to replace damaged bones, cartilage, tendons or ligaments (Gobbi et al., 2009, Grimaud et al., 2002, Cole et al., 2010).
For example, whole blood may be allogenic or autologous to the subject receiving the formulation. Preferably, the whole blood may be human whole blood, so that pharmaceutical formulations further comprising human whole blood are particularly suitable for administration to human subjects. For example, the formulation may contain whole blood and S / D plasma so that the value calculated by (volume of whole blood in the formulation) / (total blood volume in the formulation + plasma volume S / D in the formulation) formulation) is from about 0.01 to about 0.40, preferably from about 0.05 to about 0.15, more preferably about 0.10.
In preferred embodiments, the one or more of the pharmaceutical active ingredients are each independently selected from the group consisting of: a cell composition, a pharmaceutical active compound, a protein, a peptide, and a small organic molecule.
Preferably, the cell composition may comprise mesenchymal stem cells (MSCs), osteoprogenitor, osteoblast cells, osteocytes, chondroblastic cells, and / or chondrocytes. The pharmaceutical formulation thus allows the administration of such a cell composition. This viscous quality of the present pharmaceutical formulations can provide for their localized administration and a suitable support environment for the administered cells.
Particularly preferably, the cells of the cell composition may be animal cells, preferably a warm-blooded animal, more preferably mammalian cells, such as human cells or non-human mammalian cells, and most preferred way of human cells.
In other embodiments, the pharmaceutical active compound may be an anti-inflammatory substance. In preferred embodiments, the pharmaceutical active compound may be an alpha-2-adrenergic receptor agonist. Preferably, the alpha-2-adrenergic receptor agonist may be selected from the group consisting of clonidine and derivatives thereof.
In some embodiments, the alpha-2-adrenergic receptor agonist may be selected from the group consisting of clonidine and derivatives thereof, including 2,6-dimethylclonidine, 4-azidoclonidine, 4- carboxyclonidine-methyl-3,5-dichlorotyrosine, 4-hydroxyclonidine, 4-iodoclonidine, alinidine, apraclonidine, chlorethylclonidine, clonidine 4-isothiocyanate, clonidine 4-methylisothiocyanate, clonidine receptor, a substance displacing clonidine, hydroxyphenacetylaminoclonidine, N, N'-dimethylclonidine, p-aminoclonidine, and tiamenidine; imidazolidines, including imidazolines, impromidine, detomidine, medetomidine, dexmedetomidine, levamisole, losartane, lofexidine, miconazole, naphazoline, niridazole, nitroimidazoles, ondansetron, oxymetazoline, phentolamine, tetramisole, thiamazole, tizanidine, tolazoline, trimetaphan; imidazoles, including 4- (3-butoxy-4-methoxybenzyl) imidazolidin-2-one, urocanic acid, amino-imidazole carboxamide, antazoline, biotin, bis (4-methyl-1) homo-piperazinylthiocarbonyl) disulfide, carbimazole, cimetidine, clotrimazole, creatinine, dacarbazine, dexmedetomidine, econazole, enoximone, ethymizol, etomidate, fadrozole, fluspirilene, idazoxan mivazerol; guanidines, including agmatine, betanidine, biguanides, cimetidine, creatine, gabexate, guanethidine, guanethidine sulfate, guanclofine, guanfacine, guanidine, guanoxabenz, impromidine, iodine 3-benzylguanidine, methylguanidine, mitoguazone, nitrosoguanidines, pinacidil, robenidine, sulfaguanidine, zanamivir; alpha-methylnorpherphin, azepexole, 5-bromo-6- (2-imidazolidin-2-ylamino) quinoxaline, formoterol fumarate, indoramine, 6-allyl-2-amino-5,6,7 , 8-tetrahydro-4H-thiazolo [4,5-d] azepine diHCl, nicergoline, rilmenidine, and xylazine.
In certain embodiments, the pharmaceutical formulation may further comprise one or more substances having osteogenic, osteoinductive and / or osteoconductive properties. In preferred embodiments, such a substance may be selected from the group consisting of or consisting of a fibroblast growth factor (FGF), preferably FGF-2, a beta-transforming growth factor (TGFB), preferably TGFB-1, platelet derived growth factor (PDGF), interleukin 8 (IL-8), bone morphogenetic protein (BMP), for example any one or more of BMP-2, BMP-4, BMP-6 and BMP-7, parathyroid hormone (PTH), a parathyroid hormone-related protein (PTHrp), and stem cell factor (SCF).
Therefore, in some embodiments, the active pharmaceutical protein or peptide may be a growth factor, preferably a growth factor selected from the group consisting of FGF, TGFB, PDGF, IL-8, BMP. , PTH, PTHrp, and SCF, more preferably a growth factor selected from the group consisting of FGF-2, TGFB-1, PDGF, IL-8, BMP-2, BMP-4, BMP-6, BMP-7, PTH, PTHrp, and SCF.
Preferably, the pharmaceutical formulation may be configured for parenteral administration, such as parenteral injection, more preferably for intraosseous, peri-bone, intra-articular or peri-articular administration, such as intraosseous injection, periosteal -osseous, intra-articular or peri-articular, or for intra-tendinous, peri-tendinous, intra-ligamentous or peri-ligamentary administration, such as intra-tendinous, peritendinous, intra-ligamentary or peri-ligamentous injection .
A related aspect relates to the pharmaceutical formulation as described above for use in treatment (including, throughout the present specification, therapeutic and / or preventive measures) of musculoskeletal disease. Preferably, said musculoskeletal disease may be bone disease or joint disease. Alternatively or additionally, said musculoskeletal disease may affect the tendons and / or ligaments.
The use of the pharmaceutical formulation as described above for the manufacture of a medicament for the treatment of musculoskeletal disease, preferably bone disease or joint disease, is also disclosed. Alternatively or additionally, said musculoskeletal disease may affect the tendons and / or ligaments.
There is also disclosed a method for treating a musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments), in a subject in need thereof comprising administering to said subject a therapeutically or prophylactically effective amount of the pharmaceutical formulation as described above.
Based on these observations, the present inventors have made use of the aforementioned formulations as a pharmaceutical excipient, more preferably as a sustained-release or slow release pharmaceutical excipient. Accordingly, an associated aspect relates to the use of a formulation comprising S / D plasma and glycosaminoglycan as a pharmaceutical excipient, preferably as a pharmaceutical excipient in a pharmaceutical formulation configured for parenteral administration, more preferably to intraosseous, peri-bone, intra-articular, or periarticular administration, or for intra-tendinous, peritendinous, intra-ligamentary, or peri-ligament administration.
Also disclosed is a formulation comprising S / D plasma and glycosaminoglycan for use as a pharmaceutical excipient, more preferably for use as a sustained-release or slow release pharmaceutical excipient, preferably for use as an excipient. pharmaceutical for treatment of musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments). Further described is the use of a formulation comprising S / D plasma and a glycosaminoglycan for the manufacture of a pharmaceutical excipient, more preferably a sustained-release or slow-release pharmaceutical excipient, preferably for the manufacture of a pharmaceutical excipient. for treatment of musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments). There is further described a method for treating a musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments) in a subject in need of such treatment. comprising administering to said subject a formulation comprising S / D plasma and glycosaminoglycan as a pharmaceutical excipient, more preferably as a sustained-release or slow-release pharmaceutical carrier.
Preferably, the S / D plasma may be human S / D plasma, so that pharmaceutical formulations comprising human S / D plasma are particularly suitable for administration to human subjects.
It is furthermore described the use of S / D plasma as a pharmaceutical excipient, preferably as a pharmaceutical excipient in a pharmaceutical formulation configured for parenteral administration, more preferably for intraosseous, periosal administration, intra-articular, or peri-articular, or for intra-tendinous, peritendinous, intra-ligamentary or peri-ligamentary administration.
Therefore, there is further described S / D plasma for use as a pharmaceutical excipient, more preferably for use as a sustained-release or slow release pharmaceutical excipient, preferably for use as a pharmaceutical excipient for treatment of a musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments). It is furthermore disclosed the use of S / D plasma for the manufacture of a pharmaceutical excipient, more preferably a sustained-release or slow-release pharmaceutical excipient, preferably for the manufacture of a pharmaceutical excipient for the treatment of musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments). There is further described a method for treating a musculoskeletal disease, preferably bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments) in a subject in need of such treatment. comprising administering to said subject S / D plasma as a pharmaceutical excipient, more preferably as a sustained release or slow release pharmaceutical excipient.
Preferably, the S / D plasma may be human S / D plasma, so that pharmaceutical formulations comprising human S / D plasma are particularly suitable for administration to human subjects.
It is presently further described the use of the aforementioned formulations as a cell culture medium supplement. Accordingly, an associated aspect is the use of a formulation comprising S / D plasma and a glycosaminoglycan as a supplement for cell culture medium. Such formulations may advantageously make it possible to obtain cells or cell cultures having improved properties.
Throughout this aspect of the invention and any of its embodiments, the S / D plasma may preferably be S / D mammalian plasma, more preferably human S / D plasma. Also throughout this aspect of the invention and any of its embodiments, the serum may be mammalian serum, more preferably human serum. Further, preferably, the S / D plasma may be S / D mammalian plasma and the serum may be mammalian serum, or the S / D plasma may be human S / D plasma and the serum may be human serum. Plasma and / or human serum may be particularly advantageous for administration to human subjects. Also throughout this aspect of the invention and any of its embodiments, the whole blood may be whole mammalian blood, more preferably whole human blood. Further, preferably, the S / D plasma may be S / D mammalian plasma and the whole blood may be mammalian whole blood, or the S / D plasma may be human S / D plasma and the blood total can be human whole blood. Plasma and / or human whole blood may be particularly advantageous for administration to human subjects.
Further described herein are pharmaceutical formulations as described above, and the corresponding uses thereof wherein the glycosaminoglycan is omitted from the formulations. Therefore, such formulations can include, in particular, S / D plasma (preferably human S / D plasma), and any one or more components as described above, in particular, and any one or more of serum, whole blood or fractionated component thereof, one or more pharmaceutical active substances (such as each independently selected from the group consisting of cell composition, pharmaceutical active compound, protein, peptide, and small organic molecule). Such formulations may also exhibit satisfactory gelling behavior.
The above and other aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The object of the appended claims is specifically incorporated herein in this specification.
Brief description of the drawings
Figure 1 illustrates a radiographic view of bone repair / formation 4 weeks after administration of a formulation containing S / D, IL-8 and CaCl2 plasma to a bone defect site in a bone osteotomy model. cranial cap in immunocompetent mice. Bone repair can be clearly observed (mineralized zones) in treated mice (B) compared to control without IL-8 (A).
Figure 2 shows the microscopic analysis of the newly formed tissue shown in Figure 1B, at a magnification of 20 times (A) and 40 times. Four weeks after administration of a formulation containing S / D, IL-8 and CaCl2 plasma to a bone defect site in a cranial head osteotomy model in immunocompetent mice, the empty collagen matrix exhibits large mineralized and non-mineralized osteoid zones and a large number of vessels.
Description of embodiments
In the present context, the singular forms "one", "one", and "the" include both the singular and the plural unless otherwise clearly indicated in the context.
The terms "comprising", "includes" and "consisting of" in this context are synonymous with "comprising", "includes" or "containing", "contains", and are inclusive or open and do not exclude additional members, elements or process steps, not mentioned. The terms also cover "consisting of" and "essentially consisting of".
Boundary numeric ranges include all numbers and fractions that are under-inclusive in the respective ranges, as well as the limits mentioned.
The term "about" in this context, when referring to a measurable value such as a parameter, a quantity, a time duration, and the like, is intended to cover variations of and with respect to the specified value. , in particular variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and still more preferably +/- 0.1% or less of and by in relation to the specified value, insofar as such variations are appropriate in the context of the object of the invention described. It should be noted that the value to which the "about" modifier refers is also specifically, and preferably, described.
While the term "one or more", such as one or more members of a group of members, is clear as such, by means of further exemplification, the term covers, inter alia, a reference to the any of said members, or any two or more of said members, such as, for example,> 3,> 4,> 5,> 6 or> 7, etc., any of said members, and up to all said members .
All documents cited in this specification are presently incorporated by reference in their entirety.
Unless otherwise indicated, all terms used in the description of the invention, including technical and scientific terms, have the meaning commonly understood by those skilled in the art to which this invention belongs. As a further indication, definitions of terms may be included to better appreciate the teachings of the present invention.
General techniques in cell culture and media uses are described, inter alia, in Large Scale Mammalian Cell Culture (Hu et al., 1997. Curr Opin Biotechnol 8: 148); Serum-free Media (K. Kitano, 1991. Biotechnology 17: 73); or Large Scale Mammalian Cell Culture (Curr Opin Biotechnol 2: 375, 1991).
The terms "pharmaceutical formulation", "pharmaceutical composition", or "pharmaceutical preparation" may be used interchangeably presently. Similarly, the terms "formulation", "composition", or "preparation" can be used interchangeably presently.
The term "plasma" is as conventionally defined. Plasma is usually obtained from a whole blood sample, mixed or contacted with an anticoagulant (eg, heparin, citrate, oxalate or EDTA). Then, the cellular components of the blood sample are separated from the liquid component (plasma) by an appropriate technique, typically by centrifugation. Therefore, the term "plasma" refers to a composition that is not part of a human or animal body.
The terms "solvent / detergent treated plasma", "S / D treated plasma" or "S / D plasma" generally refer to decellularized plasma obtainable or obtained by a process comprising the steps of: (a) treating the plasma with a solvent and detergent and (b) filtration of solvent / detergent treated plasma.
The plasma to be treated in step (a) may be any plasma as conventionally defined such as fresh plasma, fresh frozen plasma, frozen frozen plasma, or cryoprecipitate, cryosumants or frozen plasma concentrates as well as dilution products thereof. Plasma is usually obtained from a whole blood sample, or a sample obtained by apheresis.
Solvents such as di- or trialkylphosphates and detergents are described in US 4,764,369. The solvent used to prepare S / D plasma is preferably a dialkylphosphate or trialkylphosphate, both having alkyl groups which contain 1 to 10 carbon atoms. carbon atoms, in particular 2 to 10 carbon atoms. Illustrative examples of solvents may include tri- (n-butyl) phosphate, tri- (t-butyl) phosphate, tri- (n-hexyl) phosphate, tri- (2-ethylhexyl) phosphate, or tri-n-decyl. )phosphate. A preferred solvent is tri- (n-butyl) phosphate. Mixtures of different trialkyl phosphates may also be used as well as phosphates having alkyl groups of different alkyl chains, eg, ethyl, di (n-butyl) phosphate. Similarly, the respective dialkyl phosphates can be used including those of different mixtures of alkyl dialkyl phosphate groups. In addition, mixtures of di- and trialkylphosphates can be used.
The solvent such as a di- or trialkylphosphate for use in the treatment step (a) is preferably used in an amount in the range of about 0.01 mg / ml to about 100 mg / ml, and preferably from about 0.1 mg / ml to about 10 mg / ml. In other words, di- or trialkylphosphates for use in the treatment step (a) are preferably used in an amount in the range of about 0.001% w / v to about 10% w / v, and preferably about 0.01% w / v to about 1% w / v.
The detergent used to prepare S / D plasma is preferably a non-toxic detergent. Nonionic detergents contemplated include those which disperse at room temperature at least 0.1% by weight of the fat in an aqueous solution containing it when 1 gram of detergent per 100 ml of solution is introduced therein. Illustrative examples of detergents may include polyoxyethylene derivatives of fatty acids, partial esters of sorbitol anhydrides, for example, the products sold under the name "Tween 80", "Tween 20" and "polysorbate 80" and nonionic water-soluble aqueous detergents such as that marketed under the brand name "Triton X 100" (alkylphenol oxyethyl). It is also envisaged sodium deoxycholate as well as "zwittergents" which are synthetic zwitterionic detergents called "sulfobetaines" such as N-dodecyl-N, N-methyl-2-ammonio-1-ethanesulfonate and its congeners or detergents nonionic agents such as octyl-beta-D-glucopyranoside.
The amount of detergent may be in the range of about 0.001% v / v to about 10% v / v, preferably about 0.01% v / v to 1.5% v / v.
The treatment with a solvent and a detergent is preferably carried out at a temperature between -5 ° C and 70 ° C, preferably between 0 ° C and 60 ° C. The duration of such treatment (contact) is at least 1 minute, preferably at least 1 hour and generally 4 to 24 hours. The treatment is normally effective at atmospheric pressure, but sub-atmospheric and hyper-atmospheric pressures may also be used.
Normally, after the treatment, the solvent such as a trialkylphosphate and the detergent are removed. The solvent and detergent may be removed by any technique suitable for separating solvent and detergent from the plasma. When a nonionic detergent is used with the solvent such as trialkylphosphate, these can be removed by: (1) diafiltration using microporous membranes such as TEFLON which retain plasma proteins; (2) absorption of the desired plasma components on chromatographic or affinity chromatography supports; (3) precipitation, for example, by desalting plasma proteins; (4) lyophilization, etc.
Solvents such as dialkylphosphate or trialkylphosphate can be removed as follows: (a) Antihemophilic factor (AHF) removal can be accomplished by precipitation of AHF with 2.2 M glycine and 2.0 M sodium chloride (b) The removal of fibronectin can be effected by binding fibronectin to an insolubilized gelatin column and washing the bound fibronectin free of reagent. The filtration step (b) is generally performed with a 1 μm filter to remove cells and debris, followed by sterilizing filtration using a 0.2 μm filter.
In a preferred example, as described by Horowitz et al., 1992 (Blood, 3, 826-831), S / D plasma can be prepared as follows: FFP can be rapidly thawed and can be stirred for 4 hours with 1% (v / v) tri- (N-butyl) -phosphate (TNBP) and 1% (v / v) polyoxyethylene-p-octylphenol (Triton X-100) at 30 ° C. After treatment, an edible oil such as soybean oil (5% v / v) or castor oil can be added, gently mixed for 30 minutes, and removed by centrifugation at 10,000 g for 20 minutes. The clarified plasma can be applied to a Waters Prep C18 resin column so that the volume ratio of the plasma to the column is 6 and the contact time can be 3 minutes. The column eluate can be filtered on a 0.2 μm filter.
For example, S / D plasma is marketed by Octaplas® (Octapharma AG, Lachen, Switzerland).
The term "S / D plasma" covers plasma comprising a reduced concentration or activity of plasmin inhibitor, such as a level of plasmin inhibitor less than or equal to 0.60 IU / ml or less than or equal to 0, 50 IU / ml, for example a level of plasmin inhibitor of between 0.20 and 0.30 IU / ml, more specifically between 0.22 and 0.25 IU / ml.
Compared with fresh frozen plasma (FFP), S / D plasma may comprise a reduced amount and / or activity of one or more of the plasmin inhibitor, protein S, factor XI, factor V, factor VIII, factor X, antiplasmin α2, antitrypsin, von
Willebrand (vWF), and the von Willebrand factor (VWFCP) cleaving protease also called disintegrin and metalloproteinase with a thrombospondin pattern of type 1, member 13 (ADAMTS-13), tumor necrosis factor alpha (TNFa), l interleukin 8 (IL-8), interleukin 10 (IL-10) (Benjamin and McLaughlin, 2012, Svae et al, 2007, Beeck and Hellstem, 1998, Doyle et al., 2003, Mast et al, 1999, Theusinger et al., 2011) and / or may include an increased amount and / or activity of factor VII (Doyle et al., 2003).
S / D plasma can be used directly in the present pharmaceutical formulations. It may also be appropriately stored for later use (for example, for shorter periods of time, for example, up to about 1 to 2 weeks, at a temperature above the respective freezing points of plasma or serum, but below room temperature, this temperature being generally from about 4 ° C. to 5 ° C., or for longer periods by storage in the freezer, generally between about -70 ° C. and about -80 ° C.).
S / D plasma can be heat inactivated as is known in the art, particularly for removing complement. When the present pharmaceutical formulations use autologous S / D plasma vis-à-vis the subject to be treated, it may be unnecessary to heat inactivate the S / D plasma. When the S / D plasma is at least partially allogeneic with respect to the subject to be treated, it may be advantageous to inactivate the S / D plasma by heat.
The pharmaceutical formulations of the present invention may comprise S / D plasma which is autologous to the subject to be treated. The term "autologous" with reference to S / D plasma indicates that S / D plasma is obtained from the same subject to be contacted or treated with S / D plasma. The pharmaceutical formulations of the present invention may comprise S / D plasma which is "homologous" or "allogeneic" to the subject to be treated, i.e., obtained from one or more subjects (grouped) other than the subject to be contacted or treated with S / D plasma. The pharmaceutical formulations of the present invention may also comprise a mixture of autologous and homologous (allogeneic) S / D plasma as defined above. Preferably, the pharmaceutical formulations may comprise S / D plasma which is "allogeneic" to the subject to be treated. Advantageously, the allogeneic S / D plasma is commercially available and therefore is an unlimited source of plasma.
The term "serum" is as conventionally defined. Serum can usually be obtained from a whole blood sample, initially by allowing coagulation to occur in the sample and then separating the clot thus formed and the cellular components of the component blood sample. liquid (serum) by an appropriate technique, typically by centrifugation. Coagulation can be facilitated by an inert catalyst, for example, beads or glass powder. Alternatively, serum can be obtained from plasma by removal of anticoagulant and fibrin. The term "serum", therefore, refers to a composition that is not part of a human or animal body.
The serum can be used directly in pharmaceutical formulations as described herein. It may also be appropriately stored for later use (for example, for shorter periods of time, for example, up to about 1 to 2 weeks, at a temperature above the respective freezing points of plasma or serum, but below room temperature, this temperature is generally from about 4 ° C to 5 ° C, or for longer periods by freezer storage, generally from about -70 ° C to about -80 ° C).
The serum can be heat inactivated as is known in the art, particularly for eliminating complement. When the present pharmaceutical formulations use autologous serum against cultured cells in the presence of these, it may be unnecessary to heat inactivate the serum. When the serum is at least partially allogeneic to the subject to be treated, it may be advantageous to inactivate the serum by heat.
Optionally, the serum may also be sterilized prior to preservation or use, using conventional microbiological filters, preferably with a pore size of 0.2 μm or less.
In some embodiments, the pharmaceutical formulations may use serum that is autologous to the subject to be treated. The term "autologous" with reference to serum indicates that the serum is obtained from the same subject to be contacted with the serum. In some embodiments, the pharmaceutical formulations may use serum that is "homologous" or "allogeneic" to the subject to be treated, i.e., obtained from one or more subjects ( grouped) other than the subject to be in contact with the serum. In some embodiments, the pharmaceutical formulations may use a mixture of autologous and homologous (allogeneic) sera as defined above.
The term "whole blood" is as conventionally defined. Preferably the sample can be easily obtained by minimally invasive methods, allowing removal or isolation of the subject's whole blood. Whole blood is usually mixed or contacted with an anticoagulant (eg, heparin, citrate, oxalate or EDTA).
Whole blood can be used directly in pharmaceutical formulations as described herein. Whole blood may also be suitably stored for later use (eg, for shorter periods, for example, up to about 1 to 4 weeks, at a temperature above the freezing point of whole blood, but below room temperature, this temperature is generally from about 4 ° C to 5 ° C or for longer periods by freezer storage, generally from about -70 ° C to about -160 ° C, for example between about -70 ° C and about -80 ° C, for example about -160 ° C).
In some embodiments, the pharmaceutical formulations may use whole blood that is autologous to the subject to be treated. The term "autologous" in reference to whole blood indicates that whole blood is obtained from the same subject to be in contact with whole blood. In some embodiments, the pharmaceutical formulations may use whole blood that is "homologous" or "allogeneic" to the subject to be treated, i.e., obtained from one or more subjects (grouped) other than the subject to be in contact with whole blood. In some embodiments, the pharmaceutical formulations may use a mixture of autologous and homologous (allogeneic) whole blood as defined above.
In some embodiments, the glycosaminoglycan may be selected from the group consisting of hyaluronic acid and derivatives thereof, a proteoglycan and derivatives thereof, a chondroitin sulfate, a keratan sulfate, a chitosan and derivatives thereof, chitin and derivatives thereof.
The terms "hyaluronic acid" or "HA" may be used interchangeably with "hyaluronan" or "hyaluronate". The term "hyaluronic acid" refers to an anionic, non-sulfated disaccharide polymer composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating β-1,4 and β-1,3 glycoside bonds. Hyaluronic acid derivatives include, but are not limited to, hyaluronate salts such as sodium hyaluronate or a hyaluronic acid ester with an alcohol of the aliphatic, heterocyclic or cycloaliphatic series, or a sulphated form of hyaluronic acid or a combination of agents including hyaluronic acid.
The term "proteoglycan" refers to proteins with one or more glycosaminoglycan (GAG) chain (s) covalently linked. The glycosaminoglycan may be a proteoglycan selected from decorin, biglycan, testicane, fibromodulin, lumicane, versicane, perlecane, neurocane or aggrecan.
The term "chondroitin sulfate" refers to a disaccharide polymer composed of N-acetylgalactosamine and glucuronic acid, each of which can be sulfated at varying positions and amounts. Chondroitin sulfate may be selected from chondroitin 4-sulfate, chondroitin 6-sulfate, chondroitin 2,6-sulfate, chondroitin 4,6-sulfate.
The term "keratan sulfate" can be used interchangeably with "keratosulfate" and refers to a polymer of repeating disaccharides -3Ga ^ 1 -40ΙοΝΑςβ 1 - which can be sulfated at the carbon position 6 (C6) of one or the two monosaccharides Gai or GlcNAc.
The term "chitosan" refers to a linear polymer composed of D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetyl unit) with β- (1-4) -linked distribution randomly.
The term "chitin" refers to a polymer composed of β- (1,4) -linked N-acetylglucosamine.
In some embodiments, the pharmaceutical formulation further comprises one or more pharmaceutical active ingredients.
Such pharmaceutical active substances can cover, for example, cell compositions.
In some embodiments, the cell composition may include mesenchymal stem cells (MSCs), osteoprogenitor, osteoblast cells, osteocytes, chondroblastic cells, and / or chondrocytes.
The term "mesenchymal stem cell" or "MSC" as used herein refers to an adult mesoderm-derived stem cell that is capable of generating cells of mesenchymal lineages, typically of two or more mesenchymal lineages, for example, the osteocyte lineage. (bone), chondrocytic (cartilage), myocytic (muscle), tendinocytic (tendon), fibroblastic (connective tissue), adipocyte (fat) and stromal (medullary stroma). MSC can be isolated from, for example, bone marrow, trabecular bone, blood, umbilical cord, placenta, fetal gestational sac, skin (dermis), specifically skin, periosteum and tissue fetal adipose and adolescent. Human MSCs, their isolation, in vitro expansion, and differentiation are described in, for example, U.S. Patent No. 5,486,359; U.S. Patent No. 5,811,094; U.S. Patent No. 5,736,396; U.S. Patent No. 5,837,539; or U.S. Patent No. 5,827,740. Any MSCs described in the art and isolated by any method described in the art can be adapted in the present pharmaceutical formulations.
The term MSC further encompasses MSC progeny, for example progeny obtained by in vitro or ex vivo proliferation (propagation) of MSCs obtained from a biological sample of an animal or human subject.
Preferable MSCs have the potential to generate cells of at least the osteogenic lineage (bone), such as, for example, osteoprogenitor and / or pre-osteoblasts and / or osteoblasts and / or osteocytes, etc., or at least the chondrogenic (cartilage) lineage, such as, for example, chondrogenic cells and / or chondroblasts and / or chondrocytes, etc.
The term "stem cell" usually refers to a non-specialized or relatively less specialized and proliferating cell that is capable of self-renewal, that is to say, it can proliferate without differentiation, and which or whose offspring can lead to at least one relatively more specialized cell type. The term covers stem cells capable of substantially unlimited self-renewal, i.e., the progeny of a stem cell or at least a portion thereof retains the substantially non-specialized or relatively less specialized phenotype, the potential of differentiation, and the proliferative capacity of the parent stock cell, as well as stem cells that exhibit limited self-renewal, i.e., the ability of the progeny or a portion thereof for proliferation and / or Subsequent differentiation is visibly reduced compared to the parent cell. By way of example and not limitation, a stem cell can lead to offspring that can differentiate into one or more lineages to produce more and more specialized cells, such descendants and / or cells more and more. relatively specialized may themselves be stem cells as currently defined, or even produce terminal differentiated cells, i.e., fully specialized cells, which may be post-mitotic.
The term "adult stem cell" as used herein refers to a stem cell present in or obtained from (eg, isolated from) an organism at the fetal stage or after birth, such as, for example, after have reached adulthood.
In the present context, "osteoprogenitor" may include, in particular, early and late osteoprogenitor. The "osteoblastic cells" may in particular cover pre-osteoblasts, osteoblasts and osteocytes, and the term may more preferably refer to pre-osteoblasts and osteoblasts. All of these terms are known as such and, in the present context, can typically refer to cells having an osteogenic phenotype, and which can contribute to, or are capable of developing, cells that may contribute to the formation of bone material or of bone matrix. As a further indication and not a limitation, osteoprogenitor and osteoblast as well as cell populations comprising osteoprogenitor and / or osteoblast cells may have the following characteristics: a) the cells comprise the expression of Runx2, a factor multifunctional transcriptional gene which regulates the differentiation of osteoblasts and the expression of many extracellular matrix protein genes during osteoblast differentiation; b) the cells comprise the expression of at least one of the following: alkaline phosphatase (ALP), more specifically ALP of the bone-liver-kidney type; and more preferably further include the expression of one or more additional bone markers such as osteocalcin (OCN), aminoterminal procollagen propeptide type 1 (P1NP), osteonectin (ON), osteopontin (OP). ) and / or bone sialoprotein (BSP), and / or one or more additional bone matrix protein proteins such as decorin and / or osteoprotegerin (OPG); c) the cells do not substantially express CD45 (e.g., less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45); d) the cells have a proven ability to mineralize the external environment, or to synthesize an extracellular matrix containing calcium (for example, when exposed to an osteogenic medium, see Jaiswal et al, J Cell Biochem, 1997, vol. 64, 295-312). The accumulation of calcium inside the cells and the deposition in matrix proteins can be conventionally measured, for example, by culture in 45Ca2 +, washing and re-culturing, and then determining the radioactivity possibly present inside. of the cell or deposited in the extracellular matrix (US 5,972,703), or using an alizarin red mineralization assay (see, for example, Gregory et al., Analytical Biochemistry, 2004, vol 329, 77-84 ); e) the cells do not substantially differentiate into adipocyte (e.g. adipocytes) or chondrocyte lineage (e.g., chondrocyte) lineage cells. The lack of differentiation to such cell lineages can be tested using standard differentiation induction conditions established in the art (e.g., see Pittenger et al., Science, 1999, vol 284, 143-7), and assay methods (e.g., when induced, adipocytes are typically stained with the red oil O indicating lipid accumulation, the chondrocytes are typically stained with alcian blue or safranin O). The substantial lack of propensity for adipogenic and / or chondrogenic differentiation can typically mean that less than 20%, or less than 10%, or less than 5%, or less than 1% of the cells tested exhibit signs of adipogenic differentiation or chondrogenic when evaluated in the respective test.
The cells may further comprise expression of one or more cell recruitment factors such as IL6 and / or VEGF.
In the present context, "chondroblastic cells" may include, in particular, chondroblasts, i.e., young (non-mature, immature) cartilage cells active in extracellular matrix secretion. Chondroblasts are thought to be formed by differentiation from mesenchymal stem cells. The term "chondrocyte" refers more specifically to a mature cartilage cell necessary for the maintenance of the cartilage matrix. These terms are known per se and, in the present context, can typically refer to cells having a chondrogenic phenotype, and which may contribute to, or are capable of developing into, cells that may contribute to cartilage or tissue formation. cartilaginous matrix. As a further indication and not a limitation, human articular chondrocytes may exhibit cell expression characteristics as summarized in Diaz-Romero et al. 2005 (J Cell Physiol, vol 202 (3), 731-42), for example, they can express integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51 / 61, CD54 , CD106, CD166, CD58, CD44), tetraspanines (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ecto-enzymes (CD10, CD26), and other surface molecules (CD90, CD99). During a monolayer culture, chondrocytes may upregulate certain markers considered to be distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166). Such markers can therefore also be expressed by less mature chondroblastic cells.
When a cell is said to be positive for (or expresses or understands the expression of) a particular marker, it means that one skilled in the art will conclude that a distinctive signal, for example, detectable by antibody or reverse transcription polymerase chain reaction detection, for the marker in question when conducting the appropriate measurement, relative to suitable controls. When the method allows the quantitative evaluation of the marker, the positive cells can generate on average a signal which is significantly different from the control, for example, but without limitation, at least 1.5 times higher than such a signal generated by control cells, for example, at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times higher or even higher. Expression of the above cell-specific markers can be detected using any suitable immunological technique known in the art, such as immunocytochemistry or affinity adsorption, Western blot analysis, FACS, ELISA etc., or by any suitable biochemical assay for enzyme activity (eg, for ALP), or any suitable technique for measuring the amount of the tag mRNA, for example, Northern blot, RT -PCR semi-quantitative or quantitative, etc. The sequence data for the markers mentioned in this specification are known and can be obtained from public databases such as GenBank (http://www.ncbi.nlm.nih.gov/).
The present pharmaceutical formulation may comprise one or more substances having osteogenic, osteoinductive and / or osteoconductive properties. In preferred embodiments, such a substance may be selected from the group consisting of or consisting of a fibroblast growth factor (FGF), preferably FGF-2, a transforming growth factor beta (TGFB), preferably TGFB -1, a platelet derived growth factor (PDGF), interleukin 8 (IL-8), a bone morphogenetic protein (BMP), for example any one or more of BMP-2, BMP-4, BMP -6 and BMP-7, parathyroid hormone (PTH), a parathyroid hormone-related protein (PTHrp), and stem cell factor (SCF). Any of such substances may be included in a pharmaceutical composition at a concentration sufficient to achieve its desired osteogenic, osteoinductive and / or osteoconductive effect (s) when (s) is / are are administered to a subject, while avoiding undesirable side effects, as far as possible.
Typically, but without limitation, any such substance may be included in the pharmaceutical formulation at a concentration of from 0.01 ng / ml to 1 mg / ml, for example from 0.1 ng / ml to 100 μg / ml. ml, for example from 1 ng / ml to 50 μg / ml.
The term "osteoinducer" refers to the ability of a component such as peptide growth factor to recruit immature cells such as stem cells, MSCs, and stimulate these cells to differentiate into pre-osteoblasts and mature osteoblasts to form bone tissue. The present pharmaceutical compositions may further comprise a component having osteoinductive properties such as an osteoinductive protein or peptide, for example a bone morphogenetic protein, such as BMP-2, BMP-7 or BMP-4; a hydrogel or a biopolymer such as hyaluronic acid or derivatives thereof, collagen, fibrinogen, osteonectin, or osteocalcin. Preferably, the pharmaceutical compositions may further comprise hyaluronic acid or derivatives thereof, collagen or fibrinogen.
The term "osteoconductive" refers to the ability of a component to serve as a scaffold on which bone cells can attach, migrate, grow and produce new bone. The pharmaceutical compositions may further comprise a component having osteoconductive properties, for example, an osteoconductive scaffold or matrix or a surface, such as, without limitation, tricalcium phosphate, hydroxyapatite, a combination of hydroxyapatite / tricalcium phosphate (HA / TCP), gelatin, poly (lactic acid), poly (lactic-glycolic acid), hyaluronic acid, chitosan, poly-L-lysine, or collagen.
As mentioned above, the pharmaceutical formulations of the present invention may include components useful in the repair of bone lesions and defects. The pharmaceutical formulations may comprise a scaffold or matrix having osteoconductive properties. The pharmaceutical formulations can be combined with a demineralized bone matrix (DBM) or other matrices to make the osteogenic composite as well as osteoconductive and osteoinductive. Similar methods using autologous bone marrow cells with allogeneic DBM have been successful (Connolly et al 1995. Clin Orthop 313: 8-18).
The pharmaceutical formulations according to the present invention may further comprise or be co-administered with a complementary bioactive factor or an osteoinductive protein such as a bone morphogenetic protein, such as BMP-2, BMP-7 or BMP-4, or another growth factor. Other potential auxiliary components include inorganic sources of calcium or phosphate adapted to facilitate bone regeneration (WO 00/07639). If desired, the cell preparation can be administered on a matrix or carrier material to enable improved tissue regeneration. For example, the material may be a hydrogel, or a biopolymer such as gelatin, collagen, hyaluronic acid or derivatives thereof, osteonectin, fibrinogen, or osteocalcin. Biomaterials can be synthesized according to standard techniques (eg, Mikos et al., Biomaterials 14: 323, 1993, Mikos et al., Polymer 35: 1068, 1994, Cook et al., J. Biomed, Mater Res. 35: 513, 1997).
Formulations applying the principles of the invention advantageously have a particularly satisfactory gel-forming behavior, producing advantageously viscous formulations. In some embodiments, the formulations are gelling formulations.
The terms "gelling", "one phase" or "single phase" can be used interchangeably at this time. The term "gelling formulation" as used throughout this specification refers to the ability of the formulation to form a solid material, such as a gel (gel), having a pseudoplastic behavior. For example, the present pharmaceutical formulations advantageously form a gel when their components are combined or when their components are combined with or exposed to materials and / or conditions leading to gel formation, for example, but without limitation, when they are dissolved or dispersed in synovial fluid, serum, or a cell composition.
The term "viscosity" is generally a measure of the reduction of a fluid that is deformed by shear stress or tensile stress. The present pharmaceutical formulations can have a viscosity of at least 10 Pa.s, for example the present pharmaceutical formulations can have a viscosity in the range of about 30 Pa.s to about 500 Pa.s, for example, about 50 Pa.s at about 250 Pa.s at room temperature applying a shear rate of 0.560 s -1.
The compositions embodying the principles of the invention can acquire their gelatinous consistency, particularly in the presence of divalent calcium ions (Ca 2+). Animal tissues, such as, preferably, mammalian tissues, such as, more preferably, human tissues, including body fluids such as synovial fluid, contain extracellular Ca 2+, typically at a concentration of between 1mM and 3mM. . In addition, the concentration of Ca2 + is high in bone tissue, where it is stored primarily in the form of calcium phosphate crystals in the form of hydroxyapatite.
Therefore, in some embodiments, it is not necessary to add Ca 2+ to the present pharmaceutical compositions, since after their administration, for example in bone or joint tissue, Ca 2+ present at the site of induced administration coagulation / gelation of the compositions.
In certain other embodiments, Ca 2+ can be added to the present pharmaceutical compositions, for example to facilitate their coagulation / gelation in situ (for example, when the concentration of Ca 2+ at the observed or intended administration site is insufficient to induce on its own coagulation / gelling of the compositions), or to obtain a certain degree of coagulation / gelation in vitro before their administration (for example, to improve the injection capacity and / or integrity of the product). In such embodiments, Ca 2+ may typically be added in the pharmaceutical compositions at a concentration of from about 5 mM to about 100 mM, preferably from about 10 mM to about 50 mM, more preferably from about 10 mM to about 20 mM. or between about 20 mM to about 40 mM, for example, from about 20 mM to about 30 mM or from about 30 mM to about 40 mM.
In some embodiments, the products intended for intra-articular or periarticular administration may comprise from about 20 mM to about 40 mM, for example, from about 20 mM to about 30 mM Ca 2+. In some other embodiments, the intended intraosseous or perosseous delivery products may comprise from about 10 mM to about 20 mM Ca 2+.
Ca 2+ may suitably be included in the pharmaceutical compositions by adding therein a suitable amount of pharmaceutically acceptable calcium salt (s), preferably soluble calcium salt (s). Such Ca2 + salts can be formed with inorganic or organic acids. Examples of such salts include calcium chloride (CaCl 2), calcium glycerophosphate, calcium phosphate, calcium hydrogencarbonate, calcium citrate, calcium sulfate, calcium lactate, calcium gluconate, calcium ascorbate, and mixtures thereof. It is particularly preferred CaCl 2, which advantageously has good solubility and is well tolerated in injectable solutions.
Presently provided pharmaceutical formulations may comprise from about 1 mg / ml to about 10 mg / ml CaCl 2, preferably from about 2 mg / ml to about 4 mg / ml CaCl 2. In some embodiments, the products intended for intra-articular or peri-articular administration may comprise between about 1 mg / ml and about 10 mg / ml CaCl 2, preferably between about 2 mg / ml and about 5 mg / ml, more preferably about 4 mg / ml CaCl 2. In certain other embodiments, the products intended for intraosseous or periosal administration may comprise between about 1 mg / ml and about 10 mg / ml CaCl 2, preferably between about 2 mg / ml and about 5 mg / ml. more preferably about 2 mg / ml CaCl 2.
In some embodiments, the pharmaceutical formulations may be configured for parenteral administration, such as parenteral injection, more preferably for intraosseous, peri-bone, intra-articular, or peri-articular administration, such as intravenous injection. -osseous, perosseous, intra-articular, or peri-articular, or for intra-tendinous, peritendinous, intra-ligamentary, or peri-ligamentary administration, such as intra-tendinous, peritendinous, intra-tendinous, ligamentous or peri-ligamentous.
Pharmaceutical formulations or kit components as herein described may be configured for local administration. The present pharmaceutical formulations or component kits may be configured for parenteral administration, i.e., including one or more of intraosseous, peri-bone, intra-articular, peri-articular, intramuscular, subunit administration. -cutaneous, intravenous, intrastemal, intra-tendinous, peritendinous, intra-ligamentous or peri-ligamentous, for example comprising one or more intra-osseous, peri-bone, intra-articular, peri-articular, intramuscular administrations , subcutaneous, intravenous, and intrastemal.
Preferably, the pharmaceutical formulations or kit of components as presently described are configured for intraosseous or peri-bone administration. Intraosseous delivery or delivery generally refers to a process in which treatment is administered, directly or indirectly, into the bone (trabecular or cortical). Peri-bone delivery or delivery generally refers to a process in which treatment is administered around a bone (especially around the site of fracture / injury).
It is particularly preferred that pharmaceutical formulations or kit components as herein described be configured for intra-articular or peri-articular administration. Intra-articular delivery or administration generally refers to a method in which treatment is administered, directly or indirectly, in the synovial capsule of a joint. Periarticular delivery or administration generally refers to a method in which treatment is administered around the synovial capsule of a joint and / or subchondral bone.
In addition, pharmaceutical formulations or kit components as herein described may be configured for intratendinous or peritendinous administration. In addition, pharmaceutical formulations or kit components as presently described may be configured for intra-ligament or peri-ligament administration.
An associated aspect relates to the pharmaceutical formulation as described above for use in treatment (including all therapeutic and / or preventive measures of the present specification) of musculoskeletal disease.
The term "musculoskeletal disease" in the present context refers to any type of bone disease, muscle disease, joint disease, or chondrodystrophy, the treatment of which may benefit from the administration of the present pharmaceutical formulation to a subject having disease. The term also covers diseases affecting the tendons and / or ligaments). In particular, such a disease can be characterized, for example, by reduced formation of bone and / or cartilage or excessive resorption of bone and / or cartilage, by a decrease in the number, viability or function of osteoblasts or osteocytes present in bone and / or chondroblasts or chondrocytes present in the cartilage, a decrease in bone mass and / or mass of cartilage in a subject, bone thinning, impaired strength or elasticity of bones, etc.
Non-limiting examples of musculoskeletal diseases may include local or systemic disorders, such as, any type of osteoporosis or osteopenia, for example, primary, postmenopausal, senile, corticosteroid-induced, bisphosphonate-induced, and induced by radiotherapy; any secondary osteonecrosis, mono- or multisite; any type of fracture, for example, unconsolidated, poorly consolidated fractures, with delayed consolidation or compression, conditions requiring bone fusion (for example, vertebral fusions and reconstruction), maxillofacial fractures, congenital bone defect, bone reconstruction, for example, after traumatic injury or surgery of cancer, and craniofacial bone reconstruction; traumatic arthritis, cartilaginous and / or focal articular defect, focal degenerative arthritis; osteoarthritis, degenerative arthritis, gonarthrosis, and hip osteoarthritis; osteogenesis imperfecta; osteolytic bone cancer; Paget's disease, endocrine disorders, hypophosphatemia, hypocalcemia, renal osteodystrophy, osteomalacia, adynamic bone disease, hyperparathyroidism, primary hyperparathyroidism, secondary hyperparathyroidism; periodontal disease; Gorham-Stout's disease and McCune-Albright syndrome; rheumatoid arthritis; spondyloarthropathies, including ankylosing spondylitis, psoriatic arthritis, enteropathic arthropathy, and undifferentiated spondyloarthritis and reactive arthritis; systemic lupus erythematosus and related syndromes; scleroderma and related disorders; Sjögren's syndrome; systemic vasculitis, including giant cell arteritis (Horion's disease), Takayasu arteritis, rheumatic polymyalgia, ANCA-associated vasculitis (such as Wegener's granulomatosis, microscopic polyangiitis, and Churg- Strauss), Behcet's syndrome, and other polyarteritis and related disorders (such as polyarteritis nodosa, Cogan syndrome, and Buerger's disease); arthritis accompanying other systemic inflammatory diseases, including amyloidosis and sarcoidosis; crystalline arthropathies, including gout, calcium pyrophosphate dihydrate disease, disorders or syndromes associated with joint deposition of calcium phosphate or calcium oxalate crystals; chondrocalcinosis and neuropathic arthropathy; Felty's syndrome and Reiter's syndrome; Lyme disease and rheumatic fever.
The term "prophylactically effective amount" refers to an amount of an active compound or pharmaceutical active substance that inhibits or delays the onset of a disorder in a subject as desired by a researcher, veterinarian, physician or other clinician . The term "therapeutically effective amount" as used herein refers to an amount of active compound or pharmaceutical agent that induces the biological or medicinal response in a subject that is desired by a researcher, veterinarian, physician, or other clinician which may include, among others, the alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the present pharmaceutical formulations or formulations.
In the context of the present invention, a "therapeutically effective dose" refers to an amount of a pharmaceutical active substance or formulation that, when administered, induces a positive therapeutic response with respect to the treatment of a patient with musculoskeletal disease such as bone disease or joint disease (alternatively or additionally, said disease may affect tendons and / or ligaments).
Suitable therapeutically effective doses of a pharmaceutical active compound or a pharmaceutical active substance in the present formulation may be determined by a qualified physician taking into account the nature of the pharmaceutical active compound or the pharmaceutical active substance, the condition and the severity of the disease, and the age, size and health status of the patient.
Without limitation, a typical dose, for example, of the glycosaminoglycan to be administered may be in the range of about 2 mg to 400 mg of glycosaminoglycan per injection. For example, the dose to be administered may be in the range of about 4 mg to 300 mg of the glycosaminoglycan by injection, for example, from about 8 mg to 200 mg of the glycosaminoglycan per injection. Preferably, the dose to be administered is in the range of about 8 mg to 160 mg of glycosaminoglycan per injection.
Without limitation, a typical dose of, for example, the cell composition to be administered, may be in the range of about 0.05x106 cells to 5x109 cells per injection. For example, the dose to be administered may be in the range of about 0.5 x 10 6 cells to 1 x 10 9 cells per injection. Preferably, the dose to be administered is in the range of about 4x106 cells to 250x106 cells per injection.
It is recognized that the treatments of the invention may comprise administering a single therapeutically effective dose or administering multiple therapeutically effective doses of pharmaceutical formulations or formulations.
Unless otherwise indicated, "subject" or "patient" is used interchangeably and refers to animals, preferably warm-blooded animals, more preferably vertebrates, still more preferably mammals, still more preferably primates, and specifically include human patients and mammals and non-human primates. Preferred patients are human subjects.
In the present context, a phrase such as "a subject in need of treatment" includes subjects who would benefit from the treatment of a given condition, particularly a musculoskeletal disease such as bone disease or joint disease (in particular variant or in addition, said disease may affect the tendons and / or ligaments). Such subjects may include, without limitation, those who have been diagnosed with said condition, those susceptible to develop said condition, and / or those in whom said condition is to be prevented.
The terms "treat" or "treatment" cover both the therapeutic treatment of a disease or condition already developed, as well as prophylactic or preventive measures, the objective being to prevent or reduce the risk of incidence of a condition. undesirable, for example to prevent the risk of progression of the disease or condition. Beneficial or desired clinical outcomes may include, without limitation, the attenuation of one or more symptoms or one or more biological markers, the decrease in the degree of the disease, a stabilized state (i.e., without aggravation ) of the disease, delaying or slowing the progression of the disease, ameliorating or palliating the disease state, and the like. "Treatment" can also mean the extension of survival to expected survival without receiving treatment.
The present pharmaceutical formulations may additionally include components that are particularly specified herein by one or more pharmaceutically acceptable excipients. Suitable pharmaceutical excipients depend on the pharmaceutical form and the identity of the active substances and may be selected by those skilled in the art (e.g., with reference to Handbook of Pharmaceutical Excipients 6th Edition 2009, Rowe et al. In the present context, "vehicle" or "excipient" includes any and all solvents, diluents, buffers (such as, for example, neutral buffered saline or phosphate buffered saline), solubilizers, colloids, media. dispersants, vehicles, fillers, chelating agents (such as, for example, EDTA or glutathione), amino acids (such as, for example, glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, flavorings, thickeners, deposition agents, coatings, antifungal agents, preservatives, stabilizers, antioxidants, tonicity controlling agents, agents delaying absorption, and the like. The use of such media and agents for pharmaceutical active ingredients is known in the art. Such materials must be non-toxic and must not interfere with the activity of the pharmaceutically active substances.
The precise nature of the vehicle or other material depends on the route of administration. For example, the formulation may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has a suitable pH, isotonicity and stability.
The formulations may comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, preservatives, complexing agents, tonicity adjusting agents, wetting agents and the like. for example, sodium acetate, sodium lactate, sodium phosphate, sodium hydroxide, hydrogen chloride, benzyl alcohol, parabens, EDTA, sodium oleate sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. Preferably, the pH value of the formulation is in the physiological pH range, for example, in particular, the pH of the formulation is between about 5 and about 9.5, more preferably between about 6 and about 8.5 more preferably between about 7 and about 7.5. The preparation of such pharmaceutical formulations is within the scope of those skilled in the art.
Another aspect relates to the use of the aforementioned formulations as a pharmaceutical excipient, more preferably as a sustained-release or slow release pharmaceutical excipient.
The terms "sustained release", "slow release" or "sustained release", in this context, generally refer to the release of a compound from a formulation over an extended, extended or prolonged period of time relative to the release of said formulation. compound since a reference formulation such as a formulation known in the prior art. As used herein, sustained release refers to the sustained release of one or more of the formulation components, i.e., S / D plasma, which may include beneficial biological substances such as endogenous growth factors, and a glycosaminoglycan, the cell composition, or one or more active pharmaceutical compounds, and optionally one or more additional active pharmaceutical component (s). For example, it is known in the prior art that the half-life of hyaluronic acid of high molecular weight in the joint is about 6 to 8 hours. The sustained release, in the present context, therefore refers to the sustained release of a glycosaminoglycan such as hyaluronic acid from the present formulations, for example release for one or more days, for example for 2 days, 3 days, 4 days, 5 days, 6 days, or during one or more weeks, for example for 1.5 weeks, 2 weeks, 3 weeks, or for one or more months. Therefore, these terms may further specifically cover extended release, delayed release, or controlled release.
Examples
Example 1: Comparison Between Solvent-Detergent-treated Human Plasma (S / D Plasma) and Fresh Human Frozen Plasma (FFP)
Composition characteristics of exemplary formulations
The Octapharma AG (Octapharma, Switzerland) octaplas® product monograph (Lachen, Switzerland) presents a comparison between the S / D plasma composition characteristics and the fresh frozen plasma (FFP) human plasma, as described in US Pat. According to this information, the products have to a large extent comparable plasma protein compositions; S / D plasma may have a significantly lower level of plasmin inhibitor.
Table 1: S / D plasma composition characteristics, in particular Octaplas®, and fresh frozen plasma (FFP) based on the Octaplas® product monograph
Examination of the S / D plasma composition, in particular Octaplas®, and fresh frozen plasma, Svae et al. 2007 concluded that there are no critical reductions in the activities of naturally occurring clotting factors and inhibitors caused by the manufacturing process, including S / D treatment, while protein S and plasmin inhibitors may show a decrease of 35% and 76%, respectively (Beeck and Hellstem 1998). Doyle et al. 2003 report significantly lower levels of protein S (38%), plasmin inhibitor (78%), as well as factor XI (13%) and factor V (13%), and significantly increased levels of factor VII (15%) in S / D plasma by comparing 16 lots of Octaplas® to 48 units of unpaired FFP.
Reference is made to Table 1 of Svae et al. 2007 showing the compositional characteristics of 12 consecutive lots of Octaplas® vs. 12 random units of FFP quarantined. Although most of the components are not significantly modified, Table 2 below reproduces the components of Table 1 of Svae et al. 2007 with significant differences (P value <0.05, unpaired t-test).
Table 2: Composition differences between S / D plasma, namely Octaplas®, and FFP, based on Table 1 of Svae et al. 2007
In addition, Theusinger et al. 2011 (Br. J. Anaesth., 106 (4): 505-11) investigated the relative concentrations of hemostatic factors and cytokines in S / D plasma, namely Octaplas®, and fresh frozen plasma (n = 25). ). They observed that the coagulation factor content is similar for plasma S / D and FFP, but the S / D plasma contains less factor V, von
Willebrand (vWF), and von Willebrand factor cleavage protease (vWFCP, ADAMTS-13). Cytokine concentrations (TNFa, IL-8, and IL-10) are significantly higher in FFP.
In addition, the S / D process may lead to losses in the activities of labile factor V, factor VIII, antitrypsin serine protease inhibitor (serpin) and a2-antiplasmin, but not antithrombin (Benjamin and McLaughtin 2012, Mast et al 1999). Factor VIII loss is associated with a decrease in the high molecular weight von Willebrand factor (vWF). In a study by Sachs et al. 2005 S / D plasma samples (n = 5) tested negative for specific antibodies for granulocytes as well as HLA class I and class IL
Table 3 provides an overview of demonstrable distinctions between S / D plasma, namely Octaplas®, and fresh frozen plasma, as detected in the studies mentioned above.
Table 3: Exemplary Differences Between S / D Plasma, Octaplas®, and Fresh Frozen Plasma
In conclusion, many compositional differences have been documented between human S / D plasma and fresh frozen plasma. A particularly marked characteristic of S / D plasma may be a reduced level of plasminide inhibitor. FFP, such that a level of plasmin inhibitor lower than or equal to 0.60 IU / ml or less than or equal to 0.50 IU / ml, for example a level of plasmin inhibitor between 0.20 and 0 , 30 IU / ml, more specifically between 0.22 and 0.25 IU / ml.
Coagulation behavior
The following tests use the following types of human plasma: S / D plasma, namely Octaplas®, plasma treated in a heparinized tube, plasma treated in a tube containing EDTA, or plasma treated in a citrated tube. Plasma is prepared as follows: Blood is collected directly into corresponding tubes, i.e., heparinized tubes, containing EDTA, or citrated and the tubes are centrifuged at 1500 g for 10 minutes at room temperature. ambient to collect the plasma in the form of a supernatant. Since the S / D plasma is decellularized by filtration, the other types of plasma are, as indicated, filtered on a 0.2 μm filter to ensure the absence of cells, such as platelets, or debris. The volume of the reaction mixture is 250 μΐ or 500 μΐ. Each component and the mixture are maintained at 37 ° C. The coagulation time is determined at 37 ° C for a mixture containing the respective plasma type, 10 or 20% v / v serum, and 2.5 or 5% v / v CaCl 2 (0.546 M). The coagulation time is measured by visual observation, possibly during or after the agitation of the tube. Five samples are tested. The results are summarized in Table 4. There is no distinction in the results obtained between 2.5% and 5% of CaCl2 and between 10 or 20% of serum.
Table 4: S / D plasma coagulation behavior, ie Octaplas®, compared with other types of plasma
Clot formation is not observed with heparinized, unfiltered or filtered (decellularized) plasma. S / D plasma filtration produced no significant difference in coagulation time (p = 0.7780, paired t-test) while a significant increase was observed for EDTA plasma (p = 0.0053, paired t-test) and for citrated plasma (p = 0.0143, paired t-test). However, clot formation with S / D plasma is about 50% to over 100% faster compared to citrated plasma and EDTA plasma, respectively. Therefore, S / D plasma, exemplified by Octaplas®, differs from other types of plasma in terms of coagulation properties.
A marked characteristic of the S / D plasma, in particular the decellularized S / D plasma, may therefore be a reduced clotting time. For example, the coagulation time may be less than or equal to 700 s, or less than or equal to 600 s, for example between 500 and 700 s, or between 550 and 600 s, when measured at 37 ° C in a mixture comprising decellularized human plasma S / D, 10 or 20% v / v serum and 2.5 or 5% w / v CaCl 2 (0.546 M).
Example 2: gel formation by human plasma treated with solvent / detergent (S / D) (Octaplas®) and hyaluronic acid (HA)
Clot / gel formation by Octaplas® and HA
The behavior of human S / D plasma, in particular Octaplas®, is tested in combination with synovial fluid of arthritic patients (n = 2). The synovial fluid is contacted at a ratio of 1: 1 v / v with a formulation according to an embodiment of the present invention comprising Octaplas®, hyaluronic acid (10 mg / ml sodium hyaluronate, molecular weight of 1.8 to 2.106 Da, supplied by Contipro, Czech Republic) and CaCl 2. Three concentrations of CaCl 2, namely 0, 2 and 4 mg / ml CaCl 2, are tested. Gel formation is evaluated visually after different times (20 to 30 min, 1 h, 2 h) using a timer. The viscosity is evaluated by measuring the time required for the solutions to reach a defined scale on the container wall when the reaction tube is returned. The test is performed twice on synovial fluid of two arthritic patients. Incubation of a formulation comprising 4 mg / ml of CaCl 2 with synovial fluid induces the formation of a gel which is more viscous compared to a gel obtained by contacting synovial fluid with a formulation comprising 2 mg / ml of CaCl2. After contacting a CaCl2-free formulation with synovial fluid, no clot formation is observed.
OctaplasLG AB® and HA clot / gel formation in the presence of synovial fluid
The behavior of human plasma S / D, in particular OctaplasLG AB®, is tested in combination with synovial fluid of patients with osteoarthritis (n = 5). The synovial fluid of these patients is mixed at a ratio of 1: 2 (v / v) with a formulation according to an embodiment of the present invention comprising OctaplasLG AB®, HA (10 mg / ml sodium hyaluronate, molecular weight of 2 to 3.106 Da, supplied by HTL Biotechnology, France), clonidine hydrochloride (HCl) (200 μg / ml, purchased from PC AS Laboratory, Finland) and CaCl (4 mg / ml Calciclo®, supplied by Sterop Group, Belgium) to mimic clinical conditions. Clot / gel formation is visually assessed after 30 minutes at 37 ° C (humidified incubator, 5% CO2).
Once in contact with synovial fluid, this formulation forms a clot within 30 minutes.
In another assay, three concentrations of CaCl 2 (i.e., 0, 2 and 4 mg / ml CaCl 2) are tested in the above-mentioned formulation. The test is performed twice on synovial fluid of patients with osteoarthritis (n = 2). Incubation of a formulation comprising 4 mg / ml or 2 mg / ml of CaCl 2 with synovial fluid induces the formation of a gel of the same viscosity. However, after mixing a CaCl2-free formulation with synovial fluid, no clot formation is observed. Therefore, the dose of 2 mg / ml CaCl2 (14 mM) is chosen in the final formulation.
In conclusion, in this test, Ca2 + is required for clot formation in the presence of synovial fluid. It may be necessary to add Ca 2+ to a formulation illustrating the present invention, particularly when it is observed or expected to be present at sufficient levels in the joint tissues.
Clot / gel formation by OctaplasLG AB® and HA in the presence of whole blood The gelling effect of OctaplasLG AB® with HA in combination with whole blood of a healthy donor (n = 1) is studied in order to evaluate its potential to form a matrix comprising platelet-derived growth factors. In this study, the behavior of a formulation comprising OctaplasLG AB®, HA (4 mg / ml of sodium hyaluronate, molecular weight of 2 to 3.106 Da, supplied by HTL Biotechnology, France) and CaCl2 (2 mg / ml of Calciclo ®, supplied by Sterop Group, Belgium) is tested in combination with whole blood collected in citrated tubes (No. VF054SBCS07, Venosafe, Terumo). The coagulation time is measured by visual observation after stirring of the tube / solution homogenization.
The results show that once in contact with citrated blood at a ratio of 1: 1 (v / v), this formulation forms a clot in 15 minutes at 37 ° C (humidified incubator, 5% CO2).
Clot / gel formation by Octaplas® and HA compared to other types of plasma
The consistency of several formulations is tested. The formulations contain hyaluronic acid (sodium hyaluronate, molecular weight of 2 to 3.106 Da, supplied by HTL, France), CaCL and Octaplas® or the same volume of the following human plasma types (unfiltered): plasma treated in a heparinized tube, plasma treated in a tube containing EDTA, or plasma treated in a citrated tube (see Table 5 for the composition of the formulations).
Table 5: Composition of the formulations
Human serum prepared from peripheral blood at a ratio of 1: 1 v / v is added to the product and incubated at 37 ° C with shaking. The consistency is observed visually after 30 minutes, 1 hour and 24 hours.
Table 6 presents a semi-quantitative assessment of gelation with different types of plasma. After 1 hour, the formulation containing Octaplas® has a viscous consistency, even a gelled consistency. The formulation containing heparinized plasma remains liquid after 1 hour (Table 6). The formulation containing Octaplas® is more viscous than formulations containing plasma treated in a tube containing EDTA or plasma treated in a citrated tube (Table 6). As a result, the formulation containing Octaplas® is more viscous than formulations containing the other types of plasma.
Table 6: Gelation with Octaplas® vs. other types of plasma (unfiltered) in the presence of HA
* Octaplas® is filtered plasma, that is, plasma without constituents / cellular components. ** Results obtained after agitation
Clot / gel formation by Octaplas® in the presence or absence of HA
The consistency is compared between formulations containing Octaplas®, CaCl2 and serum, with or without hyaluronic acid (sodium hyaluronate, molecular weight of 2 to 3.106 Da, supplied by HTL, France). A formulation without hyaluronic acid is prepared at 37 ° C by mixing the following components: 2 ml of Octaplas®, 222 μl of serum, and 22 μl of 30 mg / ml of stock solution of CaCl 2 (prepared from CaCl 2 salt). Sigma in water). A formulation with hyaluronic acid is prepared at 37 ° C. by mixing the following components: 8 mg of HA previously dissolved in 2 ml of Octaplas®, 222 μl of serum, and 22 μl of 30 mg / ml stock solution CaCl2 (prepared from Sigma salt of CaCl2 in water). The coagulation time is measured by visual observation and shaking of the tube. The test is performed once.
After visual inspection, a clot or gel phase formed in both formulations. The formulation with HA, however, contains a significantly lower amount of liquid phase and can be handled without change. On the contrary, the clot without HA ruptures and releases fluid after manipulation of the clot.
S / D and HA Plasma Cell Administration Formulation
An exemplary, non-limiting formulation suitable for intraosseous administration of cells to a mammalian subject comprises, consists essentially of, or consists of S / D plasma, in particular Octaplas®, 20 mg / ml HA, a composition cells comprising autologous or allogeneic mesenchymal stem cells (MSC) isolated from human bone marrow or a composition of cells comprising osteoblastic cells, and 20 mg / ml CaCl 2. The formulation is administered to a bone defect site in the patient.
Example 3: Gel Formation with Human S / D Plasma such as Octaplas® and a Cell Composition
Clot / gel formation by Octaplas® and Ca2 + -containing cell culture medium
Clot formation is tested for several cell-free formulations but including human S / D plasma, in particular Octaplas®, a conventional culture medium (which contains CaCl 2) and human serum. Different conditions are tested to verify clot formation at 37 ° C such as Octaplas® at different concentrations, for example, 5%, v / v 7.5% v / v, or 10% v / v; various conventional culture media, for example, DMEM, MEM, PBS, or PBS plus CaCl2; and the absence or presence of 5 or 10% v / v serum. Clot formation is visually observed when Octaplas®, a conventional culture medium (which contains CaCl2) and serum are combined in the absence of cells.
It can be concluded that the presence of serum as well as the presence of calcium in the culture medium seems necessary for clot formation in the absence of cells.
Clot / gel formation by Octaplas® and a cell composition
Bone marrow MSCs are deposited at 57000 cells / cm 2 in plastic vials in a conventional culture medium (DMEM) containing 5% v / v, 10% v / v, or 15% v / v plasma S / D. , in particular Octaplas®. The flasks are directly placed at 37 ° C in a 5% CO2 incubator. Gelation of the medium is observed in less than one hour at 37 ° C. The cell medium containing human plasma S / D, in particular Octaplas®, changes consistency from liquid to gel (gel) in 30 minutes after contact.
The above data demonstrate the ability of a formulation comprising S / D plasma and cells in Ca2 + -containing cell-containing culture medium to be gelated as compared to a cell culture in Ca2 + -containing cell culture medium. serum, i.e., under conventional cell culture conditions, which are generally known to remain liquid.
Clot formation / gel by Octaplas®, other types of plasma, and cell composition
The consistency of a cell culture composition with different types of human plasma -Octaplas®, plasma treated in a heparinized tube, plasma treated in a tube containing EDTA, or plasma treated in a citrated tube - is evaluated. 57000 cells / cm 2 of osteoprogenitor cells cultured from bone marrow cells are seeded in a T25 plastic vial in 6 ml of culture medium (85% v / v) supplemented with 15% v / v of the respective 37 ° plasma. vs. The results are summarized in Table 7. The medium remains liquid with heparinized plasma (Table 7). No difference is observed in this initial trial between Octaplas®, plasma-EDTA and citrated plasma (Table 7).
Table 7: Gelation / coagulation time for the different types of plasma tested in the presence of cells
* Octaplas® is filtered plasma, that is, plasma without constituents / cellular components. ** Three cell cultures are tested for each type of plasma
S / D Plasma Cell Administration Formulation
An exemplary, nonlimiting formulation suitable for intraosseous administration of cells to a mammalian subject comprises, consists essentially of, or consists of S / D plasma, in particular Octaplas®, a cell composition comprising stem cells autologous or allogeneic mesenchymal mesenchymal (MSC) isolated from bone marrow or a cell composition comprising osteoblastic cells, and 2 to 8 mg / ml CaCl 2. The formulation is administered to a bone defect site in a cranial skull osteotomy model in mice.
EXAMPLE 4 Gel Formation with Human S / D Plasma such as Octaplas®
An exemplary non-limiting pharmaceutical formulation comprising S / D plasma and a pharmaceutical active substance, the formulation being adapted for therapeutic use in bone defects, essentially consists of or consists of S / D plasma, in particular Octaplas®, growth factor (IL-8 at 30 μg / ml) and 2 to 8 mg / ml CaCl 2. The formulation is administered to a bone defect site in a cranial skull osteotomy model in nude mice. 4 weeks after the administration of the formulation, bone repair can be clearly observed in treated mice compared to control without IL-8 (see Figures 1 and 2).
List of quotes
Non-patent literature
Beeck H, Hellstem P. In vitro characterization of solvent / detergent-treated human plasma and of quarantine fresh frozen plasma. Vox Sang. 1998; 74 Suppl 1: 219-23.
Benjamin RJ, McLaughlin LS. Plasma components: properties, differences, and uses. Transfusion. May 2012; 52 Suppl. L9S-19S.
Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989; 58: 575-606.
Cole, B.J., Seroyer, S.T., Filardo, G., Bajaj, S., Fortier, L.A., 2010. Platelet-Rich Plasma. Sports Health 2, 203-210.
Doyle S, O'Brien P, Murphy K, Fleming C, O'Donnell J. Coagulation factor content of solvent / detergent plasma compared with fresh frozen plasma. Blood Coagul Fibrinolysis. April 2003; 14 (3): 283-7.
Frescaline G, Bouderlique T, Huynh MB, Papy-Garcia D, Courty J, Albanese P. Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells. Stem Cell Res. March 2012; 8 (2): 180-92.
Gobbi, A., Bathan, L., 2009. Biological Approaches for Cartilage Repair. J Knee Surg 22, 36-44.
Grimaud, E., Heymann, D., Rédini, F., 2002. Recent advances in TGF-beta effects on chondrocyte metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders. Cytokine Growth Factor Rev. 13, 241-257.
Hausser HJ, Brenner RE. Low doses and high doses of heparin have different effects on osteoblastlike Saos-2 cells in vitro. J Cell Biochem. April 1, 2004; 91 (5): 1062-1073.
Hellstem P, Solheim BG. The Use of Solvent / Detergent Treatment in Pathogen Reduction of Plasma. Transfus Med Hemother. 2011; 38 (1): 65-70.
Horowitz B, Bonomo R, Prince AM, SN Chin, Brotman B, Shulman RW. Solvent / detergent-treated plasma: a virus-inactivated substitute for fresh frozen plasma. Blood. Feb. 1, 1992; 79 (3): 826-31.
Lyon M, Rushton G, Gallagher JT. The interaction of the transforming growth factor-beta with heparin / heparan sulfate is isoform-specific. J Biol Chem. July 18, 1997; 272 (29): 18000-6.
AE Mast, Stadanlick JE, Lockett JM, Dietzen DJ. Solvent / detergent-treated plasma has decreased antitrypsin activity and absent antiplasmin activity. Blood. 1st Dec 1999; 94 (11): 3922-7.
McCaffrey TA, Falcone DJ, Brayton CF, LA Agarwal, Welt FG, Weksler BB. Transforming growth factor-beta activity is potentiated by heparin via dissociation of transforming growth factor-beta / alpha 2-macroglobulin inactive complex. J Cell Biol. July 1989; 109 (1): 441-8.
SH Park, Cui JH, SR Park, Min BH. Potential of fortified fibrin / hyaluronic acid composite gel as a cell delivery vehicle for chondrocytes. ArtifiOrgans. June 2009; 33 (6): 439-47.
Sachs UJ, Kauschat D, Bein G. White blood cell-reactive antibodies are undetectable in solvent / detergent plasma. Transfusion. Oct 2005; 45 (10): 1628-1631.
Svae TE, Frenzel W, Heger A, Romisch J. Quality differences between solvent / detergent plasmas and fresh-frozen plasma. Transfus Med. August 2007; 17 (4): 318-20.

权利要求:
Claims (15)
[1]
claims
A pharmaceutical formulation comprising solvent / detergent treated plasma (S / D plasma) and a glycosaminoglycan, wherein the pharmaceutical formulation comprises glycosaminoglycan at a concentration in the range of about 0.10 mg / ml to about 200 mg / ml ml.
[2]
The pharmaceutical formulation of claim 1, wherein the S / D plasma is human S / D plasma.
[3]
A pharmaceutical formulation according to any one of claims 1 or 2, wherein the glycosaminoglycan is hyaluronic acid or a derivative thereof.
[4]
A pharmaceutical formulation according to any one of claims 1 to 3, further comprising one or more pharmaceutical active substances.
[5]
Pharmaceutical formulation according to any one of claims 1 to 4, further comprising serum, preferably human serum.
[6]
The pharmaceutical formulation of any one of claims 1 to 4, further comprising whole blood or a fractionated whole blood component, the whole blood preferably being human whole blood.
[7]
The pharmaceutical formulation according to any one of claims 4 to 6, wherein one or more pharmaceutical active substances are each independently selected from the group consisting of: a cell composition, a pharmaceutical active compound, a protein, a peptide , and a small organic molecule.
[8]
The pharmaceutical formulation of claim 7, wherein the cell composition comprises mesenchymal stem cells (MSCs), osteoprogenitor, osteoblast cells, osteocytes, chondroblastic cells, and / or chondrocytes.
[9]
The pharmaceutical formulation according to claim 7, wherein the pharmaceutical active compound is an alpha-2-adrenergic receptor agonist, preferably the alpha-2-adrenergic receptor agonist is selected from the group consisting of clonidine and derivatives thereof. of it.
[10]
The pharmaceutical formulation of claim 7, wherein the active pharmaceutical protein or peptide is a growth factor, preferably a growth factor selected from the group consisting of fibroblast growth factor (FGF), a transforming growth beta (TGFB), platelet-derived growth factor (PDGF), interleukin-8 (IL-8), bone morphogenetic protein (BMP), parathyroid hormone (PTH), a protein related to parathyroid hormone (PTHrp), and stem cell factor (SCF); more preferably a growth factor selected from the group consisting of FGF-2, TGFB-1, PDGF, IL-8, BMP-2, BMP-4, BMP-6, BMP-7, PTH, PTHrp, and SCF.
[11]
A pharmaceutical formulation according to any one of claims 1 to 10, which is configured for parenteral administration, preferably for intraosseous, peri-bone, intra-articular, or peri-articular administration, or for intra-tendinous administration, peri-tendinous, intra-ligamentary or peri-ligamentous.
[12]
A pharmaceutical formulation according to any one of claims 1 to 11 for use in the treatment of musculoskeletal disease, wherein the musculoskeletal disease is preferably a bone disease or joint disease.
[13]
13. Use of a formulation comprising S / D plasma and a glycosaminoglycan as a pharmaceutical excipient.
[14]
The use of claim 13, wherein the S / D plasma is human S / D plasma.
[15]
The use according to any one of claims 13 or 14, wherein the pharmaceutical formulation is configured for parenteral administration, preferably for intraosseous, peri-bone, intra-articular, or peri-articular administration, or for intravenous administration. -tendinous, peritendinous, intra-ligamentous or peri-ligamentous.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

US4764369A|1983-07-14|1988-08-16|New York Blood Center Inc.|Undenatured virus-free biologically active protein derivatives|

---

EP0322786A2|1987-12-30|1989-07-05|New York Blood Center, Inc.|Extraction of process chemicals from labile biological mixtures with halogenated hydrocarbons|

---

US20020018777A1|1995-01-05|2002-02-14|Alfred M. Prince|Sterilization of virus infected plasma|

---

WO2009016451A2|2007-08-02|2009-02-05|Biotechnology Institute, I Mas D, S.L.|Method and compound for the treatment of articular diseases or articular pain, or for the treatment of skin for aesthetic or other purposes, and the method of preparation of the compound|

---

US20120171169A1|2010-12-31|2012-07-05|Jaewoo Pak|Compositions and methods for treating, preventing, or alleviating bone or cartilage diseases|

---

US5486359A|1990-11-16|1996-01-23|Osiris Therapeutics, Inc.|Human mesenchymal stem cells|

---

US5837539A|1990-11-16|1998-11-17|Osiris Therapeutics, Inc.|Monoclonal antibodies for human mesenchymal stem cells|

---

US5811094A|1990-11-16|1998-09-22|Osiris Therapeutics, Inc.|Connective tissue regeneration using human mesenchymal stem cell preparations|

---

US5972703A|1994-08-12|1999-10-26|The Regents Of The University Of Michigan|Bone precursor cells: compositions and methods|

---

US5736396A|1995-01-24|1998-04-07|Case Western Reserve University|Lineage-directed induction of human mesenchymal stem cell differentiation|

---

US5827740A|1996-07-30|1998-10-27|Osiris Therapeutics, Inc.|Adipogenic differentiation of human mesenchymal stem cells|

---

WO2000007639A1|1998-08-07|2000-02-17|Tissue Engineering, Inc.|Bone precursor compositions|

---

WO2001027623A2|1999-10-08|2001-04-19|V.I. Technologies, Inc.|Isoagglutinin-depleted blood compositions and methods of making same|

---

PT1294414E|2000-06-29|2006-07-31|Biosyntech Canada Inc|COMPOSITION AND METHOD FOR THE REPAIR AND REGENERATION OF CARTILAGE AND OTHER FABRICS|

---

IL144446D0|2001-07-19|2002-05-23|Prochon Biotech Ltd|Plasma protein matrices and methods for their preparation|

---

MXPA01011542A|2001-11-13|2003-05-22|Alcon Inc|Regeneration of articular cartilage damaged by osteoarthritis i and ii, by means of intra-articular application of sodium hyaluronate and chondroitin sulphate in a gel carrier.|

---

BRPI0511349A|2004-06-29|2007-12-04|Reliance Life Sciences Pvt Ltd|process to improve virus-proof biological fluids|

---

ITPD20050056A1|2005-03-02|2006-09-03|Fidia Farmaceutici|AMIDID DERIVATIVES OF HYALURONIC ACID IN OSTEOARTROSI|

---

US20090264477A1|2008-04-18|2009-10-22|Warsaw Orthopedic, Inc., An Indiana Corporation|Beta adrenergic receptor agonists for treatment of pain and/or inflammation|

---

JP5737860B2|2010-04-30|2015-06-17|テルモ株式会社|Gel-like cell composition and method for producing the same|

---

IL210162D0|2010-12-21|2011-03-31|Omrix Biopharmaceuticals|Viral inactivated platelet extract, use and preparation thereof|

---

US20120171182A1|2010-12-31|2012-07-05|Jae Woo Pak|Methods and compositions for the treatment, prevention, and alleviation of bone and cartilage diseases or injuries and hair loss|

---

KR101038616B1|2011-03-16|2011-06-03|박재우|Pharmaceutical composition for use in the treatment, prevention, or allevation of bone and cartilage disease|SG11201708434PA|2015-04-23|2017-11-29|Bone Therapeutics Sa|In vitro preservation of therapeutic cells|

---

CA3021764C|2016-04-27|2021-01-19|Anika Therapeutics, Inc.|Compositions for use in treating tendon degeneration via intra-osteotendinous injection|

---

CN107789647B|2016-09-05|2021-02-02|上海赛伦生物技术股份有限公司|Method for inactivating virus in animal serum or plasma|

---

KR20220008289A|2019-05-13|2022-01-20|본 테라퓨틱스 소시에테아노님|Improved lyophilized formulation comprising hyaluronic acid and plasma protein and uses thereof|

---

AU2020311591A1|2019-07-08|2022-02-03|Theravet Sa|A method for the preparation of a gel-forming composition|

---

WO2021005071A1|2019-07-08|2021-01-14|Theravet Sa|A composition, a kit and use thereof|

---

CN114173793A|2019-07-08|2022-03-11|泰瑞威公司|Composition, kit and use thereof|

法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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EP12186027|2012-09-26|

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EP121860274|2012-09-26|

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