Peritoneal dialysis fluid compositions containing complement inhibitors - Patents.com

Abstract

Provided herein is a composition for use in peritoneal dialysis (PD), the composition comprising a biologically compatible solvent, an osmotic agent, and a complement inhibitor. Methods of making and using the composition are also provided.

Description

[Technical field]

[0001] The present disclosure relates to compositions for use in and methods of peritoneal dialysis. [Background technology]

[0002] Peritoneal dialysis (PD) is one of the fastest growing treatment options for patients with kidney failure, and is a better option than hemodialysis (HD), especially for patients who have residual kidney function and cannot tolerate the rapid changes in fluid balance that accompany hemodialysis. PD offers flexibility and reduces the number of visits to the dialysis center. Choosing PD at home offers benefits such as reduced medication and dietary restrictions. Hemodialysis (HD), which requires a minimum of three hours of dialysis three times a week, limits the patient's activity possibilities, but with PD, patients can maintain an active lifestyle, pursue normal education, and continue working. PD is a dialysis method that uses the abdominal lining (peritoneum) and dialysis fluid to filter blood when the kidneys fail. Patients can perform peritoneal dialysis at home without assistance. There are two methods of peritoneal dialysis. CAPD involves three to four fluid exchanges during the day and long periods of time at night. APD or automated peritoneal dialysis requires only one fluid exchange during the day, as a machine called a cycler performs the fluid exchange at night while the patient sleeps. A new generation of connected cyclers allows for 24 / 7 patient monitoring. These advantages have made PD popular with kidney patients and clinicians, which is expected to further boost the growth of the home care segment of the peritoneal dialysis market. Summary of the Invention [Problem to be solved by the invention]

[0003] PD accounts for approximately 10% of the world's dialysis population. PD is more cost-effective than hemodialysis, has comparable outcomes, and allows for home treatment. However, the conversion rate from PD to HD is high; only approximately 15% of patients remain on PD after 5 years.

[0004] A major concern limiting long-term treatment with PD is the loss of peritoneal membrane integrity and function due to repeated and prolonged exposure to dialysate. During prolonged PD, the peritoneal membrane undergoes structural changes, including fibrosis, angiogenesis, and vasculopathy. These morphological changes are paralleled by enhanced solute transport, early loss of the glucose-induced osmotic gradient, and ultimately loss of ultrafiltration capacity.

[0005] Thus, there is a need for improved PD treatments that extend the time that PD remains an effective therapy.

[0006] At least some aspects of the present disclosure aim to address at least one of the above needs. [Means for solving the problem]

[0007] According to a first aspect, there is provided a composition for use in peritoneal dialysis (PD), the composition comprising a biologically compatible solvent, an osmotic agent and a complement inhibitor.

[0008] The biologically compatible solvent may be water, and thus the composition may be an aqueous composition.

[0009] The osmotic agent may include a soluble carbohydrate. The soluble carbohydrate may be a monosaccharide, an oligosaccharide, or a polysaccharide. In some embodiments, the soluble carbohydrate may be a sugar or a sugar derivative.

[0010] The osmotic agent may comprise an amino acid. For example, the osmotic agent may comprise one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, L-carnitine, or other suitable non-natural amino acids and / or derivatives and / or mixtures and / or polymers thereof.

[0011] Thus, the osmotic agent may comprise a protein. For example, the osmotic agent can comprise one or more proteins, such as human serum albumin or gelatin.

[0012] The composition may include one or more stabilizers. The stabilizer or stabilizers may be, for example, sodium octanoate or N-acetyl-L-tryptophanate.

[0013] As used herein, the term "osmotic agent" refers to any soluble agent that increases the osmotic potential of a composition such that the composition is hypertonic or at least isotonic when held in the peritoneal cavity, thereby drawing toxins across the peritoneal membrane and into the composition.

[0014] A typical composition used in peritoneal dialysis (PD) is an aqueous solution that includes at least one osmotic agent. To use the composition in PD, the composition is transferred into the peritoneal cavity of a patient and left there for a sufficient time to draw toxins from the patient's blood across the peritoneal membrane into the composition. The composition is then removed from the peritoneal cavity, thereby removing toxins from the patient's body.

[0015] During long-term PD treatment, in which patients repeat PD treatments over a period of time (e.g., multiple times a day, daily, or weekly), the peritoneum undergoes structural changes, including progressive fibrosis, angiogenesis, and vasculopathy. Mesothelial cells, which form a monolayer covering the peritoneum, have properties such as a distinct cobblestone-like morphology and regulation of peritoneal permeability. These morphological changes that the cells undergo are paralleled by the promotion of solute transport, the early disappearance of the osmotic gradient induced by glucose (a commonly used osmotic agent), and ultimately the loss of ultrafiltration capacity.

[0016] The underlying mechanism is the process by which mesothelial cells change from endothelial to mesenchymal type, beginning with the acquisition of fibrogenic characteristics through the disruption of cell-cell junctions and loss of polarity. Furthermore, treatment of mesothelial cells with high glucose PD solution induces the expression of transforming growth factor-β. This ultimately results in increased expression of several markers corresponding to a mesenchymal phenotype, such as vimentin, N-cadherin, VEGF-A, α-SMA, and IL-17A, while the expression of endothelial markers such as E-cadherin is significantly reduced.

[0017] The biological origin of these structural changes is unclear. Recently, it has been suggested that activation of the complement system is at least partially involved in these peritoneal changes (Non-Patent Document 1: Borceux et al. Peritoneal Dialysis International 2020, Vol. 40(2) pgs. 115-123).

[0018] The complement system is a collection of 40-50 proteins present in the blood, whose main function is to provide a first line of defense against infection, but also plays an important role in the clearance of necrotic, apoptotic, damaged cells or immune complexes. If the complement system is inappropriately regulated, it can cause damage to host or autologous cells, but also to bacterial cells. Borceux et al. (see above) state that molecules belonging to the complement system, such as C3-C9, factor B, factor D and factor H, are present in the PD effluent (i.e., the PD fluid removed from the patient at the end of PD treatment). These data may indicate either clearance of these molecules from the circulation by uptake through the peritoneum from the patient's blood, or a local intraperitoneal production of these proteins, or a combination of both phenomena.

[0019] Moreover, Bartosova et al. recently demonstrated increased activation of the complement system and deposition of proteins from the classical and alternative pathways (e.g., C1q, C3d, C5b-9) in the peritoneal parietal and peritoneal arteries of children undergoing PD and treated with low-GDPs solutions compared to end-stage renal disease and healthy controls (Non-Patent Document 2: Bartosova et al. J Am Soc Nephrol 2018, Vol 29(1) pgs 268-282). Furthermore, complement activation induced by PD solutions is closely associated with the degree of vascular disease, as is TGF-β activation.

[0020] The inventors have found that the use of complement inhibitors in PD fluids, such as the compositions of the present embodiment, at least reduces or slows the rate at which the peritoneum undergoes at least one of the structural or functional changes described above, and thus patients undergoing PD treatment may be able to continue using PD treatment for a longer period of time before the peritoneum is compromised.

[0021] In embodiments where the osmotic agent is a soluble carbohydrate, the soluble carbohydrate may be selected from the group consisting of glucose, dextrose (L-glucose), fructose, galactose, maltose, xylitol, mannitol, sorbitol, maltodextrin, icodextrin, sucrose, hyaluronic acid, or derivatives or fragments or mixtures thereof. The soluble carbohydrate may be selected from the group consisting of glucose, dextrose (L-glucose), maltodextrin or icodextrin, or derivatives or fragments or mixtures thereof.

[0022] The term "derivative" refers to a biological molecule that has been modified chemically or genetically in a manner that does not significantly reduce its biological activity. A derivative of a biological molecule may enhance the biological activity of the biological molecule. A derivative may be a functional derivative or a biologically effective analogue of the parent biomolecule.

[0023] Complement inhibitors may inhibit complement activation, or may promote complement deactivation, or may inhibit the central amplification loop, or may inhibit downstream effector functions, for example, complement inhibitors may bind to components of the complement pathway and directly inhibit complement pathway activation, or may bind to components of the complement pathway and promote complement pathway deactivation.

[0024] The complement inhibitor may be a small molecule, a peptide, a macrocyclic peptide, a monoclonal antibody, another recombinant protein, a native protein, an oligonucleotide, a hexabody, an affibody, a minibody, a nanobody, a Fab, or an equivalent antibody derivative, a biologic, or an aptamer configured to bind to and inhibit any complement pathway component. The complement inhibitor may be a monoclonal antibody, another recombinant protein, or an aptamer configured to bind to and activate or enhance one of the natural complement regulators. The complement inhibitor may be unmodified or modified, for example, with polyethylene glycol, a proline-alanine / serine rich sequence, or a lipid.

[0025] The complement inhibitor may be an inhibitor of factor D, factor B, properdin, MASPs 1-3, C1, C3, C3a, C3b, C4b, C5, C5a, C5b, C5aR1, C6, or membrane attack complex (MAC). The complement inhibitor may be a direct inhibitor of factor D, factor B, properdin, MASPs 1-3, C1, C3, C3a, C3b, C4b, C5, C5a, C5b, C5aR1, C6, or MAC. The complement inhibitor may be an indirect inhibitor of factor D, factor B, properdin, MASPs-1-3, C1, C3, C3a, C3b, C4b, C5, C5a, C5b, C5aR1, C6, or MAC.

[0026] The complement inhibitor may be an activator or activity enhancer of Factor H, C4bp, CR1, DAF, or MCP.

[0027] The complement inhibitor may be C1 inhibitor, also known as C1 inhibitor, or may be C1 esterase inhibitor.

[0028] The complement inhibitor may inhibit MAC. The complement inhibitor may be the CD59 glycoprotein, also known as MAC-inhibitory protein (MAC-IP), membrane inhibitor of reactive lysis (MIRL), or protectin.

[0029] The complement inhibitor can promote the destruction of C3 convertase. For example, the complement inhibitor can be selected from DAF, Factor H, CR1, VCP, or SPICE.

[0030] Methods for evaluating the decay-accelerating activity include those described in Non-Patent Document 3 by Biggs et al. (Invest Ophthalmol Vis Sci. 2022 Nov; 63(12): 30, Published online 2022 Nov 29. doi: 10.1167 / iovs.63.12.30, PMID: 36445700, An Evaluation of the Complement-Regulating Activities of Human Complement Factor H (FH) Variants Associated With Age-Related Macular Degeneration) and Non-Patent Document 4 by Herbert et al. (J Immunol. 2015 Nov 15; 195(10): 4986-4998, Published online 2015 Oct 12. doi: 10.4049 / jimmunol.1501388, PMID: 26459349, Complement Evasion Mediated by Enhancement There is a method described in the paper entitled "Protection of Self-Surfaces from Complement."

[0031] Complement inhibitors include Cetor, Berinert, or Cinryze, also known as C1-INH or C1 esterase inhibitor, Sanquin / CSL Behring / Takeda Pharmaceuticals (inhibits CP / LP, other serine proteases), IFX-1, also known as CaCP29, InflaRx (inhibits binding of C5a to C5aR1), Mirococept, also known as APT070, King's College London MRC (inhibits CP and AP C3 / C5 convertases), TP10, also known as CDX-1135 or soluble complement receptor 1, Avant Immunotherapeutics (inhibits CP and AP C3 / C5 convertases), Inhibition of C3 / C5 convertase), eculizumab known as Soliris, Alexion (inhibition of C5 activation), AMY-101, Amyndas (inhibition of C3 activation), ravulizumab also known as ALX1210 or ultomiris, Alexion (inhibition of C5 activation (targets same epitope as eculizumab)), clovalimab known as SKY59 or RO7112689, Hoffmann-La Roche (inhibition of C5 activation (different C5 epitope)), tesidormab known as LFG316, Novartis (inhibition of C5 activation (different C5 epitope), poselimab also known as REGN3918, Regeneron (inhibition of C5 activation (different C5 epitope)), ABP959, Amgen (biosimilar of eculizumab), SB12, Samsung Bioepis (biosimilar of eculizumab), Nomacopan, also known as rVA576 or Coversin or OmCI, Akari Therapeutics (inhibition of C5 activation), Zilcoplan, also known as RA101495, RaPharmaceuticals (ALLosteric inhibition of C5 activation), semdisilane also known as ALN-CC5, Alnylam (inhibition of hepatic expression of C5), APL-2 also known as Pegcetacoplan, Apellis (inhibition of C3 activation), LNP023, Novartis (inhibition of AP C3 convertase), Danikopan also known as ACH-4471 or ACH-0144471, Achillion (inhibition of AP C3 convertase), stimulimab also known as BIV009 or TNT009, Sanofi (CP inhibition / inhibition of C1s protease), avacopan also known as CCX168, Chemocentrix (antagonist of C5aR1 receptor), narsoplimab also known as OMS721, Omeros (LP inhibition, inhibition of MASP2 activity), Zimra also known as avacincaptad pegol or ARG 1905, IVERIC Bio (inhibition of C5 expression), Lampalizumab, Roche (inhibition of AP C3 convertase formation), CLG561 also known as NOV-7, Novartis (Properdin), IONIS-FB-LRx, Roche (inhibition of hepatic FB expression), IPH5401, Innate Pharma (blockade of C5aR1 signaling), GEN1029, GenMab (enhancing CDC against DR5+ tumors), Ruconest, Pharming (C1r / s, MASPs, C1 esterase inhibitor), ACH-5228, Achillion (FD inhibitor), ACH-5448, Achillion (FD inhibitor), APL-9, Apellis (C3 inhibitor), AAVCAGsCD59 also known as HMR59, Hemera Biosciences (soluble CD59 expression), ANX005, Annexon (C1q), ANX007, Annexon (C1q), BIVV020, Bioverativ (C1s), OMS906, Omeros (MASP3), PRO-02, Broteio (C2), AMY-103, Amyndas (C3), 5C6 also known as Compsorbin, Amyndas (FH), anti-FH.07, Sanquin, (FH), AMY-201, also known as miniFH, Amyndas (Convertases), mutant mini FH (SEQ ID NO: 1)NO.9), SOBI005, Sobi(C5), ISU305, ISU ABXIS(C5), Mubodima, Adienne(C5), IFX-2, InflaRx(C5a), IFX-3, InflaRx(C5a), ALS-205, Alsonex(C5aR1), DF2593A, Dompe(C5aR1), Regenemab, Regenesance(C6), C6-LNA, Regenesance(C6) or PspC or a functional variant or fragment thereof.

[0032] The complement inhibitor prevents complement initiation and amplification and can be selected from the group consisting of C1-INH, stimulimab / BIV009 / TNT009, narsoplimab / OMS721, ruconest, ANX005, ANX007, BIVV020, PRO-02.

[0033] The complement inhibitors impair the effector functions of complement and include IFX-1 / CaCP29, eculizumab (Soliris), ravulizumab / ALX1210 / Ultomiris, clovalimab / SKY59 / RO7112689, tesidormab / LFG316, pozelimab / REGN3918, ABP959, SB12, nomacopan / rVA576 / Coversin / OmCI, zirucoplan / RA10 1495, semidisilane / ALN-CC5, zimra / avacincaptad pegol, lampalizumab, CLG561, IONIS-FB-LRx, IPH5401, GEN1029, AAVCAGsCD59 / HMR59, SOBI005, ISU305, Mubodima, IFX-2, IFX-3, ALS-205, DF2593A, Regenemab, or C6-LNA.

[0034] The complement inhibitor attenuates complement amplification and may be selected from the group consisting of Mirococept (APT070), TP10 / CDX-1135 (soluble complement receptor 1), AMY-101, APL-2, LNP023, Danikopan / ACH-4471 / ACH-0144471, stimulimab / BIV009 / TNT009, lampalizumab, CLG561, IONIS-FB-LRx, ACH-5228, ACH-5448, APL-9, BIVV020, OMS906, PRO-02, AMY-103, 5C6 / compsorbin, anti-FH.07, AMY-201 / miniFH, mutant miniFH (SEQ ID NO:9), DAF 1-4, or PspC or a functional mutant or fragment thereof.

[0035] The mutant miniFH is a mutant of miniFH and has the sequence of SEQ ID NO:9. JPEG2025507823000002.jpg62166

[0036] DAF 1-4 is a recombinant protein consisting of complement control protein modules 1-4 from human decay-accelerating factor having the sequence of SEQ ID NO:12. JPEG2025507823000003.jpg24166JPEG2025507823000004.jpg20166

[0037] Complement inhibitors can bind to and enhance the activity of natural human complement regulators, and thus the protein can act as a complement inhibitor by enhancing the activity of the patient's own natural complement regulators.

[0038] The complement inhibitor may be capable of binding to complement factor H. The complement inhibitor may be a protein capable of binding to CFH, thereby inducing increased affinity for C3d and C3b by bound CFH compared to unbound CFH. Thus, the protein may act as a complement inhibitor of the central amplification loop at the level of C3 convertase and as an inhibitor of the alternative complement pathway.

[0039] Complement inhibitors may be derived from microbial proteins whose function is to protect the microorganism from the complement system.

[0040] The complement inhibitor may be derived from a pneumococcal surface protein. The complement inhibitor may be derived from pneumococcal surface protein C (PspC) from Streptococcus pneumoniae. Various forms of PspC are known, including mutants from different strains of Streptococcus pneumoniae. The complement inhibitor may be derived from PspC (SEQ ID NO 4) of the D39 strain (NCTC No. 7466) of Streptococcus pneumoniae. The complement inhibitor may be derived from CbpA of the TIGR4 strain (NCTC No. 7465). The complement inhibitor may comprise a fragment of PspC. The fragment of PspC may consist of a portion of the N-terminal region of PspC. The effectiveness of PspCN as a complement inhibitor has been demonstrated in an international patent application by the University Court of the University of Edinburgh (Patent Document 1: WO 2015 / 055991), the disclosure of which is incorporated herein by reference.

[0041] The sequence of the complement inhibitor PspCN is: JPEG2025507823000005.jpg14166 or a functional variant or fragment thereof, which corresponds to amino acid residues 37 to 140 of PspC. The full length sequence of PspC is set forth in Genbank accession no. AF068646.

[0042] The sequence of the alternative complement inhibitor related to PspCN is JPEG2025507823000006.jpg13166 or a functional variant or fragment thereof. This sequence is amino acids 62 to 140 of PspC.

[0043] The sequences of additional alternative complement inhibitors related to PspCN are JPEG2025507823000007.jpg22166 or a functional variant or fragment thereof.

[0044] The microbially derived complement inhibitor may be derived from the variola virus, including vaccinia virus complement control protein (VCP), or smallpox virus complement inhibitor (SPICE), or monkeypox virus complement inhibitor (MOPICE).

[0045] Thus, the microbially derived complement inhibitor may be VCP, which has the following sequence: JPEG2025507823000008.jpg35166, or a functional variant or fragment thereof.

[0046] The microbial-derived complement inhibitor may be SPICE and has the following sequence: JPEG2025507823000009.jpg35166, or a functional variant or fragment thereof.

[0047] The microbially derived complement inhibitor may be MOPICE, which has the following sequence: JPEG2025507823000010.jpg29166, or a functional variant or fragment thereof.

[0048] The term "fragment" is intended to refer to at least 3, 6, 10, 15, 30, 60 consecutive amino acids of a reference sequence, or any integer number therebetween, of polyamino acids. Preferably, the fragment is a functional fragment. A functional fragment is one that represents at least a portion of a protein necessary to bind and activate CFH, e.g., can perform this function when used alone or in the form of multiple subunits. Thus, such a functional fragment may be a functional polypeptide in itself, or may be a fragment that is functional when linked to other polypeptides to obtain chimeric proteins, etc. Such functional fragments are understood to be within the scope of the present invention. Whether a fragment is functional can be determined using various bioassays described herein.

[0049] Fragments can be produced by enzymatic cleavage of precursor molecules, particularly with restriction endonucleases for DNA and proteases for polypeptides. Other methods include chemical synthesis of fragments or production of DNA-encoded peptide fragments.

[0050] As used herein, the term "protein" may be used interchangeably with "peptide" or "polypeptide" and refers to at least two covalently attached alpha amino acid residues linked by a peptide bond. The term protein encompasses purified natural products or chemical products that may be produced in part or in whole using recombinant or synthetic techniques. The term protein may also refer to complexes of two or more polypeptides, such as dimers or other multimers, fusion proteins, protein variants, or derivatives thereof. The term also includes modified proteins, e.g., proteins modified by glycosylation, acetylation, phosphorylation, PEGylation, ubiquitination, etc. Proteins may contain amino acids not encoded by nucleic acid codons.

[0051] Proteins with minor modifications in sequence are similarly useful if functional, and a complement inhibitor may be a protein consisting of an amino acid sequence exhibiting at least 50% similarity to an amino acid sequence depicted in any of the sequences of complement inhibitors disclosed herein, or a functional fragment thereof. A protein may be a polypeptide sequence having at least 60%, preferably at least 70%, more preferably 80%, even more preferably 90%, more preferably at least 99%, and most preferably 100% similarity to a sequence of a complement inhibitor described herein, or a functional fragment thereof.

[0052] The term "similarity" refers to the degree of similarity between proteins in terms of amino acid differences, but takes into account which different amino acids are functionally similar in terms of approximately equal size, lipophilicity, acidity, etc. Percent similarity can be calculated by optimal alignment of sequences using a similarity scoring matrix such as the Blosum62 matrix described in Henikoff S. and Henikoff JG, PNAS USA 1992, 89: 10915-10919. Calculation of the percent similarity and optimal alignment of two sequences using the Blosum62 similarity matrix and the Needleman and Wunsch algorithm (J. Mol. Biol. 1970, 48: 443-453) can be performed using the GAP program from the Genetics Computer Group (GCG, Madison, Wis., USA) using the program's default parameters.

[0053] Exemplary parameters for amino acid comparison in the present invention use the Blosum62 matrix (see Non-Patent Document 5, supra) in conjunction with the following settings for the GAP program: Gap penalty: 8 Gap length penalty: 2 No penalty for end gaps.

[0054] The polymorphism of complement inhibitors described herein is included in the present disclosure. Complement inhibitor variants include natural or synthetic variants that can include amino acid residue sequence variations by deletion, substitution, insertion, inversion or addition of one or more amino acid residues in said sequence, or by changing the moiety chemically linked to protein. For example, protein variants can be changes in carbohydrate or PEG structure attached to protein. Complement inhibitors can include at least one of such protein modifications.

[0055] Substitutional variants of a protein are those in which at least one amino acid residue in the amino acid sequence has been removed and a different amino acid residue inserted in its place. Protein complement inhibitors can contain conservative or non-conservative substitutions.

[0056] The term "conservative substitution" refers to the replacement of one or more amino acid residues with amino acid residues having similar biochemical properties. In general, conservative substitutions have little effect on the activity of the resulting protein. For example, conservative substitutions are substitutions of amino acid residues that do not substantially affect the ability of the protein to inhibit complement activity. Screening of mutants of complement inhibitors of proteins can be used to identify which amino acid residues can tolerate amino acid residue substitutions. In one example, when one or more conservative amino acid residue substitutions are effected, the relevant biological activity of the modified protein is not reduced by more than 25%, preferably not more than 20%, in particular not more than 10% compared to the disclosed complement inhibitors.

[0057] Protein complement inhibitors can include one or more conservative substitutions. In one example, 10 or fewer conservative substitutions are included in the protein. Thus, protein complement inhibitors can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. Polypeptides can be produced to include one or more conservative substitutions by manipulating the nucleotide sequence that encodes the polypeptide using standard procedures, such as site-directed mutagenesis, gene synthesis, PCR, etc. Alternatively, polypeptides can be produced to include one or more conservative substitutions, such as by using peptide synthesis methods known in the art.

[0058] Examples of amino acid residues that may be substituted for the original amino acid residue in the protein and are considered conservative substitutions include Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asn, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu for Val. In one embodiment, the substitutions are between Ala, Val, Leu and Ile, between Ser and Thr, between Asp and Glu, between Asn and Gln, between Lys and Arg, and / or between Phe and Tyr. Further information on conservative substitutions can be found, inter alia, in Ben-Bassat et al. (J. Bacteriol. 169:751-7, 1987), O'Regan et al. (Gene 77:237-51, 1989), Sahin-Toth et al. (Protein Sci. 3:240-7, 1994), Hochuli et al. (Bio / Technology 6:1321-5, 1988), WO 00 / 67796 (Curd et al.), and in standard textbooks on genetics and molecular biology.

[0059] Other variants may be functional variants, such as, for example, salts, amides, esters, particularly C-terminal esters, N-acyl derivatives, etc. Also included are peptides modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation.

[0060] Protein complement inhibitors can be modified by various chemical methods to produce derivatives that have essentially the same activity as the unmodified peptides, and optionally have other desirable properties. For example, the carboxylic acid groups of the protein, whether at the carboxyl terminus or at the side chain, can be provided in the form of a salt of a pharma- ceutically acceptable cation, or can be esterified to form, for example, a C1-C6 alkyl ester, or can be converted to an amide, for example, of the formula CONR1R2, where R1 and R2 are each independently H or C1-C6 alkyl, or can be linked to form a heterocycle, such as a five- or six-membered ring. The amino groups of the peptides, whether at the amino terminus or at the side chain, can be in the form of a pharma- ceutically acceptable acid addition salt, such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid, and other organic salts, or can be modified to C1-C6 alkyl or dialkylamino, or can be further converted to an amide. Hydroxyl groups of peptide side chains can be converted to alkoxy or ester groups, e.g., C1-C6 alkoxy or C1-C6 alkyl esters, using well-known techniques. Phenyl and phenol rings of peptide side chains can be substituted with one or more halogen atoms, such as F, Cl, Br, or I, or with C1-C6 alkyl, C1-C6 alkoxy, carboxylic acids and their esters, or amides of such carboxylic acids. Methylene groups of peptide side chains can be extended to the same C2-C4 alkylene. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize how to introduce cyclic structures into the peptides of the present disclosure, selecting structures that provide improved stability and provide conformational constraints.

[0061] The composition may comprise 1 nM to 500 μM of a complement inhibitor. The composition may comprise 1 nM to 100 μM of a complement inhibitor. The composition may comprise 10 nM to 50 μM of a complement inhibitor. The composition may comprise 1 nM to 5 μM of a complement inhibitor. The composition may comprise 50 nM to 50 μM of a complement inhibitor. The composition may comprise 50 nM to 1 μM of a complement inhibitor. The composition may comprise 50 nM to 0.5 μM of a complement inhibitor. The composition may comprise 50 nM to 0.1 μM of a complement inhibitor. The composition may comprise 100 nM to 50 μM of a complement inhibitor. The composition may comprise 250 nM to 50 μM of a complement inhibitor. The composition may comprise 500 nM to 50 μM of a complement inhibitor. The composition may comprise 750 nM to 50 μM of a complement inhibitor. The composition may contain between 1 μM and 50 μM of a complement inhibitor.

[0062] The composition may comprise at least 1 nM of a complement inhibitor. The composition may comprise at least 10 nM of a complement inhibitor. The composition may comprise at least 50 nM of a complement inhibitor. The composition may comprise at least 100 nM of a complement inhibitor. The composition may comprise at least 250 nM of a complement inhibitor. The composition may comprise at least 500 nM of a complement inhibitor. The composition may comprise at least 750 nM of a complement inhibitor. The composition may comprise at least 1 μM of a complement inhibitor.

[0063] The composition may further comprise at least one biologically compatible salt. The biologically compatible salt may be a sodium salt, a calcium salt, or a magnesium salt. For example, the composition may contain sodium chloride (NaCl), calcium chloride (CaCl 2 ), or magnesium chloride (MgCl 2 The composition may include inorganic salts, including sodium lactate (NaCO), including s-sodium lactate and l-sodium lactate. 2 CH(OH)CH 3 ), sodium bicarbonate (NaHCO 3 ), Sodium Citrate (Na 3 C 6 H5 O 7 ), or sodium acetate (C 2 H 3 NaO 2 ) may be included.

[0064] The composition may further comprise a buffering agent, which may be operable to maintain the pH of the composition or at least minimize changes in the pH of the composition.

[0065] The composition may have an osmolality of at least 200 millimoles per liter (mOsmol / L). The composition may have an osmolality of at least 250 millimoles per liter (mOsmol / L). The composition can have an osmolality of at least 250, 260, 280, 300, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 450, 460, 470, 480, 490 or 500 mOsmol / L or any value therebetween.

[0066] The composition may have an osmolality of 200 mOsmol / L to 500 mOsmol / L. The composition may have an osmolality of 250 mOsmol / L to 500 mOsmol / L. The composition may have an osmolality of 280 mOsmol / L to 500 mOsmol / L. The composition may have an osmolality of 300 mOsmol / L to 500 mOsmol / L. The composition may have an osmolality of 320 mOsmol / L to 500 mOsmol / L. The composition may have an osmolality of 250 mOsmol / L to 490 mOsmol / L. The composition may have an osmolality of 250 mOsmol / L to 480 mOsmol / L. The composition may have an osmolality of 250 mOsmol / L to 470 mOsmol / L. The composition may have an osmolality of 250 mOsmol / L to 460 mOsmol / L. The composition may have an osmolality of 250 mOsmol / L to 450 mOsmol / L. The composition may have an osmolality of 300 mOsmol / L to 480 mOsmol / L. The composition may have an osmolality of 300 mOsmol / L to 380 mOsmol / L. The composition may have an osmolality of 330 mOsmol / L to 370 mOsmol / L. The composition may have an osmolality of 450 mOsmol / L to 500 mOsmol / L. The composition may have an osmolality of 450 mOsmol / L to 490 mOsmol / L. The composition may have an osmolality of 460 mOsmol / L to 490 mOsmol / L. The composition may have an osmolality of between 470 mOsmol / L and 490 mOsmol / L.

[0067] In a second aspect, there is provided a composition for use in inhibiting mesothelial cell transformation, said composition comprising a complement inhibitor.

[0068] Preferred and optional features of the complement inhibitor of the first aspect are the preferred and optional features of the complement inhibitor of the second aspect.

[0069] According to a third aspect there is provided a method of producing an enhancement composition for use in peritoneal dialysis, the method comprising: Providing a base composition comprising an osmotic agent dissolved in an aqueous solvent; Providing a complement inhibitor; adding said complement inhibitor to a base composition to form an enhanced composition; Equipped with.

[0070] Preferred and optional features of the complement inhibitor and osmotic agent of the first aspect are the preferred and optional features of the complement inhibitor and osmotic agent of the third aspect.

[0071] In a fourth aspect, a method of peritoneal therapy is provided, the method comprising: Providing a composition according to a first aspect; transferring the composition to the peritoneal cavity of a patient; maintaining the composition within the peritoneal cavity of the patient for a treatment period; removing the composition from the peritoneal cavity after the treatment period has expired. Equipped with The composition removed from the peritoneal cavity contains toxins that have been drawn into the composition from the patient's blood across the peritoneal membrane.

[0072] The treatment time may be 1 hour, 2 hours, 3 hours or more, or any value in between. The treatment time may be overnight.

[0073] According to a fifth aspect, there is provided a composition for use in peritoneal dialysis (PD), the composition comprising an osmotic agent and a complement inhibitor.

[0074] The composition may be a dry composition and thus may be free of a solvent such as, for example, water.

[0075] The composition may be a lyophilized composition.

[0076] Before use, the user can add the composition to a solvent, such as water, to form a peritoneal dialysis solution or fluid. The peritoneal dialysis solution or fluid thus formed may be a composition according to the first aspect. Thus, the characteristics of the composition of the first aspect are the characteristics of the composition of the fifth aspect when added to a solvent.

[0077] It is understood that any feature, integer, property, compound, chemical moiety or group described in connection with a particular aspect, embodiment or example of the invention is also applicable to other aspects, embodiments or examples described herein, except where inconsistent therewith. All of the features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention extends to any novel or novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings), or any novel or novel combination of steps of the method or process so disclosed.

[0078] Embodiments of the invention will now be described, by way of non-limiting examples, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0079] [Figure 1] 1 is a schematic example of a patient undergoing peritoneal dialysis. [Diagram 2] It shows the three pathways of the complement system, the various proteins involved, and where therapeutic intervention can occur in the complement cascade (Ricklin D et al. The renaissance of complement therapeutics. Nat Rev Nephrol 14(1), 26-47 (2018)). [Diagram 3]FIG. 1 is a schematic diagram of the plasmid pE-SUMO Kan used for expression of SUMO fusion proteins according to the present invention (Non-Patent Document 12: http: / / www.lifesensors.com). [Figure 4] These red blood cells were treated with AET to generate "paroxysmal nocturnal hemoglobinuria (PNH)-like" cells that were susceptible to acidified serum lysis similar to PNH red blood cells. Addition of high concentrations of PspCN inhibited the lysis of PNH-like red blood cells with an IC50 of 27 nM. PspCN activates FH already present in serum, effectively blocking hemolysis. [Diagram 5] Fluorescence-based confocal microscopy images showing activation of complement by PD solution on human mesothelial cells (HMC). [Figure 6] Graph of C3d levels measured as a marker of complement activation in pHUVECs exposed to complex solutions containing 2.3% glucose PD solution (see Table 3 below) with and without increasing concentrations of the complement inhibitor FH. Results are shown for different cell passages (P2-P6) and seeding times on 24-well plates. A significant decrease in C3d levels was observed in all conditions except P6, consistent with primary cells likely changing their genetic and phenotypic properties. [Figure 7] Graph of C3d levels measured as a marker of complement activation in pHUVECs exposed to complex solutions containing 2.3% glucose PD solution (see Table 3 below) with or without increasing concentrations of the complement inhibitor FH+PspCN complex. Results are shown for different cell passages (P2-P6) and seeding times on 24-well plates. A significant decrease in C3d levels was observed at P2 but not at P6, suggesting that primary cells have altered genetic and phenotypic characteristics. [Figure 8]This is a graph of C3d levels measured as a marker of complement activation in pHUVECs exposed to a complex solution containing 2.3% glucose PD solution (see Table 3 below) in the presence or absence of PspCN, an activity enhancer of the complement inhibitor FH. Results are shown for different cell passages (P3-P5) and seeding times on 24-well plates. A significant decrease in C3d levels was observed in all conditions. It is noteworthy that the complex solution contains 5% NHS. NHS serves as a source of complement, but also contains FH, which PspCN can bind and activate, thus increasing its potency. [Figure 9] Figure 1 shows graphs of C3d levels measured as a marker of complement activation in pHUVEC exposed to complex solutions containing 2.3% glucose PD solution (see Table 3 below) with and without increasing concentrations of complement inhibitors DAF 1-4. Results are shown for different cell passages (P3-P6) and seeding times in 24-well plates. A significant decrease in C3d levels was observed at P3, less at P5, and no decrease at P6. [Figure 10] Graph of C5b-9 levels measured as a marker of complement activation in pHUVEC exposed to a complex solution containing 2.3% glucose PD solution (see Table 3 below) with or without increasing concentrations of the complement inhibitor FH and the FH+PspCN complex. Results are shown at cell passage P2, seeding time 1 day in 24-well plates. A significant decrease in C5b-9 levels was observed in both FH and FH+PspCN complexes compared to samples without added inhibitors. [Figure 11] 1 is a graph of C3d levels measured as a marker of complement activation in pHUVEC exposed to complex solutions containing a 2.3% glucose PD solution (see Table 3 below). An approximately 30% increase in C3d levels was observed in 95% PD solution (see Table 3 below) vs. 5% NHS and in 95% cell culture medium vs. 5% NHS. [Figure 12]Microscopic images of pHUVECs exposed to the combined solutions for 3 days. Cells exposed to 85% medium-10% PBS-5% NHS maintain the characteristic cobblestone-like endothelial appearance, whereas cells exposed to 35% medium-40% PD solution (see Table 3 below)-10% PBS-5% NHS begin to lose their endothelial appearance and become more elongated and spindle-shaped. [Figure 13] Graph of lactate dehydrogenase assay (LDH) data for measuring the toxicity of complement inhibitors to pHUVEC. The LDH assay is a means of measuring either cell number by total cytoplasmic LDH or membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. The displayed LDH corresponds to the amount of LDH released normalized to total LDH / cell number. Little difference is observed between the values ​​obtained for cells cultured in normal growth medium and those cultured in growth medium supplemented with inhibitors. [Figure 14] To identify genes involved in the complement system and epithelial-mesenchymal transition (EMT), we used Gene Set Enrichment Analysis (GSEA) (A). Of these 38 identified genes, 28 were found in transcriptome and proteome datasets (B) obtained from microdissected fetal membrane arterioles (Bartosova et al. JASN 2018), including complement factors (highlighted in red) that control the alternative pathway. [Figure 15] Figure 15A shows images of early EndMT in primary human umbilical vein endothelial cells (pHUVECs) exposed to TGFβ for 72 hours, resulting in more spindle-shaped cells in the culture. Figure 15B shows increased expression of myofibroblast markers Col13A and αSMA compared to media control. Mean ± SD, Scale bar = 200 μm, *p<0.05, ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] Although the making and using of various embodiments of the invention are described in detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0081] To facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings commonly understood by those of ordinary skill in the art relevant to the present invention. Terms such as "a", "an", and "the" are not intended to refer to a singular entity only, but include general classes of which specific examples may be used for illustration. While the terms herein are used to describe specific embodiments of the present invention, their use is not intended to limit the present invention, except as outlined in the claims.

[0082] Exemplary compositions suitable for use in peritoneal dialysis, compositions according to the present disclosure, uses of the compositions according to the present disclosure, and therapeutic methods using the compositions are described below.

[0083] Comparative Example 1 A first exemplary exemplary peritoneal dialysis composition has the composition according to Table 1.

[0084] [Table 1]

[0085] The theoretical osmolality of the composition is 284 millimoles per liter (mOsmol / L) and the pH is 5-6.

[0086] The composition is used in peritoneal dialysis therapy as part of continuous ambulatory peritoneal dialysis (CAPD) or automated peritoneal dialysis (APD) for the treatment of chronic renal failure.

[0087] A schematic diagram of a typical peritoneal dialysis treatment is shown in Figure 1. Typically, during treatment, a composition is transferred into the patient's peritoneal cavity via a catheter placed in the patient's abdomen. The composition is retained in the peritoneal cavity overnight (6-12 hours) for CAPD treatment and 14-16 hours for APD treatment.

[0088] A typical volume of peritoneal dialysis composition used in treatment is up to 2 L of fluid administered over 10-20 minutes for an average sized patient.

[0089] After the usual treatment time, the fluid is removed from the abdominal cavity.

[0090] Comparative Example 2 A typical peritoneal dialysis composition of the second example has a composition according to Table 2.

[0091] [Table 2]

[0092] The composition has an osmolality of 395 millimoles per liter (mOsmol / L) and a pH of 7.4.

[0093] The compositions are used in peritoneal dialysis therapy as part of continuous ambulatory peritoneal dialysis (CAPD) or automated peritoneal dialysis (APD) for the treatment of chronic renal failure.

[0094] Typically, during treatment, the composition is delivered into the patient's peritoneal cavity via a catheter placed in the patient's abdomen. The composition is retained in the peritoneal cavity for up to four cycles per day for CAPD treatment, or four to five cycles at night and up to two cycles during the day for APD treatment.

[0095] A typical volume of peritoneal dialysis composition used in treatment is up to 2 L of fluid administered over 10-20 minutes for an average sized patient.

[0096] After the usual treatment time, the fluid is removed from the abdominal cavity.

[0097] (Role of the alternative complement pathway in mesothelial cell transformation) It has been suggested that mesothelial cell transformation is involved in the degradation of the peritoneum. Therefore, we investigated the expression of C3, CFB, C5b9 and C1q in human mesothelial cells (HMC).

[0098] Immortalized HMCs were cultured in monolayer and incubated for 8 hours in BicaVera PD solution (Fresenius) containing 2.5% glucose. After incubation, cells were exposed to fetal bovine serum (FBS) for 1 hour and incubated with antibodies. Specific fluorescence indicating expression of C3 (Figure 5A), CFB (Figure 5B), C5b9 (Figure 5C) and C1q (Figure 5D) by HMCs was assessed by confocal microscopy. For each of Figures 5A-D, the top three images (basal) show blue staining corresponding to cell death markers, the middle three images show colocalization (merged) of blue and green staining, where green staining indicates expression of the corresponding complement activation marker after incubation with BicaVera solution, and the bottom three images show colocalization of cell death markers and complement activation markers after incubation with BicaVera solution in the presence of FBS. Staining for C3, CFB and C5b9 after incubation of HMCs with BicaVera PD solution shows that simple contact with glucose induces local expression and activation of the alternative pathway of complement. This occurs even in the absence of FBS, indicating that complement markers are made locally by the cells and not by FBS. As can be seen in Figure 5D, activation of the classical pathway is less evident. These results demonstrate the importance of local complement pathway activation in mesothelial cell transformation and highlight the need for complement control. EXAMPLES

[0099] Example 1 Referring to Figure 2, the complement system is activated by three pathways: classical pathway (CP), mannose-binding lectin pathway (LP), and altered pathway (AP). All three pathways converge with the cleavage of C3 to C3b, amplifying the initial reaction through the AP, where C3b interacts with factor B (FB) and factor D (FD) to form new C3 convertase. In the absence of regulator of complement activation (RCA), C3 convertase further reacts with C3b molecules to form C5 convertase. These cleave C5 and initiate events that lead to the formation of the membrane attack complex (MAC). C3 and C5 convertase release the anaphylatoxins C3a and C5a, which trigger downstream inflammatory responses. RCA inhibits the assembly of the convertase and serves as a coenzyme for factor I, which cleaves C3b into iC3b and C3d, which remain attached to the surface to stimulate phagocytosis and immune signaling. Complement therapeutics act by blocking initiation, suppressing amplification, or affecting downstream effector functions. Complement inhibition can target FB, FD, properdin, MASP1-3, C1, C3, C3a, C3b, C5, C5a, C5b, C5aR1, C6, or MAC. Alternatively, enhancers or activators of RCA can be used.

[0100] An example of a composition according to the present disclosure is the composition of Comparative Example 1, which contains 0.5 μM of the complement inhibitor PspCN (SEQ ID NO 1), characterized below.

[0101] PspCN is known to be a particularly effective inhibitor of C3b and C3d, as shown below. Thus, compositions containing PspCN in peritoneal dialysis (PD) treatment inhibit activation of complement immune responses in the peritoneal cavity and membrane, thereby suppressing the structural changes of the peritoneum that have been reported over long-term PD treatment.

[0102] A further example composition is the composition of Comparative Example 2, which contains 1.5 μM of the complement inhibitor DAF 1-4 (SEQ ID NO:12).

[0103] Exemplary Complement Inhibitor PspCN The complement inhibitor PspCN has been described and its ability to inhibit the alternative complement pathway demonstrated in an international patent application by the University Court of Edinburgh (International Publication No. WO 2015 / 055991), the contents of which are incorporated herein by reference. A summary of the findings presented therein is provided below.

[0104] (Protein Preparation) A gene representing residues 37-140 of PspC(D39) was optimized for expression in E. coli (see SEQ ID NO 4 below) and purchased from GeneArt-LifeTech. The resulting construct was cloned into the pE-SumoProKan E. coli expression vector (LifeSensors, Malvern, PA; see FIG. 3) and expressed in BL21(DE3) E. coli in lysis broth (LB). Protein production was induced by the addition of 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 25°C. The resulting hexaHis-SUMO tagged protein was named "sPspCN" and was captured on a HisTrap immobilized Ni2+ affinity column (GE Healthcare) and eluted with a linear gradient of 0-0.5 M imidazole. Samples of sPspCN were further purified by size-exclusion chromatography on a HiPrep Superdex 75 column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS). A similar strategy was used to prepare a longer construct, called sPspCNR1, encompassing the adjacent R1 domain (see Figure 2) (residues 37-292).

[0105] In most experiments, the catalytic domain of the SUMO-specific protease ULP1 was used to remove the hexaHis-SUMO tag from sPspCN (or sPspCNR1) (without vector-derived residues). The cleaved material - PspCN (or PspCNR1) - was further purified on a HiPrep Superdex 75 column in PBS as described above. The protein was judged to be homogeneous by SDS-PAGE, and the integrity and identity of the protein was confirmed by mass spectrometry (not shown).

[0106] (Demonstration that PspCN can activate factor H present in serum and effectively prevent cell lysis) Referring to Figure 4, it is shown that addition of PspCN (SEQ ID NO.1) to PNH-like cells inhibits cell lysis with an IC50 of 27 nM. PspCN activates factor H, which is already present in the serum along with the cells, thereby inhibiting cell lysis by complement.

[0107] Therefore, PspCN is a suitable complement inhibitor for use in such PD compositions.

[0108] (Examples of complement inhibitors: DAF 1-4) (- Protein Preparation) The gene consisting of complement control protein modules 1-4 from human decay accelerating factor (DAF 1-4) was optimized for expression in Komagataella phaffii (commonly known as Pichia Pastoris) and inserted into Thermofisher Scientific's pPICZalphaB expression vector. A proprietary KM71 Komagataella phaffii (designated KM71PDI) was transformed by electroporation to express DAF 1-4, and successfully transformed colonies were selected on Zeocin® agar plates.

[0109] Selected colonies were grown to high cell numbers in buffered complex glycerol medium (BMGY) for 64-72 hours in baffled shaker flasks (30°C, 225 RPM). To promote protein production / secretion, cells were reconstituted in buffered complex methanol medium (BMMY). Growth in BMMY was carried out in baffled shaker flasks (20°C, 225 RPM) for 96 hours, with 0.5% (v / v) neat methanol added twice daily until harvest.

[0110] The resulting clarified supernatant was adjusted to 1 mM phenylmethylsulfonyl fluoride (pmsf) with 5 mM ethylenediaminetetraacetic acid (EDTA) and buffer exchanged into Dulbecco's phosphate buffered saline (DPBS) by continuous volumetric diafiltration. Protein purification was performed using Capto™ SP ImpRes cation exchange medium, a citrate buffer system at pH 4.5 with a linear salt gradient of 0 to 500 mM sodium chloride. Final polishing was performed by size exclusion chromatography (SEC) using Superdex 75 medium and eluted isocratically into DPBS.

[0111] Analysis of the intermediates was performed by SDS-PAGE and analytical SEC (TOSOH g3000SWXL column), and confirmation of protein integrity and identity was confirmed by the same methods, including mass spectrometry (not shown).

[0112] (-Description of methods for cell culture and exposure to PD solutions and complement inhibitors) Primary human umbilical vein endothelial cells (pHUVEC, PromoCell) were cultured in medium supplemented with 1% penicillin / streptomycin at 37 °C for 24 h at 5% CO 2 The cells were cultured in an incubator according to the manufacturer's instructions. Cells at passages 2-6 (P2-P6) were used for the C3d assay and were seeded in 24-well ELISA plates overnight or for 5-7 days. The number of passages is important because after P5 the cells lose key characteristics (genetic and phenotypic properties). The cells were then cultured in a commercial 2.3% glucose PD solution (see Table 3 below)-cell medium complex + 5% *Exposed to NHS (normal human serum, used as complement source) + 6-20% PBS (to keep PBS content constant from inhibitor stock solution) ± complement inhibitors (e.g. DAF 1-4, FH, PspCN) for 20-24 hours.

[0113] [Table 3]

[0114] Cells were fixed with -20°C methanol for 7–10 min, washed with PBS, permeabilized with 0.2% TritonX-100 for 8–10 min, blocked with Superblock-PBS, and incubated with primary C3d antibody (Abcam). Alexa594 (Abcam) secondary antibody + DAPI stain (SIGMA; nuclear stain, indicator of cell number) was added, and fluorescence measurements were performed using a CLARIOstar Plus plate reader. Data were normalized to DAPI staining and then normalized to maximum value within each experiment minus assay background. C3d is the final surface-attached portion of C3b after complement activation and was used as a biomarker to measure complement activation.

[0115] The results are shown in Figures 6 to 9.

[0116] In Figure 6, a significant decrease in C3d levels was observed in all conditions except P6, consistent with the primary cells likely having altered their genetic and phenotypic characteristics. In Figure 7, a significant decrease in C3d levels was observed in P2 but not P6, consistent with the primary cells likely having altered their genetic and phenotypic characteristics. In Figure 8, a significant decrease in C3d levels was observed in all conditions. Of note is the inclusion of 5% NHS in the complex solution. This NHS serves as a source of complement, but also contains FH, which PspCN can bind and activate, thus increasing its potency. In Figure 9, a significant decrease in C3d levels was observed in P3, a decrease in P5, and no decrease in P6.

[0117] C5b-9 levels were measured using the same assay as for C3d above. The results for cells passage number P2, seeded on 24-well plates for 1 day, are shown in Figure 10. A significant decrease in C5b-9 levels was observed in both FH and FH+PspCN complexes compared to samples without the addition of inhibitor.

[0118] As shown in FIG. 11, C3d levels are increased by about 30% in a 95% balanced 2.3% glucose-5% NHS-containing solution versus a 95% cell culture medium-5% NHS-containing solution.

[0119] Physiological changes in pHUVEC cells have been shown to be associated with exposure to PD solutions. Figure 12 shows microscopic images at two magnifications (4x and 10x) of pHUVECs exposed to the composite solutions for 3 days. Cells exposed to 85% medium-10% PBS-5% NHS maintain the characteristic cobblestone-like endothelial appearance (Figure 12A), whereas cells exposed to 35% medium-40% balanced 2.3% glucose-10% PBS-5% NHS begin to lose their endothelial appearance and become more elongated and spindle-shaped (Figure 12B).

[0120] The toxicity of complement inhibitors to pHUVEC cells was measured using a lactate dehydrogenase (LDH) assay. The LDH assay is a means of measuring cell number via the total amount of cytoplasmic LDH or membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. The displayed LDH corresponds to the amount of LDH released normalized by total LDH / cell number. The results are shown in Figure 13. There was little difference between the values ​​obtained for cells cultured in normal growth medium and those cultured in growth medium supplemented with inhibitors. Thus, the tested complement inhibitors showed no clear toxicity to pHUVEC cells.

[0121] Upon exposure to low glucose degradation products (GDP) PD solution, 604 genes were found to be upregulated in cells, of which 38 genes are involved in the complement system, suggesting a link between complement and epithelial-mesenchymal transition (EMT) (see Figure 14). Factor H (FH), factor B (FB), and properdin (factor P) were among the genes identified, highlighting the involvement of the alternative pathway of complement. Therefore, the use of complement inhibitors in PD is expected to mitigate the epithelial-mesenchymal transition of cells exposed to PD solution during PD treatment.

[0122] Exposure of pHUVECs to TGFβ for 72 hours resulted in early EMT (TGFβ is used to stimulate EMT, see FIG. 15A). Expression of myofibroblast markers Col13A and αSMA was increased compared to medium controls (see FIG. 15B). This increase is associated with the acquisition of a mesenchymal phenotype. The inclusion of complement inhibitors in the setting is expected to reduce early EMT and reduce the corresponding levels of these markers compared to systems without complement inhibitors.

[0123] Thus, exposure to peritoneal dialysis fluid has been shown to induce physiological changes in cells similar to those associated with EMT. Furthermore, it has been shown that complement inhibitors such as FH, PspCN, and DAF1-4 can inhibit this transition. Therefore, in clinical applications, peritoneal dialysis fluid containing complement inhibitors is expected to reduce EMT in at least peritoneal endothelial cells, thereby extending the period during which peritoneal dialysis can be used effectively.

[0124] The amino acid sequence of PspC from S. pneumoniae strain D39 (NCTC no 7466) is shown below (SEQ ID NO 7), with the sequence of PspCN (amino acids 37 to 140) in bold.

[0125] JPEG2025507823000014.jpg64166

[0126] The codon-optimized synthetic DNA sequence used to express PspCN is shown below (SEQ ID NO 8).

[0127] JPEG2025507823000015.jpg30166

[0128] While approved embodiments of the invention have been described herein, it will be readily apparent that many various changes and modifications in the shape, design, construction and arrangement of parts of other embodiments may be made without departing from the invention, and it will be understood that all such changes and modifications are contemplated as embodiments that are part of the invention as defined in the appended claims.

Claims

1. A composition for use in peritoneal dialysis (PD), comprising a biologically compatible solvent, an osmotic agent, and a complement inhibitor.

2. 10. The composition of claim 1, wherein the biologically compatible solvent is water, and the composition is an aqueous composition.

3. 10. The composition of claim 1, wherein the osmotic agent is selected from the group consisting of glucose, dextrose (L-glucose), fructose, galactose, maltose, xylitol, mannitol, sorbitol, maltodextrin, icodextrin, sucrose, hyaluronic acid, or derivatives, fragments, or mixtures thereof.

4. 4. The composition of claim 3, wherein the osmotic agent is selected from the group consisting of glucose, dextrose, icodextrin, or derivatives or fragments or mixtures thereof.

5. 2. The composition of claim 1, wherein the complement inhibitor is an inhibitor of factor D, factor B, properdin, MASPs 1-3, C1, C3, C3a, C3b, C4b, C5, C5a, C5b, C5aR1, C6, or MAC.

6. 6. The composition of claim 5, wherein the complement inhibitor promotes the destruction of C3 convertase.

7. 6. The composition of claim 5, wherein the complement inhibitor is selected from the group consisting of C1-INH, IFX-1 / CaCP29, Mirococept or APT070, TP10 / CDX-1135, eculizumab, AMY-101, ravulizumab or ALX1210 or ultomiris, crovalimab / SKY59 / RO7112689, tesidolumab / LFG316, pozelimab / REGN3918, ABP959, SB12, nomacopan / rVA576 / Coversin / OmCI, zilucoplan / RA101495, semdisilane / ALN-CC5, APL-2, and LNP. 023, Danicopan / ACH-4471 / ACH-0144471, stimulimab / BIV009 / TNT009, avacopan / CCX168, narsoplimab / OMS721, Zimra / avacincaptad pegol, lampalizumab, CLG561, IONIS-FB-LRx, IPH5401, GEN1029, Ruconest, ACH-5228, ACH-5448, APL-9, AAVCAGsCD59 / HMR59, ANX005, ANX007, BIVV020, OMS906, PRO-02, AMY-103, 5C6 / Compsorbin, anti-FH. 07, AMY-201 / miniFH, mutant mini FH (SEQ ID NO: 9), DAF1-4, SOBI005, ISU305, Mubodima, IFX-2, IFX-3, ALS-205, DF2593A, Regenemab, C6-LNA, or PspC or a functional variant or fragment thereof.

8. 8. The composition of claim 7, wherein the complement inhibitor prevents complement initiation and amplification and is selected from the group consisting of C1-INH, stimulimab / BIV009 / TNT009, narsoplimab / OMS721, ruconest, ANX005, ANX007, BIVV020, and PRO-02.

9. In the composition of claim 7, the complement inhibitor attenuates complement amplification and is selected from the group consisting of Mirococept (APT070), TP10 / CDX-1135 (soluble complement receptor 1), AMY-101, APL-2, LNP023, Danicopan / ACH-4471 / ACH-0144471, stimulimab / BIV009 / TNT009, lampalizumab, CLG561, IONIS-FB-LRx, ACH-5228, ACH-5448, APL-9, BIVV020, OMS906, PRO-02, AMY-103, 5C6 / Compsorbin, and anti-FH. 07, AMY-201 / miniFH, DAF 1-4, mutant miniFH (SEQ ID NO: 9), or PspC or a functional variant or fragment thereof.

10. 10. The composition of claim 9, wherein the complement inhibitor is a protein capable of binding to complement factor H.

11. In the composition of claim 7, the complement inhibitor impairs the effector function of complement and is selected from the group consisting of IFX-1 / CaCP29, eculizumab (Soliris), ravulizumab / ALX1210 / Ultomiris, crovalimab / SKY59 / RO7112689, tesidolumab / LFG316, pozelimab / REGN3918, ABP959, SB12, nomacopan / rVA576 / Coversin / OmCI, and Zirconia. A composition selected from the group consisting of Pran / RA101495, Semidisilane / ALN-CC5, Zimra / Avacincaptad pegol, Lampalizumab, CLG561, IONIS-FB-LRx, IPH5401, GEN1029, AAVCAGsCD59 / HMR59, SOBI005, ISU305, Mubodima, IFX-2, IFX-3, ALS-205, DF2593A, Regenemab, and C6-LNA.

12. 2. The composition of claim 1, wherein the complement inhibitor is a vaccinia virus complement control protein (VCP), or a smallpox complement inhibitor (SPICE), or a monkeypox virus complement inhibitor (MOPICE), or a functional fragment or variant thereof.

13. 10. The composition of claim 1, further comprising at least one biologically compatible salt.

14. 14. The composition of claim 13, wherein the biologically compatible salt is a sodium salt, a calcium salt, or a magnesium salt.

15. 10. The composition of claim 1, wherein the composition has an osmolality of at least 250 millimoles per liter (mOsmol / L).

16. 16. The composition of claim 15, wherein the composition has an osmolality of at least 260, 280, 300, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 450, or 500 mOsmol / L.

17. A composition for use in inhibiting mesothelial cell transformation, the composition comprising a complement inhibitor.

18. 18. The composition of claim 17, further comprising an osmotic agent selected from the group consisting of glucose, dextrose, icodextrin, sucrose, or derivatives or fragments or mixtures thereof.

19. 20. The composition of claim 17, wherein the composition is an aqueous composition.

20. 18. The composition of claim 17, wherein the complement inhibitor is an inhibitor of Factor D, Factor B, properdin, MASPs 1-3, C1, C3, C3a, C3b, C4b, C5, C5a, C5b, C5aR1, C6, or MAC.

21. 21. The composition of claim 20, wherein the complement inhibitor promotes the destruction of C3 convertase.

22. 21. The composition of claim 20, wherein the complement inhibitor is C1-INH, IFX-1 / CaCP29, Mirococept or APT070, TP10 / CDX-1135, eculizumab, AMY-101, ravulizumab or ALX1210 or ultomiris, crovalimab / SKY59 / RO7112689, tesidolumab / LFG316, pozelimab / REGN3918, ABP959, SB12, nomacopan / rVA576 / Coversin / OmCI, zilucoplan / RA101495, semidisilane / ALN-CC5, APL-2, LNP023, mitochondrial inactivator ... Copan / ACH-4471 / ACH-0144471, stimulimab / BIV009 / TNT009, avacopan / CCX168, narsoplimab / OMS721, Zimra / avacincaptad pegol, lampalizumab, CLG561, IONIS-FB-LRx, IPH5401, GEN1029, Ruconest, ACH-5228, ACH-5448, APL-9, AAVCAGsCD59 / HMR59, ANX005, ANX007, BIVV020, OMS906, PRO-02, AMY-103, 5C6 / Compsorbin, AMY-201 / miniFH, mutants A composition selected from the group consisting of miniFH (SEQ ID NO. 9), DAF1-4, SOBI005, ISU305, Mubodima, IFX-2, IFX-3, ALS-205, DF2593A, Regenemab, C6-LNA, or PspC or a functional variant or fragment thereof.

23. 23. The composition of claim 22, wherein the complement inhibitor prevents complement initiation and amplification and is selected from the group consisting of C1-INH, stimulimab / BIV009 / TNT009, narsoplimab / OMS721, ruconest, ANX005, ANX007, BIVV020, and PRO-02.

24. In the composition of claim 22, the complement inhibitor attenuates complement amplification and is selected from the group consisting of Mirococept (APT070), TP10 / CDX-1135 (soluble complement receptor 1), AMY-101, APL-2, LNP023, Danicopan / ACH-4471 / ACH-0144471, stimulimab / BIV009 / TNT009, lampalizumab, CLG561, IONIS-FB-LRx, ACH-5228, ACH-5448, APL-9, BIVV020, OMS906, PRO-02, AMY-103, 5C6 / Compsorbin, and anti-FH. 07, AMY-201 / miniFH, DAF 1-4, mutant mini FH (SEQ ID NO: 9), or PspC or a functional variant or fragment thereof.

25. 25. The composition of claim 24, wherein the complement inhibitor is a protein capable of binding to complement factor H.

26. In the composition of claim 22, the complement inhibitor impairs the effector function of complement and is selected from the group consisting of IFX-1 / CaCP29, eculizumab (Soliris), ravulizumab / ALX1210 / Ultomiris, crovalimab / SKY59 / RO7112689, tesidolumab / LFG316, pozelimab / REGN3918, ABP959, SB12, nomacopan / rVA576 / Coversin / OmCI, and Zirconia. A composition selected from the group consisting of Pran / RA101495, Semidisilane / ALN-CC5, Zimra / Avacincaptad pegol, Lampalizumab, CLG561, IONIS-FB-LRx, IPH5401, GEN1029, AAVCAGsCD59 / HMR59, SOBI005, ISU305, Mubodima, IFX-2, IFX-3, ALS-205, DF2593A, Regenemab, and C6-LNA.

27. 20. The composition of claim 17, further comprising at least one biologically compatible salt.

28. 28. The composition of claim 27, wherein the biologically compatible salt is a sodium salt, a calcium salt, or a magnesium salt.

29. 18. The composition of claim 17, wherein the composition has an osmolality of at least 250 millimoles per liter (mOsmol / L).

30. 30. The composition of claim 29, wherein the composition has an osmolality of at least 260, 280, 300, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 450, or 500 mOsmol / L.

31. 1. A method of making an enhancement composition for use in peritoneal dialysis, comprising: providing a base composition comprising an osmotic agent dissolved in an aqueous solvent; providing a complement inhibitor; adding said complement inhibitor to a base composition to form an enhanced composition; A method comprising:

32. 32. The method of claim 31, wherein the complement inhibitor is an inhibitor of Factor D, Factor B, properdin, MASPs 1-3, C1, C3, C3a, C3b, C4b, C5, C5a, C5b, C5aR1, C6, or MAC.

33. 33. The composition of claim 32, wherein the complement inhibitor promotes the destruction of C3 convertase.

34. 32. The method of claim 31, wherein the complement inhibitor is C1-INH, IFX-1 / CaCP29, Mirococept or APT070, TP10 / CDX-1135, eculizumab, AMY-101, ravulizumab or ALX1210 or ultomiris, crovalimab / SKY59 / RO7112689, tesidormab / LFG316. Pozelimab / REGN3918, ABP959, SB12, nomacopan / rVA576 / Coversin / OmCI, zilucoplan / RA101495, semidisilane / ALN-CC5, APL-2, LNP023, danicopan / ACH-4471 / ACH-0144471, stimulimab / BIV009 / TNT009, avacopan / CCX168, narsoplimab / OMS721, Zimra / Abasi Ncaptad pegol, lampalizumab, CLG561, IONIS-FB-LRx, IPH5401, GEN1029, Ruconest, ACH-5228, ACH-5448, APL-9, AAVCAGsCD59 / HMR59, ANX005, ANX007, BIVV020, OMS906, PRO-02, AMY-103, 5C6 / Compsorbin, AMY-201 / miniFH, mutant mini FH (SEQ ID NO. 9), DAF1-4, SOBI005, ISU305, Mubodima, IFX-2, IFX-3, ALS-205, DF2593A, Regenemab, C6-LNA, or PspC or a functional variant or fragment thereof.

35. 1. A method of peritoneal treatment, comprising: Providing a composition according to any one of claims 1 to 30 or producing using a method according to any one of claims 31 to 34; transferring the composition to the peritoneal cavity of a patient; maintaining the composition within the peritoneal cavity of the patient for a treatment period; removing the composition from the peritoneal cavity after the treatment period has expired; Equipped with The method, wherein the composition removed from the peritoneal cavity comprises toxins that have been drawn into the composition from the patient's blood across the peritoneal membrane.

Images