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WO2018078626A1 - Polythérapies comprenant une chitinase humaine (chit1) pour le traitement d'une infection fongique systémique - Google Patents

Polythérapies comprenant une chitinase humaine (chit1) pour le traitement d'une infection fongique systémique Download PDF

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WO2018078626A1
WO2018078626A1 PCT/IL2017/051171 IL2017051171W WO2018078626A1 WO 2018078626 A1 WO2018078626 A1 WO 2018078626A1 IL 2017051171 W IL2017051171 W IL 2017051171W WO 2018078626 A1 WO2018078626 A1 WO 2018078626A1
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Prior art keywords
chitinase
combination
echinocandin
use according
enzyme
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Chen Katz
Itai BLOCH
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Gavish-Galilee Bio Applications Ltd
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Gavish-Galilee Bio Applications Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)

Definitions

  • the present invention relates in general to treatment of fungal infections by using improved forms of human chitinase.
  • the present invention further relates to combination therapies including human chitinase for treatment of fungal infections.
  • Chitotriosidase (CHITl) and other human chitinases
  • Chitin is a naturally occurring P-(l,4)-linked polymer composed of N-acetylglucosamine repeats. Following cellulose, chitin is the second most abundant polysaccharide in nature, functioning as a major structural polymer in many life forms including the cell walls of bacteria and fungi, the shell of crustaceans, and the exoskeletons of arthropods. In addition to this key structural function, chitin is also an important nutritional source for many organisms (Ober et al., 2009).
  • Chitinases are a family of evolutionarily conserved hydrolases characterized by their ability to cleave chitin. These enzymes have been studied extensively in lower life forms where they function to control chitin homeostasis in the organism and degrade chitin present in the surrounding environment. Recent studies have identified functional mammalian chitinase genes and several chitinase-like genes, coding for chitin-binding proteins (Kzhyshkowska et al., 2007).
  • Glyco_18 domain-containing proteins The common feature of mammalian chitinases and chitinase-like proteins is the presence of highly conserved Glyco_18 domain. These evolutionary conservative chitinases belong to the glycosyl hydrolase family 18. Family 18 chitinases are distributed in a wide range of organisms, including bacteria, plants, fungi, insects, viruses, protozoan parasites and mammals. These chitinases catalyze the hydrolysis of chitin via substrate-assisted mechanism. An additional activity of these enzymes is irans-glycosylation resulting in the formation of chitin oligomers longer than the initial substrate. There are six Glyco_18 domain-containing proteins identified in human up to date.
  • CHITl chitotriosidase
  • AMCase acidic mammalian chitinase
  • CHITl is able to hydrolyse the artificial substrate 4-MU- chitotrioside as well as chitin (Renkema et al., 1995). Under conditions of excess of substrate, CHIT1 exhibits irans-glycosylation activity toward substrate molecules (Aguilera et al., 2003).
  • AMCase is extremely acid-stable chitinase with pH optimum around 2 which is capable of cleaving both natural chitin and artificial chitin-like substrates (Boot et al., 2001).
  • CBD chitin-binding domain
  • the other four of these highly homologous members of the chitinase family contain amino acid substitutions at their active sites, rendering these proteins non-catalytic.
  • chi-lectins or chitinase-like proteins include chitinase- 3 -like protein 1 (CHI3L1, also known as YKL-40), stabilin- interacting chitinase-like protein (SI-CLP), YKL-39 (chitinase 3-like protein 2), and oviductin (Kzhyshkowska et al., 2007).
  • the major cell types producing mammalian chitinases and chitinase-like proteins are macrophages, neutrophils, epithelial cells, chondrocytes and synovial cells, as well as tumor cells (Kzhyshkowska et al., 2007). Expression of these proteins is regulated at the mRNA level by various cytokines and hormones. Monocyte-derived macrophages start to produce CHIT1 approximately after a week of culturing in vitro. CHIT1 production is strongly stimulated by GM-CSF and inhibited by IFNy and IL-4 (Van Eijk et al. 2005). At the same time several reports indicate the stimulatory effect of proinflammatory agents (IFNy, TNFa, LPS) on the expression of CHIT1 in monocyte-derived macrophages.
  • IFNy, TNFa, LPS proinflammatory agents
  • CHIT1 mRNA for CHIT1 is found in lymph nodes, lung, and bone marrow, and its expression is restricted to the professional phagocytes (Boot et al. 2005). In addition to macrophages, neutrophils have been shown to be a source of CHIT1 in human (Van Eijk et al. 2005).
  • Fungal infections are a major disease concern, in immunosuppressed individuals such as recipients of solid organs and hematopoietic stem cells, AIDS patients and burn victims. Fungal infections are also on the rise in intensive care settings, likely due to a growing use of procedures with invasive medical devices and long-term use of antibiotics. In all cases, the most common etiological agents are Candida albicans and Aspergillus fumigatus. Antifungal drugs, which target ergosterol or glucan biosynthetic pathways, can have more severe side effects, or low efficacy. In recent years other components of pathogenic fungi such as chitin were tested as potentially promising drug targets and the role of human chitinases in antifungal therapy have been investigated.
  • US5928928 discloses human CHITl, its recombinant production, its use in therapy or prophylaxis against infectious disease.
  • the shorter 39 kDa form (which does not include the chitin binding domain) is suggested for production in bacteria, since it was not glycosylated.
  • production of recombinant enzyme was only attempted in the eukaryotic COS cells.
  • the present invention provides improved chitinases which overcomes issues of stability and sensitivity to physiological conditions for treating fungal infections, as well as providing improved methods for using chitinases in treating fungal infections.
  • the present invention provides a combination comprising chitinase and at least one echinocandin for use in treating a fungal infection.
  • the present invention provides a pharmaceutical composition comprising chitinase and at least one echinocandin, and a pharmaceutically acceptable carrier, for use in treating a fungal infection.
  • the present invention provides a method for treating a fungal infection, comprising administering to a subject in need the combination or the pharmaceutical composition of the invention.
  • the present invention provides a method for treating a fungal infection, comprising administering to a subject in need chitinase and at least one echinocandin.
  • the present invention provides a a kit for treating a fun infection, said kit comprising chitinase and at least one echinocandin.
  • Fig. 1 shows the sequence of the mature human CHIT1 enzyme (SEQ ID NO: 1). Dashed underline - amino acid 1 : a methionine residue that replaces the 22 amino acids signal sequence present in the pro-enzyme. Gray font color: amino acids 2-366, the catalytic domain. Italics: amino acids 367-395: a proline rich linker between the catalytic domain and the CBD, predicted to be a proteolytic site for human prolyl endopeptidase, in bold: the PKP sequence at positions 379-381. Underlined: amino acids 396-445: the chitin-binding domain (CBD).
  • CBD chitin-binding domain
  • Figs. 2A-2B show that the absence of a chitin binding domain (CBD) affects degradation of insoluble substrate (A) but not of soluble substrate (B).
  • A CHIT1 - red line (thick, bottom); CHIT1 w/o CBD - green line (thick, middle); no enzyme - purple line (X-shape, top).
  • B CHIT1 - purple X (top); CHIT1 w/o CBD - light blue asterisk (middle); control (no enzyme) - dark blue diamond (bottom).
  • Figs. 3A-3B show that increased concentration of a reducing agent (DTT) affects degradation of insoluble substrate by CHIT1 (A), but not soluble substrate (B).
  • DTT a reducing agent
  • A oxidized - blue line (bottom); ImM DTT - red line (second from bottom); 2mM DTT - green line (second from top); 5mM DTT - purple line (top);
  • B CHIT1 - light blue asterisk; CHIT1 ImM DTT - orange line; CHIT1 5mM DTT - gray line; control (no enzyme) - dark blue diamond (on the X axis).
  • Figs. 4A-4B show the effect of reducing agent on the CBD domain of CHIT1.
  • Fig. 5 shows the effect of the TRX system on degradation of insoluble chitin by CHIT1.
  • Fig. 6 shows the effect of oxidation on degradation of insoluble chitin by CHIT1.
  • Human chitinase was incubated with the thioredoxin system before insoluble chitin was added. The ability of the enzyme to degrade chitin was monitored for 2.5 h before NADP + (2mM) or dehydroascorbate (DHA) (2mM) were added (marked by an arrow and "Add").
  • Figs. 7 A and B show microscopy images of the effect of CHITl on the minimal effective concentration (MEC) of caspofungin on Aspargillus fumigatus.
  • MEC was determined by the M38-A reference method for broth dilution antifungal susceptibility testing of filamentous fungi, and defined microscopically as the lowest concentration of caspofungin that produced small and stubby hyphae (0.031 ⁇ g/ml in A, 0.0019 ⁇ g/ml in B).
  • the most important group of drugs that shows synergistic effect with chitinase is the echinocandins. These drugs are used for the treatment of systemic fungal infections in humans.
  • the mechanism of action of the echinocandins is inhibition of the synthesis of beta-glucans sugar chains, which are additional component of the fungal cell wall.
  • the present invention provides a combination comprising chitinase and at least one echinocandin for use in treating a fungal infection.
  • the chitinase is CHIT1.
  • the CHIT1 is of human origin.
  • the chitinase may be produced by recombinant DNA technologies in various protein expression systems such as bacteria, insect cells, plants cells or mammalian cells according to methods known in the art (see e.g. Structural Genomics Consortium et al., 2008, Protein production and purification, Nature Methods 5, 135 - 146).
  • the chitinase may be produced by other methods, such as e.g. isolated from cells.
  • the human CHIT1 is a wild-type human chitinase.
  • the human CHIT1 comprises at least one mutation in any one of the amino acids at the proline-rich linker at positions 367-395 of the mature CHIT1 enzyme (SEQ ID NO: 1) or in any one of the amino acids at the chitin binding domain (CBD) at positions 396-445 of the mature enzyme.
  • SEQ ID NO: 1 the proline-rich linker
  • CBD chitin binding domain
  • the at least one echinocandin is selected from pneumocandins, caspofungin, micafungin, anidulafungin, VER-002, V-echinocandin, LY303366, and eraxis. In some embodiments, the at least one echinocandin is selected from caspofungin, micafungin, and anidulafungin. In some embodiments, the echinocandin is caspofungin or Anidulafungin. In some embodiments, the echinocandin is caspofungin.
  • the fungal infection is caused by a fungal species selected from Candida sp. (causing Candidiasis) Aspergillus sp. (causing aspergillosis), Paracoccidioides brasiliensis (causing Blastomycosis), Coccidioides sp. (causing Coccidioidomycosis), Cryptococcus sp.
  • the fungal infection is resistant to treatment with at least one echinocandin.
  • the fungal infection is selected from Candidiasis, aspergillosis Blastomycosis, Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Mucormycosis, Paracoccidioidomycosis, penicilliosis and Sporotrichosis.
  • the fungal infection is caused by at least one fungus selected from Candida albicans, Candida Parapsilosis, Candida Glabrata, and Aspergillus fumigatus .
  • the fungal infection is caused by Candida albicans or Aspergillus fumigatus. In some embodiments, the fungal infection is caused by Candida albicans
  • the infection is a systemic infection.
  • the chitinase and the at least one echinocandin are formulated in separate formulations for administration separately or together.
  • the chitinase and the at least one echinocandin are formulated in a single formulation.
  • the chitinase and the at least one echinocandin are administered at the same time. In some embodiments, the chitinase and the at least one echinocandin are administered in a single composition. In some embodiments, the chitinase and the at least one echinocandin are administered separately.
  • the chitinase is provided at a plasma concentration of about 50 ⁇ g/ml. In some embodiments, the chitinase is provided at a plasma concentration of less than 50 ⁇ g/ml. In some embodiments, the plasma concentration of the chitinase is between 5 and 50 ⁇ g/ml. In some embodiments, the plasma concentration of the chitinase is between 5 and 25 ⁇ g/ml. In some embodiments, the plasma concentration of the chitinase is below 25 ⁇ g/ml. In some embodiments, the plasma concentration of the chitinase is below 15 ⁇ g/ml.
  • the minimal inhibitory concentration (MIC) of the echinocandin is reduced by at least 10-fold compared to its MIC when used alone. In some embodiments, the minimal inhibitory concentration (MIC) of the echinocandin is reduced by at least 50-fold compared to its MIC when used alone. In some embodiments, the MIC of the echinocandin is reduced by at least 100-fold compared to its MIC when used alone. In some embodiments, the minimal inhibitory concentration (MIC) of the echinocandin is reduced by at least 500-fold compared to its MIC when used alone. In some embodiments, the MIC of the echinocandin is reduced by at least 1000-fold compared to its MIC when used alone.
  • Minimal inhibitory concentration means the lowest concentration of a chemical which causes growth of a microorganism to be 10% or less of its growth without the chemical.
  • the MIC of the echinocandin is reduced for both the yeast form and the hyphae form of the fungus.
  • the MIC of the echinocandin is reduced only for the yeast form of the fungus.
  • the MIC of the echinocandin is reduced only for the hyphae form of the fungus.
  • the minimal effective concentration (MEC) of the echinocandin is reduced by at least 10-fold compared to its MEC when used alone. In some embodiments, the minimal effective concentration (MEC) of the echinocandin is reduced by at least 15-fold compared to its MEC when used alone. In some embodiments, the minimal effective concentration (MEC) of the echinocandin is reduced by at least 20-fold compared to its MEC when used alone. In some embodiments, the minimal effective concentration (MEC) of the echinocandin is reduced by at least 50-fold compared to its MEC when used alone. In some embodiments, the minimal effective concentration (MEC) of the echinocandin is reduced by at least 100-fold compared to its MEC when used alone.
  • MEC minimal effective concentration
  • the inventors have studied the recombinant human chitinase and found that the enzyme may be regulated in-vivo by two distinct mechanisms that inactivate the CBD of the enzyme, thus preventing the enzyme from binding to insoluble substrates such as chitin molecules and fungi cell wall. These mechanism do not affect the catalytic domain of the enzyme and therefore the ability of the enzyme to degrade soluble substrates such as synthetic fluorogenic substrate (4- Methylumbelliferyl P-D-N,N',N"-triacetylchitotriose) is not compromised.
  • synthetic fluorogenic substrate (4- Methylumbelliferyl P-D-N,N',N"-triacetylchitotriose) is not compromised.
  • Example 2 describes an in silico analysis of the human CHIT1, which identified a putative binding site for a prolyl endopeptidase.
  • Fig. 2 shows the inability of an enzyme lacking CBD to degrade an insoluble substrate, while the ability to degrade a soluble substrate remains intact.
  • the other mechanism is reduction of the CBD of the enzyme by natural reduction systems such as the thioredoxin system, which has the potential to inactivate at least part of the recombinant therapeutic enzyme by disabling its ability to effectively bind chitin (see Fig. 5).
  • a modified version of the recombinant enzyme with introduced mutations which increase stability of the oxidized (substrate binding) form and reduce sensitivity to endogenous proteases, or that prevent the cleavage of the enzyme by endogenous proteases, is expected to confer the recombinant enzyme with favored features that better suit its purpose as a therapeutic agent.
  • the present application provides an improved human CHITl, said CHITl containing at least one mutation in any one of the amino acids in the proline-rich linker between positions 367-395 of the mature CHITl enzyme or in any one of the amino acids at the CBD between positions 396-445 of the mature enzyme, thereby rendering the human CHITl with increased stability due to being resistant to cleavage by endopeptidases and/or being less sensitive to the redox state of the environment.
  • Mutations are generated by any method known in the art for site-directed mutagenesis or using de-novo DNA synthesis for synthesizing the new mutated sequence.
  • random mutagenesis techniques known in the art can be applied in order to generate an enzyme with altered regulation by randomly changing one or more of the positions described above.
  • the sequence of the human CHITl enzyme was analyzed in silico for proteolytic sites and it was found that the proline-rich linker immediately upstream of the CBD (see Fig. 1) contains a natural unique proteolytic site for endopeptidases, specifically the PKP sequence found at positions 379-381 of the mature CHITl sequence, suggesting that the enzyme can be regulated in-vivo by the action of endopeptidases.
  • the two most likely candidates peptidases are the prolyl-endopeptidases Fibroblast Activation Protein (FAP), and the Prolyl endopeptidase PREP. The cleavage presumably occurs at the C-terminal side of the PKP sequence.
  • FAP Fibroblast Activation Protein
  • PREP Prolyl endopeptidase PREP
  • the present invention provides an improved human
  • said CHIT1 containing at least one mutation in any one of the amino acids at the proline- rich linker at positions 367-395 of the mature CHIT1 enzyme which abolishes a recognition site for an endopeptidase, thereby rendering the human chitinase resistant to cleavage by endopeptidases.
  • the endopeptidase is a prolyl-endopeptidase.
  • the mutations are in any one of the amino acids in the PKP sequence at positions 379-381 of the mature CHIT1.
  • CBD chitin binding domain
  • CHIT1 includes cysteines, which in the reduced state remain as -SH (causing the enzyme to be inactive), and in an oxidative state form disulfide bridges (whereby enzyme is activated).
  • Fig. 3 shows the effect of a reducing agent on the ability of the enzyme to degrade an insoluble substrate, while the ability of the enzyme to degrade a soluble substrate is not affected. This redox switch is presumably a control element of the enzyme activity that controls the binding and release of the enzyme from insoluble chitin molecules. The inventors have demonstrated that under reduction conditions, the CBD undergoes a significant structural change that leads to its inability to bind and degrade chitin (Fig. 4).
  • This redox switch was found to have no effect on the catalytic domain of the enzyme, as its ability to degrade a soluble synthetic substrate remains intact (Fig. 3).
  • the oxidized state may occur naturally as a result of inflammation, but may be suppressed in people with compromised immune system.
  • enzymatic machinery found in humans namely the thioredoxin system found both in the cells and in the plasma, can either reduce or oxidize the chitinase enzyme, depending of the redox state of the environment (Fig. 5- 6).
  • the inventors therefore hypothesized that the natural enzyme circulates in the blood in an inactive (reduced) form, and during a fungal infection, the inflammatory conditions result in oxidative conditions that activate the CBD of the enzyme thereby activating the enzyme itself.
  • a reduction/oxidation cycle causes alternating binding and release of the enzyme from its target pathogen substrate, which amplifies its antifungal activity.
  • a redox switch is known for various proteins (e.g. tissue factor) but not for chitinases and cellulases, in which the affinity of the enzyme to the substrate is thought to be constant. This appears to be the first mechanism ever described for environment-dependent binding and release of insoluble substrate in glycosylhydrolases such as chitinases or cellulases. Usually, the binding affinity of enzymes that contain polysaccharide-binding domains (chitin-binding domain or cellulose-binding domain) to their substrates is known to be constant. Design of a redox switch-insensitive CHITl chitin-binding domain.
  • the human CHITl CBD includes six cysteines that are able to form three disulphide bridges. Three of these cysteines are found in a CXCC sequence (at positions 439-442, see Fig. 1) which is typical of enzymes which mobilize disulfide bridges (e.g. thioredoxin, glutaredoxin, or protein disulfide isomerase), and therefore at least the cysteines at positions 439, 441, or 442 are likely to participate in the redox switch.
  • CXCC sequence at positions 439-442, see Fig. 1
  • thioredoxin thioredoxin, glutaredoxin, or protein disulfide isomerase
  • the CBD also includes three additional cysteines (at positions 399, 419, and 429) which are extremely conserved across species (data now shown) and also participate in relevant disulfide bridges. Based on Fadel el al. (2016) who published the crystal structure of the full length human chitinase, the pairs of cysteines forming disulfide bridges are predicted to be at positions 399 + 419; 429 + 442; and 439 + 441 of the mature CHITl sequence shown in Fig. 1
  • cysteines pairs especially those involving positions 439, 441 or and 442 of the mature CHITl sequence with a different pair of amino acids which form a different type of interaction, such as two Serines, which form a hydrogen bond, or a positively-charged amino acid (e.g. Arginine or Lysine) and a negatively charged amino acid (such as Aspartic or Glutamic acid) that form a salt bridge
  • a positively-charged amino acid e.g. Arginine or Lysine
  • a negatively charged amino acid such as Aspartic or Glutamic acid
  • the present invention provides an improved human CHITl, said CHITl including at least one mutation in the chitin binding domain (CBD) at positions 396-445 of the mature CHI 1, thereby causing said CHIT1 to maintain a substrate- binding state regardless of the environmental redox state.
  • CBD chitin binding domain
  • the conformation of the CBD is close to the conformation of the oxidized state, independently of the redox state of the environment.
  • the chitinase enzyme is less sensitive to redox state, or is even completely redox state-insensitive, and its ability to bind insoluble substrates such as chitin is not affected by the redox state.
  • the at least one mutation includes a substitution of at least one pair of cysteines found at the CBD with a different pair of amino acids that form a different type of bond.
  • the at least one mutation includes at least two substitutions of cysteines selected from cysteines at positions 399, 419, 429, 439, 441, and 442 of the mature CHIT1 sequence. In some embodiments, at least one of the cysteines is selected from cysteines at positions 439, 441, and 442 of the mature CHIT1 sequence. In some embodiments the at least one pair of cysteines is selected from cysteine pairs at positions 399 + 419, 429 + 442, and 439 + 441.
  • the different pair of amino acids is a pair of Serines forming a hydrogen bond.
  • the different pair of amino acids is pair made from a negatively-charged amino acid such as Aspartic or Glutamic acid and a positively-charged amino acid such as Arginine or Lysine, together forming a salt bridge.
  • the present invention provides an improved human CHIT1 as described above, for use in treating fungal infections. Combination of CHIT1 and an oxidizing agent.
  • an additional way of maintaining the active state of the enzyme regardless of local redox state would be to deliver the enzyme during therapy together with a mild oxidizing agent (e.g. an electron acceptor such as oxidized vitamin C (dehydroascorbate - DHA) or oxidized glutathione (GSSG).
  • a mild oxidizing agent e.g. an electron acceptor such as oxidized vitamin C (dehydroascorbate - DHA) or oxidized glutathione (GSSG).
  • the present invention provides a combination comprising chitinase and a mild oxidizing agent such as dehydroascorbate (DHA) or oxidized glutathione (GSSG) for treating a fungal infection.
  • a mild oxidizing agent such as dehydroascorbate (DHA) or oxidized glutathione (GSSG) for treating a fungal infection.
  • the mild oxidizing agent is DHA.
  • the oxidizing agent and the chitinase are administered together. Both the oxidizing agent and the chitinase may be included in the same composition or each may be provided in a separate composition. According to some embodiments, the oxidizing agent and the chitinase are administered separately at different times.
  • the present invention provides a pharmaceutical composition for treating a fungal infection comprising the improved human CHIT1 of the invention as described above, or a combination of human chitinase with an antifungal agent or with an oxidizing agent as described above.
  • the pharmaceutical composition comprises chitinase and at least one antifungal agent.
  • the present invention provides a method for treating a fungal infection comprising administering to a subject in need the improved human CHIT1 of the invention as described above, the combination of the invention as described above, or the pharmaceutical composition of the invention as described above.
  • the method comprises administering to a subject in need the combination or the pharmaceutical composition of the invention as described above.
  • the present invention provides a method for treating a fungal infection comprising administering to a subject in need chitinase and at least one echinocandin.
  • treating refers to means of obtaining a desired physiological effect.
  • the effect may be therapeutic in terms of partially or completely curing a disease and/or symptoms attributed to the disease.
  • the term refers to inhibiting the disease, i.e. arresting its development; or ameliorating the disease, i.e. causing regression of the disease.
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers or excipients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • the following exemplification of carriers, modes of administration, dosage forms, etc. are listed as known possibilities from which the carriers, modes of administration, dosage forms, etc., may be selected for use with the present invention. Those of ordinary skill in the art will understand, however, that any given formulation and mode of administration selected should first be tested to determine that it achieves the desired results.
  • Methods of administration include, but are not limited to, intravenous, intraperitoneal, or intramuscular.
  • the pharmaceutical composition is adapted for intravenous administration.
  • carrier in the context of a pharmaceutical composition refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered.
  • the carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.
  • a binder such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate
  • a disintegrating agent such as alginic acid, maize starch and the like
  • compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.
  • the exact dosage for use will be determined by the practitioner, based on factors related to the subject, such as weight and the severity of the infection.
  • Caspofungin acetate (Cat no. SML0425) was purchased from Sigma-Aldrich and Anidulafungin (LY303366, Cat no. S4286) was purchased from Selleckchem. Species of fungi were obtained as follows: all C. albicans strains (ATCC 18804, ATCC MYA-2876, ATCC 76485) and the Aspergillus fumigatus strain (ATCC MYA-4609) were purchased from the ATCC collection. Two C. Parapsilosis (1, 2), and two C. Glabrata (1, 2) are clinical isolates from lab collections.
  • CHITl Human coding sequence for CHITl was optimized for expression in E. coli and cloned into pET expression system. Recombinant CHITl was expressed in E. coli cells and purified from inclusion bodies by oxidative refolding techniques, essentially as described in Moghadam et al., 2015, Refolding process of cysteine-rich proteins :Chitinase as a model . Reports of Biochemistry & Molecular Biology, 4(1), 19-24.
  • the activity of the catalytic domain of the recombinant human chitinase (CHITl) was determined by its ability to degrade the soluble synthetic substrate 4-Methylumbelliferyl ⁇ -D- ⁇ , ⁇ ', ⁇ ''-triacetylchitotriose (Sigma cat. no. N8638).
  • CBD chitin-binding domain
  • Degradation of colloidal chitin was used to determine the activity of the chitin-binding domain (CBD) of the enzyme, as its ability to bind and degrade insoluble chitin depends on a functional CBD.
  • Enzyme was added to a final concentration of 50 ⁇ g/ml and the plates were incubated at 37°C under agitation for several hours as indicated. Incubation was done in a tecan infinite m200 plate reader, and turbidity was determined automatically every 15 min by 600nm absorbance spectra.
  • NADP + , dehydroascorbate (DHA), or oxidaized glutathione (GSSG) were added when indicated to a final concentration of 2mM.
  • the solution was further diluted -1:2000 into the assay growth medium (RPMI supplemented with glutamine and MOPS buffer) to a final concentration of about 500-2000 fungal cells per ml.
  • the cell suspensions were divided into 13ml culture tubes (1ml in each tube) or 24 wells plates (with same results), caspofungin was added in doubling dilutions, and enzyme (CHIT1) was added to a final concentration of 50 ⁇ g/ml where indicated. All tubes were incubated for 48 hours at 35°C before turbidity (OD 600nm) was recorded. Minimal inhibitory concentration was determined as the lowest concentration of caspofungin in which the final turbidity was lower than 10% of the control tube (fungi only).
  • albicans ATCC MYA-2876 and tested the activity of the human chitinase with combination of caspofungin on both the yeast form of the strain and the hyphae form of the strain (differentiated by incubation of the fungi in the presence of 20% serum at 37°C for 24h). Both the yeast form and the hyphae forms demonstrated similar sensitivity to the human chitinase, reducing the minimal inhibitory concentration (MIC) of caspofungin by -130 fold. From Table 2 it can be seen that concentrations of CHIT1 as low as 5 ⁇ g/ml still had the same effect.
  • MIC minimal inhibitory concentration
  • MEC minimal effective concentration
  • Fig. 1 shows the sequence of CHIT1, with the proline rich linker between the catalytic domain and the CBD (in italics).
  • the sequence of the enzyme was analyzed for proteolytic sites in-silico , using "PeptideCutter” software at the Expasy bioinformatics portal.
  • the analysis has revealed that the proline-rich linker contains a natural unique proteolysis site for proline- endopeptidases at position 381 of the mature enzyme, with the predicted recognition sequence for prolyl endopeptidase being PKP (proline-lysine-proline), highlighted in bold at positions 379-381 (Fig. 1).
  • the predicted proteolysis site is after the second (c-terminal) proline.
  • Example 3 Degradation of an insoluble substrate (colloidal chitin) by a recombinant human chitinase.
  • the recombinant human chitinase was obtained by expression of the protein in E. coli ER2566 followed by inclusion-bodies separation and oxidative protein refolding as previously described (Nelson et al., 2014), and incubated with insoluble chitin molecules.
  • the ability of the chitinase to degrade the insoluble substrate was determined by the decline in turbidity indicating degradation of large insoluble molecules into small soluble products.
  • the ability of the enzyme to degrade chitin is dependent on a functional Chitin-Binding Domain (CBD) as a mutant enzyme lacking CBD in unable to degrade insoluble chitin, while its ability to degrade the soluble (fluorogenic) substrate was not affected (Figs. 2A-2B).
  • CBD Chitin-Binding Domain
  • Degradation of insoluble chitin but not of soluble substrate is controlled by the redox state of the environment. Incubation of the enzyme with growing concentration of the reducing agent DTT (up to 5mM) inhibits the ability of the enzyme to degrade insoluble chitin, but not soluble substrate (Figs. 3A-3B).
  • Example 4 The structure of the human chitinase CBD, is changed by the redox state of the environment.
  • Example 5 The human thioredoxin system can reduce the human chitinase and abolish its ability to degrade insoluble chitin.
  • thioredoxin system contains a thioredoxin enzyme that can reduce disulphide bonds on substrate proteins.
  • the oxidized thioredoxin is recycled (reduced) by an additional enzyme named thioredoxin reductase, which uses NADPH as an electron donor.
  • TRX The thioredoxin system
  • TRX can reduce the human enzyme, abolishing its ability to degrade insoluble chitin (Fig. 5).
  • Reactivation of the enzyme can be achieved by changing the balance of the redox state by changing the balanced between NAPDH and NADP + in the environment into more oxidizing conditions.
  • Renkema GH Boot RG, Strijland A, et al. Synthesis, sorting, and processing into distinct isoforms of human macrophage chitotriosidase. Eur J Biochem. 1997;244:279-285.

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Abstract

La présente invention concerne des associations comprenant de la chitinase et au moins une échinocandine destinées à être utilisées en traitement d'une infection fongique, et des procédés pour le traitement d'infections fongiques par administration de compositions comprenant de la chitinase et au moins une échinocandine.
PCT/IL2017/051171 2016-10-27 2017-10-26 Polythérapies comprenant une chitinase humaine (chit1) pour le traitement d'une infection fongique systémique Ceased WO2018078626A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025108507A1 (fr) * 2023-11-25 2025-05-30 Bioinova, A.S. Chitinase acide humaine mutée à usage médical

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034781A2 (fr) * 1999-11-12 2001-05-17 Biomarin Pharmaceuticals Enzymes lytiques utiles pour le traitement des infections fongiques
US6372212B1 (en) * 1996-06-14 2002-04-16 Icos Corporation Chitinase materials and methods

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372212B1 (en) * 1996-06-14 2002-04-16 Icos Corporation Chitinase materials and methods
WO2001034781A2 (fr) * 1999-11-12 2001-05-17 Biomarin Pharmaceuticals Enzymes lytiques utiles pour le traitement des infections fongiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VERWER P.: "Chitinases in Invasive Fungal Infections: Novel diagnostic and therapeutic approaches", DISS. ERASMUS MC : UNIVERSITY MEDICAL CENTER ROTTERDAM, 8 June 2016 (2016-06-08), XP055480039, Retrieved from the Internet <URL:https://repub.eur.nl/pub/80125> *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025108507A1 (fr) * 2023-11-25 2025-05-30 Bioinova, A.S. Chitinase acide humaine mutée à usage médical

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