Patent Application
20240415940
The present invention relates to a hyaluronidase peptide for use in the treatment or prophylaxis of a neurodegenerative disease and/or a dysfunctional blood-brain barrier associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis, a pharmaceutical composition for use in the treatment or prophylaxis of the neurodegenerative disease, and a method of treating subjects exhibiting the respective neurodegenerative diseases and/or dysfunctional blood-brain barrier.
Hyaluronic acid is an essential component of the extracellular matrix and a quantitatively significant constituent of the interstitial barrier.
Hyaluronic acid is a large, negatively charged, unbranched polymer consisting of repeated disaccharides of glucuronic acid and N-acetylglucosamine. It supports the activity of neuronal voltage-dependent Ca2+ channels of the L-type.
Hyaluronic acid, a central extracellular matrix (ECM) glycosaminoglycan, is represented in neuronal cells by a class synthesized from integral membrane proteins. It is transported through the cell membrane in the extracellular space, where it serves as a framework for the cohesion of the ECM.
The glycosaminoglycan hyaluronan (HA), the scaffolding base of the extracellular matrix, is attached to the regulation of neuronal differentiation, survival, proliferation, migration and cell signaling in central nervous system (CNS). HA is found throughout the CNS as part of proteoglycans, especially in perineural networks, which regulate neuronal activity. HA also occurs in the white matter, where it is diffusely distributed around astrocytes and oligodendrocytes.
Hyaluronidase is a hydrolytic enzyme that cleaves hyaluronic acid in D-glucuronic acid and N-acetyl glucosamine, increasing the permeability of the interstitial matrix. Hyaluronidase is widely distributed in nature. In the human, six different hyaluronidases, HYAL1-4, HYAL-P1 and PH-20, have been identified, wherein PH-20 is regarded to exert the strongest biologic activity. Today, animal-derived bovine or ovine testicular hyaluronidases as well as synthetic hyaluronidases are clinically applied as adjuncts to increase the bioavailability of drugs, for the therapy of extravasations, or for the management of complications associated with the aesthetic injection of hyaluronic acid-based fillers.
While hyaluronidase derived from animal origin imparts a risk of transmitting animal diseases, such spongiform encephalopathy, human and bacterial recombinant hyaluronidase exhibit a higher purity, which reduces pharmaceutical risks.
In order to meet the clinical need, it is an aim of the present invention to provide a hyaluronidase polypeptide that is suitable for pharmaceutical application and in particular exhibits a suitable high purity degree, and/or a suitable specific activity, suitable stability and solubility and at the same time exhibits a time and cost effective production process.
The aforementioned aim is solved at least in part by means of the claimed inventive subject matter. Advantages (preferred embodiments) are set out in the detailed description hereinafter and/or the accompanying figures as well as in the dependent claims.
Accordingly, a first aspect of the invention relates to a hyaluronidase peptide for use in the treatment or prophylaxis of a neurodegenerative disease associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis or a dysfunctional blood-brain barrier.
A second aspect of the present invention relates to a pharmaceutical composition for use in the treatment or prophylaxis of a neurodegenerative disease associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis, characterized in that the pharmaceutical composition comprises the hyaluronidase peptide according to the first inventive aspect in a therapeutically effective amount and one or more pharmaceutically acceptable excipients or a dysfunctional blood-brain barrier.
A method of treating a neurodegenerative disease and/or a dysfunctional blood-brain barrier associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis disease comprising or consisting of administering the hyaluronidase peptide according to the first inventive aspect, or the pharmaceutical composition according to the second inventive aspect to a subject in need thereof.
The inventive aspects of the present invention as disclosed hereinbefore can comprise any possible (sub-) combination of the inventive aspects and preferred inventive embodiments thereof as set out in the dependent claims or as disclosed in the following detailed description and/or in the accompanying figures, provided the resulting combination of features is reasonable to a person skilled in the art.
Further characteristics and advantages of the present invention will ensue from the accompanying drawings, wherein
As set out in more detail hereinafter, the inventors of the different aspects of the present invention have found out that hyaluronidase may be used in the treatment or prophylaxis of a neurodegenerative disease and/or a dysfunctional blood brain barrier associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis, can lead to an accumulation of gycosaminoglycans, such as chondroitin sulphate (CS) and hyaluronic acid, in the extracellular matrix (ECM) and/or the synaptic cleft of the central nervous system (CNS). Furthermore, hyaluronidase may be used to regenerate a dysfunctional blood-brain barrier.
These glycosaminoglycans are either organized as diffuse or condensed ECM, wherein the diffuse ECM is distributed throughout the CNS and fills the perisynaptic spaces, while the condensed ECM selectively surrounds parvalbumin expressing inhibitory neurons (PV cells) as network-like structures so called perineural networks (PNN). The cerebral ECM functions as a non-specific physical barrier that protects the neural plasticity and modulates axon regeneration.
In particular in case hyaluronic acid is present in the ECM or other physiologic systems, such as the blood-brain barrier, the long chain molecule of hyaluronic acid generally forms a three dimensional molecular sieve using nanogaps. Short and middle chain hyaluronic acid fragments as degradation products may block such nanogaps and may decrease the respective function of the ECM or the blood brain barrier.
An increased accumulation of proteoglycans, in particular hyaluronic acid and its fragments may also be due to the aforementioned diseases and, thus, can significantly deteriorate the function of the plasticity of the ECM and the synaptic plasticity of the synaptic cleft and/or the blood-brain barrier.
A reduction of these increased proteoglycan levels by the inventive use of hyaluronidase, thus, can assists restoration of the homeostasis of the constituents, which is significant for the functionality of the ECM and/or the synaptic cleft and/or blood-brain barrier.
As an example, hyaluronidase may be used according to the present invention in Alzheimer's disease in order to “de-camouflage” amyloid ß-peptide, which is deposited in the ECM due to the increased hyaluronan levels. Hyaluronan level is in particular elevated in the grey matter of patients with Alzheimer's disease (AD). According to the present invention, the hyaluronan depository is lysed due to the inventive use of hyaluronidase. Accordingly, e.g., the amyloid ß-peptide gets again accessible to physiological cerebral self-repair and transport processes so that the amyloid ß-peptide can be removed from the ECM.
Furthermore, hyaluronan level is in particular elevated in the white substance from patients with vascular dementia. A lysis of the increased hyaluronan level may improve the cognitive performances of a vascular dementia patient.
Alternatively, hyaluronidase is according to the present invention also applicable for treatment or prophylaxis of Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis, which all show an increased glycosaminoglycan level, in particular hyaluronan level in the respectively degenerated or inflamed areas.
In case of a dysfunctional blood-brain barrier, the use of hyaluronidase may regenerate the endothelial glyclocalix by further degradation of the short and middle chamin hyaluronic acid fragments so that the bidirectional function of the blood-brain barrier is regenerated.
When lysing high molecular weight glycosaminoglycan, in particular hyaluronan into smaller fragments, so called hyaluronan cleavage products, these fragments can have unique biological activities, such a relating to regulation of precursor cell differentiation and proliferation. Accordingly, the inventive use of hyaluronidase can open up a new path to promote repair of central nervous system injuries/damages.
Demyelising diseases such as multiple sclerosis, and autoimmune encephalomyelitis, have also been shown to increase hyaluronan content in the lesions in question. The lysis of hyaluronan may improve the physiological signaling performances. In other words, remyelination may be promoted through hydrolytic degradation of inhibitory hyaluronic acid and, thus, reversing the blockade of oligodendrocyte maturation.
Thus, the inventive use of hyaluronidase for the treatment or prophylaxis of neurodegenerative diseases facilitates transformation of the neurodegenerative deformed ECM/synaptic cleft back to a degree of increased plasticity of the ECM and/or the synaptic cleft and/or blood-brain barrier, which in turn facilitates retaining physiological processes, formation of new synapses, increased lateral diffusion, synaptic exchange of receptors, and/or cognitive performances. The modifiability of synaptic connections, is considered a decisive prerequisite for learning and memory.
Thus, according to the first inventive aspect a hyaluronidase peptide for use in the treatment or prophylaxis of a neurodegenerative disease associated with an increased cerebral glycosaminoglycan level is provided, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis.
According to an additional or alternative embodiment, the inventive hyaluronidase peptide is selected from a bacterial hyaluronidase.
According to a further additional or alternative embodiment, the inventive hyaluronidase peptide is a modified bacterial hyaluronidase comprising or consisting of at least 90% sequence identity to SEQ ID No. 1, SEQ ID No. 9 or SEQ ID No 10. The inventive hyaluronidase peptide may comprise a C-terminal HIS tag of SEQ ID No. 7 and/or an N-terminal Strep tag of SEQ ID No. 5. Alternatively, the hyaluronidase may be free of a C-terminal HIS tag of SEQ ID No. 7 and/or an N-terminal Strep tag of SEQ ID No. 5. According to a further additional or alternative embodiment, the inventive hyaluronidase peptide is a modified bacterial hyaluronidase comprising or consisting of at least 90% sequence identity to SEQ ID Nos. 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
According to a further additional or alternative embodiment, the inventive hyaluronidase peptide is a modified hyaluronidase peptide with increased lipophilic constituents, and/or increased positively charged constituents, and/or an amino acid sequence binding to at least part of a blood-brain barrier transporter system, such as to large neutral amino acid receptor, in particular L-type Amino acid transporter (LAT1), and/or with an amino acid sequence binding to at least part of a transferrin receptor, such as transferrin receptor 1 or transferrin receptor 2.
According to a further additional or alternative embodiment, the inventive hyaluronidase peptide is simultaneously or sequentially administered with one or more blood-brain barrier efflux inhibitory agents, preferably selected from the group consisting of p-glycoprotein inhibitory agents, such as Cyclosporine or a Cyclosporine derivative, such as Valspodar, Elacridar, Zosuquidar; calcium antagonistic agents, such as Verapamil; procyanidine or procyanidine derivatives; etc., In case of sequential administration, the blood-brain barrier efflux inhibitory agent is administered prior to the hyaluronidase peptide.
According to a further additional or alternative embodiment, the inventive modified bacterial hyaluronidase polypeptide comprises or consists of at least 90% sequence identity to SEQ ID No. 1, SEQ ID No. 9 or SEQ ID No. 10, characterized in that the hyaluronidase polypeptide exhibits a high purity of >95% or >98.8% (see example section 1.4.1) and a high Specific Activity of 1,500,000 U/mg (see example section 1.4.2). The inventive modified bacterial hyaluronidase polypeptides of SEQ ID No. 9 or 10 may respectively include one or two remaining residues of a C-terminal and/or N-terminal tag.
In addition or alternatively, the inventive modified bacterial hyaluronidase polypeptide comprises or consists of at least 90% sequence identity to SEQ ID Nos. 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
In contrast thereto, comparative hyaluronidases, such as bovine hyaluronidases exhibit a wide range of lower Specific Activities, namely in the range of 300 to 15,000 U/mg (see example section 2.3). PH20, regarded as the most active out of the human hyaluronidases, also exhibits a lower Specific Activity, namely in the range of 40,000 and 50,000 U/mg. The Specific Activity of bacterial hyaluronidase derived from Streptomyces koganeiensis is comparable to the Specific Activity of PH20 and, thus, is also lower than the Specific Activity of the inventive modified bacterial hyaluronidase.
Moreover, the inventive modified bacterial hyaluronidase exhibits suitable stability and solubility, which is shown in example section 1.4.5 below. The increased stability, including stability against (exo-) peptidases (half life) may be due to the use of the respective C-terminal HIS tag of SEQ ID No. 7 and/or the respective N-terminal Strep tag of SEQ ID No. 5. The respective tags may also increase the solubility of the inventive modified bacterial hyaluronidase in comparison to the wild-type hyaluronidase (see SEQ ID No. 3, DNA encoding wild-type hyaluronidase see SEQ ID. No. 4). Due to the increased stability and solubility properties, the inventive modified bacterial hyaluronidase is preferred for formulating pharmaceutical compositions, in particular parenteral injection compositions.
In the context of the present invention, the expression “inventive modified bacterial hyaluronidase polypeptide comprising or consisting of at least 90% sequence identity to SEQ ID No. 1” means that the inventive modified bacterial hyaluronidase of SEQ ID No. 1 comprises or consists of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%, of the sequence of SEQ ID No. 1. In case that the sequence of the inventive hyaluronidase consists of 100% of the SEQ ID No. 1, the inventive hyaluronidase is also synonymously called “d016tag”. In the context of the present invention, the expression “inventive modified bacterial hyaluronidase polypeptide comprising or consisting of at least 90% sequence identity to SEQ ID No. 9 or SEQ ID No. 10” means that the inventive modified bacterial hyaluronidase of SEQ ID No. 9 or SEQ ID No. 10 respectively comprises or consists of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%, of the sequence of SEQ ID No. 9 or 10. In the context of the present invention, the expression “inventive modified bacterial hyaluronidase polypeptide comprising or consisting of at least 90% sequence identity of any one of SEQ ID Nos. 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38” means that the inventive modified bacterial hyaluronidase of any one of SEQ ID Nos. 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 comprises or consists of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%, of the sequence of SEQ ID Nos. 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
The inventive modified bacterial hyaluronidase polypeptide may comprise or may be free of a C-terminal HIS tag of SEQ ID No. 7 and/or an N-terminal Strep tag of SEQ ID No. 5. Optionally, the SEQ ID No. 9 or SEQ ID No. 10 respectively comprise one or two remaining residues of a previously comprised C-terminal and/or N-terminal tag. A sequence of the inventive hyaluronidase consists of SEQ ID Nos. 9, 10, 20 and 30 including no, one, two or three amino acid residues of the HIS tag and no, one, two, three, or more residues of the Strep tag is also synonymously called “d016”. A sequence of the inventive hyaluronidase of at least 90% sequence identity of SEQ ID Nos. 22, 24, 26, 28, 32, 34, 36, or 38 is also synonymously called “d016 variant”. The inventive modified bacterial hyaluronidase polypeptide is preferably suitably adapted for passing the blood-brain barrier and/or is simultaneously or sequentially applied with active ingredients increasing the blood-brain barrier efflux inhibitory agents. For passing the blood-brain barrier, preferably smaller hyaluronidase sequences are administered, such as preferably SEQ ID Nos. 22, 24, 26, 28, 32, 34, 36, or 38.
Thus, the inventive modified bacterial hyaluronidase, preferably d016tag or d016 or d016 variants or their respective adaptions to increase passing the blood-brain barrier, is suitable for use in the inventive pharmaceutical composition according to the second inventive aspect.
In view of the presented comparatively high Specific Activity, purity, stability, solubility and safety profiles, the inventive modified bacterial hyaluronidase, preferably d016tag or d016 or d016 variant or their respective adaptions to increase passing the blood-brain barrier is suitable for use in the treatment or prophylaxis of the neurodegenerative diseases disclosed above and/or a dysfunctional blood-brain barrier.
In addition, the present invention discloses a nucleic acid encoding the inventive modified bacterial hyaluronidase polypeptide of the first aspect. The nucleic acid preferably comprises or consists of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% sequence identity to SEQ ID No. 2. The nucleic acid sequence encoding the C-terminal HIS tag of the inventive hyaluronidase is preferably SEQ ID No. 8 and the nucleic acid sequence encoding the N-terminal Strep tag of the inventive hyaluronidase is preferably SEQ ID No. 6. The nucleic acid may be prepared according to any suitable method. An example method is described in the example section 1.1.1 below. Alternatively, the nucleic acid encoding the inventive modified bacterial hyaluronidase polypeptide may comprise or consist of at least comprises or consists of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% sequence identity to SEQ ID Nos. 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
In the context of the present application, the expression “nucleic acid” refers to a nucleic acid encoding the inventive modified bacterial hyaluronidase, preferably d016tag or d016 or d016 variant. The nucleic acid preferably comprises or consists of at least 90% sequence identity to SEQ ID. 2, preferably the inventive nucleic acid consists of 100% SEQ ID No. 2.
In addition, the present invention discloses a recombinant expression vector comprising the nucleic acid encoding the inventive bacterial modified hyaluronidase of at least 90% sequence identity to SEQ ID Nos. 1, 9, 10, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38. The recombinant expression vector may be prepared according to any suitable method. An example method is described in example section 1.1.1 below. The recombinant expression vector comprises the nucleic acid comprising or consisting of at least 90% sequence identity of SEQ ID Nos. 2, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, any suitable vector, such as a pET-28a DNA vector, in particular pET28a using NcoI/BlpI restriction sites (N-terminal cloning sequence NcoI, e.g. SEQ ID No. 13, skipped sequence in sequence listing consisting of CCATGGGC, and C-terminal cloning sequence BlpI, e.g. SEQ ID No. 14, skipped sequence in sequence listing consisting of GCTGAGC, for the respective expression vectors). After removing the C-terminal purification tag, such as the HIS tag, the resulting inventive modified bacterial hyaluronidase may comprise the N-terminal purification tag, e.g. such as SEQ ID No. 11, and remaining C-terminal cleavage residues, e.g. of SEQ ID No. 12). The inventive recombinant expression vector may be of any suitable form, such as in form of a plasmid.
In addition, the present invention discloses a host cell transformed with the recombinant expression vector comprising the nucleic acid encoding the inventive bacterial modified hyaluronidase of SEQ ID No. 1. The host cell may be prepared according to any suitable method. An example method is described in example section 1.1.2 below. The inventive host cell may be selected from any suitable host cells. Preferably, the host cell is selected from E. coli cells, more preferably E. coli BL21 (DE3) competent cells, which provide a comparatively high yield of inventive modified bacterial hyaluronidase, preferably d016tag or d016 or d016 variant.
In addition, the present invention discloses a process of production of a purified inventive bacterial hyaluronidase polypeptide (see also
The production process comprises or consists of the following steps:
Step a) Culturing a transformed host cell according to the fourth inventive aspect in a suitable growth medium under suitable growth conditions to express the inventive modified bacterial hyaluronidase polypeptide according to the first inventive aspect. As an example, terrific broth (TB) media can be used. The respective broth media may be supplemented by suitable antibiotics, such as kanamycin, and/or buffer constituents, such as potassium phosphate buffer. The growth media also comprises a suitable expression inducer, such as Isopropyl β-D-1-thiogalactopyranoside (IPTG). According to laboratory scale, cell growth and protein expression may be conducted in a shaker culture flask at a suitable temperature, preferably at 28 to 30° C. and shaking, preferably 180 rpm, for 18 to 20 hours.
Step b) Harvesting the cultured transformed host cell of step a). After culturing the transformed host cells under suitable growth conditions, the host cells are harvested with suitable methods. According to laboratory scale, preferably pyrogen-free, sterile tubes are used to collect the harvested host cell containing growth media. This harvested growth medium is preferably centrifuged, preferably at 4 000 rcf at a reduced temperature, preferably 4° C., for at least 30 minutes in order to separate the medium from the host cells, which aggregate to so called pellets after centrifugation. The supernatant is to be discarded and the harvesting tubes are preferably sealed and stored at reduced temperature, preferably below 0° C., more preferably at −80° C.
Step c) Lysing the harvested host cells of step b) and separating resulting host cell fragments from resulting host cell content comprising the inventive bacterial hyaluronidase polypeptide. According to laboratory scale, the host cells, which after centrifugation form aggregated pellets, are generally resuspended and mixed, preferably by vortexing, in suitable medium, preferably a suitable buffer medium, such as phosphate buffered saline (PBS; containing 280 mM NaCl, 6 mM KCl, 15.1 mM Na2HPO4, 4.9 mM NaH2PO4, PH=7.4 at room temperature). The host cells are lysed by any suitable methods, such as sonication. In order to maintain suitable temperatures within the medium, the sonication takes preferably place at reduced temperatures, more preferably wherein the tubes are surrounded by ice while sonicating the medium. The resulting host cell fragments are separated from the inventive bacterial hyaluronidase polypeptide with suitable methods, preferably by centrifugation, more preferably centrifugation at reduced temperature, e.g. 4° C., for at least 30 minutes at e.g. 4 000 rcf. The supernatant comprising the inventive bacterial hyaluronidase is preferably transferred to a new tube and optionally one or more centrifugation steps are further conducted. The resulting supernatant comprising the inventive bacterial hyaluronidase is then used for the purification step d).
Step d) Purifying the resulting host cell content of step c) with HIS affinity chromatography and STREP affinity chromatography to result in a purified inventive bacterial hyaluronidase polypeptide of the first aspect. Preferably the HIS- and STREP affinity chromatography takes place subsequently, wherein the order is interchangeable, i.e, wherein the HIS affinity chromatography purification is conducted first followed by STREP affinity chromatography or vice versa. In the following the subsequent purification conducting first HIS affinity chromatography and then STREP affinity chromatography is described as one example embodiment. In view that HIS affinity chromatography columns are generally cheaper than STREP affinity chromatography columns, the high purity yield may be achieved in a more cost effective way in case the HIS affinity chromatography is conducted first.
According to the present disclosure, any suitable HIS affinity chromatography can be used in order to bind to the C-terminal HIS tag (amino acids 735 to 740 of SEQ ID NO. 1, see also SEQ ID NO. 7) of the inventive modified bacterial hyaluronidase. According to laboratory scale, as an example one or more suitable gravitational HIS-purification columns are equilibrated to general PBS downstream buffer. The content of each tube resulting after step c) is distributed to the one or more HIS-columns. Optionally the HIS purification procedure is repeated one, two or more times, preferably two times. The loaded one or more HIS-columns are then preferably washed with a suitable washing medium, e.g. with 10 mM imidazole PBS solution, before being eluted in a suitable eluting medium, e.g. 150 mM imidazole PBS solution. Generally, the eluate of the one or more HIS-columns originating from the same production flask (tube) are combined and preferably diluted to 35 ml with general downstream buffer PBS. Preferably, the purified eluate samples comprising the inventive modified bacterial hyaluronidase is then stored under reduced temperature, preferably on ice until the following STREP purification.
According to the present disclosure, any suitable STREP affinity chromatography can be used in order to bind to the N-terminal STREP tag (amino acids 3 to 10 of SEQ ID NO. 1, see also SEQ ID NO. 5) of the inventive modified bacterial hyaluronidase. According to laboratory scale, one or more suitable syringe-based STREP affinity chromatographic columns can preferably be used. As an example, one or more syringe-based STREP-purification steps, more preferably with a flowrate <5 ml/min, are performed. STREP-columns (5 ml bed volume) are generally washed and equilibrated with 2×25 ml general downstream buffer PBS. A suitable amount of the eluate sample resulting from the HIS purification is applied and runs through the STREP column. The loaded STREP column is then preferably washed with a suitable amount of general downstream PBS buffer to remove any remaining contaminant proteins. By applying a suitable eluting medium, e.g. 2.5 mM d-Desthiobiotin containing PBS buffer, the protein of interest, namely the inventive modified bacterial hyaluronidase, is eluted, preferably into a fresh, sterile and pyrogen-free tube. This tube is either stored at reduced temperature, e.g. on ice, or the lyophilized to result in a dry storable product of the purified inventive modified bacterial hyaluronidase. The STREP column can be reused after suitable regeneration according to the prior art.
Optionally, the buffer of the eluate may be exchanged by suitable methods including centrifugation of the eluate to aggregate the inventive bacterial hyaluronidase, discarding the supernatant and resuspending the aggregated inventive bacterial hyaluronidase into a different buffer medium, such as Tris-HCL NaCl. According to a preferred embodiment the resuspended inventive bacterial hyaluronidase is stored under reduced temperature, e.g. on ice for further post processing, such as polishing.
According to a preferred embodiment, the inventive purification step additionally comprises one or more suitable polishing steps to remove last impurities of the inventive bacterial hyaluronidase polypeptide in step d) and, thus, to increase purity thereof. According to laboratory scale, one or more endotoxin removal steps, such as Polymyxin B based endotoxin removal steps; one or more sterile filtration steps, and one or more particle removal steps can be performed.
The laboratory scale protein yield results in 0.09 to 0.13 mg/ml after HIS purification and 0.04 to 0.06 mg/ml after dual HIS/STREP purification and subsequent polishing purification step (see example section item 1.3.4 below). It is expected that this yield significantly increases upon fermentation scale production.
In addition, a modified bacterial hyaluronidase polypeptide consisting of an adapted SEQ ID No. 1 which comprises suitable enzymatic or non-enzymatic cleavage sites, such as protease cleavage sites, e.g. SEQ ID No. 16 or 18 (encoding DNA sequence SEQ ID No. 15 or 17 respectively) for subsequent removal of the STREP-tag and/or removal of the HIS-tag. In case both tags are subsequently removed from this modified bacterial hyaluronidase polypeptide, the inventive modified bacterial hyaluronidase polypeptide of SEQ ID Nos. 9, 10, at least 90% sequence identity to SEQ ID Nos. 1, 9, 10, 30, 32, 34, 36, or 38 is produced.
If removal of the HIS-Tag and/or STREP-Tag, preferably only HIS-Tag is necessary, specific suitable recognition sites are incorporated to allow subsequent enzymatic cleavage using suitable enzymes, such as Carboxapeptidase A for removal of C-terminal HIS-Tags. Alternatively, suitable non-enzymatic sequence-specific nickel-assisted cleavage (SNAC)-tag may be used (see Dang, B., Mravic, M., Hu, H. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat Methods 16, 319-322 (2019). https://doi.org/10.1038/s41592-019-0357-3).
According to the present invention, a modified bacterial hyaluronidase polypeptide, preferably wherein the inventive modified bacterial hyaluronidase polypeptide consists of at least 90% sequence identity to SEQ ID Nos. 1, 9, 10, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 is obtainable according to the production process as disclosed above.
All features of the different embodiments of the first inventive aspect are combinable with each other as long as the resulting feature combination is reasonable to a skilled person.
According to the second inventive aspect a pharmaceutical composition for use in the treatment or prophylaxis of a neurodegenerative disease and/or a dysfunctional blood-brain barrier associated with an increased cerebral glycosaminoglycan level is provided, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis, characterized in that the pharmaceutical composition comprises the hyaluronidase peptide according to the first inventive aspect in a therapeutically effective amount and one or more pharmaceutically acceptable excipients.
According to an additional or alternative embodiment, the inventive the pharmaceutical composition forms an intranasal formulation; an intracerebral formulation, in particular an intraventricular or an intrathecal formulation; or a parenteral formulation. According to a further additional or alternative embodiment, the inventive pharmaceutical composition forms an oral formulation, a rectal formulation or a sub-cutaneous formulation, wherein the hyaluronidase is adapted to pass the blood-brain barrier.
According to a further additional or alternative embodiment, the inventive pharmaceutical composition is selected from a solid, semi-solid or liquid application form.
All features of the different embodiments of the first inventive aspect are combinable with each other as long as the resulting feature combination is reasonable to a skilled person.
In general, the therapeutically effective amount of the inventive hyaluronidase, in particular the inventive bacterial hyaluronidase depends on the therapeutic application of the pharmaceutical composition. According to the present invention, the term “therapeutically active amount” means that the amount of inventive hyaluronidase peptide, in particular the inventive modified bacterial hyaluronidase polypeptide, preferably the inventive modified bacterial hyaluronidase polypeptide with or without tags, preferably comprising or consisting of at least 90% sequence identity to SEQ ID Nos. 1, 9, 10, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 in the pharmaceutical composition or in a pharmaceutical unit dose thereof is suitable for treatment or prophylaxis of the neurodegenerative diseases.
According to the present invention, the dosage regimen for the aforementioned diseases, and in particular for ALS and/or multiple sclerosis, may be a high dose regimen, wherein hyaluronidase polypeptide may be applied in an amount of 200 U per kg/per day to 30,000 U per kg/per day. As an example 10,000 U hyaluronidase or more, in particular 15,000 U hyaluronidase may respectively applied once a day or may be applied twice a day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive days, wherein in case the treatment is conducted for 2 or more days, the treatment may be interrupted by 1, 2, or 3 days. Using such a dosage scheme, the regeneration of the physiological state of the neurodegenerative diseases and/or the dysfunctional blood brain barrier may be improved. Furthermore, when comparing the application of bovine hyaluronidase over the application of inventively modified bacterial hyaluronidase, it may follow that the inventively modified bacterial hyaluronidase may be applied in a lower dosage due to its increased specific activity and/or its optimized blood-brain-barrier crossing. The unit dose of the inventive pharmaceutical composition may comprise the modified bacterial hyaluronidase polypeptide in an amount of 100 U per kg/per day to 30,000 U per kg/per day. In order to increase the half-life of the inventive bacterial hyaluronidase, the dosing scheme may preferably comprise a suitable bolus amount of the inventive hyaluronidase in order to saturate the exo- and/or endo-proteinases followed by a subsequent administration of the therapeutically effective unit dose amount. Preferably, in case of intravenous administration, the subsequent unit dose is administered up to 1 hour, alternatively up to 30 minutes or up to 15 minutes or up to 5 minutes after the bolus administration of the inventive bacterial hyaluronidase. Alternatively, in case the inventive modified bacterial hyaluronidase sequences shall cross the blood-brain barrier, the hyaluronidase may be administered in two consecutive applications, wherein the second unit dose is administered up to 12 hours, preferably 1 to 6 hours after the first administration of the inventive modified bacterial hyaluronidase. More preferably, the unit dose may comprise 10,000 U, 11,000 U, 12,000 U, 13,000 U, 14,000 U, 15,000 U or more inventive modified bacterial hyaluronidase. It is presently believed, that the first administration of the inventively obtainable/inventive hyaluronidase, in particular of a high dose of inventively obtainable/inventive hyaluronidase may condition the blood-brain barrier in such a way that it the second subsequent administration of hyaluronidase shows an increased uptake.
According to the third inventive aspect, a method of treating a neurodegenerative disease and/or a dysfunctional blood-brain barrier associated with an increased cerebral glucosaminoglucan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis comprising or consisting of administering the hyaluronidase peptide according to the first inventive aspect, or the pharmaceutical composition according to the second inventive aspect to a subject in need thereof.
The present invention is described in the following on the basis of exemplary embodiments, which merely serve as examples and which shall not limit the scope of the present protective right.
Further characteristics and advantages of the present invention will ensue from the following description of example embodiments of the inventive aspects with reference to the accompanying drawings.
All of the features disclosed hereinafter with respect to the example embodiments and/or the accompanying figures can alone or in any sub-combination be combined with features of the aspects of the present invention including features of preferred embodiments thereof, provided the resulting feature combination is reasonable to a person skilled in the art.
1.1.1 Gene Synthesis and Subcloning—pET28a(+)
The d016tag gene sequence of SEQ ID No. 2 encoding the inventive modified bacterial hyaluronidase of SEQ ID No. 1 was designed by truncation of the wildtype sequence of Streptococcus pneumonia of SEQ ID No. 4 and the addition of C-terminal HIS-Tag gene sequence of SEQ ID No. 8 and N-terminal Strep-Tag gene sequence of SEQ ID No. 6 for a two-step affinity chromatography purification. By design, both tags were designated to remain on the final enzyme product for increased solubility and protection against (exo-) peptidases in the vascular space, which is the designated drug compartment. Synthesis of the gene construct, cloning into pET28a using NcoI/BlpI restriction sites leads to a recombinant expression vector (syn.: plasmid comprising pET28a (+) vector including d016tag insert). The Vector Map Plasmid of pET28a (+) and insert d016tag is displayed in
1.1.2 Plasmid Transformation into—E. coli BL21 (DE3)
The plasmid was transformed into host cells of E. coli BL21 (DE3) competent cells via heat-shock at 42° C. according to supplier protocol (New England Biolabs). Selection for positive transformants of inventive host cells was performed on lysogeny broth (LB) agar plates with 50 μg/ml kanamycin antibiotics in accordance with pET28a encoded resistance. The plates were grown for 48 h at room temperature.
A single clone of inventive host cells was picked from the plate and cultivated overnight at 37° C. in LB media with 50 μg/ml kanamycin. A sterile, pyrogen-free tube was prepared with sterilized 50% glycerol solution. Under sterile laminar flow conditions, the overnight grown liquid culture (12-14 h) was diluted in the master clone storage tube containing 50% liquid culture volume and 50% of prepared glycerol solution. The master clone was stored at −80° C. All batches of d016tag inventive host cell cultivation originate from this master clone.
Two 250 ml culture flask (autoclaved) were prepared with (vegetal) LB media (autoclaved). 50 μg/ml kanamycin was added to prevent growth of potential bacterial contaminants. The preparation/handling of the pre-culture was conducted under sterile laminar flow conditions at all times. One flask with 65 ml of kanamycin-containing (vegetal) LB-media was inoculated by a single pipette tip (autoclaved) of sample from the master clone storage tube. A second flask was used as negative control with a pipette tip not containing cells of the master clone. The preculture tubes were sealed by an air-permeable membrane under laminar flow and delivered to the incubation shaker. Cells were grown at 37° C. overnight (12-14 h) at 180 rpm. The master clone storage tube was tightly sealed throughout the process and only opened under laminar flow conditions. After inoculation, the master clone tube was sealed and stored at −80° C.
Fourteen 500 ml baffled flasks with air-permeable screwcaps were autoclaved and each was filled with 200 ml of (vegetal) TB-media (autoclaved, vegetal peptone 12 g/l, yeast extract 24 g/l, glycerol 8 ml/l) containing potassium phosphate buffer (autoclaved separately, final concentration of 9.4 g/l K2HPO4 and 2.2 g/l KH2PO4). 50 μg/ml kanamycin was added to prevent growth of potential bacterial contaminants. 12 flasks were individually inoculated by 4 ml of the preculture using 5 ml pipette tips (autoclaved). The two remaining flasks containing 4 ml of the control preculture were used as controls. The preparation of the main cultures was performed under sterile laminar flow conditions at all times. All flask caps were tightly screwed before flasks were transferred to the incubation shaker, where growth conditions were set to 37° C., 180 rpm. After 3-4 h and a growth OD600 of 0.7-1.1, all flasks were equilibrated to room temperature. Protein production was induced with 200 μl of a 100 mM IPTG stock solution. Finally, all flasks (including contamination controls) were placed inside a second incubator shaker at 28-30° C. (180 rpm) for 18-20 h.
After the defined growth period of 18-20 h, culture flasks were taken out of the incubation shakers. Control flasks were checked to ensure that no growth of contaminants had occurred within the batch. Controls were not processed further. All twelve culture flasks were harvested using pyrogen-free, sterile 50 ml tubes. 3×50 ml of each flask were harvested and the remaining volume was discarded. All tubes were centrifuged at 4000 rcf, 4° C. for 30-45 min. The supernatant was discarded, and all tubes containing pellet comprising the inventive modified bacterial hyaluronidase were tightly sealed and stored at −80° C. for use in downstream processing.
Pellets were resuspended in 10 ml of the general downstream buffer PBS (phosphate buffered saline, containing 280 mM NaCl, 6 mM KCl, 15.1 mM Na2HPO4, 4.9 mM NaH2PO4, PH=7.4 at room temperature) by thorough vortexing. The three tubes of the same flask origin were combined for sonication (36 tubes were combined into 12 tubes) and stored on ice until processing. The sonicator was set to 60% amplitude, 2 sec on 4 sec off cycle. Each tube was individually sonicated, surrounded by ice-water in a 100 ml glass bottle, to ensure constant low temperatures for all samples. After sonication of all samples, the tubes were centrifuged for 45 min at 4000 rcf and 4° C. Supernatant was transferred to new sterile, pyrogen-free 50 ml tubes while ensuring that no significant amounts of cell fragment material was transferred. A further round of centrifugation for 45 min at 4000 rcf and 4° C. was performed to remove residual cell fragments. The resulting supernatant comprising the inventive modified bacterial hyaluronidase was directly used for HIS purification.
18 gravitational HIS-purification columns were equilibrated to general PBS downstream buffer. Contents of each centrifuged tube were distributed to 3 HIS-columns. The purification procedure was repeated twice for a single batch (2×18 columns=36 column purifications per batch). Unprocessed tubes were stored on ice at all times. Of each tube, 3×9 ml supernatant samples were carefully transferred via pipetting into 3 independent HIS-columns so as to not perturb any residual cell pellet remaining within the tube. The loaded columns were then washed by 10 ml of 10 mM imidazole PBS solution before being eluted in 6 ml 150 mM imidazole PBS solution prepared in new sterile, pyrogen-free tubes. The eluate of the 3 columns originating from the same production flask were combined (36 total purification eluates combined into 12 tubes) and diluted to 35 ml by general downstream buffer PBS. Those purified samples of the inventive modified bacterial hyaluronidase were stored on ice until the following STREP purification. The HIS-purification columns were cleaned by applying 12 ml of 300 mM imidazole PBS solution and prepared for the second run by 12 ml ultrapure water followed by 12 ml of general downstream buffer PBS.
All tubes containing HIS-purified protein samples of the inventive modified bacterial hyaluronidase were stored on ice until completion of the entire STREP-purification process. For each combined eluate sample from HIS-purification, two iterations of syringe-based STREP-purification steps with a flowrate <5 ml/min were performed in each process (12 combined tubes from HIS-purification were processed by 24 STREP-columns). STREP-columns (5 ml bed volume) were washed and equilibrated by 2×25 ml general downstream buffer PBS. 17.5 ml of eluate sample was applied and run through the column. The loaded column was then washed by 50 ml of general downstream PBS buffer to remove any remaining contaminant proteins. By applying 20 ml of 2.5 mM d-Desthiobiotin containing PBS buffer, the protein of interest was eluted into a fresh, sterile and pyrogen-free tube, which was stored on ice at all times. The column was regenerated by 15 ml ultrapure water, followed by 15 ml 0.5 M NaOH, followed by 15 ml ultrapure water and finally by 25 ml of general downstream PBS buffer. At this stage, the next sample was loaded and purified in the same manner. A single STREP-column was used for 12 purification runs, i.e. half of the batch. After purification of the whole batch, buffer exchange was performed via Sartorius VivaSpin20 50 kDa centrifugal concentrators. Each VivaSpin was loaded by 20 ml of STREP-eluate. By centrifugation at 4000 rcf at 4° C. for 45 min, the samples were concentrated below 1 ml. Purified protein of the inventive modified bacterial hyaluronidase was collected in the VivaSpins and was buffer-exchanged by adding Tris-HCl NaCl (50 mM Tris, 154 mM NaCl, pH=7.4 at room temperature) to 20 ml. This procedure of centrifugal concentration and re-diluting to 20 ml by Tris-HCl NaCl was performed from that point on 3 times to meet specifications of the CoA. After the last centrifugation, the VivaSpins were not filled to 20 ml but to 5 ml. Each VivaSpin was then resuspended carefully and the 5 ml were taken out into a sterile, pyrogen-free tube. Two more resuspensions by 5 ml Tris-HCl NaCl were performed to ensure good recovery of the desired protein from the VivaSpins. All 12 tubes containing each 15 ml buffer exchanged protein of the inventive modified bacterial hyaluronidase were kept on ice for the polishing step.
For polishing, 2× Polymyxin B based endotoxin removal steps, 2× sterile filtration steps and 1× particle removal step were performed. In preparation, all buffer-exchanged proteins were run through sterile, pyrogen-free 0.22 μm PES filters. The filtrates were stored on ice in fresh, sterile and pyrogen-free tubes. The following steps were performed under sterile laminar flow conditions. Each sample was subject to two sequential endotoxin removal runs on (two independent) immobilized Polymyxin B columns (supplier GenScript). Each column was cleaned by a total of 15 ml of supplied regeneration buffer to remove any residual endotoxins, followed by a total of 18 ml of supplied equilibration buffer (phosphate-based) to remove all of the regeneration buffer. Additionally, a total of 15 ml of Tris-HCl NaCl was applied to further remove the phosphate buffer. The 15 ml of sterile filtered sample was loaded on the first column and gathered in a new sterile, pyrogen-free tube. To gather the remaining protein of the inventive modified bacterial hyaluronidase and enhance the yield, 5 ml of Tris-HCl/NaCl was applied to the column, and the flow-through was gathered in the same tube as well. The process was repeated using a fresh second column. Both endotoxin-removal columns were shortly regenerated multiple times within the batch run by a total of 10 ml regeneration buffer, followed by a total of 10 ml of equilibration buffer and finally followed by a total of 15 ml of Tris-HCl NaCl. A set of two endotoxin removal columns was used in total (for the whole batch). The endotoxin removal columns were regenerated after 3 sample runs. These endotoxin removal procedures were performed for all samples, and the resulting samples were stored on ice. Samples were then collected in fresh, sterile, pyrogen-free tubes and transferred under laminar flow into fresh Sartorius VivaSpin20 100 kDa centrifugal concentrators for centrifugation at 3000 rcf at 10° C. for 20 min. Under laminar flow conditions, the VivaSpin flow-through was resuspended and gathered in fresh, sterile and pyrogen-free tubes. After having gathered all the flow-through (individually for each sample), another round of sterile filtration through sterile, pyrogen-free 0.22 μm PES filters was performed inside the laminar flow. Using pyrogen-free pipette tips, a sample aliquot was made to measure the concentration of protein in the final sample via 280 nm absorption. Based on this concentration data, the combined product was diluted by Tris-HCl NaCl to a final concentration of approx. 0.1 mg/ml. Multiple small aliquots from the final product of a batch were prepared for batch analysis in sterile, pyrogen-free tubes. The final product comprising the inventive modified bacterial hyaluronidase d016tag and all aliquoted batch analysis samples were tightly screwed and sealed by parafilm before storage at −15° C.
Protein yields [mg/ml] from E. coli BL21 (DE3)/pET28a (+) shaker flask expression were determined by measuring amount of protein [mg] after HIS- & STREP purification via absorption at 280 nm (with a calculated absorption coefficient of 132590 mol-1 cm-1 and a calculated mass of 84516 Da) in reference to the culture volume [ml] during protein expression.
Thus, attained yields were approx. 0.09-0.13 mg/ml (after HIS-purification). Final yield after HIS-Purification, STREP-Purification and polishing was approx. 0.04-0.06 mg/ml.
1.3.5 Optional Removal of C-Terminal and/or N-Terminal Tag Using Enzymatic or Non-Enzymatic Cleavage
Alternatively, the inventive modified bacterial hyaluronidase polypeptide may subsequently be adapted by removing one or both HIS- and STREP-tags, preferably at least the HIS-Tag.
If removal of the HIS-Tag and/or STREP-Tag, preferably only HIS-Tag is necessary, specific suitable recognition sites are incorporated to allow subsequent enzymatic cleavage using suitable enzymes, such as Carboxapeptidase A for removal of C-terminal HIS-Tags. Alternatively, suitable non-enzymatic sequence-specific nickel-assisted cleavage (SNAC)-tag may be used (see Dang, B., Mravic, M., Hu, H. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat Methods 16, 319-322 (2019). https://doi.org/10.1038/s41592-019-0357-3).
According to the present invention, a modified bacterial hyaluronidase polypeptide, preferably wherein the inventive modified bacterial hyaluronidase polypeptide consists of SEQ ID No. 1, SEQ ID No. 9 or SEQ ID No. 10, is obtainable according to the production process as disclosed above.
For analysis of protein purity, a single batch analysis aliquot was thawed, and the concentration was checked via 280 nm absorbance. Based on this data, samples with concentrations of 0.1 μg/25 μl up to 16 μg/25 μl were created to analyze purity on the SDS-PAGE. To each dilution 25 μl 2×LAMMELI buffer was added and gently mixed by pipetting. To improve homogeneous transmission through the gel matrix, samples were then denatured by 60° C. incubation for 20 min time before loading. A following centrifugation at 20° C. and 6000 rcf for 1 min was performed to restore condensate into the sample volume. A GenScript GelBox was filled half-way with the GenScript Tris-MOPS SDS running buffer and ready-to-use SurePage gels were installed after removal of the safety strip. The inner space was then fully filled by Tris-MOPS SDS running buffer and the combs were gently removed. Each individual loading chamber (12 per gel) was washed by pipetting 100 μl of Tris-MOPS SDS multiple times to remove glycerol. All denatured and prepared samples (50 μl) were loaded (11 per gel), and an additional chamber was used to apply 5 μl of NEB Prestained Protein ladder. The GelBox was sealed and connected to the Power Supply (120 V) until the smallest band of the ladder was close to running out of the gel (60-90 min). The power was turned off, and the gel(s) were removed from the chamber. After opening the gel cassette(s), the gels were placed inside staining boxes that were filled up to 1 cm levels with staining solution (50% MeOH, 40% ultrapure water, 10% acetic acid, 1.0 g/l Comassie R-250). Gels were stained overnight on a rocker shaker. The staining solution was removed the next morning.
The gels were washed with demineralized water and heated in a microwave to est. 70-90° C. The gels were then transferred to the shaker for 20 min. The process was repeated until bands were clearly visible and the background signal had decreased significantly. As a final step, destaining scans (1200 dpi) were made (see
No contamination band was detectable in any of the loaded concentrations of the inventive modified bacterial hyaluronidase (0.1-16 μg), which indicates a theoretical purity of >98.8% for single contamination proteins. This finding is based on a limit-of-detection at 0.2 μg per lane, although the analyzed protein & BSA can already be detected at 0.1 μg per lane as well.
According to US Pharmacopoeia (USP), the unit activity of hyaluronidase is determined by calibration to the USP National Formulary Reference Standards. The unit activity was defined as: “One unit is based on the change in absorbance at 600 nm (change in turbidity) of a USP reference standard hyaluronidase which is assayed concurrently with each lot of this product.”
As this standard is no longer available for purchase, supplier Sigma-Aldrich derived a method using previously calibrated reference enzymes. The new unit definition is: “One unit will cause a change in A600 nm of 0.330 per minute at pH 5.7 at 37° C. (45-minute assay).”
And furthermore: “The change in absorbance value of 0.330 in the new unit definition was chosen in order to most closely match the results found using the USP hyaluronidase standard defined activity. As a result, the discontinued USP-based unit definition and the new unit Sigma-Aldrich unit definition will give a conversion factor of approximately 1:1 (One old unit will equal approximately one new unit).” The source on the definition of activity unit is also derivable under: https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=111679355
The turbicity assay to measure activity units for the inventive modified bacterial hyaluronidase was conducted in accordance to Sigma Aldrich protocols (which includes identical preparation of all assay buffers, reaction parameters and measurement procedures). Any changed procedures are described below:
The thus described USP-based specific activity analysis was furthermore calibrated against a “reference” hyaluronidase from Sigma-Aldrich, to remove deviations arising from individual measurements. This reference enzyme (Sigma-Aldrich bovine Hyaluronidase Typ I-S [H3506]) was used in different concentrations to allow generating a standard curve (linear fit) to convert measured transmittance (blank A600-sample A600) into U/mg. Sigma-Aldrich has determined the specific activity according to the above described protocol.
The standards (3.16 U/1.5 ml reaction-6.55 U/1.5 ml reaction) were prepared by dilution procedures. To measure the specific activity, each sample of the inventive modified bacterial hyaluronidase (syn: d016) was diluted (if necessary) into Tris-HCl NaCl to a concentration of 0.1 mg/ml. Concentrations were checked by A280 measurement, and any identified deviations were used to correct the corresponding specific activity measurement.
Subsequently, the samples were diluted 400× into enzyme dilution buffer of the Sigma-Aldrich assay-11.7 μl diluted sample was used per 1.5 ml reaction, resulting in 2.93 ng d016tag per 1.5 ml reaction (see Table 1 below). The dilution procedure for 2.93 ng enzyme was determined empirically to result within the absorbance of the standard range described above.
Thus, the amount of inventive modified bacterial hyaluronidase d016tag in final reaction (11.7 μl used for each reaction) corresponds to 2.93 ng/reaction.
For absorbance measurement, 250 μl of the 45 min reaction were combined with 1.25 ml precipitation buffer (pH=3.75) and measured for A600 in a spectrophotometer. This value was used to calculate transmittance and convert the value to specific activity according to aforementioned protocol.
Details on the turbicity method are described on the following website of Sigma Aldrich: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General Information/2/hyaluronidase.pdf
In reference to Sigma-Aldrich bovine Hyaluronidase, which has been determined according to USP Unit definition, the inventive modified bacterial hyaluronidase d016S resulted in the respective specific activity as displayed in Table 2 as follows:
Thus, the general specific activity for the inventive modified bacterial hyaluronidase ranges between 1.5 Mio. USP U/mg+/−150 k USP U/mg. This equals 25.000 katal/kg+/−2.500 katal/kg.
For Endotoxin quantification, the testing kit ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (L00350) by supplier GenScript was used in accordance with the manufacturer protocol. The kit included endotoxin standards, endotoxin-free water, Limulus Amebocyte Lysate, chromogenic substrate, color-stabilizers, endotoxin-free pipette tips and tubes, as well as a tube rack. All packaging were cleaned by ultrapure water and components were sterilized under laminar flow. The powder/lyophilized components were reconstituted and stored according to the manufacturer protocol. By diluting the endotoxin standard of the supplied kit, four standards for endotoxin measurement were prepared (0.1 EU/ml, 0.25 EU/ml, 0.5 EU/ml, 1.0 EU/ml). The standards were used to generate a reference curve, by which the absorption of each sample can be converted into a concentration in EU/ml. The respective standards were analyzed in parallel during all batch sample measurements. For batch analysis, a single batch analysis aliquot was thawed on ice, the tube was disinfected and washed by ultrapure water and transferred to laminar flow. 100 μl of an undiluted batch sample, 100 μl of endotoxin-free water as blank and four standards with endotoxin concentrations between 0.1 EU/ml and 1.0 EU/ml were used for a single analysis run. All sample handling was performed under sterile laminar flow conditions. The following steps were performed:
1. Incubation of well-mixed standards, blank and sample(s) with LAL for 12 min at 37° C.
2. Addition of substrate solution for chromogenic reaction and incubation at 37° C. for 6 min
3. Stepwise addition of the three color-stabilizer solutions to each measurement vial with gentle mixing
4. Transfer of the final reacted solutions to cuvettes for 545 nm absorption measurement on a spectrophotometer. Background from blank was subtracted from all batch sample measurements, and the standard curve was plotted. Endotoxin concentration of the sample(s) in EU/ml were extrapolated from the standard curve. By taking into account the final batch product/aliquot concentration of 0.1 mg/ml the endotoxin level was further described as EU/mg product.
Vegetal LB-Agar (10 g/l Soya-Peptone, 10 g/l NaCl, 5 g/l Yeast Extract, 15 g/l Agar-Agar) was prepared with ultrapure water and autoclaved. The hot bottle was placed inside the ethanol-sterilized laminar flow together with sterile petri dishes. Plates were poured without addition of antibiotics to the growth media. For sterility testing, a single batch analysis aliquot was taken and thawed on ice. After the plates were solidified and cooled down, a single batch analysis aliquot was pipetted on a single plate with sterile, pyrogen-free pipette tips and spread out by sterile Lazy-L-Spreaders. A plate containing sterile filtered Tris-HCl NaCl served as control to test for environmental contaminations. A plate containing untransformed E. coli BL21 (DE3) served as positive control. The plates were closed but not sealed to allow air exchange. To ensure environmental sterility, the plates for the product sterility test were kept inside the laminar flow for 4 days to check for any growth (timeframe may be adapted depending on environmental contamination speed during processing in laminar flow). No visible growth is confirmation for product sterility.
Accordingly, the inventive modified bacterial hyaluronidase d016tag can be used for pharmaceutical compositions, in particular for pharmaceutical compositions requiring a sterile quality, such as intravenous application.
To test stability & solubility of inventive samples, specific activity measurements of multiple concentrations of purified protein were conducted over time. Multiple sample dilutions were prepared in sterile-filtered Tris-HCl NaCl at 0.2 mg/ml (200% final stock solution of product), 0.1 mg/ml (targeted final stock solution of product) and 0.01 mg/ml (low concentration dose for targeted application). Inventive protein concentrations were measured using 280 nm absorption. Respective dilutions were stored at −15° C., 2-8° C. and 25° C. representing relevant storage and handling conditions. Aliquots for Specific Activity measurements were taken at the start of stability tests, after 1 day, 2 days, 3 days, 1 week, 2 weeks and 4 weeks. All aliquots were stored in Tris-HCl NaCl with 154 mM NaCl concentration, which equals the ionic strength of 0.9% medicinal saline solution. Aliquots were stored on ice until measurement. Measurements were performed according to the activity measurement protocol.
Furthermore, freeze-thaw tests were conducted to benchmark how product solution withstands ice crystal formation during freeze-thaw processes. Samples were frozen 5× at −80° C. and thawed again, while aliquots were taken after each cycle to measure activity. General stability & solubility results demonstrated that Specific Activity overall does not drop within 7 days if the sample concentration is above 0.1 mg/ml for −15° C., 2-8° C. and 25° C. The freeze-thaw stability of a −80° C. freeze-thaw cycle does not reduce Specific Activity if the sample is frozen less than 2 times. Sample storage concentration showed loss of protein, most likely due to adsorption, within a single freeze-thaw cycle and within a single day of storage at −15° C. and 2-8° C. of 5-15%. At 25° C. samples with a concentration above 0.2 mg/ml seem to show no concentration loss over 7 days.
The results indicate that the final product should preferably be stored in protein-low-bind tubes, at concentrations above 0.2 mg/ml and frozen not more than once to ensure absolute stability. The measurements have been conducted in small microtubes (1.5 ml) with a low filling volume of 150-1500 μl. This results in a much smaller volume-to-surface quotient, compared to product storage in 50 ml tubes. Therefore, the results above are very likely to be more dependent on adsorption effects than a commercialized product.
“The rHyal_Sk in the periplasmic soluble portion was thus produced at a final concentration of approximately 2 g/L of culture medium with very high functional activity (more than 40 000 units/mg), 670- to 750-fold higher than the autologous Hyal produced by fermentation.” (see Messina et al., “Identification and characterization of a bacterial hyaluronidase and its production in recombinant form”, Federation of European Biochemical Societies (FEBS) Letters, Volume 590, Issue 14, July 2016, pp. 2180-2189)
Accordingly, the Specific Activity per mg for the bacterial Messina hyaluronidase derived from Streptomyces koganeiensis is: >40 000 U/mg, i.e. between 40 000 U/mg and 50 000 U/mg.
Specific Activity of inventive modified bacterial hyaluronidase d016tag as set out in Example 1.4.2 is: 1 500 000 U/mg.
Thus, the Specific Activity of the inventive modified bacterial hyaluronidase d016tag per mg protein is appr. 30× to 37.5× of the Specific Activity of the comparative Messina hyaluronidase.
2.2 Comparison of Human PH20 Hyaluronidase and Inventive Modified Bacterial Hyaluronidase d016
“The activity of Human PH20, His Tag (Cat. No. PH0-H5225) is measured by its ability to hydrolyze HA in turbidimetric assay (45 minute assay). The specific activity is >40,000 U/mg. (Unit Definition: One unit of Hyaluronidase activity will cause a change in A600 of 0.330 per minute at pH 5.35 at 37° C. in a 2.0 mL reaction mixture)”—ACRObiosystems, https://www.acrobiosystems.com/P563-Human-PH20-SPAM1-Protein-His-Tag.html
Accordingly, the Specific Activity per mg for the human PH20 hyaluronidase is: >40 000 U/mg, i.e. between 40 000 U/mg and 50 000 U/mg.
Specific Activity of inventive modified bacterial hyaluronidase d016tag as set out in Example 1.4.2 is: 1 500 000 U/mg.
Thus, the Specific Activity of the inventive modified bacterial hyaluronidase d016tag per mg protein is appr. 30× to 37.5× of the Specific Activity of the comparative human PH20 hyaluronidase.
2.3 Comparison of Bovine Hyaluronidase and Inventive Modified Bacterial Hyaluronidase d016
“Hyaluronidase degrades hyaluronan and has been found to be inappropriately regulated during cancer progression. These enzymes randomly cleave β-N-acetylhexosamine-[1→4] glycosidic bonds in hyaluronic acid, chondroitin, and chondroitin sulfates. Unit Definition: One unit will cause a change in % transmittance at 600 nm of 0.330 per minute at pH 5.35 at 37° C. in a 2.0 mL reaction mixture (45 minute assay).”-Sigma-Aldrich, Supplier of Hyaluronidase from bovine testes
Hyaluronidase from bovine testes, Type I-S, lyophilized powder 400-1 000 U/mg solid
Hyaluronidase from bovine testes, Type IV-S, powder, suitable for mouse embryo cell culture 750-3 000 U/mg solid,
Hyaluronidase from bovine testes, Type IV-S, lyophilized powder (essentially salt-free), 750-3 000 U/mg,
Hyaluronidase from bovine testes, Type VIII, lyophilized powder, 300-1 000 U mg,
Hyaluronidase from bovine testes, Type VI-S, lyophilized powder, 3 000-15 000 U/mg.
Accordingly, the Specific Activity of bovine hyaluronidases widely range between 300 and 15 000 U/mg.
Specific Activity of inventive modified bacterial hyaluronidase d016tag as set out in Example 1.4.2 is: 1 500 000 U/mg.
Thus, the Specific Activity of the inventive modified bacterial hyaluronidase d016tag per mg protein is approximately 100× to 5 000× of the Specific Activity of the comparative bovine hyaluronidases.
3: Treatment Regimen for, e.g., Treatment of Multiple Sclerosis or Amyotrophic Lateral Sclerosis (ALS) in a Patient
Hyaluronidase and in particular inventive modified bacterial hyaluronidase may be used for treatment of multiple sclerosis and/or amyotrophic lateral sclerosis (ALS).
Accordingly, hyaluronidase, in particular bovine hyaluronidase 1,500 IU, e.g., from Wockhardt UK Ltd, marketing authorisation number UK PL 29831/0113 can be used. Alternatively, any other of the inventive modified bacterial hyaluronidase can be used, wherein in particular the inventive modified bacterial hyaluronidase comprising or consisting of at least 90% sequence identity to SEQ ID Nos. 1, 9, 10, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 can be used.
Potential contraindications for treatment: allergy, hypersensitivity to hyaluronidase or bovine proteins, bacterial or viral infection, venous congestion or shock symptoms, serum proteins <5.5 g %.
Day 1:2 intravenous infusions at intervals of approx. 6 hours with 5 vials 1,500 IU bovine hyaluronidase each, each vial (lyophilisate as dry substance) is dissolved with 2 ml 0.9% NaCl solution ad infusionem each, filled up to e.g. 50 ml 0.9% NaCl solution ad inf. in the perfusor, and the mixture is preferably applied within 30 minutes. Alternatively, the dosis of hyaluronidase is increased on day 1 to 6, 7, 8, 9 or 10 vials 1,500 IU bovine hyalase each.
Days 2 to 10 (consecutive, but also possible with interruptions of 1, 2 or 3 days, e.g. weekends or bank holidays): 2 intravenous infusions at intervals of approx. 6 h with 10 vials of 1,500 bovine hyaluronidase each, each vial (lyophilisate as dry substance) is dissolved with 2 ml of 0.9% NaCl solution ad infusionem each, filled up to e.g. 50 ml of 0.9% NaCl solution ad infusionem in a perfusor, and the mixture is preferably applied within 30 minutes.
Due to the increased Specific Activity of the inventive modified bacterial hyaluronidase, the inventively modified bacterial hyaluronidase may be used at the same dosage and may impart an increased effect or the dosage of the inventively modified bacterial hyaluronidase may be lowered in comparison to the bovine hyaluronidase due to its increased specific activity in order to impart a comparable effect.
According to an alternative embodiment, the hyaluronidase, in particular the inventive modified bacterial hyaluronidase may be applied intra-nasal, which may be beneficial in order to cross the blood-brain barrier, e.g. via the filae olfactoriae in the lamina cribrosa.
Have necessary medications/emergency equipment ready in case of allergic (shock) symptoms. During infusion: ECG monitoring, pulse oximetry, RR control.
Follow-up after each infusion for approximately 30 minutes.
Depending on the individual need of a patient, the treatment can be repeated after a break, e.g. a break of about 4 weeks each time.
Treatment of an individual off label treatment trial of a 58-year-old patient (male, 80 kg) with confirmed ALS diagnose since appr. 4 years. Increasing limitations of motoric abilities in the shoulder-arm area and in swallowing/speaking in the last weeks prior to treatment. High-dose intravenous therapy of hyaluronidase as set out above with twice-daily administration for 10 days was tolerated without side effects. Treatment control after 6 weeks: first signs of positive changes in relation to clearer articulation and less swallowing while eating and drinking. Further treatment controls are to be conducted, e.g. 12, 24, 36, 48 and 60 weeks after application.
Embodiment 1 (first inventive aspect) A hyaluronidase peptide for use in the treatment or prophylaxis of a neurodegenerative disease associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis.
Embodiment 2: The hyaluronidase peptide according to embodiment 1, wherein the hyaluronidase peptide is selected from a bacterial hyaluronidase.
Embodiment 3: The hyaluronidase polypeptide according to embodiment 1 or 2, wherein the hyaluronidase peptide is a modified bacterial hyaluronidase comprising or consisting of at least 90% sequence identity to SEQ ID No. 1, SEQ. ID No. 9 or SEQ ID No. 10.
Embodiment 4: The hyaluronidase polypeptide according to any one of embodiments 1 to 3, wherein hyaluronidase peptide is a modified hyaluronidase peptide with increased lipophilic constituents, and/or increased positively charged constituents, and/or an amino acid sequence binding to at least part of a blood-brain barrier transporter system, such as to large neutral amino acid receptor, in particular L-type Amino acid transporter (LAT1), and/or with an amino acid sequence binding to at least part of a transferrin receptor, such as transferrin receptor 1 or transferrin receptor 2.
Embodiment 5: The hyaluronidase polypeptide according to any one of embodiments 1 to 4, wherein hyaluronidase peptide is with one or more blood-brain barrier efflux inhibitory agents, preferably selected from the group consisting of p-glycoprotein inhibitory agents, such as Cyclosporine or a Cyclosporine derivative, such as Valspodar, Elacridar, Zosuquidar; calcium antagonistic agents, such as Verapamil; and procyanidine or procyanidine derivatives.
Embodiment 6: A pharmaceutical composition for use in the treatment or prophylaxis of a neurodegenerative disease associated with an increased cerebral glycosaminoglycan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis, characterized in that the pharmaceutical composition comprises the hyaluronidase peptide according to any one of embodiments 1 to 5 in a therapeutically effective amount and one or more pharmaceutically acceptable excipients.
Embodiment 7: The pharmaceutical composition according to embodiment 6, wherein the pharmaceutical composition forms an intranasal formulation; an intracerebral formulation, in particular an intraventricular or an intrathecal formulation; or a parenteral formulation.
Embodiment 8: The pharmaceutical composition according to embodiment 6, wherein the pharmaceutical composition forms an oral formulation, a rectal formulation or a sub-cutaneous formulation, wherein the hyaluronidase is adapted to pass the blood-brain barrier.
Embodiment 9: The pharmaceutical composition according to any one of embodiments 6 to 8, wherein the composition is selected from a solid, semi-solid or liquid application form.
Embodiment 10: A method of treating a neurodegenerative disease associated with an increased cerebral glucosaminoglucan level, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), cerebral insult, brain trauma, cerebral inflammations, and autoimmune encephalitis comprising or consisting of administering the hyaluronidase peptide according to any one of embodiments 1 to 5, or the pharmaceutical composition according to embodiments 6 to 9 to a subject in need thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2021 127 065.0 | Oct 2021 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/079102 | 10/19/2022 | WO |
