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Full description

Background deception

The invention relates to alkaline phosphatases with improved properties, pharmaceutical compositions comprising alkaline phosphatases with improved properties and the use of alkaline phosphatases with improved properties for the prevention, treatment or cure of diseases.

5 Phosphatase enzyme is an enzyme that removes phosphorylates from its substrates? That is, it hydrolyzes the monoesters of phosphoric acid into a phosphate ion and a molecule with a free hydroxyl group. This effect is directly opposite to that of phosphorylases and kinases, which connect phosphate groups to their substrates by using active molecules such as

10 ATP. Phosphatases can be classified into two main classifications: Phosphatases

Cysteine-Dependent Phosphatases (CDPs).

Mineral phosphatase 0105001565-0.

Metal phosphatases typically coordinate two catalytically essential metal ion(s) into their active site. There is currently some confusion regarding the identity of these metal ions resulting from

15 successive attempts to determine it have different answers. There is currently evidence that these minerals may be magnesium, manganese, iron, zinc, or any combination thereof. We believe that the hydroxyl ion, which forms a bridge bond between two metal ions, participates in the nucleophilic attack on the phosphate group.

.phosphate

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Phosphatases function in opposition to kinases/phosphorylases, which add phosphate groups to proteins. The addition or removal of a phosphate group may activate or inhibit an enzyme (eg a kinase signaling pathway) or enable a protein-protein interaction: phosphatases are therefore integrated with many cross-signal transduction pathways. It should be noted that phosphate addition and removal do not necessarily correspond to enzyme activation or inactivation and that many enzymes contain independent phosphorylation sites for activation or inhibition of functional regulation. For example, its inhibition or inhibition depends on the specific amino acid residue undergoing phosphorylation. Phosphate compounds are important in transient signal transduction because they regulate the proteins they contact. To reverse the regulating effect the phosphate can be removed. It happens spontaneously

10 itself by hydrolysis or mediated by protein phosphatases.

A type of phosphatase enzyme is alkaline phosphatase (ALP) or alkaline phosphatase.

Responsible for the removal of hydrolase in the hydrolase enzyme, (EC 3.1.3.1) (“AP” phosphatase

Phosphate groups of many types of molecules including, for example, nucleotides, proteins, and alkaloids. Until recently we believed 15 that alkaline phosphatases were most effective in an alkaline environment, as the name suggests.

It is common in the field that one possible physiological role for AP may be its interaction with inflammatory molecules. Firstly it has been hypothesized that AP dephosphorylates endogenous endotoxins and as such reduces the inflammatory response to these highly inflammatory molecules. 20 Since then, other mechanisms of influence have been postulated and pursued. However, until now, despite its beneficial role in many inflammatory and other diseases, the mechanism of action has not been completely clear. In the past, the enzyme alkaline phosphatase of bovine origin has been used in animal models and clinical trials to treat, for example, sepsis, acute kidney injury, inflammatory bowel disease, enterocolitis, and local anemia due to

25 Ischemia reperfusion damage, or inflammatory diseases

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Riggle et al, for Surg Res. 2013) Other inflammatory diseases Mar;180(1):21-6; Peters et al, for Pharmacol Exp Ther. 2013 0,344(1):2-7; Martinez-Moya et al, Pharmacol Res. 2012 9,66(2):144-537 Pickkers et al, Crit Care. 2012 Jan 23,16(1): Ramasamy et al, Inflamm Bowel Dis. 2011 00,17(2):532-42: Lukas et al, Inflamm Bowel Dis. 2010 5

,16(7):1180-67 Bol-Schoenmakers et al, Eur for Pharmacol. 2010 May 10,633(1-3):71-77 Heemskerk et al, Crit Care Med. 2009 00,37(2):417-23; Tuin et al, Gut. 2009 Mar;58(3):379-87; Su et al, Crit Care Med. 2006 49,34(8):2182-7) van Veen et al, Br L Surg. 2006 00,93(4):448-56: van Veen, Infect Immun. Verweij et al, Shock. 2005, 73(7):4309-14. 2004 10

(Aug:22(2):174-9

Although currently isolated alkaline phosphatase enzymes are available from natural sources as well as genetically engineered recombination products useful in both diagnosis and treatment of disease, there is a need for replacement phosphatases with, for example, transgene activity (e.g.

15 developed) a stability determinant (eg in vivo half-life Τ1/2, or stability to light storage (shelf life)) or substrate specificity.

The present invention provides said modified phosphatases having advanced properties compared to the gene recombination chimeric alkaline phosphatase enzyme described extensively in International Application 0133511/2008.

20 Working description of the invention

In a first embodiment, the invention provides an isolated protein with phosphatase activity wherein said protein comprises an amino acid sequence having at least 2,000 amino acid sequences having at least 90% sequence identity with a sequence with identity number: 5, sequence An amino acid with at least 50 consecutive amino acids that has 90% sequence identity.

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At least with a sequence of identity number: 6, and an amino acid sequence having at least 40 amino acid sequences having at least 90% sequence identity with a sequence of identity number: 7, wherein the full-length protein includes an amino acid sequence having at least 90% sequence identity. At least with the full-length amino acid sequence of a sequence with identity number: 1, provided that the amino acid at position 5 279 is leucine, the amino acid at position 328 is valine (7), and the amino acid at position 478 is Leucine (L).

In a preferred embodiment, the full-length protein includes an amino acid sequence that has at least 90%, at least 95%, or at least 98% sequence identity with the full-length amino acid sequence of a sequence with identity number: 1, provided that the amino acid The acid 10 corresponding to position 279 is leucine, the amino acid corresponding to position 328 is valine, and the amino acid corresponding to position 478 is leucine.

The expression “corresponding to” in the present application is intended to mean the particular position with respect to the N-terminal amino acid of the mature protein that is identified at “position 1”. The expression "paralogous to" does not explicitly refer to the residue of the particular amino acid at that particular position.

15 The expressions protein and polypeptide refer to compounds containing amino acids linked by peptide bonds and can be used interchangeably.

As used in the present application, where “amino acid sequence” is mentioned it refers to the amino acid sequence of a protein or peptide molecule. 0 “amino acid sequence” can be inferred from the amino acid sequence that codes for the protein. However, expressions such as “polypeptide” or “protein”20 are not intended to restrict the amino acid sequence to the inferred amino acid sequence. However, it can include genetic post-translational modifications of deduced amino acid sequences such as amino acid deletions, additions, and modifications such as glycosylations and the addition of lipid moieties. There is also the use of unnatural amino acids such as amino acids -

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D to improve stability or pharmacokinetic properties within the “amino acid sequence” expression domain unless otherwise stated.

Part of the said protein should preferably include an amino acid sequence of 5 - 65, preferably 60 - 65, preferably more 62 - 65, preferably 64 - 65, preferably 65 amino acid sequences 5 that have at least an identity of 9%, preferably at least. More preferably 95% At least 98% sequence identity to the full-length mitral domain of human PLAP. For the purposes of the present invention the amino acid sequence corresponding to the reference sequence of the full-length crown domain of human PLAP is underlined in sequence ID number: 3, depicted in Figure 1 and corresponding to positions 366-430 therein.

10 Preferably, a portion of the aforementioned protein should include an amino acid sequence of »»2 - 365, preferably more »25 - 365, preferably more »»3 - 365, preferably more »35 - 365, preferably more At least 360 - 365 Best 365 amino acid sequences It has a 90%, preferably at least 95%, more preferably at least 98% sequence identity with the N-terminal region flanking the crown domain of human ALPI. The N-terminal region flanking the crown domain is a two-part region of the catalytic domain.

15 Preferably, a portion of the aforementioned protein should include an amino acid sequence of 40 - 54, preferably 45 - 54, preferably more 5 - 54, preferably more 52 - 54 best 54 amino acids having a sequence of 9%, preferably at least 95%, preferably The sequence is at least 98% identical to the C-terminal region flanking the coronary domain of human ALPI. The aforementioned C-terminal region flanking the coronary domain is the second of two parts of the catalytic domain.

»2 For the purposes of the present invention the amino acid sequence of the reference sequence of a mature human PLAP protein is depicted in Figure 1 (sequence ID number: ). For the purpose of identifying secret areas

The N-terminus and C-terminus, together referred to as the catalytic domain, are underlined in the present application.

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In a certain preferred embodiment the invention provides a protein according to the invention wherein said protein comprises the amino acid sequence of “5 - 65, more preferably 60 - 65, more preferable 62 - 65, more preferably 64 - 65, Best 65 Amino acid sequences that have at least 90% identity Preferably at least 95% Most preferable At least 98% sequence identity with the full-length crown domain 5 of human PLAP Part of said protein includes an amino acid sequence of 200 - 365 Most preferred 250 - 365 Most preferred 300 - 365 Most preferred 350 - 365 Most preferred 360 - 365 The best 365 amino acid sequences that have an identity of at least 90% Preferably at least 95%, More preferably at least 98% sequence identity with the N-terminal region flanking the crown domain of human ALPI and that a portion of said protein includes the 10 amino acid sequence of 40 - 54, preferably 45 - 54, more preferably 50 - 54, more preferably 52 - 54. , preferably 54 amino acid sequences that have at least 90% identity, preferably at least 95%, most preferably at least 98% sequence identity with the C-terminal region flanking the crown domain of human ALPI.

A region adjacent to the N terminus of the crown domain means a contiguous amino acid stretch (i.e., 15 preferably less than 20 amino acids, more preferably less than 15, more preferable less than 10, more preferable less than 5, more preferable less than 3, more preferable less than 2, It is best not to diverge any amino acid) into the coronal sequence (the corresponding amino acids are underlined in Figure 1) at the left side of the coronary domain, where the left side is defined as that part of the peptide chain that bears the amino radical (NH2). The first amino acid.

20 What is meant by a region adjacent to the N-terminus of the crown domain is an adjacent amino acid stretch (i.e., less than 20 amino acids are preferred, less than 15 are preferred, more are preferred, less than 10, more are preferred, less than 5, more are preferred, less than 3, more are preferred, less than 2, it is best not to diverge any amino acid) into the coronal sequence (the corresponding amino acids are underlined in Figure 1) at the left side of the coronary domain, where the left side is defined as that part of the 25-peptide chain that carries the amino group (NH2). Of the first amino acid.

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What is meant by a region adjacent to the C terminus of the crown domain is a stretch of amino acids corresponding to adjacent positions (i.e. less than 20 amino acids, preferably less than 15, preferably more, less than 10, preferable more, less than 5, preferably more, less than 3, preferable more, less than 2. It is preferable not to diverge. any amino acid) to the coronal domain sequence (the corresponding amino acids are underlined in Figure 5 1) at the right-hand side of the coronary domain, where the right-hand side is defined as that portion of the peptide chain bearing the free alpha carboxyl group of the last amino acid.

In humans, four isoforms of alkaline phosphatase have been characterized to date. It is the intestinal alkaline phosphatase enzyme (ALPI), and the placental (ALPP).

placental, and placental-like (GCAP), taken from the liver/bone/kidney (or tissue non-specific). The first three are found together on chromosome 2, while the non-tissue-specific form is found on chromosome 1. The exact physiological functions of APs are not known, but APs appear to be involved in a large number of physiological processes.

The sequences of human alkaline phosphatase enzymes are known in the art and can be easily found in 15 relevant databases. Determines the % match of the sequence to the mitral band of PLAP

and the catalytic domain of ALPI, it is preferable to use the corresponding reference sequences 0 The reference sequence for human ALPI is depicted as sequence ID number: 02 The reference sequence for human ALPP is depicted as sequence ID number: 03 Within those reference sequences in Figure 1, the sequence that is identified is underlined. Commonly known as mitral band.

20 Placental alkaline phosphatase is abbreviated ALPP or PLAP. The abbreviations ALPI or IAP stand for intestinal alkaline phosphatase enzymes. Placental alkaline phosphatase 2 is abbreviated in the present application as ALPP2, ALPG or GCAP and the abbreviations ALPL, TNAP, TNSALP or BLK are used in the present application to indicate non-liver/tissue specific alkaline phosphatase. Different abbreviations for an alkaline phosphatase enzyme and the enzyme itself may be used interchangeably in the present application.

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From a conformational point of view, alkaline phosphatase consists of barely two domains: a crown domain and a locus domain

Mammalian Alkaline Phosphatases: From Biology to Applications in Medicine and Biotechnology. Jose Luis Millan; Wiley, 2006.

The active site is divided into separate parts such as the catalytic building block and three metal ion sites (Zinc1(20), Zinc2(20), Magnesium39). From the point of

Initial basic consideration It is clear that the corona domain is partitioned by the amino acids that form the active site domain. Hence, in a preferred embodiment the catalytic domain does not consist of a contiguous amino acid sequence but which is adjacent to the crown domain. By reference to a sequence with ID number: 1 refers to the amino acid sequence of one alkaline phosphatase enzyme according to the invention which does not in any way limit the present invention 10 The crown domain preferably includes the amino acid at position 366

<p dir="rtl">— 430 While the catalytic domain preferably refers to the remaining sequences before position 366 and after position 0 3 4 in the mature protein sequences as depicted in Figure 1. The amino acid sequence of alkaline phosphatase enzymes and the relative positions of the catalytic domain and the coronary domain are known by the person</p>

Mammalian Alkaline Phosphatases: From Biology to Applications ) experienced

15.(in Medicine and Biotechnology. Jose Luis Millan; Wiley, 2006).

In some embodiments a protein according to the invention in this manner comprises a sequence having at least 90% sequence identity with the catalytic domain of the human ALPP protein and a sequence having at least 90% sequence identity with the catalytic domain of human intestinal alkaline phosphatase. It is preferable to have the sequence J

Said identically to a sequence of the ALPP crown domain found in a protein according to the invention at approximately 20 identical positions as the ALPP protein crown domain in the original ALPP protein, i.e. approximately

At position 366 - 430 relative to position 1 as shown in Figure 1 (the sequence representing the coronal band is underlined).

A sequence that has sequence identity with the ALPP protein coronary domain preferably has at least 9%, preferably at least 98%, and preferably 100% sequence identity with the original sequence of the domain.

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The crown of the ALPP protein is represented by underlined amino acids at positions 366 - 430 in the sequence with ID number: 03.

The percentage match of an amino acid or nucleic acid sequence, or the expression "% sequence match" in the present application, is defined as the percentage of residues in a candidate amino acid or nucleic acid sequence

5 nuclei are identical to residues in a reference sequence after aligning the two sequences and insertion spaces if necessary to achieve maximum sequence matching. In a preferred embodiment a calculation of at least said percentage matching of a sequence without insertion spaces is performed. Alignment methods and computer programs are well known in the field, for example “2Align” or the National Center’s BLAST service.

0(NCBI) for Biotechnology Information

10 It is preferable for the aforementioned sequence to have a sequence that is at least 90% identical.

The N-terminal portion adjacent to the ALPI crown domain is located in the protein by order at approximately the same position as that portion of ALPI in the native ALPI protein, i.e., as represented by positions 1-365 in the sequence with ID number: 1 in Figure 01.

In addition to the above, a sequence that has a sequence identity with the catalytic domain of ALPI 15 preferably has at least 95%, and more preferably at least 98% sequence identity with the N-terminal portion next to

The ALPI crown domain is represented by positions 1 - 365 in Figure 1, a sequence with identity number: 2, provided that the amino acid at position 279 is L and the amino acid corresponding to position 328 is V. It is preferable that the sequence that matches it is a sequence with the part of that end. The N of the ALPI catalytic domain is identical to the original sequence of the ALPI catalytic domain, except for the amino acid at the position

20 279 is L and the amino acid is at position 328 V.

It is preferable that the mentioned sequence has a sequence that is at least 90% identical

The C-terminal portion adjacent to the crown domain of ALPI is located in a protein according to the invention at approximately the same position as that portion of ALPI of the original ALPI protein, i.e., as represented by positions 431 - 484 in the sequence with ID number: 01

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A sequence that has sequence identity with the C-terminal portion that is adjacent to the ALPI crown domain will preferably have at least 95%, more preferably at least 98% sequence identity with the C-terminal sequence, which is adjacent to the ALPI crown domain, and is represented by positions 431 - 484. In the sequence with identity number: 2, provided that the amino acid at position 478 is L. It is preferable that the sequence 5 that has a sequence match with the C-terminal part of the catalytic domain of ALPI is identical to the original sequence of the band.

The catalytic effect of ALPI, except for the amino acid at position 478 L.

Previously, it was shown that alkaline phosphatase has a crown domain sequence that matches the ALPP catalytic domain and a catalytic domain that matches the ALPI catalytic domain (referred to in the present application as 00/0000) and has retained its initial specific activity in low Zn2+ medium. 10. These results showed that the activity in vivo is independent of Zn2+. In comparison it has ALPI

Its activity rapidly under the influence of the same reduction conditions in Zn2+. The inventors conclude that such an enzyme whose activity is independent of 202 could be useful in disease where Zn2+ depletion is part of the disease (e.g. nutritional deficiencies, alcoholism and associated intestinal damage, chronic infections including sepsis or inflammatory diseases in general) or Where the addition of Zn2+ (as a stabilizing agent in manufacturing) may interfere 15 (eg acute phase of sepsis)

autoimmune diseases). Apart from production and application advantages catALPI/crownALPP also has advantages regarding stability during storage. The properties of catALPI/crown ALPP are described in detail in International Application No. 08/133511 0 2.

The present application surprisingly shows that the protein of the present invention is also more stable at 20 lower concentrations of Zn2+. It has been shown here that the specific modification that includes three positions in relation

The catALPI/crownALPP sequence described above increases the unreliability of 212 even further. In short, it turns out that the original AP protein, such as ALPI, secretly loses its enzymatic activity

Environments with low concentrations of catALPI/crownALPP, Zn2+ (as described in 2008/133511) Retains its activity in environments with low Zn2+ but loses its activity when secreted.

25 For example, adding chelating agents to Zn2+, while retaining the protein according to the present invention

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It is highly active even in the presence of a zinc chelator such as EDTA. Therefore, in diseases where acute Zn2+ depletion is part of the pathology, the original AP protein is unable to unfold its enzymatic activity at the site where it is thought to be most beneficial, for example at the site of inflammation. In contrast, the AP protein is the product of a genetic combination that is not exposed to very low concentrations of 212+T

5 In particular a protein according to the invention retains its activity in a medium with a very low concentration of 212+, for example in a site of inflammation. Such an enzyme would therefore be very useful for treating diseases that are due to or accompanied by low levels of 202. Such low levels of Zn2+ can make other alkaline phosphatase enzymes less effective. Comparable to a protein according to the invention.

10 Many enzymes in the human body depend on Zn2+ for their activity. For example, immune responses are more effective if sufficient levels of Zn2+ are present. 0 It is known that inactive parts as well as specific parts of the immune system are affected by zinc, and it has been determined that proteins containing zinc accumulate in Sites of inflammation. In a healthy individual, the reference values for serum Zn2+ range between 10 and 20 microM. For example, in the case of alcoholism or malnutrition, it can occur

15 That these levels fall to less than 10 micromolar or even less than 1 micromolar. Alaw: 'Based on the above, it is accompanied by sub-chronic inflammation. Such as rheumatoid arthritis, sepsis, and Crohn's disease. Serum zinc deficiency. In such media lacking 202+ the protein of the invention is nevertheless highly active while other known alkaline phosphatase enzymes lose more or less rapidly their phosphatase activity. The invention therefore presents a view that a protein according to...

20 The invention is particularly useful in treating a disease accompanied by a local or systemic deficiency in Zn2+. Compared to other known alkaline phosphatase enzymes, the protein according to the invention is more effective under low Zn2+ conditions. Therefore, in a preferred embodiment, the invention provides a protein according to the invention for use in the prevention or treatment of a disease accompanied by a deficiency in Zn2+. Said disease preferably includes an inflammatory disease, preferably selected from the group consisting of immune diseases.

<p dir="rtl">25 autoimmune diseases, rheumatoid arthritis,</p>

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Asthma, chronic obstructive pulmonary disease, atherosclerosis, inflammatory disease of the gastro-intestinal tract, infection, sepsis, neurodermatitis, and inflammatory liver disease 5. Inflammatory lung disease and inflammatory kidney disease

.kidney disease

What is meant by the expression “zinc deficiency” or “Zn2+ deficiency” is that the amount of (topically) available zinc is insufficient to allow unimpeded cellular and/or enzymatic activity. Zinc deficiency can be absolute or relative. Absolute zinc deficiency can be easily determined by reference to 10 reference values in healthy individuals known in the art. Relative zinc deficiency can occur on

An example is when zinc concentrations are still within limits set by reference values in healthy individuals but the required zinc concentrations are higher than normal, for example during inflammation. In some embodiments, zinc deficiency means that the subject's zinc concentration (topically) is less than 10 micromolar, preferably more, less than • micromolar, preferable more, less than 2 micromolar, 15, more preferred, less than 1 micromolar, preferable more, less than 0.1 micromolar. Molar, preferably more, less than 0.05 micromolar, preferably more, less than 0.02 micromolar, preferably more, less than 0.01 micromolar, preferably more, less than 0.005 micromolar, and preferably less than 0.02 micromolar. In some models. Zinc deficiency means that the concentration of (topical) zinc is lower than necessary for unimpaired cellular and/or enzymatic activity.

<p dir="rtl">20 What is meant by the expression “inflammatory disease” is any disease that is due to or is accompanied by a protective tissue response to injury or tissue destruction that leads to the destruction of, relief from, or resistance to both the agent causing the injury and the affected tissue. The classic signs of inflammation are pain (ache) and heat ( Heat, redness (flushing of the skin), swelling (tumor), and loss of function(s). Inflammation is typically characterized by an increase in inflammatory variables such as C-reactive protein, leucocytes, IL-6, etc.</p>

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Although inflammation is primarily protective in nature, dysfunctional inflammatory disease such as rheumatoid arthritis and (other) autoimmune diseases also fall within the definition of “KD.”

Inflammatory. In a preferred embodiment the protein according to the present invention is useful for use in the treatment of dysfunctional or deleterious inflammatory disease.

<p dir="rtl">5 Furthermore, during standard pharmacokinetic analysis the present inventors unexpectedly observed that the protein according to the invention is targeted to several organs in particular the skin, kidneys, spleen, liver, lungs, brain, fat, bones, and colon. Using a radioactive iodine-conjugated protein according to the invention it was observed that the organ/blood ratio of the protein according to the invention relative to that of catALPI/crownALPP is particularly favorable to the skin and kidneys.</p>

10 And the spleen, liver, lungs, brain, fat, bones, and colon, that is, the protein according to the invention is targeted relatively more than the known catALPI/crownALPP protein for these organs. In particular, the protein according to the invention is stabilized at lower concentrations in the blood than catALPI/crownALPP and is stabilized at higher concentrations in the said organs. When it is necessary to treat a condition related to these organs such as liver neurodermatitis, kidney injury, cirrhosis, and liver deficiency

<p dir="rtl">15 Phosphatases or similar Less protein is required for treatment, which reduces cost and/or increases effectiveness. Trials that include diseases related to these organs (hypophosphatase 000000500205) that are on the rise (kidney disease) have already been conducted or are planned. Therefore, the invention provides a protein that shows improved independence from zinc and pharmacokinetic properties at Cr</p>

Comparison with well-known alkaline phosphatase enzymes.

<p dir="rtl">20 The inventors have shown in various working embodiments that the protein according to the invention is useful as a drug. Diseases or conditions that can be treated with a protein according to the invention include: reduced renal function, kidney injury, renal failure, and hypophosphatasia. Moreover, due to specific features of the protein enzyme alkaline phosphatase</p>

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According to the invention which constitutes improvements to the known alkaline phosphatase enzyme proteins that have been used Ser C

<p dir="rtl">25 According to the invention, protein is useful not only for decreased kidney function, kidney injury, and kidney failure</p>

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Phosphatase deficiency but also the prevention or treatment of autoimmune diseases such as rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, inflammatory diseases of the gastrointestinal tract, infections, sepsis, neurodermatitis, sepsis, neurodermatitis, and hepatitis. Pneumonia and kidney inflammatory disease. And all of these

<p dir="rtl">• Diseases affecting organs targeted by a protein according to the invention and/or accompanied by zinc deficiency</p>

zinc deficiency (relative).

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The expression “prevention” or “protective” is defined in the present application as administering (producing an effect of) a secreted protein

According to the invention to a subject with the intention of preventing said subject from contracting a certain disease. Although the treatment is intended to prevent the aforementioned subject from contracting the aforementioned disease, it is not necessary that the disease condition be completely prevented after one or several administrations of a protein according to the invention since the subjects are not, for example, necessarily exposed to a treatment protocol. In any case, it is preferable that the aforementioned prevention efficacy be achieved in that group of subjects that received a protein according to the invention for the prevention of a specific disease that shows at least a low increase in the severity of the disease or, for example, fewer complications of the said disease. When compared to a group of subjects that did not receive the aforementioned protein. For example, in the case of kidney disease, it is preferable that subjects at risk of developing kidney disease after treatment with a protein according to the invention will show less decline in kidney function than subjects who were not treated with said protein. In the event that a subject is afflicted with a disease for which a protein has been administered to prevent said disease, any additional administration may be provided (referred to in the present application as treatment) to prevent worsening of the incidence of said disease or as a treatment with the intention of treating.

20 The expression “treat” or “treat” is defined in the present application as the procedure of (producing the effect of) administering a protein according to the invention to a subject with the intent of curing the subject of a disease (anticipated) or improving the reduction or elimination of the symptoms of a disease in the subject. Although the intention of the treatment is to treat the aforementioned subject, it is not necessary for the aforementioned subject to be treated after one or several administrations of a protein.

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According to the invention where subjects are not, for example, necessarily subject to a treatment protocol.

<p dir="rtl">25 However, it is preferable that the effectiveness of the above treatment be achieved in those subjects who have been treated</p>

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With a protein according to the invention, it shows at least an improvement in the disease - their condition, for example, fewer complications from the aforementioned disease when compared to an untreated group (or treated with a satisfactory treatment). For example, in the case of kidney disease, it is preferable that subjects after treatment with a protein in accordance with the invention will show improved kidney function or less deterioration in kidney function relative to subjects who were not treated with the protein.

<p dir="rtl">5 mentioned.</p>

The invention also provides a polynucleotide comprising a nucleotide sequence encoding a protein according to the invention.

and

As used in the present application the expressions “nucleic acid sequence” and “polynucleotide” also include unnatural molecules based on and/or derived from nucleic acid sequences such as, for example, artificially modified nucleic acid sequences, peptide nucleic acids

<p dir="rtl">10 nucleic acids, as well as nucleic acid sequences containing at least one modified nucleotide and/or an unnatural nucleotide, such as, for example, LNA, inosine, morpholino, and -e-methyl 2-0- 0610 RNA</p>

.RNA

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A vector comprising said polynucleotide is also provided in accordance with the invention. Said vector preferably includes additional nucleic acid sequences such as elements necessary for genetic transcription/translation of the amino acid sequence encoding the phosphatase (e.g. promoter and/or terminator sequences). Said vector may also include nucleic acid sequences that encode selection markers (e.g., an antibiotic) to select or maintain host cells transformed with said vector. Examples of suitable vectors are cloning or expression vectors. Any suitable mediating vector can be used

In expression in a selected host cell according to the invention, it is either incorporated or transcribed chromosomally into a host cell. The vector may be a plasmid, a virus (e.g. retrovirus, adenovirus, adeno-associated virus 000-550000 virus, baculovirus and/or derivatives thereof), cosmid, phage or phage assembly 0 019000 episomal vector Or artificial chromosome

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chromosome. Said polynucleotide or vector is particularly useful for protein production according to the invention but can also be used for gene therapy. gene

The invention therefore provides a protein according to the invention for use as a drug. It is also possible to treat a patient who suffers from or is at risk of suffering from any of the above-mentioned diseases who is required to be treated with polyurethane.

<p dir="rtl">5 nucleotides according to the invention, or with a vector according to the invention, to express a protein according to the invention in a living organism. E£ e</p>

The invention therefore further provides a polynucleotide according to the invention or a vector according to the invention for use as a drug preferably for the prevention or treatment of decreased kidney function, kidney injury, kidney failure, autoimmune diseases related to genophosphatase deficiency, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, and inflammatory diseases of the ducts. Gastrointestinal, infection, sepsis, dermatitis

<p dir="rtl">10 Neurological disease, hepatitis, pneumonia and kidney inflammatory disease.</p>

A polynucleotide protein or carrier according to the invention is also provided for use in a method for preventing or treating inflammatory disease of the kidneys or phosphatase deficiency.

Also provided is the use of a polynucleotide protein and/or carrier for use in accordance with the invention to manufacture a drug preferably for the prevention or treatment of decreased kidney function, kidney injury, renal failure, hypophosphatase.

<p dir="rtl">15 Autoimmune diseases Rheumatoid arthritis Asthma, chronic obstructive pulmonary disease, atherosclerosis, inflammatory diseases of the gastrointestinal tract, infection, sepsis, neurodermatitis, hepatitis, inflammatory lung disease, and inflammatory kidney disease.</p>

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In another embodiment, the invention provides for the use of a protein alkaline phosphatase according to the invention, a polynucleotide according to the invention, or a vector according to the invention in the preparation of a drug for the treatment of a disease accompanied by a local deficiency or

20 systemic in 202+ The aforementioned disease is preferred to be an inflammatory disease, kidney disease or hypophosphatase deficiency. The aforementioned disease is preferred to include an inflammatory disease. The disease is most preferred to be selected from the group consisting of autoimmune diseases, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, and atherosclerosis. inflammatory diseases of the gastrointestinal tract, infection, sepsis, neurodermatitis, hepatitis, inflammatory pneumonia, and nephritis.

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In yet another embodiment, the invention provides a method for treating a subject (preferably a human) to treat a disease, preferably a free person

Accompanied by a deficiency of 22+ It includes the administration of an effective amount of the enzyme phosphatase according to the invention, where it is preferable that the aforementioned disease include an inflammatory disease, and it is more preferable for it to be selected from the group consisting of autoimmune diseases, rheumatoid arthritis, asthma, and obstructive pulmonary disease.

<p dir="rtl">5 Chronic, atherosclerosis, inflammatory diseases of the gastrointestinal tract, infection, sepsis, neurodermatitis, hepatitis, inflammatory pneumonia and kidney inflammatory disease.</p>

The invention thus provides a polynucleotide protein and/or carrier according to the invention for use in a variety of diseases. The protein according to the invention is particularly useful because of its organ distribution for use in the treatment of a disease including the digestive tract, kidney, skin, liver, lung, brain, adipose tissue, or bone.

<p dir="rtl">10 In one preferred embodiment the invention provides a polynucleotide protein and/or carrier according to the invention for use in the treatment of kidney disease.</p>

Although there is continuing insight into the pathophysiology of kidney injury and there are treatments

Lameire 11, Acute kidney injury: an increasing global 0 Improvement of kidney function

170-9:(9887)13:382 Money 2013.concern. Lancet], but it is still needed

<p dir="rtl">15 Alternative therapies to treat kidney injury and/or improve kidney function. The present invention provides such an alternative treatment by providing a polynucleotide protein and/or vector for use in accordance with the invention.</p>

In a preferred embodiment the invention provides a polynucleotide protein and/or carrier for use in accordance with the invention wherein said kidney disease is selected from the group consisting of kidney failure, acute kidney injury, chronic kidney disease, and ischemia-related kidney disease.

20 The expression acute kidney injury (AKI), previously called acute kidney failure, means that kidney function is rapidly lost. AKI is diagnosed on the basis of characteristic laboratory findings such as urea nitrogen and high blood creatinine, or no blood creatinine.

The ability of the kidneys to produce sufficient amounts of urine. AKI can be subdivided into prerenal AKI, endogenous AKI and postrenal AKI.

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Prerenal AKI arises due to reduced effective blood flow to the kidney. Typical laboratory findings for prerenal AKI are: Uosm: &gt; 500, UNa: &lt; 10, FeNa: &lt; 1% and BUN/Cr ratio: &gt; 020

The term endogenous AKI is used when sources of damage to the kidneys themselves are the cause. 5 Typical laboratory results for endogenous AKI are: Uosm: 350, UNa: &gt;20, FeNa: &gt; 2% and %

BUN/Cr: &lt; 015

Postrenal AKI expression is limited to those cases in which urinary tract obstruction is the cause of AKI. Typical laboratory findings for postrenal AKI are: Uosm: 350, UNa: &gt;40, FeNa: &gt;4% and BUN/Cr ratio: &gt;015.

<p dir="rtl">10 Across the world, there are several guidelines for the classification of chronic kidney disease. As an example, the National Kidney Foundation (2002) “K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease” classification criteria are described below. An experienced person can select different patient groups, based on other guidelines.</p>

Typically, all individuals with a glomerular filtration rate (GFR) &lt; 60 mL/min/1.73 m2 for 3 months are classified as having chronic kidney disease, regardless of the presence or absence of kidney damage. The rationale for inclusion of these individuals is that a decline in renal function to this level or less represents a loss of half or more of the adult level of normal renal function, which can be related to a number of complications. Individuals with chronic kidney damage are generally classified as having chronic kidney disease, regardless of their 20 GFR level. The rationale for inclusion is individuals with GFR &gt; 60 mL/min/1.73

It states that GFR can be maintained at normal or increased levels regardless of the underlying kidney damage and that patients with kidney damage are at increased risk for two of the cardinal outcomes of chronic kidney disease: loss of kidney function and the development of cardiovascular disease, which can lead to death. Or death.

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Loss of protein in the urine is an independent predictor of worsening kidney function and cardiovascular disease. Hence, the British guidelines attach a 'P' to the stage of chronic kidney disease if there is significant protein loss.

• The five stages of chronic kidney disease are typically classified as follows:

<p dir="rtl">• Stage 1: slightly decreased function; Kidney damage with a normal or relatively high GFR level (90 ml/min/1.73 m). Kidney damage is defined as pathological abnormalities or markers of damage, including abnormalities in blood or urine testing or imaging studies.</p>

Stage 2: Moderate reduction in GFR (60-89 ml/min/73.1 m) with kidney damage. Kidney damage is defined as pathological abnormalities or markers of damage, including abnormalities on blood or urine tests or imaging studies.

Stage 3: Moderate reduction in GFR (30-59 ml/min/1.73 m). British guidelines distinguish between stage 3a (59—45 GFR) and stage 3b (44—30 GFR) for screening and reference purposes.

Stage 4: Acute reduction in GFR (15-29 ml/min/1.73 m). Preparing for 15 kidney replacement therapy.

Stage 5: scheduled renal failure (GFR 15 mL/min/1.73 m ), renal replacement therapy (RRT), or end-stage renal disease.

(ESRD) renal disease

The expression kidney failure now refers to a medical condition in which the kidneys fail to efficiently filter waste products from the blood. Its two main forms are acute kidney disease, which is often treatable with adequate treatment, and chronic kidney disease, which is often intractable.

Secret

Ischemia-related kidney disease is reserved for those cases in which a clinically significant reduction results

A; A. A

In the renal glomerular filtration rate or loss of renal parenchyma due to renal artery stenosis hemodynamically on

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Significant or other causes that result in low blood pressure in the kidney, for example, hemodynamic shock or the use of an artery clamp to temporarily stop blood flow.

The inventors also explained that the protein according to the invention is useful in treating phosphatase deficiency

HPP (hypophosphatasia). This was completely unexpected given the failure of previous trials5 using TNAP to treat hypophosphatasia to demonstrate the effectiveness of enzyme replacement therapy. Accordingly, it was done

Whyte et al, N ) and bone shell peptide TNAP developed an artificial protein fusion between an enzyme

904-13:(10)366;8 Med. 2012 Mar for Engl). Surprisingly, the protein does not require application0 of the present invention to the aforementioned bone shell peptide and can effectively reduce signs of hypophosphatasia symptoms in a mouse model. In another preferred embodiment the invention thus provides a 10-nucleotide polyprotein and/or carrier according to the invention for use in the treatment of hypophosphatasia.

The basis of hypophosphatase metabolism stems from a molecular defect in a gene encoding an unidentified alkaline phosphatase enzyme.

It is a tissue non-specific alkaline phosphatase (TNAP).

On the outer surface of osteoblasts and the membrane of chondrocytes. TNAP naturally hydrolyzes many substances, including inorganic pyrophosphate (PPi) pyrophosphate 15 and pyridoxal 1 phosphate (PLP) 010050188-5 The main form of vitamin 96.

When TNAP is low, inorganic pyrophosphate (PPi) accumulates outside the cells and effectively inhibits the formation of hydroxyapatite (mineralization), causing rickets in infants and children and osteomalacia (soft bones) in adults. PIP is a major form of vitamin 86 and can be dephosphorylated by TNAP 20 to pyridoxal (PL) to cross the cell membrane. Vitamin 86 deficiency in the brain hinders the synthesis of nerve transmitters, causing seizures. In some cases, the decomposition of calcium pyrophosphate dehydrate (CPPD) crystals in the joints can cause pseudogout.

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There are no approved treatments for HPP today. Current management methods consist of explaining symptoms, monitoring calcium balance, using physical and occupational interventions, and dental and orthopedic treatment as necessary. Administration of bisphosphonate (a synthetic pyrophosphate analog) to one infant had no significant effect on the skeletal structure and disease progression of the infant.

5 infant until death at 14 months.

Bone marrow cell transplantation into two severely affected infants resulted in advances in radiological and clinical imaging, although the mechanism of action is not fully understood and a significant incidence of disease persists. Enzyme replacement therapy with normal serum or ALP-enriched serum from patients was not recommended

Whyte MP, Valdes R, Ryan LM, McAlister WH] Useful Paget's Disease of Bone (September 1982). “Infantile hypophosphatasia: enzyme replacement 10 therapy by intravenous infusion of alkaline phosphatase-rich plasma from patients with Paget bone disease.” L. Pediatr. 101 (3): 379-86][Whyte MP, McAlister WH, Patton LS, et al. (December 1984). "Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions

in three additional results alkaline phosphatase-rich Paget plasma: 15

Enzyme replacement therapies mentioned were not [patients]. Pediatr. 105 (6): 926-33.

It is effective because the alkaline phosphatase enzyme is supposed to function at the surface of the bone and not in the blood. Moreover, attempts to develop the effectiveness of enzyme replacement therapy were based on the TNAP enzyme, which...

Whyte et al. N Engl J Med. 2012 Mar 1 Targets bone with promising results

20 904-13:(10)8,366]. Surprisingly, the present invention presents without providing an artificial bone-targeting moiety a protein according to the invention that is useful in a murine model of phosphatase deficiency. Lay one

to. Bone Miner. ] Millan et Wah Whyte et al Advantages of the present protein over that described in

Res 777-787:(6) 2008, 23. Because due to the absence of an artificial bone targeting cleft the present protein is expected to be less immunogenic. The present protein consists of a protein with a high 25 sequence identity with naturally occurring human alkaline phosphatase proteins. In addition to the above, enjoy

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The protein of the present invention has an advantage over the bone-targeting enzyme alkaline phosphatase with respect to ease and cost of production.

In a preferred embodiment a polynucleotide protein and/or carrier according to the invention is provided for use in the treatment of hypophosphatasia where said hypophosphatasia is selected from hypophosphatasia in the surrounding period.

<p dir="rtl">5 By birth, hypophosphatase in children Hypophosphatase in childhood and hypophosphatase in adults.</p>

Perinatal hypophosphatase is the most malignant form of hypophosphatase. In utero, profound hypermineralization of the dome membrane results in deformity or shortening of the limbs during pregnancy and at birth and rapid death due to respiratory failure. Phosphatase deficiency appears in children in the first 6 years

<p dir="rtl">10 months old. Postnatal development appears normal until malnutrition, inadequate weight gain, clinical symptoms and rickets begin to be recognized. Hypercalcemia and hypercalciuria are also common and may explain nephrocalcinosis, renal disease, and episodes of recurrent vomiting. Mortality rates are estimated at 0% in the first year of life.</p>

Childhood hypophosphatasia has a variable clinical expression. As a result of aplasia deficiency

<p dir="rtl">15 aplasia. Or dysplasia of the cementum layer of the teeth, causing premature loss of baby teeth (i.e. before the age of 10 years). Often, incisors fall out first. Sometimes almost the entire primary teeth are erupted prematurely. Dental x-rays sometimes show widened pulp chambers and root canals (shell teeth) characteristics of rickets. Patients may also experience delayed walking, a characteristic sway in the way they walk, and complain of pain, stiffness, and weakness</p>

20 Appendiceal muscles (particularly in the thighs) consistent with non-progressive myopathy. Typically, radiographs show floccular deformities and characteristic bony defects near the ends of the major long bones (ie, “tongues” radiolucent projections from the floccular growth plate of the metaphysis). Delay is common. Development Frequent fractures and fragility In severely affected infants and young children despite the wide, “open” appearance of the fontanel on radiographic study, fusion is uncommon.

25 Functional bones from the sutures in the skull. The illusion of an "open" fontanelle results from large areas of the dome

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Hypermineralized. Later, premature osseointegration of the sutures into the skull may raise intracranial pressure.

Phosphatase deficiency in adults may be associated with rickets, premature loss of baby teeth or premature loss of teeth in adults followed by relatively good health. Osteomalacia appears in painful feet caused by poor foot pain

5 Healing of recurrent metatarsal fractures and discomfort in the thighs or buttocks due to pseudofemoral fractures, which, when seen on radiographic study, can be distinguished from most other types of osteomalacia (which occur medially) by their location in the lateral cortices of the proximal thighs. Some patients suffer from dissolution of calcium pyrophosphate dihydrate crystals with occasional attacks of arthritis (pseudogout).

10 Which appears to be a result of high levels of endogenous inorganic pyrophosphate

(PPi) pyrophosphate

These patients may also suffer from articular cartilage erosion

may be . Pyrophosphate arthropathy and pyrophosphate degeneration

Radiology reveals false fractures in the lateral cortices of the proximal thighs

15 proximal femora, stress fractures, and patients may suffer from osteopenia, chondrocalcinosis, features of pyrophosphate arthropathy, and calcified periarthropathy.

Calcific periarthritis

In a preferred embodiment a polynucleotide protein and/or vector is provided for use in treating deficiency -

20 Phosphatase according to the invention where the treatment results in prolongation of survival; Increase in body weight;

Improved skeletal phenotype (e.g. induction of ossification centers).

Development of bone mineralization, induction of secondary ossification centres

improvement of bone mineralization, for example in trabecular and/or cortical bone, or induction of osteoid); Attenuation of defects

shape (such as shape deformities) attenuation of craniofacial defects 25 craniofacial

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abnormalities and coronal suture fusion); Development of the dental phenotype

Such as the development of the alveolar molar (improvement of dento-alveolar phenotype).

improvement of molar height, dentin-thickness, and bone mineralization); And/or low levels of 01 in plasma in patients.

<p dir="rtl">• Also provided is the use of a polynucleotide protein and/or carrier according to the invention to prepare a drug for the prevention or treatment of hypophosphatase deficiency, where the treatment results in prolongation of survival Increase in body weight: Development of a skeletal phenotype (e.g. induction of secondary ossification centers Development of bone mineralization For example in trabecular and/or cortical bone or induction of osteoclasts): attenuation of craniofacial defects (e.g. shape abnormalities and fusion of the coronal suture): Development of a dento-alveolar phenotype (e.g. development of molar 10, tooth thickness and bone mineralization) and/or decreased plasma PPi levels in patients.</p>

A method is also presented for treating a subject who suffers from or is at risk of suffering from hypophosphatasia, whereby treatment results in prolongation of survival and increased body weight; Development of the skeletal phenotype (e.g. induction of secondary ossification centers Development of bone mineralization e.g. in trabecular and/or cortical bone or induction of osteoclasts): Attenuation of craniofacial defects (e.g. shape abnormalities and fusion of the coronal suture): Development of the dento-alveolar phenotype ( Such as molar development, tooth profile and thickness, and bone mineralization); And/or decreased plasma PPi levels in the subject.

In a more preferable embodiment a polynucleotide protein and/or vector is provided for use in the treatment of D deficiency

Phosphatase according to the invention, wherein said deficiency of phosphatase is selected from in infants during childhood 20 or deficiency of phosphatase in adults. Hypophosphatasia is more likely to be hypophosphatasia in children.

Standard Crescent Examples and References in the Field Another nomenclature is used to designate isotypes of the enzyme alkaline phosphatase. For the purpose of clarity, names and abbreviations commonly used or used in this application are listed in Table 1.

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Table 1: Synonyms and abbreviations used in the present application or generally known for the various types of alkaline phosphatase enzymes

<tr><td><p dir="rtl">Abbreviations</p></td><td><p dir="rtl">Alkaline phosphatase enzymes</p></td></tr><tr><td><p>ALPP, PLAP,</p></td><td><p dir="rtl">Placental alkaline enzyme</p><p>phosphatase</p></td></tr><tr><td><p>shPLAP, sALPP</p></td><td><p dir="rtl">Secretable placental alkaline phosphatase</p><p>Placental alkaline phosphatase</p></td></tr><tr><td><p>ALPI, IAP hlAP</p></td><td><p dir="rtl">Intestinal alkaline phosphatase</p><p>phosphatase</p></td></tr><tr><td><p>shlAP, sALPI</p></td><td><p dir="rtl">Secretable intestinal alkaline phosphatase</p><p>Intestinal alkaline phosphatase</p></td></tr><tr><td><p>GCAP</p></td><td><p dir="rtl">Placental-like alkaline phosphatase</p><p>alkaline phosphatase</p></td></tr><tr><td><p>140,3,ALPL,</p><p>TNSALP</p></td><td><p dir="rtl">Tissue-specific alkaline phosphatase enzyme</p><p>nonspecific alkaline phosphatase</p></td></tr><tr><td><p>catALPI/crownALPP, RecAP, Xinplap, sALPI-ALPP-CD</p></td><td><p dir="rtl">Alkaline phosphatase recombination enzyme includes the catalytic domain of ALPI and the crown domain of the ALPP protein.</p><p>Recombinant alkaline phosphatase</p><p>Comprising the catalytic domain of ALPI and the crown domain of ALPP</p></td></tr>

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<tr><td><p>RecAP</p></td><td><p>,LVLRecAP</p></td><td><p dir="rtl">Protein according to the invention</p></td></tr><tr><td></td><td><p dir="rtl">developed</p></td><td></td></tr>

What is meant by the expression “secretable” is that the glycosylphosphatidylinositol anchor (glycosylphosphatidylinositol) is not attached to a mature protein at a later stage. This helps the protein to be secreted and not bind to the membrane. The GPI anchor is a sugar lipid that can be attached to the C terminus of the protein during subsequent stage modification. It consists of a phosphatidylinositol group linked through a 5-carbohydrate bond (glucosamine and mannose to the inositol residue through a glycosidic bond) and via an ethanolamine phosphate (EtNP) bridge bond to the amino acid at the C terminus of the mature protein. Two fatty acids within the hydrophilic phosphatidyl-inositol group anchor the protein to the cell membrane. It is useful for production and post-processing and therefore preferably the protein according to the invention does not include a GPI stabilizer.

10 It is shown that any of the described modified secretable phosphatases (and thus also the secretable protein according to the invention) can be produced, for example, by introducing into a host cell a nucleic acid that can encode said secretable phosphatase in an operational association with the regulatory sequences and allowing the host cell to mentioned by expressing the said secretory phosphatase enzyme and optionally isolating and/or preserving the produced phosphatase enzyme from the medium in which the host cell grows.

<p dir="rtl">15 However, apart from the mutants in the previously mentioned GPI anchoring sequences, there are other methods that create non-GPI anchored secreted proteins, e.g.</p>

<p dir="rtl">1) After expression as membrane-anchored proteins, phospholipase enzymes can be used to cleave the GPI anchor. The invention then also provides a method for producing a secreted phosphatase enzyme comprising culturing a host capable of expressing the membrane-anchored phosphatase enzyme, allowing said host cell to secretly produce said phosphatase enzyme and optionally incubating the cells obtained with the phospholipase enzyme.</p>

Isolation of the released phosphatase enzyme.

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<p dir="rtl">2) Interfering with the production of a GPI stabilizer or using a cell (type) that is also deficient in producing a GPI stabilizer can be used to manufacture a secretable form of another GPI stabilized protein. Examples of cell lines that have been generated to be biochemically deficient in GPIing are for example CHO, S49, BW 084, B-AM, Jurkat and Raji. In yet another embodiment of the invention based on 5 thereof a method for producing secretory phosphatase enzyme is provided comprising culturing a host cell capable of expressing a secretory (alkaline) phosphatase enzyme (e.g. a host cell comprising a nucleic acid sequence encoding any of the modified secretory phosphatase enzymes (e.g. Alkaline (mentioned) and allowing the aforementioned host Cr</p>

By producing said secretory phosphatase enzyme and optionally isolating said phosphatase enzyme produced where said host cell is unable to biosynthesise functional GPIed stabilizer proteins. However, a family 10 cell can also produce a phosphatase using a functional GPI signal sequence.

<p dir="rtl">3) Interfering with or using a cell deficient in transamidases can be used to inhibit the attachment of a GPI anchor to a protein, rendering the protein unfixable and secretable. The aforementioned deficiency cell was obtained through mutagenesis</p>

.CHO

<p dir="rtl">15 The vector according to the invention may preferably include additional nucleic acid sequences such as elements necessary for genetic transcription/translation of the amino acid sequence encoding the phosphatase (e.g. enhancer and/or terminator sequences). Said vector may also include nucleic acid sequences encoding Selection markers (eg an antibiotic) to select or maintain host cells transformed with the vector. Preferably, a nucleotide sequence encoding the 20 GPI anchor sequences should not be present in a polynucleotide and/or vector according to the invention. Examples of suitable vectors include</p>

On cloning or expression vectors. Any vector suitable for mediating expression in a suitable host cell may be used according to the invention either as an integrated or chromosomally transcribed particle into a host cell. The vector may be a plasmid, a virus (e.g. retrovirus, adenovirus, adeno-associated virus 000-550,000 virus, baculovirus 25 and/or derivatives thereof), a cosmid, a phage or a phage assembly.

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M••, artificial vector

.chromosome

In addition to the above, the invention also provides a family cell comprising a protein, nucleic acid sequence or vector according to the invention as described. The cell may be a eukaryotic cell, preferably a mammalian cell, a plant cell, or a yeast cell that is suitable for producing proteins resulting from genetic recombination. Cells of a family of suitable yeasts include, for example, Saccharomyces cerevisiae and Pichia pastoris. The preferred host cells are mammalian (or more preferably human) derived cells e.g

HEK293, BHK, Chinese hamster ovary (CHO) or PerC6TM. In a particular preferred embodiment a family cell is a Chinese hamster ovary cell

.hamster ovary (CHO) 10

and

A protein-coding nucleic acid sequence according to the invention comprises a vector comprising said nucleic acid sequence and/or a host cell containing said nucleic acid sequence that is extremely useful in producing a protein according to the invention. The protein according to the invention includes glycosylation sites and thus the protein is preferably produced in cells that provide the desired glycosylation mode. In a preferred model production system

<p dir="rtl">15 The user is a mammalian (e.g. human) laboratory production platform, preferably the production also involves large-scale production. In another preferred embodiment, the production system used is a plant, yeast, or mammalian (preferably non-human) platform in which an artificial human-like glycosylation mode is provided.</p>

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In one embodiment the invention thus provides a method for producing a protein according to the invention. The method includes culturing a host cell comprising a polynucleotide according to the invention or a vector according to the invention and allowing a host cell to produce said protein. The preferred host cells are mammalian (or more preferably human) derived cells such as HEK293, BHK, Chinese hamster ovary (CHO) or not. In a particular preferred embodiment, a host cell is a Chinese hamster ovary (CHO) cell. . Preferably a method for producing a protein according to the invention further comprises aggregation and optionally purification of said protein from said culture.

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According to the already mentioned protein described in the present application according to the invention is useful in treatment. In one embodiment the invention provides a composition preferably a pharmaceutical composition comprising a protein according to the invention. Said pharmaceutical composition optionally includes a pharmaceutically acceptable carrier, diluent or excipient

.excipient

<p dir="rtl">5 The composition may be presented in any form, for example in the form of a tablet, an injectable fluid, an infusion fluid, etc. In addition to the above, the nucleotide protein composition and/or carrier according to the invention can be administered via different routes, for example intravenously, in the rectum, in the bronchial tubes, or orally. Another appropriate route of administration is using a duodenal drip</p>

. duodenal drip

10 In a preferred embodiment the route used for administration is an intravenous route. It is apparent to an experienced person that it is preferable to deliver an effective amount of protein in accordance with the invention. As a starting point, 1-50,000 units/kg/day can be used. Another suitable route, for example for HPP, is the subcutaneous route. If the intravenous route of administration is used the protein according to the invention may be administered (at least for a specified period of time) by continuous infusion.

This is a secret

15 Said composition according to the invention may optionally include pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeators, propellants, desinfectants, diluents and preservatives.

.preservatives

Suitable excipients are commonly known in the field of pharmaceutical formulation and can be quickly found20 and used by the skilled reference eg. Remmington's.

Pharmaceutical Sciences, Mace Publishing Company, Philadelphia PA,

17th ed. 1985

For oral administration, protein can be administered e.g. in solid dosage forms such as tablet capsules (eg with enteric coating) and powders or in liquid dosage forms

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Such as elixirs, juices, and suspensions. AP may be encapsulated in softgels along with inactive ingredients and powdered carrier materials such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, and sodium saccharin. Talcum, magnesium carbonate and the like.

Examples of additional inactive ingredients that may be added to provide the desired color, taste, stability, buffering ability, dispersibility or other known desired attributes include red iron oxide, silica gel, sodium lauryl sulphate 10, sodium lauryl sulphate, etc. Titanium dioxide, white edible ink and the like.

Similar diluents can be used to manufacture compressed tablets. Both tablets and capsules can be formulated as sustained release products to provide sustained release of a drug over a period of hours. Compressed tablets may be sugar or film coated to mask any unpleasant taste and protect the tablet from the atmospheric climate 15 or enteric coated for selective disintegration in the gastrointestinal tract. Images may contain liquid dosage

For oral administration on color and flavor to increase patient acceptance.

and

In a preferred embodiment the composition comprises a protein according to the invention that is suitable for oral administration and includes an enteric coating to protect the AP from the adverse effects of gastric juice and the pH of

The low. Enteric coating and controlled release formulations are well known in the industry. The 20 enteric coating formulations in the art may comprise a solution of a water-soluble enteric coating polymer mixed

With the active ingredients and other excipients which are dispersed in an aqueous solution and which can subsequently be dried and/or made into pellets. The enteric coating formed provides resistance to protein attacks according to the invention by atmospheric moisture and oxygen during storage and by intestinal fluids and low pH after digestion while being easily broken down under alkaline conditions found in the intestinal tract25.

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The invention provides a composition according to the invention for use as a medicine preferably for the treatment of a disease accompanied by a local or systemic deficiency of zinc, inflammatory diseases, kidney disease, or phosphatase deficiency. The inflammatory disease should preferably be selected from the group consisting of autoimmune diseases, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, and inflammatory diseases of the ducts.

5 gastro-intestinal tract, infection, sepsis, neurodermatitis, sepsis, neurodermatitis, inflammatory liver disease, inflammatory lung disease^^ inflammatory kidney disease. Renal disease should preferably be selected from the group consisting of kidney failure, acute kidney injury, chronic kidney disease, and kidney disease

10 related to ischemia. The hypophosphatase deficiency should preferably be selected from the group consisting of perinatal hypophosphatase deficiency in children Hypophosphatase deficiency in childhood 10000050885 childhood, and adult hypophosphatasia

.hypophosphatasia

In addition to the fact that the protein according to the invention can be incorporated into a pharmaceutical composition, said enzyme phosphatase 15 may also be part of a nutritional composition or nutritional food.

So. Yes

The protein according to the invention can be added to a food substance (e.g. milk) and can also be produced within said food substance (e.g. by molecular engineering). In addition to the above, tablets and/or capsules can be prepared which are later added to a nutritional substance or which can be taken directly by a person.

20 Furthermore, a method for treating a subject suffering from inflammatory kidney disease or hypophosphatase deficiency is presented which includes administering an effective amount of protein according to demand, for example, an isolated protein or gene recombination product having phosphatase activity, where a portion of said protein includes An amino acid sequence of at least 50 amino acids The sequence has at least 90% sequence identity with the full-length crown domain of human PLAP comprising a portion of the protein

25 mentioned in an amino acid sequence in which at least 0 0 2 amino acids in the sequence are 90% identical.

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A sequence with the N-terminal region adjacent to the crown domain of human ALPI, and a portion of said protein includes an amino acid sequence containing at least 40 consecutive amino acids having 90% sequence identity with the C-terminal region adjacent to the crown domain of human ALPI, where the protein with The full-length amino acid sequence has a sequence identity of at least 9% (5) with the full-length amino acid sequence of a sequence with identity number: 1, provided that the amino acid

At position 279 is leucine, the amino acid at position 328 is valine, and the amino acid at position 478 is leucine (L), or an effective amount of a polynucleotide according to the demand or an effective amount of a vector according to the demand.

The invention can be explained in more detail in the following non-limiting examples.

10 Brief explanation of the drawings

Figure 1: Amino acid sequences of mature protein 500-1/hIAP and hPLAP. Putative crown domains of different proteins are underlined.

Figure 2: Organ distribution for 05000-/1 compared to catALPI/crownALPP. The organ-to-blood distribution of LVL-RecAP is depicted divided by the organ-to-blood distribution of catALPI/crownALPP.

15 (y-axis ratio) 0 A higher value indicates that LVL-RecAP targets a corresponding organ when comparing

.catALPI/crownALPP b

Figure 3: Survival of 4502-/- mice treated with either 1 mg LVL-RecAP/kg/day^ or 16 mg LVL-RecAP/kg/day^ or 16 mg LVL-RecAP/kg/day.

Figure 4: Serum creatinine concentrations in piglets with kidney ischemia/reperfusion damage 20. Sham animals underwent a surgical procedure during which the left kidney was removed. And with

Therefore, renal embolization and reperfusion were not performed. In the group that received 0.32 mg/kg/day on day 0, half the dose was given before reperfusion and the remaining half dose was given at 8 ± 2 hours after reperfusion. The full dose was given daily for the remainder of life.

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Figure 5: AP concentrations in piglets with renal ischemia/reperfusion damage. Sham animals underwent a surgical procedure during which the left kidney was removed. However, renal embolization and reperfusion were not performed. In the group that received 0.32 mg/kg/day (200 units/kg/day) on day 0, half the dose was given before reperfusion and the remaining half dose was given at 8 ± 2 • hours after reperfusion. The full dose was given daily for the remainder of life.

Figure 6: Evolution of survival and body weight of Alpl-/- mice by LVL-RecAP treatment. A) 0500016-1/1 survived until the end of the experiment. Median survival was p22.019 and p42.5 for LVL-RecAP1 and LVL-RecAP8 carriers, respectively. b) Vector-treated Alpl −/− mice are lighter than WT pups from the same litter: treatment improves

10 LVL-RecAPl body weight is close to that of WT at 0Ρ18. Vector-treated mice did not survive longer and mice treated with 08-11 were still lighter than WT littermates from the same litter at 0ρ53, while 60016-/1-treated mice were Body weight

It is not significantly different from the small WT of the same litter at 0ρ53

Figure 7: LVL-RecAP treatment improves the skeletal phenotype of Alpl-/- mice. 15 x-rays of the spine, fore limbs, rib cage, paws and hind limbs of mice

-402/ Treatment with vector 00008, LVL-RecAPl-71, LVL-RecAP16, and untreated WT control groups (magnification Χ5(0) -402/ mice showed severe osteomalacia (arrows) in the vertebrae, long bones, and rib cage. Secondary ossification centers Missing in 02-/- mice (asterisk). Treatment with LVL-RecAPl slightly corrects the 20 phenotype (arrowheads) compared to untreated WT at 0ρ18. B) Treatment with LVL-recAPl slightly corrects the 20-recAPl phenotype (arrowheads) compared to untreated WT at 0ρ18.

RecAP8 and LVL-RecAP16 clearly show an osteogenic phenotype in the long bone vertebrae and rib cage. Specifically, most of the distal extremities improve, with the development of secondary ossification centers in the metatarsal region becoming clear (arrowheads).

Figure 8: Advanced mineralization in LVL-RecAP-treated Alpl-/- mice. A) Histological analysis 25 of the thighs of Alpl (018) —/— mice treated with LVL-RecAP16 (p51) vector LVL-RecAP8.

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p53, and p53-untreated WT mice. Von Kossa staining revealed better bone mineralization with increasing doses of 560408-/1 8004916-/. The secondary ossification centers and cortical bone area show a surprising development in mineralization (black) compared to the untreated. There is less trabecular bone in LVL-RecAP8 and LVL-RecAP16 compared to WT, but increased mineralization in the trabecular region.

• As well as more osteoblasts are expected to become trabecular bone. B/C) BV/TV OV/BVj analysis of 50008-1 and LVL-RecAP16 had lower bone volume and higher osteoid volume than age-matched control groups.

Figure 9: Development of osteomalacia and plasma PPi levels in Alpl-/- mice treated with -LVL RecAP. A) Histological analysis of the thighs of Alpl-/- treated with vector (50008), (p18-471).

10(051)0500016 p53-171 and p53-untreated WT mice. Goldner's Trichrome staining of femoral segments demonstrated severe osteomalacia in Alpl—/—, and development of mineralization in the cortical zone and in secondary ossification with increasing doses of 86008-/1 8604016-. The presence of large areas of osteoid bone, which suggests bone decomposition rather than mineralization. 8/0) PPi concentrations in the plasma of WTj-02/ mice receiving 88001-7 880016-/WTj. Performs PT-LVL treatment

15 80091 to a significant reduction in elevated PPi levels in Alpl-/- mice at p18. At p53 LVL-RecAP16 treatment led to correction of plasma PPi levels compared to the WT control groups.

Figure 10: Absence of craniofacial defects in Alpl-/- mice treated with LVL-RecAP. Countertop pCT images of WT (a, d), vector-treated Alpl-/- (b, e) and LVL-RecAP1620 (c, f) mouse skulls at p21 and p53, respectively. Neither the frontal bones nor the parietal bones differed

treatment of Alpl-/- mice than that of WT mice. Adult skulls of Alpl-/- mice treated with 004016-/1 did not appear different from WT in terms of size and shape.

Figure 11: LVL-RecAP treatment partially rescued the dentoalveolar phenotype in Alpl-/- mice.

(8) Radiographic and (d) pCT analysis Main type comparisons at 26-025 (B, 25 3) Mandibular hypermineralization in untreated Alpl-/- mice and decreased alveolar bone (AB),

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-٣٦-

Short molars (M2, M1, and M3) with thin dentin (0), wide pulp chambers, and defective dentin (white asterisk) in incisor tooth (0). Compare (B) Histological analysis of comparative gingival tissue (G) Ama-/ mice do not show acellular cementum layer (40) and alveolar osteoblasts invade the PDL space, forming sclerotic dentin (asterisk). (H,C) LVL-RecAP8-treated 0 −/− mice show development

5 Radiographic appearance of molar height, dentin thickness and bone mineralization despite the persistence of incisor defects. (I) 500-1/5 does not histologically restore the acellular cementum layer to the root surface. Compared to control groups (L, L) at 053 (M, K), Alpl-/- mice treated with 0500016-11 show decreased alveolar bone mineralization around grinding teeth. Molar formation and mineralization appear relatively normal in treated Alpl-/- mice while 10 incisor teeth remain severely affected on the analog root (white asterisk). Histological analysis of (N) molars of comparison (P) and WT) Alpl-/- mice treated with 500016-471 depicts a combination of mineralized alveolar bone and osteoclasts (stars) and decreased PDL space with its remainder. Poor attachment to the gums due to the lack of cementum layer is manifested by disorganization of the PDL, its separation and decreased growth of junctional epithelium. Small areas of PDL (badge) connection to the tooth are noted. Compared to organization

15 Strong, parallel PDL collagen fibrils in (O) comparative tissue shown by picrosirius staining under polarized light (Q) Alpl-/- mice treated with 80016-/1 present a less organized PDL although areas of organization and conduction are present adjacent to penetrating zones for tooth root conduction. .

Figure 12: RecAP treatment attenuates LPS-stimulated cytokine production in human proximal tubule epithelial cells.

(ciPTEC). CiPTEC were pre-treated with recAP (1-5-10 U/ml) followed by LPS incubation (10 μg/ml) for 24 h and thus the production of IL-6, TNF-α and IL-8 (IL-8) was measured at the gene level. In cells by qPCR (10 U/ml recAP) and ( ) at the protein level in supernatant solution by ELISA (C). recAP (10 U/ml) was administered 2 hours before exposure.

25 to LPS, simultaneously with LPS or 2 hours after exposure to LPS, followed by content measurement

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-٣٧-

IL-6' TNF-α and 8-protein. (9) ciPTEC were pretreated with inactive AP for 2 h. It lacks hydrolysis properties, followed by incubation with 10 μg/ml LPS for 24 hours, after which the protein content of IL-6, TNF-α, and IL-8 was measured. The control cells were incubated with culture media. Data were expressed as mean ±SEM (h) # 0.05 h compared to control group, * 0.05 h compared to LPS.

Figure 13 The effects of recAP are not limited to LPS-stimulated inflammation and are kidney-specific. CiPTEC were pretreated with recAP (10 U/ml) for 2 h followed by incubation for 24 h with (B) TNF-α (10 ng/ml) or (B) supernatant lysate of peripheral blood mononuclear cells (PBMCs) mimicked with LPS. , 1 ng/ml LPS), and then the production of IL-6 and IL-8 was measured in C

10 Protein level by ELISA (C). PBMCs were pre-incubated for 2 h with recAP (10 U/ml) followed by exposure to LPS (1 ng/ml) for 24 h. IL-6 and TNF-α production were measured by ELISA. The control cells were incubated with culture media. Data were expressed as mean ±SEM (h5) # p&lt;5”,0 compared to comparator * ρ&lt;05,0 compared to LPS.

15 Figure 14: Effect of recAP on LPS-stimulated ATP release in vitro. ciPTEC were pretreated with recAP (10 U/ml) followed by LPS incubation (10 μg/ml or 100 μg/ml). After 30 minutes, the supernatant solution was collected to determine cellular ATP release by biofluorescence. The control cells were incubated with culture media. Data were expressed as mean ± SEM (h5), #ρ&lt;0.05 compared to the comparator.

20 Figure 15: RecAP prevents LPS-stimulated decline in renal function in vivo. Renal function was assessed by transdermal measurement of FITC—sinistrin tl/2 sinistrin. AKI in rats was induced by LPS (0.3 mg/kg 0b0w &lt; t=0)' followed by treatment with recAP (1000 U/kg b0w.) at =. 2/Tl measurements were made at 4h and 4h=21h (B' A: Examples of FITC—sinestrin kinetics obtained for a single rat at two consecutive time points).

25 Urine was collected between t=5 and t=16, and a plasma sample was obtained at t=5' 1 unit24

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An hour and allow the calculation of (C) Fractional Urea excretion and () Creatinine clearance with the average plasma value = 5 1/24. Data were expressed as mean ±SEM (placebo LPS n=6? LPS+recAP n=5) # 0.05 compared to placebo.

5 Figure 16: RecAP prevents kidney injury during LPS-stimulated AKI in vivo. (A) Excretion of 1-KIM in urine and () Excretion of NGAL, C) NGAL levels in plasma (1=24) and () The content of 1-KIM protein in the kidneys was determined by ELISA. Kidney sections were embedded in paraffin. Paraffin kidney sections using anti-1-KIM to view expression of the 1-KIM protein. Scale bars: 0 0 4 μM (left panel) 200 μM (right panel). Expressed

10 Data are presented as mean ± SEM (Placebo, LPS, p=6? LPS+recAP, p=5: Urine variables: Placebo, p=e), #p&lt;05, 0 compared to placebo.

Figure 17. There is no effect of recAP on LPS-stimulated cytokines in supernatant. A supernatant solution of LPS was incubated for 24 h (10 μg/ml) or incubated in medium (comparator). ciPTEC were collected, and incubated with or without recAP (10

15 units/ml) for another 24 hours followed by measurement of IL-6, TNF-α and IL-8 by ELISA. Data were expressed as mean ± p # , SEM &lt; 5 0, 0 compared to comparator * p &lt; 05, 0 compared to LPS.

Figure 18. Relationship between enzyme activities of RecAP in human serum at 25°C and 37°C.

20 Figure 019 Relationship between enzyme activities of LVL-RecAP in human serum at 25°C and at 37°C.

Figure 20 displays activity/μg protein at 25°C and at 37°C for RecAP and 0LVL-RecAP. The values represent the average activity Δ between 25°C and 37°C for a given protein concentration.

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Detailed description:

Examples

Example 1

Stability of LVL-RecAP in buffer solution

5 Materials:

Alkaline phosphatase enzymes, human recombinant alkaline

:phosphatases

01 sALPI-ALPP-CD (sequence ID number: 4) (2008-9-8-04:00)

02 LVL-recAP (Series ID: 1) (DOM: 14-Oct-20H)

10 ways:

Zinc dependency:

Determine the enzyme activity and protein content of each batch of alkaline phosphatase enzyme resulting from human genetic recombination. Protein solutions in the next step, 100 μg/ml, were prepared for each condition for determination

Zinc dependence for batches sALPI-ALPP-CD and LVL-recAP as shown in Table 02

15 The prepared samples were stored at room temperature and analyzed for enzyme activity at =1. 1=2h24h0

Table 2: Conditions for batches of alkaline phosphatase enzyme recombination products sALPI-ALPP-CD and LVL-recAP to determine their stability (retained activity) in the presence and absence of zinc and the effect of a chelating agent.

0(EDTA)

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— ٤٠ —

<tr><td><p>mM EDTA</p><p dir="rtl">Molar</p></td><td><p dir="rtl">Mannitol(%)</p></td><td><p dir="rtl">79 A (Milli Mola</p></td><td><p>BSA</p><p>(%)</p></td><td><p dir="rtl">Zn micro</p><p dir="rtl">Molar</p></td><td><p dir="rtl">the condition</p></td></tr><tr><td></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">١</p></td></tr><tr><td></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠١٠٠</p></td><td><p dir="rtl">٢</p></td></tr><tr><td></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">١٠</p></td><td><p dir="rtl">٣</p></td></tr><tr><td></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">١٠٠</p></td><td><p dir="rtl">٤</p></td></tr><tr><td><p dir="rtl">٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">١٠٠٠</p></td><td><p dir="rtl">H</p></td></tr><tr><td><p dir="rtl">٢</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">H</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٧</p></td></tr><tr><td><p dir="rtl">١٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٨</p></td></tr><tr><td><p dir="rtl">١٠٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٩</p></td></tr><tr><td><p dir="rtl">٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠٢٥٠٠</p></td><td><p dir="rtl">0 e</p></td><td><p dir="rtl">١٠</p></td></tr>

About - — — - -

Determinations of enzyme activities were according to standard procedures, as outlined in 500 PC001.

Protein concentrations were determined by 00280 measurements (srecAP 1.01 mL/mg/cm 00280)

Results

Table 3:

and

5 Activity of different alkaline phosphatase enzymes under conditions as specified in Table 2.

<tr><td colspan="3"><p>LVL-RecAP</p></td><td colspan="3"><p>sALPI-ALPP-CD</p></td><td></td></tr><tr><td><p dir="rtl">-٢٤1</p></td><td><p dir="rtl">-٢1</p></td><td><p>0t-</p></td><td><p dir="rtl">-٢٤1</p></td><td><p>2t=</p></td><td><p>0t-</p></td><td><p dir="rtl">Diluted</p></td></tr>

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-٤١-

<tr><td><p dir="rtl">٤.٥٧</p></td><td><p dir="rtl">٨.٥٩</p></td><td><p dir="rtl">٧٠٦٠</p></td><td><p dir="rtl">٥٠</p></td><td><p dir="rtl">٣.٥٣</p></td><td><p dir="rtl">٧.٤٥</p></td><td><p dir="rtl">١</p></td></tr><tr><td><p dir="rtl">١.٥٨</p></td><td><p dir="rtl">٢.٥٧</p></td><td><p dir="rtl">٧٠٥٧</p></td><td><p dir="rtl">٦.٥١</p></td><td><p dir="rtl">٨.٥٣</p></td><td><p dir="rtl">٢.٤٨</p></td><td><p dir="rtl">٢</p></td></tr><tr><td><p dir="rtl">٩.٥٢</p></td><td><p dir="rtl">٢.٥٤</p></td><td><p dir="rtl">٧٠٥٧</p></td><td><p dir="rtl">٦.٥١</p></td><td><p dir="rtl">٤.٥٤</p></td><td><p dir="rtl">٣.٤٧</p></td><td><p dir="rtl">٣</p></td></tr><tr><td><p dir="rtl">٧.٥٥</p></td><td><p dir="rtl">٧.٥٣</p></td><td><p dir="rtl">٣.٥٥</p></td><td><p dir="rtl">٤.٥٣</p></td><td><p dir="rtl">٥٢</p></td><td><p dir="rtl">١.٤٦</p></td><td><p dir="rtl">٤</p></td></tr><tr><td><p dir="rtl">٧.٥٤</p></td><td><p dir="rtl">٣.٥٦</p></td><td><p dir="rtl">٧.٥٦</p></td><td><p dir="rtl">٢.٥٢</p></td><td><p dir="rtl">٢.٥١</p></td><td><p dir="rtl">٢.٤٨</p></td><td><p dir="rtl">٥</p></td></tr><tr><td><p dir="rtl">٧.٣٧*</p></td><td><p dir="rtl">٢.٥٤*</p></td><td><p dir="rtl">٦.٥٧</p></td><td><p dir="rtl">٧.١٨</p></td><td><p dir="rtl">٢.٢٤</p></td><td><p dir="rtl">٩.٤٦</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">٩.٣٨*</p></td><td><p dir="rtl">١.٥٢*</p></td><td><p dir="rtl">٦.٥٧</p></td><td><p dir="rtl">٤.٢٠</p></td><td><p dir="rtl">٦.٢٣</p></td><td><p dir="rtl">٤٧</p></td><td><p dir="rtl">٧</p></td></tr><tr><td><p dir="rtl">٨.٣٥*</p></td><td><p dir="rtl">٣٠٥٠*</p></td><td><p dir="rtl">٨.٥٦</p></td><td><p dir="rtl">٢.١٧</p></td><td><p dir="rtl">٢.٢٣</p></td><td><p dir="rtl">٤٥</p></td><td><p dir="rtl">٨</p></td></tr><tr><td><p dir="rtl">٢.٣١*</p></td><td><p dir="rtl">٥.٤٦*</p></td><td><p dir="rtl">٦.٥٥</p></td><td><p dir="rtl">٢.١٦</p></td><td><p dir="rtl">٥.٢٢</p></td><td><p dir="rtl">٩٠٥٠</p></td><td><p dir="rtl">٩</p></td></tr><tr><td><p dir="rtl">٤.٥٥</p></td><td><p dir="rtl">٥٠٥٧</p></td><td><p dir="rtl">٧.٦٢</p></td><td><p dir="rtl">٦.٥٣</p></td><td><p dir="rtl">١.٥٥</p></td><td><p dir="rtl">٤٧-٣</p></td><td><p dir="rtl">١٠</p></td></tr>

As is clearly evident in Table 3:

LVL-RecAP is significantly more stable (i.e. displays more residual enzyme activity: indicated by asterisk values) in the presence of an metal chelating agent (EDTA) than sALPI-ALPP CD, indicating less Zn2+ dependence of its activity.

5 Example 2

Stability of LVL-RecAP in buffer solution

Materials:

Alkaline phosphatase enzymes produced by human genetic recombination:

(DOM: O4-Aug-2OO8) sALPI-ALPP-CD 01

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-٤٢-

(00:14-Oct-2011) LVL-recAP .2

Roads

In a second independent experiment, the stability of sALPI-ALPP-CD and LVL-recAP was tested under several conditions as shown in Table 4.

5 For each of the AP lots, the result of genetic recombination. In this experiment, 0.01 µg/ml protein solutions were prepared in 0.025 M glycine buffer, pH 9.6, human serum and human citrate plasma for each condition buffer solution. To determine stability.

All AP gene recombination samples that were prepared were incubated at 37°C and their enzyme activity was analyzed at:

10 1= 0, T= 0.5 hours, T= 1 hour, T= 2 hours and T= 24 hours.

Table 4: Alkaline phosphatase conditions for sALPI-ALPP-CD and LVL-recAP batches. Recombination product in glycine buffer solution to determine its stability (retained activity) in the presence/absence of zinc and the effect of a chelating agent (EDTA or citrate).

<tr><td><p>EDTA</p><p dir="rtl">(millimolar)</p></td><td><p dir="rtl">Mannitol</p><p>Mannitol</p><p>(%)</p></td><td><p dir="rtl">Mg (milli mola</p></td><td><p>BSA%</p></td><td><p dir="rtl">Zn (micro mola</p></td><td><p dir="rtl">the condition</p></td></tr><tr><td><p dir="rtl">٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">١</p></td></tr><tr><td><p dir="rtl">٢</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٢</p></td></tr><tr><td><p dir="rtl">١٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٣</p></td></tr><tr><td><p dir="rtl">١٠٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">٤</p></td></tr><tr><td><p dir="rtl">٠</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td><td><p dir="rtl">٠٢٥٠٠</p></td><td><p dir="rtl">٥٠</p></td><td><p dir="rtl">٨</p></td></tr>

٦٣١٣

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Results

and

Table 5 - Activity of different alkaline phosphatase enzymes under conditions as specified in Table 4.

<tr><td colspan="5"><p>sALPI-ALPP-CD</p></td><td colspan="5"><p>LVL-recAP</p></td><td><p dir="rtl">adverb</p><p dir="rtl">H</p></td></tr><tr><td><p dir="rtl">Z241</p><p dir="rtl">٠</p></td><td><p>2t=</p></td><td><p>1t=</p></td><td><p>00t=</p><p dir="rtl">٥</p></td><td><p>0T=</p></td><td><p>24t=</p></td><td></td><td><p>1t=</p></td><td><p>.0t=</p><p dir="rtl">٥</p></td><td><p>0t=</p></td><td></td></tr><tr><td><p dir="rtl">٢٠٥٥</p></td><td><p dir="rtl">٠٥٣</p><p dir="rtl">٨</p></td><td><p dir="rtl">٠٥٣</p><p dir="rtl">٨</p></td><td><p dir="rtl">٥٠٥٢</p></td><td><p dir="rtl">٦٣</p></td><td><p dir="rtl">٨.٥٥</p></td><td><p dir="rtl">٢.٥٥</p></td><td><p dir="rtl">٠٥٤</p><p dir="rtl">٩</p></td><td><p dir="rtl">٧٠٥٧</p></td><td><p dir="rtl">٠٥٥</p><p dir="rtl">١</p></td><td><p dir="rtl">١</p></td></tr><tr><td><p dir="rtl">٣.٢٣</p></td><td><p dir="rtl">٠٣٦</p><p dir="rtl">٣</p></td><td><p dir="rtl">٠٥١</p><p dir="rtl">٦</p></td><td><p dir="rtl">٤٨</p></td><td><p dir="rtl">٠٥٦</p><p dir="rtl">٥</p></td><td><p dir="rtl">٧٠٣٧</p><p dir="rtl">*</p></td><td><p dir="rtl">٦.٥١</p><p dir="rtl">*</p></td><td><p dir="rtl">٥٢</p></td><td><p dir="rtl">٥٠٥٢</p></td><td><p dir="rtl">٠٥٩</p><p dir="rtl">٥</p></td><td><p dir="rtl">٢</p></td></tr><tr><td><p dir="rtl">٩.١٨</p></td><td><p dir="rtl">٠٣٢</p><p dir="rtl">٩</p></td><td><p dir="rtl">٠٤٣</p><p dir="rtl">٧</p></td><td><p dir="rtl">٤٤ 7</p></td><td><p dir="rtl">٣٠٣</p></td><td><p dir="rtl">٢.٣٥</p><p dir="rtl">*</p></td><td><p dir="rtl">٢٠٤٩</p><p dir="rtl">*</p></td><td><p dir="rtl">٠٥١</p><p dir="rtl">٦</p></td><td><p dir="rtl">٥.٥٣</p></td><td><p dir="rtl">٠٥٠</p><p dir="rtl">٩</p></td><td><p dir="rtl">٣</p></td></tr><tr><td><p dir="rtl">٣.١٣</p></td><td><p dir="rtl">٠٢٩</p><p dir="rtl">٤</p></td><td><p dir="rtl">٠٣٤</p><p dir="rtl">٦</p></td><td><p dir="rtl">٩.٣٧</p></td><td><p dir="rtl">٠٤٩</p><p dir="rtl">٨</p></td><td><p dir="rtl">٢٠٢٤</p><p dir="rtl">*</p></td><td><p dir="rtl">٣.٤١</p><p dir="rtl">*</p></td><td><p dir="rtl">٠٤٨</p><p dir="rtl">٧</p></td><td><p dir="rtl">٩.٤٣</p></td><td><p dir="rtl">٠٤٩</p><p dir="rtl">٣</p></td><td><p dir="rtl">٤</p></td></tr><tr><td><p dir="rtl">٥٦</p></td><td><p dir="rtl">٠٥٧</p><p dir="rtl">٥</p></td><td><p dir="rtl">٠٥٧</p><p dir="rtl">٩</p></td><td><p dir="rtl">٥٦</p></td><td><p dir="rtl">٠٥٥</p><p dir="rtl">٩</p></td><td><p dir="rtl">١.٥٢</p></td><td><p dir="rtl">١.٥٣</p></td><td><p dir="rtl">٠٥٣</p><p dir="rtl">٩</p></td><td><p dir="rtl">٣.٤٩</p></td><td><p dir="rtl">٠١٢</p><p dir="rtl">٢</p></td><td><p dir="rtl">٨</p></td></tr>

Determinations of enzyme activities were performed according to SOP PC001.

Protein concentrations were determined by 00280 measurements (1.01 mL/mg/cm 09280).

Secret

5 As clearly shown in Table 5 and in line with the results in Table 3, 47-80 is significantly more stable in the presence of an metal chelating agent (EDTA) than sALPI-ALPP CD (as indicated by asterisk values), indicating lower reliability. For Zn2+ for its activity.

Example 3

٦٣١٣

-٤٤-

Thermal stability and PLP kinetics of LVL-RecAP

Roads

Protein expression

Expression plasmids containing 09-4-5 and secreted LVL-RecAP tagged Sr were generated.

Kozlenkov et al. For Biol Chem 277,) according to the previously described FLAG epitope 22992-22999 (2002) and Kozlenkov et al. Bone Miner. Res. 19, 1862

(2004) 1872). FLAG-tagged enzymes were transfectionally transfected into COS cells

1 By electrophoresis and then cultured in DMEM medium for 24 hours according to the conditions

Narisawa et al. Am.l. Physiol. Gastrointest. Live Physiol. ) as described previously

Serum-free Opti-MEM 10 (2007) 1077-651068, 293) when the medium was replaced with

(Life Technologies) Secreted proteins containing Opti-MEM were collected 60 h posttransfection and subsequently filtered through a 2 μM cellulose acetate filter and dialyzed against TBS containing 1 mM MGCl2 and 20 μM ZnCl2.

Enzyme kinetics

15 To measure the relative catalytic activities of FLAG-tagged enzymes, microtiter plates were coated with the Sigma-Aldrich antibody (FLAG M2) at 0.2-0.6 μg mL-1. These plates were incubated with saturating concentrations of FLAG-tagged LVL-RecAP or sALPI-PLAP for 3 hours at room temperature after which the plates were washed with 085 containing 0.008% 80-Tween and the relative activities of PLP to M2-saturated enzymes were compared.

20 Hydrolysis of the physiological substrate pyridoxal-phosphate 00050186-5-0100 (Sigma-Aldrich) (PLP) was measured at pH 7.4 in a standard buffer solution for the experiment (0 mM Tris-HCl buffer solution, 0 1 mM NaCl). , 1 mM MGCl2 and 20 μM ZnCl2). The concentration of phosphate released using the Innova (Pi ColorLock Gold Biosciences) was determined by measuring absorbance at 0 nM (4650). It was the curves

٦٣١٣

-٤٥-

The standard set was created to increase linear phosphate concentrations between 0-50 μM and all experiments were designed to fall within this range of hydrolyzed phosphate concentrations. Molar reaction rates expressed as [1-Pi]s were calculated for the given substrate concentration range and were fitted to a single binding site model (GraphPad Prism) versus [substrate] to calculate Km (no schemes applied

5 Lineweaver-Burk, due to lack of accuracy of cross-conversions at very low substrate concentrations).

A substrate concentration for PLP of 0.4 μM was used. The concentration of soluble enzyme was approximately 1 nM and incubation times ranged from 15-30 minutes depending on the catalytic efficiency of each enzyme. To ensure steady state conditions and to correct unidentified substrate signals in the Pi ColorLock method

10 Gold, the early reading (at • minutes) was subtracted from the last reading and Α650□ was measured on a corresponding standard curve Pi, generated for each experiment separately. All experiments were performed three to five times and the derived constants were recorded as mean ± 59.

To measure thermal stability. FLAG-tagged enzymes were incubated at 65 mM in 1 M DEA (pH 9.8) containing 1 mM MGCl2 and 20 μM ZnCl2. Done

15 Samples were removed at different time points and placed on ice, after which residual activity was measured with pNPP using the following method: Enzymes associated with activity were measured as absorbance at 405 nM (4405) as a function of time at 25 °C using pNPP (10 mM molar) as the substrate at pH 7.4 in 50 mM Tris-HCl buffer solution, 100 mM NaCl, containing 1 mM MGCl2 and 20 μM

20 2002. Enzymes were also incubated in this buffer solution for 10 minutes at increasing temperatures (25-100°C) and residual activity was measured in the same manner.

Results

Production of FLAG-tagged enzymes

٦٣١٣

-٤٦-

To compare the kinetic properties of 500-71 4b sALPI-PLAP, a FLAG-tag sequence was added to both cDNAs, as was previously done to comparatively study PLAP and TNAP.

Kozlenkov et al. For Biol Chem 277, 22992-22999 (2002) and Kozlenkov)

Expressing cDNAs 1)). al.l. has been restored. Bone miner. Res. 19, 1862-1872 (2004

5 about it in cells 1-005 and the culture supernatant solution containing the secreted enzymes. Successful expression and recovery were confirmed by Western blot analysis of anti-FLAG antibody.

Kinetics variables using physiological substrates

The phosphohydrolase properties of sAIPI-PIAPj LVL-RecAP were investigated in relation to the substrates.

Physiological factors involved in inflammation and seizures, specifically vitamin 96 vitamer PLP. LVL 10 Km showed lower RecAP than sALPI-PLAP (30.1 μM vs. 60.7 μM) at no.

Physiological pH (7,4) indicating that the developed LVL-RecAP has a higher binding affinity for PLP than sALPI-PLAP at physiological pH.

Enzyme stability

The effect of amino acid mutations in LVL-RecAP on the overall stability of the enzyme was investigated by thermal immobilization studies. Although sALPI-PLAP includes...

Advanced heat resistance was already high (% inhibition at 77.8°C) and LVL-RecAP was even more resistant to thermal inhibition (50% inhibition at 80.6°C).

Example 4

Pharmacokinetic distribution of LVL-RecAP in rats

20 Part A Radiocoding using Iodine-H21

repeat

The sALPI-PLAP-CD protein and the LVL-RecAP protein were radiolabeled with iodine-125 using the T-chloramine procedure as described by Greenwood et al.* The coding principle is based on in-site oxidation of iodine to atomic iodine and nucleophilic substitution.

٦٣١٣

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To the phenol rings in the ortho position of the hydroxyl group of the tyrosine residues

.tyrosine

The sALPI-PLAP protein was subjected to radioiodine labeling using the chloramine technique, in order to obtain -5 mAh/mg final specific activity and ~1 mg/mL (NaCl 9.0%) final concentration.

*FC Greenwood, VM Hunter, HG Glover, The preparation of 13111 5 why growth hormone of high specific radioactivity, Biochem.114 (1963) 89. l-

. 123

A.1 Materials

LVL-496.7 units/mg f) sALPI-PLAP Alkaline Phosphatase 10 - Alkaline phosphatase enzyme

RecAP (4 62 units/mg) was supplied by AM-Pharma at 4 6.3 mg/mL concentration in 25% glycerol v/w 5 mM Tris, 2 mM MGCl2, 50 μM ZnCl2, pH 8. ,0. The sALPI-PLAP protein and the LVL RecAP protein were stored at 4 mM.

15 - Iodine-125 radionuclide was purchased from Perkin Elmer in

Image of sodium iodide in 10-5 p sodium hydroxide (specific activity: 643.8 GBq/mg - radionuclide purity: 99.95%).

<p dir="rtl">-T-Chloramine was purchased (la-chloro-m-toluene sulfonamide -0-00000-20 MW, toluenesulfonamide 227.6 g/mol) trichloroacetic acid</p>

acid, sodium metabisulfite (MW) 190.1 g/mol) and tyrosine from Sigma.

5 mM Tris buffer solution, pH 8, was prepared in the Chelatec laboratory.

٦٣١٣

-٤٨-

<p dir="rtl">-NaCI 0.9% is provided by Versol®.</p>

a. method

0 85 µg of protein approximately 0 0 6 µCi of Na125I, 50 µL of Tris buffer and 10 µL of chloramine (404.8 nmol 50 equivalents/protein) • were added sequentially into a 1.5 mL tube. 0-800 eppendorf. The reaction was left to stir for 1 minute at °C

Room temperature. Two microliters of radiocoding medium was mixed with 5% MBS solution, and radiocoding efficiency was evaluated by instant thin layer chromatography (110) using 10% TCA as a sequential filtration product (alkaline phosphatase was filtered out at the bottom of the strip and 25 1- Free iodine sequentially at the top of the strip.

10 After adding 30 μL of tyrosine solution (10 mg/mL in water), the raw mixture was

G10, iodinated by gel filtration LVL-RecAP iodinated and sALPI-PLAP purification

GE Healthcare) and sequentially filtered with 0.9% NaCl. Portions of 0.2 ml/L were collected in test tubes. The radioactivity in each fraction was measured in an automated Gamma counter calibrated for the radionuclide Iodine-H12 (Wallace Wizard 2470 - Perkin Elmer). The 15 parts containing the required radioactive iodinated product have been collected. Certified radiochemical purity verified

Radiochemical purity of the radiolabeled compound radiolabelled by ITLC.

<p dir="rtl">A.3 Results and characteristics of radiolabeled sALPI-PLAP protein</p>

The radiocoding efficiency determined by ITLC exceeded 85% for both proteins. After G10 purification, the radiopurity of the coding reaction was above 97% for both proteins.

<p dir="rtl">20 The properties of the radiolabeled sALPI-PLAP solution after 6510 purification are summarized in Table 6.</p>

Table 6: Properties of [14-51-125] solution after purification

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<tr><td><p>125I-LVL-RecAP</p></td><td><p>1251-sALPI-</p><p>PLAP</p></td><td></td></tr><tr><td><p dir="rtl">0٦٤.٨٥</p></td><td><p dir="rtl">0٣٦.٨٦</p></td><td><p dir="rtl">Coding Efficiency (ITLC)</p></td></tr><tr><td></td><td colspan="2"><p dir="rtl">purification 510)</p></td></tr><tr><td><p dir="rtl">١٤٥٠١</p></td><td><p dir="rtl">١٤٩٠١</p></td><td><p dir="rtl">Concentration (mg/mL)</p></td></tr><tr><td><p dir="rtl">٥٤٤٠٠</p></td><td><p dir="rtl">٦٦٩٠٠</p></td><td><p dir="rtl">Specific activity (mCi/mg^</p></td></tr><tr><td><p dir="rtl">٦٣٧٠٠</p></td><td><p dir="rtl">٧٩١٠٠</p></td><td><p dir="rtl">Volumetric activity</p><p dir="rtl">(mCi/mL)</p></td></tr><tr><td><p dir="rtl">٨.٩٧</p></td><td><p dir="rtl">٢٣.٩٧</p></td><td><p dir="rtl">Radiological purity</p><p>(%) Radiopurity</p></td></tr>

a. 4 Alkaline phosphatase enzyme activity

Secret

The enzymatic activity of radiolabeled sALPI-PLAP was evaluated by ELISA.

Materials

<p dir="rtl">- Alkaline phosphatase colorimetric experiment kit (Ref. ab83369 - Batch number: -GR118166 E3.</p>

<p dir="rtl">— Human non-coding recombination enzyme human alkaline phosphatase diluted at 0.25 mg/ml</p>

0.9% NaCl in

and

<p dir="rtl">— Human alkaline phosphatase enzyme resulting from radio-encoded gene recombination diluted at 0.25 mg/</p>

ml in NaCl 0.9%

٦٣١٣

— ٥٠ —

Protocol

The Abcam kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate that turns yellow (Lamda max = 405 nM) when dephosphorylated by AP. The kit can monitor

5 10-250 micro AP units in samples.

A colorimetric alkaline phosphatase assay experiment was performed on non-coding iodinated sALPI-PLAP.

Radiant. The experiment was performed as described in the protocol provided by Abcam.

Briefly a standard curve was generated from 0 to 20 nmol/eye of standard pNPP (final volume:

<p dir="rtl">0 2 1 microliter). 10 μL of AP enzyme solution was added into each eye. It was done in parallel</p>

10. Dilute the test sample 15,000 sALPI-PLAP 8,000 times in buffer solution for the experiment. 10 and 20 μL of each dilution were added and the final volume was reached at 80 μL with the experimental buffer solution. Then 50 μL of pNPP solution was added to each eye containing the test sample.

Standard reactions and sample reactions were incubated 60 min at 25°C in the dark. 15 All reactions were then stopped using 20 μL of stop solution. A.0 was measured. when

5 0 4 nanomolar in a small plate reader.

The pNP standard curve was plotted. The sample readings were applied to the standard curve to obtain the amount of pNP generated by the 0AP sample. The AP activity of the test samples can be calculated:

sALPI-PLAP activity (U/mL) = XT / V / A test sample dilution factor

20 8: Amount of pNP generated by samples (in micromol)

<p dir="rtl">7: The volume of the sample added to the experimental eye (in milliliters)</p>

<p dir="rtl">1: Reaction time per minute</p>

٦٣١٣

-٥١-

sALPI-PLAP activity (U/mg) sALPI-PLAP activity (U/mL) / sALPI-PLAP concentration

mg of amal per litre

Results

Table 7 summarizes the results.

5 Table 7: Enzymatic activity of sALPI-PLAP and 1251-non-coding ALPI-PLAP

<tr><td colspan="3"><p dir="rtl">sALPI-PLAP activity (U/mg)</p></td><td></td></tr><tr><td><p dir="rtl">Average</p></td><td><p dir="rtl">Dilution 1/</p><p dir="rtl">٨٠٠٠</p></td><td><p dir="rtl">Dilution 1/</p><p dir="rtl">١٥٠٠٠</p></td><td></td></tr><tr><td><p dir="rtl">١٠٤٧٣</p></td><td><p dir="rtl">٦٠٤٧١</p></td><td><p dir="rtl">٦٠٤٧٤</p></td><td><p>sALPI-PLAP</p></td></tr><tr><td><p dir="rtl">٠٠٤٧٩</p></td><td><p dir="rtl">٣٠٤٧٥</p></td><td><p dir="rtl">٦٠٤٨٢</p></td><td><p>125I-sALPI-</p><p>PLAP</p></td></tr><tr><td><p dir="rtl">٨٠٥١٥</p></td><td><p dir="rtl">٢٠٥٣٠</p></td><td><p dir="rtl">٣٠٥٠١</p></td><td><p>LVL-RecAP</p></td></tr><tr><td><p dir="rtl">٨٠٥٦٣</p></td><td><p dir="rtl">٥٠٥٨٨</p></td><td><p dir="rtl">٢٠٥٣٩</p></td><td><p>125I-LVL-</p><p>RecAP</p></td></tr>

Secret

The enzymatic activity of non-coding and radiolabeled sALPI-PLAP is similar at about 475 units/mg. Therefore, the activity of sALPI-PLAP is not compromised by radiation treatment with chloramine - in the form of an oxidant.

10 Part B: Study of the biodistribution of protein 83•-51-125 in healthy rats

<p dir="rtl">B. 1 Materials</p>

٦٣١٣

-٥٢-

The characteristics of the rat strain used in this study are presented below:

Species: Sprague Dawley's rats

Breed: Crl CD® (SD) IGS BR

Source: Charles River France

<p dir="rtl">• Number and gender: 15 males</p>

Cr

Body weight/age range: approximately 250 g at the beginning of the study

Adaptation period: five days before treatment

Discrimination method: Distinguish the cage using the group (recite time).

Animal treatment

<p dir="rtl">10 Breeding: Accommodating the rats in animal facilities before treatment and in the radioactivity room after treatment.</p>

Food: Diet freely available to mice. There is no fasting period before treatment.

Water: Automatically deliver tap water via PP bottle.

Accommodation: Before treatment, the animals were housed in groups of three in polycarbonate cages under standard conditions and identified by distinguishing the study card number, animal number, species, and the 15 start and end dates of the study. Animals selected for excretory balance were transferred to metabolic cages (one rat in each cage) after

treatment.

Environmental environment: recording temperature daily. The room temperature ranged between 22 and 24°C. The artificial light cycle was controlled using an automatic timer (10 h light, 14 h dark).

Staff: Participants are suitably qualified and trained.

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-٥٣-

Selection: Animals were examined on arrival by the study director. Only healthy animals are selected. Particular attention was paid to any indication of an inflammatory reaction in the animals (eg abscess? dermatitis, etc.).

<p dir="rtl">B. Dosage solution of A•-51-125 and 125I-LVL-RecAP</p>

5 Iodinated LVL-RecAPj sALPI-PLAP solutions were diluted at 0.25 mg/mL in 0.9% NaCl before in vivo administration.

<p dir="rtl">B. 3 Study design</p>

The study design is presented in Tables 8 and 9.

Table 8: Study design for the distribution of A8A3-A3A58-A125 in the organism

<tr><td><p dir="rtl">Biodistribution</p></td><td><p dir="rtl">A time of sacrifice</p></td><td><p dir="rtl">Time taken</p><p dir="rtl">Blood sample</p></td><td><p dir="rtl">number</p><p dir="rtl">the animals</p></td><td><p dir="rtl">the group</p></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">30 million</p></td><td><p dir="rtl">2 nanomolar,</p><p dir="rtl">10 million</p></td><td><p dir="rtl">3 males</p></td><td></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">2 s</p></td><td><p dir="rtl">5 nanomolar,</p><p dir="rtl">45 million</p></td><td><p dir="rtl">3 males</p></td><td></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">6 s</p></td><td><p dir="rtl">15 nano</p><p dir="rtl">Mola, 4 s</p></td><td><p dir="rtl">3 males</p></td><td></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">24 hours</p></td><td><p dir="rtl">1s, 3s,</p><p dir="rtl">18 s</p></td><td><p dir="rtl">3 males</p></td><td></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">48 s</p></td><td><p>NA</p></td><td><p dir="rtl">3 males</p></td><td></td></tr>

10 * Were housed separately in metabolic cages with urine and feces collected at 24h and 48h

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-٥٤-

Table 9: Study design for in vivo distribution of 604-7-125

<tr><td><p dir="rtl">Biodistribution</p></td><td><p dir="rtl">A time of sacrifice</p></td><td><p dir="rtl">Time taken</p><p dir="rtl">Blood sample</p></td><td><p dir="rtl">Number of animals</p></td><td><p dir="rtl">the group</p></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">30 million</p></td><td><p dir="rtl">2 nanomolar,</p><p dir="rtl">10 million</p></td><td><p dir="rtl">3 males</p></td><td><p>81</p></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">2 s</p></td><td><p dir="rtl">5 nanomolar,</p><p dir="rtl">45 million</p></td><td><p dir="rtl">3 males</p></td><td><p>B2</p></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">6 s</p></td><td><p dir="rtl">15 nano</p><p dir="rtl">Molar, 4 s</p></td><td><p dir="rtl">3 males</p></td><td><p>83</p></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">24 hours</p></td><td><p dir="rtl">١، ٣،</p><p dir="rtl">18 s</p></td><td><p dir="rtl">3 males</p></td><td><p>84</p></td></tr><tr><td><p dir="rtl">Blood and organs</p></td><td><p dir="rtl">48 s</p></td><td><p>NA</p></td><td><p dir="rtl">3 males</p></td><td><p>B5*</p></td></tr>

. 4 Giving

At the time of the experiment, the average weight of Sprague Dawley rats was approximately 2.4 g. Unanesthetized rats were injected intravenously into the lateral tail vein (left) at a dose level of 0.04 μg/kg.

5 grams corresponds to a protein amount of 96 micrograms per rat and an activity of about 58 micrograms per rat. The injection volume was approximately 380 microliters.

Individual dose volumes were calculated using the individual body weight of each rat on the day of treatment. Rats were placed in the content device. In order to enable vascular dialysis in the tail, the tail was immersed in warm water (45°C) and then disinfected with alcohol.

10 The dose solution was slowly injected into the tail vein. In order to calculate the actual dose received by each rat, the syringes were weighed before and after treatment and a portion of the dose solution was counted on a Gamma counter.

٦٣١٣

-٥٥-

B.5 Distribution in various organs

At the time of sacrifice, the animals were anesthetized by intraperitoneal injection of 2.5 ml/kg body weight of a mixture of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (20 mg/ml) in PBS.

<p dir="rtl">• The rats were then killed quickly by exsanguination via intracardiac puncture. The concerned organs were collected, followed by cutting into parts for organs weighing more than 2 grams, such as the liver, stomach, small intestine, and colon.</p>

Each piece was then rinsed with physiological serum before wiping with soft tissue paper, weighing and counting

Separately. The selected organs were the liver, kidneys, heart, lungs, spleen, skeleton, 10th femur, brain, thyroid, stomach, small intestine with content, colon with skin content, and fat surrounding the kidney.

Tissue radioactivity counting was performed in a Wallace Wizard 2470 Gamma Perkin Elmer automated counter calibrated for radionuclide iodine-125 (efficiency: 74% - counting time: 10 seconds).

The concentration of radioactivity in organs/tissues was expressed as a percentage of the dose 15 injected per gram of tissue (9% A/g).

Data analysis includes the percentage dose injected (%ID) and the amount of protein equivalent per organ or tissue. For specifically defined organs, the above was calculated using the radioactivity counted in the entire organ.

Regarding blood, this was achieved by assuming blood counts of 6.4% of total body weight.

<p dir="rtl">20 Additionally, the ratio between the radioactivity retained in the tissues and the radioactivity in the blood (organ/blood ratio) was calculated. Finally, the ratio between the organ/blood ratio sALPI-PLAP and the organ/blood ratio LVL-RecAP was calculated (Figure ).</p>

٦٣١٣

-٥٦-

<p dir="rtl">B. Distribution in rat blood and serum</p>

Radioactive level in blood and serum

At the time of sacrifice, blood samples from the exsanguination times were obtained by intracardiac puncture in a rat that had been anesthetized by intraperitoneal injection of a mixture of ketamine hydrochloride.

<p dir="rtl">5 hydrochloride and xylazine hydrochloride in PBS.</p>

At other time points indicated in the study design (Tables 8 and 9), blood was drawn from the lateral tail vein (right) using a 23-gauge butterfly needle without anesthesia.

Each blood sample was collected in preweighed Microvette tubes with a coagulation activator (Sarstedt). The tubes were weighed and radioactivity was measured in a Gamma counter.

<p dir="rtl">10 Blood samples were incubated at room temperature for 30 minutes and then centrifuged for 10 minutes at 10,000 g to prepare serum. Serum was pooled into preweighed tubes and counted on a Gamma counter.</p>

The concentration of radioactivity in blood and serum was expressed as a percentage of the injected dose and an equivalent amount of 51-14[ per.

<p dir="rtl">15 Data analysis includes the percentage of injected calculated for total blood and serum.</p>

Tables 10 and 11 present the ratio of radioactivity measured in various organs with respect to radioactivity in serum for sALPI-PLAP and LVL-RecAP, respectively.

Figure 2 displays the organ/blood LVL-RecAP ratio to the organ/blood sALPI-PLAP ratio. A ratio of 2, for example, indicates that LVL-RecAP is twice as targeted to the 20 organs shown as sALPI-PLAP.

Higher ratios furthermore indicate that relatively greater activity of LVL-RecAP is detected in a specific organ when the same dose is used.

٦٣١٣

-٥٧-

Table 10 - Organ/blood ratio for sALPI-PLAP

Table 10 - Organ/blood ratio for sALPI-PLAP

<tr><td colspan="2"><p>H 48</p></td><td colspan="2"><p>H 24</p></td><td colspan="2"><p>H 6</p></td><td><p>H2</p></td><td colspan="2"><p>H 5.0</p></td><td><p dir="rtl">Members</p></td></tr><tr><td colspan="2"><p dir="rtl">٧٣٣,٨٥+</p><p dir="rtl">٧٩,١٦</p></td><td colspan="2"><p dir="rtl">٢٠٨,١٦±</p><p dir="rtl">٢٣,٧٤</p></td><td colspan="2"><p dir="rtl">٣١,٧٤٥+</p><p dir="rtl">٥,١٧٥</p></td><td><p dir="rtl">٤,٦٧٧١</p><p dir="rtl">٠,٤٤٣٣</p></td><td colspan="2"><p dir="rtl">٠,١٦٢٤</p><p dir="rtl">٠,٠١٥٧</p></td><td><p dir="rtl">Thyroid/</p><p dir="rtl">Trachea</p></td></tr><tr><td><p dir="rtl">١±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٧٨٧</p><p dir="rtl">٣٦٨٦</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٩٤٤٨</p><p dir="rtl">٢٥٦٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤٦٦١</p><p dir="rtl">١٦١٧</p></td><td><p dir="rtl">٠,٢٨٤٢±</p><p dir="rtl">٠,٠١٣٤</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٣٠٧</p><p dir="rtl">٠٠٣٥</p></td><td><p dir="rtl">Skin</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤٢٩٩</p><p dir="rtl">٠٢١٩</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٢٥٠</p><p dir="rtl">٠٤٦٧</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٥٣٥</p><p dir="rtl">٠٢١٩</p></td><td><p dir="rtl">٠,٣٤٣٨±</p><p dir="rtl">٠,٠٣١٠</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٠٧٦</p><p dir="rtl">٠٠١١</p></td><td><p dir="rtl">Kidney</p></td></tr><tr><td><p dir="rtl">٣</p><p dir="rtl">٠</p></td><td><p dir="rtl">٥٨١٩</p><p dir="rtl">٩٠١٨</p></td><td><p dir="rtl">١±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤٤٧٨</p><p dir="rtl">١٢٠٠</p></td><td><p dir="rtl">٣±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٦٢٢</p><p dir="rtl">٦٩٧٧</p></td><td><p dir="rtl">١,٠٦٩٩±</p><p dir="rtl">٠,١٦٦٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٤٤٦</p><p dir="rtl">٠٠١٦</p></td><td><p dir="rtl">Stomach</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٢٧٣</p><p dir="rtl">٠٢٥٤</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٢٥٦</p><p dir="rtl">٠٢٧٠</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٨٨٠</p><p dir="rtl">٠٢٨٣</p></td><td><p dir="rtl">٠,٤١٣٣±</p><p dir="rtl">٠,٠٣٢٢</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٥٣٨٨</p><p dir="rtl">٠٧٩٦</p></td><td><p dir="rtl">Spleen</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤٦٧٦</p><p dir="rtl">٠٢٩٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٢١٩</p><p dir="rtl">٠٣٢٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤٨١١</p><p dir="rtl">٠٨٧٧</p></td><td><p dir="rtl">١,٩٩١٧±</p><p dir="rtl">٠,٠٣٤٧</p></td><td><p dir="rtl">٥±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٤٠٣</p><p dir="rtl">١٩٤٣</p></td><td><p dir="rtl">Liver</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٨٠٩</p><p dir="rtl">٠١٦٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٧٩١</p><p dir="rtl">٠٣٨٧</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٦٠٥</p><p dir="rtl">٠٢١٧</p></td><td><p dir="rtl">٠,٢٢٥٩±</p><p dir="rtl">٠,٠١٩٨</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٨٢٤</p><p dir="rtl">٠١٣٦</p></td><td><p dir="rtl">the heart</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٩٣٧</p><p dir="rtl">٠٢٥١</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٨٣٣</p><p dir="rtl">٠٠٩١</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٣٩٧</p><p dir="rtl">٠٧٣٠</p></td><td><p dir="rtl">٠,٢٧٦٧±</p><p dir="rtl">٠,٠٣١٨</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٧٧٧</p><p dir="rtl">٠١١٩</p></td><td><p dir="rtl">Lungs</p></td></tr>

٦٣١٣

-٥٨-

<tr><td><p dir="rtl">٠±</p><p dir="rtl">»</p></td><td><p dir="rtl">٠٩٤٧</p><p dir="rtl">٠١٥٠</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٨٧٦</p><p dir="rtl">٠٠٣٨</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٨٤٢</p><p dir="rtl">٠١٧٨</p></td><td><p dir="rtl">٠,٠٩٧٨±</p><p dir="rtl">٠,٠١٩٣</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٣٣٤</p><p dir="rtl">٠٠٣٣</p></td><td><p dir="rtl">The skeleton</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">.</p></td><td><p dir="rtl">٠٨٧٤</p><p dir="rtl">٠١٥٣</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٦١٥</p><p dir="rtl">٠٠٦٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٥٠٩</p><p dir="rtl">٠١١١</p></td><td><p dir="rtl">٠,٠٢٦١±</p><p dir="rtl">٠,٠٠٧٠</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٠٩٥</p><p dir="rtl">٠٠٢٣</p></td><td><p dir="rtl">Fats</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">.</p></td><td><p dir="rtl">٠٤٠٨</p><p dir="rtl">٠٠٦٣</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٢٥٣</p><p dir="rtl">٠٠٢٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٢١٣</p><p dir="rtl">٠٠١١</p></td><td><p dir="rtl">٠,٠٢٥٧±</p><p dir="rtl">٠,٠٠١١</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠١٦٠</p><p dir="rtl">٠٠٣١</p></td><td><p dir="rtl">The brain</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">'</p></td><td><p dir="rtl">١٦١٢</p><p dir="rtl">٠١٣٢</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٦٠٢</p><p dir="rtl">٠١١٩</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٩٠١</p><p dir="rtl">٠١٥٥</p></td><td><p dir="rtl">٠,١٩٩١±</p><p dir="rtl">٠,٠١٣١</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٨٨٦</p><p dir="rtl">٠٢١٧</p></td><td><p dir="rtl">Thigh bone</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">,</p></td><td><p dir="rtl">٦٣٢٩</p><p dir="rtl">١٠٤٤</p></td><td><p dir="rtl">١±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٧٢٩</p><p dir="rtl">١٢٦٨</p></td><td><p dir="rtl">١±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٠٥٥</p><p dir="rtl">٠٨٢٧</p></td><td><p dir="rtl">٠,٨٤٤٠+</p><p dir="rtl">٠,١٨٩٦</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٨٨٢</p><p dir="rtl">٠٢٤٧</p></td><td><p dir="rtl">Small intestine</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">,</p></td><td><p dir="rtl">٣٩٩١</p><p dir="rtl">٠٩٦٤</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٤٧٣</p><p dir="rtl">٠٥٩٦</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤١٥٠</p><p dir="rtl">٠٤٨٩</p></td><td><p dir="rtl">٠,٠٧٧٧±</p><p dir="rtl">٠,٠٠٧٧</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٢٢٧</p><p dir="rtl">٠٠٢٨</p></td><td><p dir="rtl">Colon</p></td></tr>

Table 11 - Organ/blood ratio for LVL-RecAP

<tr><td><p dir="rtl">٦٤٨</p></td><td><p>H24</p></td><td><p>H6</p></td><td><p>H2</p></td><td><p>H 015</p></td><td><p dir="rtl">The doubles</p></td></tr><tr><td><p dir="rtl">١٠٧٥,٢±</p></td><td><p dir="rtl">٤٦٢,٨٣±٣</p></td><td><p dir="rtl">٥٢,٩٣٤±</p></td><td><p dir="rtl">٧,٣٦٣٨±</p></td><td><p dir="rtl">٠,٥٩٣٤±</p></td><td><p dir="rtl">Thyroid/</p></td></tr><tr><td><p dir="rtl">١١٨,٦٥</p></td><td><p dir="rtl">٤,٣٩٥</p></td><td><p dir="rtl">١٣,٤٧٥</p></td><td><p dir="rtl">١,٥٠٤٩</p></td><td><p dir="rtl">٠,٠٨٩٢</p></td><td><p dir="rtl">Trachea</p></td></tr><tr><td><p dir="rtl">١,٩٢١٣±</p></td><td><p dir="rtl">١,٢١٣٠±٠</p></td><td><p dir="rtl">٠,٦٨٤٣±</p></td><td><p dir="rtl">٠,٤١١٠±</p></td><td><p dir="rtl">٠١٠٩٤٢±</p></td><td><p dir="rtl">Skin</p></td></tr><tr><td><p dir="rtl">٠,٨٩٦٥</p></td><td><p dir="rtl">١٢٤٣,</p></td><td><p dir="rtl">٠,٠٨١٠</p></td><td><p dir="rtl">٠,٠٤٨٥</p></td><td><p dir="rtl">٠,٠٠١٢</p></td><td></td></tr><tr><td><p dir="rtl">١,٢٤٨١ +</p></td><td><p dir="rtl">٠,٨٢٣٨±٠</p></td><td><p dir="rtl">٠,٦٤٢٨</p></td><td><p dir="rtl">٠,٦٤٢٥+</p></td><td><p dir="rtl">٠١٥٧٩٣±</p></td><td><p dir="rtl">Kidney</p></td></tr><tr><td><p dir="rtl">٠,١٠٨٩</p></td><td><p dir="rtl">٠٨٢٦,</p></td><td><p dir="rtl">٠,٠٠٩٨</p></td><td><p dir="rtl">٠,٠٥٥٥</p></td><td><p dir="rtl">٠,٠١٨٧</p></td><td></td></tr>

٦٣١٣

-٥٩-

<tr><td><p dir="rtl">٠±</p><p dir="rtl">»</p></td><td><p dir="rtl">٩٩٣٠</p><p dir="rtl">٣٢٧٩</p></td><td><p dir="rtl">٣,٠٠٤٢±١</p><p dir="rtl">٢٠١٢,</p></td><td><p dir="rtl">٦±</p><p dir="rtl">٣</p></td><td><p dir="rtl">٢٩١٧</p><p dir="rtl">٥٦٩٨</p></td><td><p dir="rtl">١,٥٩٨٣±</p><p dir="rtl">٠,٣٠٩٩</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٢٦٢</p><p dir="rtl">١٠٢١</p></td><td><p dir="rtl">Stomach</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٦٢٣</p><p dir="rtl">٠٤٢٩</p></td><td><p dir="rtl">٠,٣٦٧٣±٠</p><p dir="rtl">٠٧٩٦,</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٥٨٣١</p><p dir="rtl">٠٢٩٢</p></td><td><p dir="rtl">١,٠٩٧٧±</p><p dir="rtl">٠,١١٩٠</p></td><td><p dir="rtl">٢±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٧٧١</p><p dir="rtl">١١٧٢</p></td><td><p dir="rtl">Spleen</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٧٩٤٠</p><p dir="rtl">٠١٣٣</p></td><td><p dir="rtl">٠,٥٨٦٩+٠</p><p dir="rtl">٠٩٦٧,</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٦١٣١</p><p dir="rtl">٠٩٠٠</p></td><td><p dir="rtl">٣,٨٤٠٩+</p><p dir="rtl">٠,٠٢٧٧</p></td><td><p dir="rtl">٧±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٦٥١٣</p><p dir="rtl">٩٢٩٥</p></td><td><p dir="rtl">Liver</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">»</p></td><td><p dir="rtl">٣١٤٤</p><p dir="rtl">٠٠٢٣</p></td><td><p dir="rtl">٠,٢٦٣٠±٠</p><p dir="rtl">٠١٣٨,</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٧٨٠</p><p dir="rtl">٠٣١٦</p></td><td><p dir="rtl">٠,٢٧٦٢±</p><p dir="rtl">٠,٠٠٩٦</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٠٣٠</p><p dir="rtl">٠١٠٥</p></td><td><p dir="rtl">the heart</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">»</p></td><td><p dir="rtl">٤٥١٥</p><p dir="rtl">٠٢٢٠</p></td><td><p dir="rtl">٠,٣٩٦٦±٠</p><p dir="rtl">٠٢٣٥,</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٤٣٣٢</p><p dir="rtl">٠١١١</p></td><td><p dir="rtl">٠,٣٧٤٠+</p><p dir="rtl">٠,٠٤٤٦</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٠٠٧</p><p dir="rtl">٠٢٠١</p></td><td><p dir="rtl">Lungs</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">»</p></td><td><p dir="rtl">٠٩٥٣</p><p dir="rtl">٠٠٨٠</p></td><td><p dir="rtl">٠٦٠,٠٧٨٤</p><p dir="rtl">٠١١٣,</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٠٣٦</p><p dir="rtl">٠٢٥١</p></td><td><p dir="rtl">٠,٠٩٩٧±</p><p dir="rtl">٠,٠١٤٢</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٤٦٢</p><p dir="rtl">٠٠٧٢</p></td><td><p dir="rtl">The skeleton</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">,</p></td><td><p dir="rtl">٠٩٧٠</p><p dir="rtl">٠٢٤٥</p></td><td><p dir="rtl">٠٦٠,٠٧٨١</p><p dir="rtl">٠١٥٥,</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٤٩٥</p><p dir="rtl">٠٠٣٤</p></td><td><p dir="rtl">٠,٠٣٨٠±</p><p dir="rtl">٠,٠٠٦٣</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٢٩٨</p><p dir="rtl">٠٠٧٨</p></td><td><p dir="rtl">Fats</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">»</p></td><td><p dir="rtl">٠٦٤٦</p><p dir="rtl">٠١٠٠</p></td><td><p dir="rtl">٠,٠٢٨٨±٠</p><p dir="rtl">٠٠١٦</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٢٥٣</p><p dir="rtl">٠٠٢٨</p></td><td><p dir="rtl">٠,٠٣٨٩±</p><p dir="rtl">٠,٠٠٨٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٢٦١</p><p dir="rtl">٠٠٥٢</p></td><td><p dir="rtl">The brain</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">.</p></td><td><p dir="rtl">١٧٤٦</p><p dir="rtl">٠٣٧٠</p></td><td><p dir="rtl">٠,١٨٠٤±٠</p><p dir="rtl">٠٣٢٥</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٢٤٦٠</p><p dir="rtl">٠١٤٩</p></td><td><p dir="rtl">٠,٢٩٧٣±</p><p dir="rtl">٠,٠٠٩٨</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٣٧٦٤</p><p dir="rtl">٠١٠٢</p></td><td><p dir="rtl">Thigh bone</p></td></tr><tr><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٦٦١٢</p><p dir="rtl">١٠٠٣</p></td><td><p dir="rtl">٠,٩٤٠٢±٠</p><p dir="rtl">٠٩٣٥</p></td><td><p dir="rtl">١±</p><p dir="rtl">٠</p></td><td><p dir="rtl">٠٣٠٨</p><p dir="rtl">١١٣٤</p></td><td><p dir="rtl">٠,٥٦٢٥±</p><p dir="rtl">٠,٠٣٩٣</p></td><td><p dir="rtl">٠±</p><p dir="rtl">٠</p></td><td><p dir="rtl">١٥٧٣</p><p dir="rtl">٠٠٤٩</p></td><td><p dir="rtl">Small intestine</p></td></tr>

٦٣١٣

-٦٠-

<tr><td><p dir="rtl">٠,٤٧٨٧+</p><p dir="rtl">٠,٠٧١٢</p></td><td><p dir="rtl">3H3H,0±0</p><p dir="rtl">١٩٦٧,</p></td><td><p dir="rtl">8H0.36±</p><p dir="rtl">٠,٠٦٩٣</p></td><td><p dir="rtl">٠,١٠٤٩±</p><p dir="rtl">٠,٠٢٧١</p></td><td><p dir="rtl">٠,٠٣٦١±</p><p dir="rtl">٠,٠٠٤٧</p></td><td><p dir="rtl">Colon</p></td></tr>

Example e

Akp2—/—mouse model of pediatric phosphatase deficiency

The 002-/- mouse model of pediatric hypophosphatase deficiency is known in the art (Dent Res 2011, 470-476:90(4). Briefly, 402-/- mice were generated by inserting a Neo cassette into exon 6 of the mouse TNALP gene (02) via homozygous recombination to inhibit

Towards a functional Akp2 gene, resulting in TNALP mRNA or undetectable protein.

Animal use and tissue collection procedures followed Sanford-Burnham-approved protocols

.Medical Research Institute Animal Ethics Committee

Animals were treated with either vector (N = 10) 1 mg •-A/A/kg/day (N = 10) 8 10 mg •mm-1// (N = 8) or 16 mg LVL-RecAP/kg. /day (N= ).

Survival was measured and skeletal development assessed. Kidney mineralization was assessed. Plasma pyridoxal PPi levels, plasma calcium phosphate levels, and femur and/or tibia length were measured for the various treatment groups. MicroCT data were collected to analyze residual bone overdose at each dose.

<p dir="rtl">E1 Results</p>

<p dir="rtl">— Improved long-term survival of animals treated at 16 mg/kg/day (Figure 3).</p>

<p dir="rtl">— There is (incomplete) rescue of skeletal defects in long bones even at the highest dose (data not shown)</p>

20 — There is some rescue in the dental phenotype (data not shown).

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<p dir="rtl">— There appears to be rescue of craniosynostosis (data not shown, confirmation required)</p>

<p dir="rtl">- Work on the kidneys is still ongoing. There is an indication of mineralization in the kidneys of untreated mice and less mineralization in the kidneys of treated mice (data not shown).</p>

5 Example 6: Reperfusion of local anemia in a porcine kidney model

Materials and Methods: Comparative vector material and test material information

Preparing the comparative material and the test material

New Comparator LVL-RecAP Solution Diluent (placebo) was prepared for use in the study before each dose administration and kept refrigerated at 2 to 8°C when not in use.

<p dir="rtl">10 The test material LVL-RecAP was used, as received. No purity adjustment was made when preparing the test substance formulas. Test substance formulas were prepared by mixing with an appropriate volume of sterile saline to achieve nominal concentrations of 0, 0.96, or 4.8 mg/mL. Formulas were prepared before each dose administration under a laminar flow hood using sterile equipment and aseptic techniques. Formulas were decanted in appropriate amounts into amber glass serum bottles and kept on ice before use</p>

<p dir="rtl">15 and use it to administer the dose within 2 hours of preparation. Additional preparations are sometimes made as necessary during the course of the study.</p>

Analysis of dosage formulations: Double 5.0 mL samples of the final dosage formulation were collected before dose administration on each day of preparation on day 0 (groups 3 to 7) and day 7 (group 7). Samples were collected from the middle layers and cryopreserved (−50 to −90°C) for possible future analysis.

<p dir="rtl">20 Test system information: Animal acquisition and acclimatization</p>

Yes

An experimental, domestic male Yorkshire crossbred (farm pigs) (approximately 8 to 10 weeks old at receipt) was received from Midwest Research Swine,

٦٣١٣

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Minnesota gibbon. During the acclimation period of 10 to 28 days, animals were observed daily regarding general health and any signs of disease. Evaluation of eggs and parasites in stool samples was performed and all results were negative for the animals participating in the study.

Randomized allocation to study and follow-up

5 Using simple separate randomization procedures animals (weighing 12.5 to 25.0 kg when randomized) were assigned to the control and treatment groups and are identified in the following Table 12.

<tr><td colspan="4"><p dir="rtl">Table 12: Group assignments</p></td></tr><tr><td rowspan="2"><p dir="rtl">Group number</p></td><td rowspan="2"><p dir="rtl">Dose level</p></td><td colspan="2"><p dir="rtl">Number of male animals</p></td></tr><tr><td><p dir="rtl">first</p></td><td><p dir="rtl">Rated</p></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td><p dir="rtl">١</p></td><td><p dir="rtl">Comparative</p></td><td><p dir="rtl">٧</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">٢</p></td><td><p>Shamb</p></td><td><p dir="rtl">٧</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">H</p></td><td><p dir="rtl">3200 mg/kg</p></td><td><p dir="rtl">٧</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">٦</p></td><td><p dir="rtl">6.1 mg/kg</p></td><td><p dir="rtl">٧</p></td><td><p dir="rtl">٧</p></td></tr><tr><td><p dir="rtl">٧</p></td><td><p dir="rtl">٣٢٠٠</p><p dir="rtl">mg kg/day</p></td><td><p dir="rtl">٧</p></td><td><p dir="rtl">٧</p></td></tr><tr><td colspan="4"><p dir="rtl">On the day, the animals underwent a surgical procedure during which the left or right kidney was removed and the renal artery on the opposite side was covered for 45 minutes. Following capping, the vessel was allowed to reperfusion. Seven animals were subjected to surgery in each group with the aim of achieving six animals in the study. Sham animals were subjected to</p></td></tr>

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A surgical procedure during which the left kidney was removed. However, renal embolization and reperfusion were not performed. On day 0, half the dose was given before reperfusion and the remaining half dose was given at 8 ± 2 hours after reperfusion. A dose of ointment was given

Completely daily for the rest of your life.

The animals selected for the study were as homogeneous in age and weight as possible. A veterinarian assessed the health of the animals before enrolling them in the study. Additional animals obtained in the study and not enrolled in the study were transferred to the colony.

Each animal was assigned an animal number used in the Provantis™ data collection system and implanted

It has a microchip that carries a unique identification number. Each animal is also identified by a specific ear tag from the supplier. An individual animal number, implant number, ear tag number, and study number included a unique identifier for each animal. Each cage was labeled with animal number, study number, group number, and species.

Animals were housed individually in raised-floor runs or stainless steel movable cages with plastic-coated floors. This type of accommodation provides adequate room

10 for training these animals. Animal enrichment was provided in accordance with the MPI Research SOP. Fluorescent lighting was provided for approximately 12 hours per day. The dark cycle was interrupted intermittently due to study-related activities. Temperature and humidity were continuously monitored, recorded and maintained to the greatest extent possible within the range values designed in the protocol of 61 to 81 F and 30 to 70%, respectively. Actual temperature and humidity results are not recorded, but are maintained

<p dir="rtl">15 in the study file.</p>

Certified Lab Diet® 5699, PMI Nutrition International, ) diet has been shown

Inc·) via limited meals, except during designated periods of time. Nutritional enrichment including fiber portions or tablets has been offered as needed.

Surgical procedures

<p dir="rtl">20 Amendments related to the procedure</p>

٦٣١٣

-٦٤-

The following Table 13 presents the procedure-related modifications and dose levels used during the course of the study.

<tr><td colspan="3"><p dir="rtl">Table 13: Modifications related to the procedure</p></td></tr><tr><td colspan="3"><p dir="rtl">Interval for dose levels Dose level, and route</p></td></tr><tr><td><p dir="rtl">Daily</p><p dir="rtl">After surgery</p></td><td><p dir="rtl">Surgery (day 0)</p></td><td><p dir="rtl">Medicine</p></td></tr><tr><td></td><td></td><td></td></tr><tr><td><p>-</p></td><td><p dir="rtl">100mg beauty 1</p></td><td><p dir="rtl">Acepromazine maleate</p></td></tr><tr><td><p>-</p></td><td><p dir="rtl">0500 mg in total</p></td><td><p dir="rtl">Atropine sulfate</p></td></tr><tr><td><p>-</p></td><td><p dir="rtl">• 5-1 8 mg kg1</p></td><td><p dir="rtl">Telazol</p></td></tr><tr><td><p>-</p></td><td><p dir="rtl">Effect by inhalation</p></td><td><p dir="rtl">Isofluorane</p></td></tr><tr><td><p dir="rtl">0200 mg/kg IMTID</p><p dir="rtl">3x days</p></td><td><p dir="rtl">0200 mg/kg IM</p></td><td><p dir="rtl">Buprenorphine</p></td></tr><tr><td><p dir="rtl">3 mg/kg IM SID x</p><p dir="rtl">3 days</p></td><td><p dir="rtl">2mg sentences 1</p></td><td><p dir="rtl">Ketoprofen</p></td></tr><tr><td><p>—</p></td><td><p dir="rtl">25 mg/kg IV</p></td><td><p dir="rtl">Cefazolin</p></td></tr><tr><td><p dir="rtl">202 mg/kg IM SID</p><p dir="rtl">3x days</p></td><td><p dir="rtl">202 mg/kg7a1</p></td><td><p>Ceftiofur</p></td></tr>

٦٣١٣

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<tr><td colspan="2"><p>-</p></td><td colspan="2"><p dir="rtl">10 to 15 ml/kg/</p><p dir="rtl">IV hour</p></td><td><p dir="rtl">Ringer's solution treated with lactate</p><p>(LRS)</p></td></tr><tr><td colspan="2"><p>-</p></td><td colspan="2"><p dir="rtl">2 mg/kg INF</p></td><td><p>Marcaine</p></td></tr><tr><td colspan="2"><p>-</p></td><td colspan="2"><p dir="rtl">As needed for irrigation</p></td><td><p>%NaCl9.0</p></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2"><p dir="rtl">TID — twice daily</p><p dir="rtl">(every 6 to 9 hours)</p></td><td colspan="2"><p dir="rtl">IM - Intramuscular INF -Infusion into the incision</p><p dir="rtl">Secret</p><p dir="rtl">SID — once daily</p></td><td><p dir="rtl">IV - Intravenously</p></td></tr><tr><td colspan="5"></td></tr>

H

Before and after surgical procedures were performed according to the MPI Research SOP. The animals were starved for at least 8 hours before surgery, and anesthesia was induced and maintained as shown in Table 013. Body temperature was maintained at 37°C. Before surgery. Ultrasound was performed to determine the presence of renal cysts. If no cysts were observed, the left kidney was removed and the right kidney was treated as described below. If cysts are present in one kidney, that kidney is removed and the opposite side kidney undergoes a covering procedure. If cysts are present in both kidneys, the animal is removed from the study without undergoing a surgical procedure.

Surgical procedure

Renal ischemia/reperfusion injury was induced using the procedure published by Lee et al

to)). Once anesthetized all animals are placed in a position. Vet. Med. Sci 72(1): 127-130 10

Lie on the back and prepare the surgical sites by alternating swabs with chlorine scrub wipes

٦٣١٣

-٦٦-

Hexidine and solution. A midline laparotomy was performed to expose both kidneys. Based on the ultrasound results, the left or right kidney was removed.

For group 2 animals (Sham), the inserted sponge roll was then removed and examined and the abdomen was washed with warm sodium chloride. The abdomen was closed in the routine manner and the skin was closed using staples.

5 Leather and textile glue. The animals were then left to recover.

All other animals after removal of the selected kidney had the remaining renal vessels isolated and retracted using vessel loops. Vessel rings were used to occlude the vessels for 45 (+ 1) minutes after which the vessels were left for reperfusion. An intravenous bolus dose of comparator or test substance was given at a dose volume of 0.333 mL/kg (a half dose of 0.167 mL/kg was given to group 7 of

10 animals) immediately before reperfusion. For all group 1, 6 and 6 animals (control 0.32 and 1.6 mg/kg) the implanted sponge roll was then removed, examined, and the abdomen was washed and sealed.

As previously described, the animals were left awake. For all group 7 animals (0.32 mg/kg/day), an incision was made in the thigh and the left or right femoral vein was isolated. A catheter was inserted into the vessel and a catheter was passed in a subcutaneous and internal-to-external tunnel through an incision in the rib cage 15. A port was connected and fixed to the muscle using a non-absorbable suture. Done afterwards

Lap sponges were implanted and examined. The abdomen was washed and closed as previously described, and the animals were left awake.

Giving the test material or comparison material

On day 00, the control or test substance was administered intravenously to all groups 1, 5, and 6 of 20 animals immediately before reperfusion at a full dose of 0, 0.32, and 1.6 mg/kg, respectively. It was completed

Administer all doses at a dose volume of 0.333 ml/kg. Also on day 0, the test substance was administered intravenously to all group 7 animals at a combined dose level of 0.32 mg/kg in two separate half doses of 0.16 mg/kg at a dose volume of 0.167 mL/kg. . The first dose was given immediately before reperfusion and the second dose was given approximately 8 (±

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2) Hours after reperfusion. Total doses of 0.32 mg/kg/day (0.333 mL/kg) were administered to all 7 animals on days 1 to 7 at approximately the same time of day as the initial day 0 dose (+ 2 hours).

statistics

Table 14 below identifies a set of comparisons used in the statistical analyzes described in this section.

<tr><td colspan="2"><p dir="rtl">Table 14: Statistical comparisons table</p></td></tr><tr><td><p dir="rtl">Reference group</p></td><td><p dir="rtl">Comparison groups</p></td></tr><tr><td></td><td></td></tr><tr><td><p dir="rtl">١</p></td><td><p dir="rtl">2, e, 6, 7</p></td></tr><tr><td><p dir="rtl">٢</p></td><td><p dir="rtl">E,6,7</p></td></tr><tr><td><p dir="rtl">H</p></td><td><p dir="rtl">٧</p></td></tr><tr><td></td><td></td></tr>

١٠

Unprocessed data were tabulated within each interval and the mean and standard deviation were calculated for each endpoint and group. For serum creatinine concentrations, the treatment groups were compared to the reference groups using standard analysis of covariance

.(RMANCOVA)

Multi-parametric analysis of covariance (RMANCOVA)

For end points measured at three or more post-test intervals a parametric analysis (mixed model) was performed. For each final point. The tested model for time treatment effects was included

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The interaction of treatment and time. Before the test data (the last measurement before the dose is given) is entered into the model as the independent variable.

If there was no (0.,0) significant treatment by time interaction, the main effect of treatment was evaluated. If the treatment effect is not significant (p&gt;5 0'')', the results are considered non-significant and further analyzes are not performed 5 on the variable. If the treatment effect is large (0.,0), linear contrasts are generated to pairwise compare each treatment group with the reference group. If the interaction is significant (0.0), each treatment group is compared to the appropriate reference group by the simple effect of “treatment” for each time point. These simple effect pairwise comparisons were obtained from the “treatment by time” interaction.

The results of all pairwise comparisons were reported at the 0.05 and 0.01 observed levels. All 10 tests were two-tailed tests.

Results

Serum creatinine

As can be seen in Figure 4, there were moderate to moderate creatinine elevations at all time intervals relative to pre-test values. Creatinine tends to increase to a maximum at 24 SR

15 hours after reperfusion and then gradually decreased over subsequent time intervals. Changes in creatinine are consistent with decreased glomerular filtration secondary to kidney injury related to the surgical procedure (data not shown). In most treatment groups. And at most time intervals. Administration of the test substance tends to attenuate elevations in creatinine relevant to the surgical procedure demonstrating the protective effect of LVL-RecAP.

20 As can be seen in Figure 5, there were dose-dependent increases in alkaline phosphatase (ALP) activity in all treatment groups that received the test substance at 24 hours after reperfusion. and

For pre-test values. ALP activity gradually decreased in the treatment groups receiving &lt;6 1 mg/kg but continued to gradually increase in animals receiving 2 3' 0 mg/kg/day.

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Example 7

Safety tests in humans

Material and methods

Objectives:

5 To evaluate the safety and tolerability of single and multiple doses of advanced alkaline phosphatase recombination product (LVL-recAP) administered by intravenous infusion (7.a.) in healthy subjects.

To determine the pharmacokinetics (PK) of LVL-recAP after intravenous infusion of single and multiple doses of LVL-recAP in healthy subjects.

10 Design and treatments

A two-part concentric study in a planned number of 50 healthy subjects. Part A will be a randomized, double-blind, placebo-controlled, single-escalating dose study in up to 4 consecutive groups of 8 healthy males and females.

In all of them (6 in LVL-recAP and 2 in placebo). An attempt will be made to include in every 15 treatment groups an equal number of male and female subjects with a minimum of 2 and a maximum of 4 females for each.

group. A single dose of LVL-recAP or placebo will be given by a 1-hour intravenous infusion. Part B will be a randomized, double-blind, placebo-controlled, multiple ascending dose (49,000) study in up to 2 groups of 9 healthy male and female subjects each (6 in LVL-recAP 20 and 3 in placebo). An attempt is made to include in each treatment group an equivalent number of males and females, with a minimum of 2 and a maximum of 4 females per group. Subjects received a 1-hour intravenous infusion of LVL-recAP or placebo on days 1, 3 and 1. The following treatments will be given:

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Part A

Group 1: 1-hour infusion of 200 units/kg LVL-recAP

Group 2: - 1 hour infusion of 500 units/kg LVL-recAP

Group 3: - 1 hour infusion of 1000 units/kg LVL-recAP

Group 4: - 1 hour infusion of 2000 units/kg LVL-recAP

Part B

Group E: 1-hour infusions of 100 U/kg LVL-recAP on days 1, 2, and 3

Group 6: 1-hour infusions of 1000 units/kg LVL-recAP on days 1, 2 and 3

After day 9 of group 1 and day 4 of group 2 of Part A are completed, a PK assessment is performed

10 temporary.

Based on the results, infusion and PK sample schedules for the remaining SAD and MAD groups can be adjusted.

In this first-in-human study, subjects at the lowest dose level in Part A (Group 1) are dosed according to a warning dosing design to ensure minimal risk. This means giving the dose to 2 subjects initially. One of these subjects will receive the active drug

15 LVL-recAP and the other subject will receive placebo. If the safety and tolerability results for the first 24 hours after dosing for the initial subjects are acceptable to the principal investigator, 6 additional subjects from the lowest dose level will receive the placebo-controlled dose in a randomized manner (5 active and 1 placebo).

Monitoring period

20 Part 8: From day 1 to 48 hours (day 3) after drug administration. Short outpatient visits to the clinical research center on days 4, 6, 9 and H1

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Part 9: From day 1 to 48 hours (day 7) after the last administration of the drug. Short outpatient visits to the clinical research center on days 8, 10, 13 and 19

Subjects will be screened for eligibility within 3 weeks prior to the (first) administration of the drug in each study arm.

Follow-up tests are done on day 15 (Part 8) and Day 19 (Part B).

Submissives

Part 8: 32 A healthy male and female submissive

Part B: 18 healthy male and female submissives

The main criterion for inclusion

10 Gender: male or female

Age: 18-55 years inclusive

Body Mass Index (BMI): 18.0-30.0 kg/m2 inclusive

Study drug

Active drug

15 Active ingredient: LVL-recAP, a form of genetic recombination product developed from the enzyme alkaline phosphatase

(AP) human alkaline phosphatase

Activity: Hydrolase enzyme responsible for dephosphorylation treatment

mono-esters of phosphoric dephosphorylation

acid

20 Indicator: Acute kidney injury

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Strength: 600, 1500, 3000 and 6000 units/ml

Dosage form: Intravenous infusion

Manufacturer: PRA Pharmacy

Placebo

Material: 20 mM citrate, 250 mM sorbitol, mM MGCI2, 50 μM ZnCI2, pH 7.0.

Activity: none

Indicator: Not applicable

Strength: Not applicable

10 Dosage form: Intravenous infusion

Nova Laboratories Ltd, Gloucester Crescent, Wigston, Leicester, Manufacturer: LE18 4YL, UK

Evaluation Criteria

Safety: Adverse events (AEs), vital signs (including weak blood pressure, systolic and diastolic blood pressure, pulse, body temperature, respiratory rate) and graphic image.

Electrocardiogram (ECG), continuous cardiac monitoring (telemetry), clinical laboratory tests (including clinical chemistry [AP is a PK variable], hematology and urinalysis), physical examination and antibodies

(ADA) anti-drug antibodies for drugs

20 Pharmacokinetics: PK parameters based on analysis of LVL-recAP and AP activity concentrations in serum.

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Results

Dose administration and observation time were completed for all dose groups and no serious adverse events were observed in any of the groups. Analyzes of all measured variables continue.

Example 8

5 Materials and methods

Mice

and

The generation and characterization of Alpl-/- mice has been reported previously (Narisawa et al., 1997). Alpl-/- mice exhibit an infant HPP phenotype, including a complete TNAP deficit, PPi accumulation and mineralization defects.

Fedde et al., 19997 Narisawa et al., 2001; Anderson et al., 2004)

10, 2008, et al. (A nutritional supplement with vitamin 86 briefly suppresses seizures and extends lifespan up to days after exercise.

Birth 18-22, but hypermineralization and osteoclast accumulation continue to worsen with age

Narisawa et al., 1997; Fedde et al., 19997 Narisawa et al., 2001; Milan)

2008 Accordingly, all animals (breeders, lactating mothers, pups, and weanlings) in this study were provided free access to a laboratory-modified rodent diet 5001 15 containing increasing levels (325 ppm) of pyridoxine. SCR was performed

Genotyping on genomic DNA was as described previously (Yadav et al., 2011). All animal studies were approved by the Institutional Animal Care and Use Committee (400).

Soluble human chimeric alkaline phosphatase (LVL-RecAP)

A solution of LVL-RecAP was used at 10.1 mg/mL in 25% glycerol v/w, 20 5 mM Tris/HCl, 2 mM 165012, 50 μM 20012, and at pH repeat.

8.0. The enzyme had &gt;99.99% purity as detected by high-pressure liquid chromatography.

Dose-response study using LVL-RecAP

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Alpl-/- mice were divided into 5 model groups: Vector-treated: Alpl-/- mice treated with vector (0 = 14) only: LVL-RecAPl: Alpl-/- mice treated with 00-/- at 1 mg/ kg/day (0 = 14) 800408-7: Alpl-/- mice treated with 000-/1 at 8 mg/kg/day (n = 12) and LVL-RecAP16^ Alpl-/- mice treated with 800-/1. At 5 16 mg/kg/day (π = 10)0 represents young primate mice from the same litter of mice.

Alpl-/- reference animals did not receive injections (π = 14). Model groups had vector or LVL-RecAP injected daily SC into the shoulder region. Injections were given between 8 and 11 a.m. The volumes administered to the 3 infants were calculated based on the body weight measured before injection. All treatments were started on p.m

Day 1 after birth and repeated daily for up to 53 days or until the time of autopsy.

<p dir="rtl">“1 Collect the sample</p>

Necropsy was performed on postnatal day 53 (p53), 24 hours after the final injection of LVL RecAP for those animals that completed the experimental protocol or sooner for those animals that appeared to have diseased extremities. Avertin was administered intraperitoneally before anesthesia. Blood was collected in lithium heparin tubes by cardiac puncture. The autopsy consisted of a comprehensive pathology examination and X-ray.

<p dir="rtl">15 Radiography and microcomputed tomography</p>

(pCT)

Radiographic images of the skeleton were acquired with a Faxitron MX-20 DC4 (Chicago, IL, USA), using an energy of 20 kV. The mandible halves were imaged at 30 kV. Whole dissected skulls from P21 mice were mounted, and then scanned at 18 resolution

<p dir="rtl">20 microM isotropic voxels using the GE Explore Locus SP pCT imaging system</p>

Obtained from Healthcare Before Clinical Imaging, London, ON, Canada

Measurements at an operating voltage of 80 kV and a current of 8 mA with an exposure time of 1600 ms using the Parker scanning technique, which rotates the sample 180 degrees plus a fan angle of 20 degrees. The scan was calibrated to a hydroxyapatite phantom

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Three-dimensional images were created at an effective voxel size of 18 μM3. A fixed cut-off value of 1400 Hounsfield units was used to distinguish mineralized tissue. Regions of interest (ROI's) were determined from the frontal and parietal bones, 1 mM in length and 1 mM in width, depth equivalent to K thickness.

Bone and start position at 0.75 mm from the sagittal and coronal sutures. As described

<p dir="rtl">• Previously (Ah Liu, 2014). The variables of bone volume, density and structure were measured using software</p>

(GE Healthcare before Clinical Imaging, London, ON) Microview version 2.2

Existing algorithms and algorithms (Meganck et al., 2009; Umoh et al., 2009)

Conduct Student's t-tests to compare quantitative results to determine statistically significant differences between genotypes. Bone pCT data were analyzed and recorded according to the recommendations of 2010. Bouxsein et al

·(Bouxsein et al., 2010 10

For dental imaging, the anatomy of the lower jaw was scanned on a Scanco Medical

At 10 μM volume (Scanco Medical AG, Bruttisellen, Switzerland) 01 50

voxel. Mandibular z-packs were imported as DICOM files and reoriented using ImageJ 1.48 software, comparing the coronal, sagittal, and transverse planes of the selected section.

15 for comparison. For qualitative analysis the mandible was scanned on a 35 01 Scanco Medical at 6 μM voxel size hKV 145 mA with 0.36° rotation step (180° angular range) and 400 ms exposure for each view. HP software (Scanco pCT 1.6 DECwindows Motif) was used to recreate 3D and view the image. After 3D reconstruction, the volumes of the crown, enamel, roots, and alveolar bone were segmented using a global cutoff value of 6.0.

20 g/cubic centimeter. Total volume (TV), bone volume (mineralized tissue BV), and tissue mineral density (TMD) were measured for the entire crown and separately for enamel, root dentin, and alveolar bone in the crotch area. For enamel and roots, thickness was also measured.

Histological analyses

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Bone specimens were cleaned and fixed in 4% paraformaldehyde/phosphate buffered saline for 3 days at 4°C. They were then transferred to 70% ethanol for storage at 4°C. Plastic sections were prepared

According to previously described (Yadav et al., 2012). Von Kossa and Van Gieson staining was performed on d

Trichrome on plastic sections according to previously described (Narisawa et al., 1997). has been erased

Aperio, ScanScopeXT, scanned using the Van Gieson or Von Kossa system. • Bioquant Osteo sections. The images were analyzed using Vista, 0, USA. The half of the left lower jaw used was fixed. Osteoanalysis Co., Nashville, TN, USA

The tissue was analyzed in Bouin's solution for 24 hours, and then it was demineralized in AF S solution (acetic acid, formaldehyde, sodium chloride).

10 chloride), and embedded in paraffin for serial sectioning as previously described (Foster, 2012). For picrosirius red staining, sections of demineralized tissue were stained with 0.2% aqueous solution of gusmolybdic acid hydrate, 0.4% Direct Red 80 and 1.3% 2,4,6-trinitrophenol 1001000600-2,4,6 ( Polysciences, Inc. Warrington, PA, as previously described (Foster, 2012). Spotted sections are noted

15 in Picrosirius red under polarized light for microscopic imaging.

Biochemical tests

After removing the muscle tissue, the lengths of the femur, tibia, humerus and radius were measured using calipers. The bones were frozen and wrapped in gauze containing saline to avoid dehydration. Isolated femurs and tibias were evaluated using a three-point flexion test using the RS Sr testing material machine

20 Instron 1101 universal according to previously described (Huesa et al., 2011). Bones were slowly thawed and fixed at room temperature before tests. The intact femur and tibia were placed in the testing machine on two supports spaced 15 mM apart and the load was applied to the middle of the strand, thus creating a three-point bending test at a speed of 2 mm·min—1.

PPi experience

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Plasma concentrations of PPi were determined by differential adsorption onto activated charcoal to UDP-D-glucose.

UDP- [from the reaction produced 6-6-13 (Amersham Pharmacia) [[6-3Η]

Cr

(Hessle et al., 2002; Yadav et al., 2014) As described, D-[6-3H]glucose

5 Statistics

Given that different LVL-RecAP concentrations resulted in different survival rates, it was not possible to compare the three typical treatment groups in an age-matched manner. For this reason a standard two-tailed unpaired Student's t test was performed to compare the AMA-/treated mice to the WT group. Differences were significant when p&lt;5 0.0 in order to compare differences in survival curves

10 survival among typical treatment groups (90850-500 test was performed)

.Wilcoxon

Results

Increased survival and body weight in Alpl-/- mice treated with LVL-RecAP

Survival in mice receiving 8 mg/kg/day (8608-17) or 16 mg/kg/day (500016-) of LVL-RecAP was significantly improved compared to treatment with

Carrier and 1 mg/kg/day (00001-1) group (p = 0.001) (Figure 6A(0) Differences were statistically significant when comparing survival curves for the treated vs.

Dr

each other (h 0.0001 for all comparisons). Median survival 44 was 22 and 19 days in model groups treated with 88041, LVL-RecAP8-7 1 and vector, respectively.

20 The average survival of group 0016-1 could not be calculated because the animals remained alive until the end of the experiment on day 053.

Alpl—/— animals weigh less than WT pups from the same litter beginning at about age 07. Treatment with 1 mg/kg/day LVL-RecAP resulted in a statistically significant increase in body weight compared to mice

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-٧٨-

Treatment with a carrier substance. There is no significant difference compared to WT pups from the same litter at 18 days of treatment (Figure 6B). Alpl-/- mice died by 24-Ρ18 on a vitamin 86 diet, so the long-lived M-47 model groups were not compared 00016-47 Secret.

in mice treated with a possible vector, and alternatively compared to WT mice. Weigh the animals in a group

5 LVL-RecAP8 was significantly lower than WT pups from the same litter of Ρ18 (Figure August) while mice in the 000016-1 group showed parity in body weight and were indistinguishable from WT mice at Ρ53 (Figure 6B).

LVL-RecAP treatment improved the skeletal phenotype in radiographs of untreated Alpl-mice (Figure 7A) demonstrating profound skeletal abnormalities including decreased Cr.

10 Tissue mineral density and bone fracture according to previously described (Yadav et al. 2011). We found improvement in skeletal pathology in Alpl-/- mice that received 1 mg/kg/day -LVL RecAP for 18 days (Figure 7A), and the benefit was more profound in the -LVL 50008 and LVL-RecAP16 model groups (Figure 7B). The distal extremities showed normal morphology regardless of dose while partial correction was observed in the spine, extremities

15 forelimbs, hindlimbs and cage ribs for all model treatment groups. 1-80008 and LVL-RecAP16 mice also exhibited secondary cracks or fractures in the femurs and tibia. The joint contour of the knees and elbows displays 88008-/1 ^LVL-RecAP anomalies at Ρ44 and Ρ53, respectively (Figure ).

To evaluate the degree of progression of osteomalacia we performed histomorphometric analysis of embedded plastic unclassified sections

20 hindlimbs of mice treated with LVL-RecAP and the WT comparator (Figures 8a and a). Bone and osteoclast volumes were measured (Figures 8b8c) and serum PPi levels were also analyzed (Figures 9b9c). Consistent with previous findings (Yadav et al., 2011), von 1055 staining revealed that Alpl-/- mice display severe defects in thin cortical bone mineralization, reduced trabecular bone, and weak ossification centers (Figure 8A), while both animals treated with

25 08008-471 ^LVL-RecAP Cortical bone improves significantly and ossification centers improve

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high school. Histomorphometry (Figures 8b and 8c) revealed that in both the 86008-/1 and LVL-RecAP16 model groups, the BV/TV ratio (Figure 8b) was still significantly lower in age-matched comparator-treated WT mice (0.0321). and 0.0302 N=9) while the percentage of OV/BV (Figure 8c) was significantly higher for both treatment groups modeled compared to WT mice treated with comparator (0.0001 and 0.0175 Lat9). She was not pleased

The differences between the 6008-/1 and 8604016-/1 treatment groups were statistically significant. Trichromatic histological mottling (Figure 9a) in plastic sections of the hindlimbs confirmed these findings. Bone mineralization mass in Alpl—/— mice is severely decreased (green speckled areas) with defects in cortical and trabecular bone defined by osteoclast accumulation (red speckled areas). In contrast, WT mice treated with comparator at 53 days of age had stronger cortical bone and trabecular bone present.

In secondary ossifications and few ossicles at the bone surface. Alpl—/— mice in the 880408-/1 and LVL-RecAP16 model groups display significant improvement in cortical bone especially in the 8804016-7 group where no major differences were observed compared to WT mice. While trabecular bone formation in secondary ossification centers in Alpl—/— mice was improved by treatment with NO

15 008-1 and LVL-RecAP16 mice still retain larger than normal regions of osteoclasts (red). Secret

Consistent with the skeletal findings we detected correction for serum PPi concentrations in all model treatment groups. 80091-/1 animals (Figure 9B) harbored significantly lower PPi levels when compared to comparator-treated animals. The 20 animals treated with 0560016-7 4 had PPi levels that were difficult to distinguish statistically from those of mice.

Little WT from the same birth.

Craniofacial absence abnormality in LVL-RecAP-treated Alpl-/- mice

Alpl-/- mice present with craniofacial shape abnormalities and coronal suture fusion (Liu et al., 2014).

To determine the extent to which the craniofacial skeleton was affected by treatment in Alpl-/- mice, we performed μCT-based analyzes on frontal bones and cranial parietal bones. The results at p21 show that both...

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Frontal bones and cranial parietal bones in vector-treated mice had a significant decrease in bone fracture volume, bone mineral content, tissue mineral density, and tissue mineral density when compared to WT or either-/treated mice (Table 15). In contrast to Neither the frontal nor the cranial bones of treated 0-/- mice differed significantly from those

5 in mice of the main type. The adult skulls of B-80016-treated Alpl-/- mice did not appear to differ from those of WT in terms of size and shape (Figure 10).

Table 15. pCT analyzes of cranial bones. Frontal and cranial bones were analyzed in WT, Alpl-/- mice (vector) and Alpl-/- mice treated with 80016 at Ρ21· Values are reported as averages ± 80.

<tr><td><p dir="rtl">density</p><p dir="rtl">Minerals in fabric</p><p dir="rtl">mg name)</p></td><td><p dir="rtl">Content</p><p dir="rtl">Mineral in fabric (mg)</p></td><td><p dir="rtl">density</p><p dir="rtl">Minerals mg name)</p></td><td><p dir="rtl">Content</p><p dir="rtl">Bone mineral (mg)</p></td><td><p dir="rtl">Bone size</p></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">In front of me</p></td></tr><tr><td><p dir="rtl">٥٩٥ ±</p><p dir="rtl">٢٨ *</p></td><td><p dir="rtl">٠٠٠٠٤ ±</p><p dir="rtl">٠,٠٠١ *</p></td><td><p dir="rtl">٤٦٤ ±</p><p dir="rtl">٢٧ *</p></td><td><p dir="rtl">٠,٠٠٨ ±</p><p dir="rtl">٠,٠٠١ *</p></td><td><p dir="rtl">٠,٤١ ±</p><p dir="rtl">٠,٠٦ *</p></td><td><p>Alpl-/-</p></td></tr><tr><td><p dir="rtl">٦٩٧ ±</p><p dir="rtl">٥٧</p></td><td><p dir="rtl">٠,٠١٥ ±</p><p dir="rtl">٠,٠٠٦</p></td><td><p dir="rtl">٦١٥ ±</p><p dir="rtl">٨٤</p></td><td><p dir="rtl">٠,٠١٨ ±</p><p dir="rtl">٠,٠٠٥</p></td><td><p dir="rtl">٠,٧٢ ±</p><p dir="rtl">٠,١٥</p></td><td><p>1\Ι1-</p><p>50016</p></td></tr><tr><td><p dir="rtl">٧٠٩ ±</p><p dir="rtl">٥٦</p></td><td><p dir="rtl">٠,٠١٦ ±</p><p dir="rtl">٠,٠٠٨</p></td><td><p dir="rtl">٦١٥ ±</p><p dir="rtl">٧٩</p></td><td><p dir="rtl">٠,٠١٩ ±</p><p dir="rtl">٠,٠٠٨</p></td><td><p dir="rtl">٠,٧٠ ±</p><p dir="rtl">٠,١٢</p></td><td><p>WT</p></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">cranial</p></td></tr>

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<tr><td><p dir="rtl">٥٩٧ ±</p><p dir="rtl">١٢ *</p></td><td><p dir="rtl">٠,٠٠٥ ±</p><p dir="rtl">٠,٠٠١*</p></td><td><p dir="rtl">٤٩٤ ±</p><p dir="rtl">٢١ *</p></td><td><p dir="rtl">٠,٠١٨ ±</p><p dir="rtl">٠,٠٠١ *</p></td><td><p dir="rtl">٠,٥٢ ±</p><p dir="rtl">٠,٠٥ *</p></td><td><p>Alpl-/-</p></td></tr><tr><td><p dir="rtl">٦٩١ ±</p><p dir="rtl">٦١</p></td><td><p dir="rtl">٠,٠١٥ ±</p><p dir="rtl">٠,٠٠٦</p></td><td><p dir="rtl">٦٠٨ ±</p><p dir="rtl">٧٤</p></td><td><p dir="rtl">٠,٠١٨ ±</p><p dir="rtl">٠,٠٠٧</p></td><td><p dir="rtl">٠,٧٢ ±</p><p dir="rtl">٠,٠٨</p></td><td><p>LVL-</p><p>[50016</p></td></tr><tr><td><p dir="rtl">٧٢٦ ±</p><p dir="rtl">٥١</p></td><td><p dir="rtl">٠,٠١٧ ±</p><p dir="rtl">٠,٠٠٧</p></td><td><p dir="rtl">٦٤٥ ±</p><p dir="rtl">٧٣</p></td><td><p dir="rtl">٠,٠٢٠ ±</p><p dir="rtl">٠,٠٠٧</p></td><td><p dir="rtl">٠,٧٦ ±</p><p dir="rtl">٠,١٠</p></td><td><p>WT</p></td></tr><tr><td><p dir="rtl">density</p><p dir="rtl">Minerals in fabric</p><p dir="rtl">mg name)</p></td><td><p dir="rtl">Mineral content in fabric (mg)</p></td><td><p dir="rtl">density</p><p dir="rtl">Minerals mg name)</p></td><td><p dir="rtl">Content</p><p dir="rtl">Bone mineral (mg)</p></td><td><p dir="rtl">Bone volume fraction</p></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">In front of me</p></td></tr><tr><td><p>595 ±</p><p>28 *</p></td><td><p>0.004 ±</p><p>0.001 *</p></td><td><p>464 ±</p><p>27 *</p></td><td><p>0.008 ±</p><p>0.001 *</p></td><td><p>0.41 ±</p><p>0.06 *</p></td><td><p>Alpl-/-</p></td></tr><tr><td><p>697 ±</p><p>5</p></td><td><p>0.015 ±</p><p>0.006</p></td><td><p>615 ±</p><p>84</p></td><td><p>0.018 ±</p><p>0.005</p></td><td><p>0.72 ±</p><p>0.15</p></td><td><p>WIL-</p><p>RecAP16</p></td></tr><tr><td><p>709 ±</p><p>56</p></td><td><p>0.016 ±</p><p>0.008</p></td><td><p>615 ±</p><p>79</p></td><td><p>0.019 ±</p><p>0.008</p></td><td><p>0.70 ±</p><p>0.12</p></td><td><p>WT</p></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">cranial</p></td></tr><tr><td><p>597 ±</p><p>12 *</p></td><td><p>0.005 ±</p><p>0.0)01 *</p></td><td><p>494 ±</p><p>21 *</p></td><td><p>0).()180 ±</p><p>0.0)01 *</p></td><td><p>0.52 ±</p><p>0.05 *</p></td><td><p>Alpl-/-</p></td></tr>

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<tr><td><p>691 ±</p><p>61</p></td><td><p>0.015 ±</p><p>0.006</p></td><td><p>608 ±</p><p>74</p></td><td><p>0.018 ±</p><p>0.00)7</p></td><td><p>0.72 ±</p><p>0.08</p></td><td><p>LVL-</p><p>RecAP16</p></td></tr><tr><td><p>726 ±</p><p>51</p></td><td><p>0.017 ±</p><p>0.00)7</p></td><td><p>645 ±</p><p>13</p></td><td><p>0.020 ±</p><p>0.00)7</p></td><td><p>0.76 ±</p><p>0.10</p></td><td><p>WT</p></td></tr>

* Indicates statistical significance between genotypes and between typical treatment groups.

LVL-RecAP treatment partially rescues dentoalveolar defects

-/-Alpl in Ablation

Ablation of Alpl in mice results in advanced mineralization defects in cementum, dentin, and alveolar bone

Foster et al., 2014a; Foster et al., 2014b; McKee et al. 2011 Yadav) 5 and enamel

2012 (et al), consistent with case reports in human subjects with HPP. The absence of acellular cementum results in loss of gingival attachment to the tooth root surface and premature tooth exfoliation as a hallmark of HPP. Alpl-/- mice treated with 50008-471 and WT treated with a comparator at 26-025 when the formation of the molar tooth is nearly complete

10 Radiographic and pCT imaging, compared for comparisons (Figures 11d) The mandible of untreated Alpl—/— mice depicts hypermineralized bone, short molars with thin dentin, wide pulp chambers, and severely defective maxillary incisors (Figure 11b, f). By histological analysis, Alpl—/— mouse teeth present a lack of acellular cementum layer and invasion of alveolar osteoblasts into the periodontal ligament (PDL) space, leading to bone fusion and loss of functional periodontium (Figure 11g vs. 11e).

15 Administration of 8 mg/kg/day LVL-RecAP improves the radiological appearance of molar height, dentin thickness and bone mineralization at p26, despite the persistence of the incisor defect (Figure 11C-H). Histologically, although this dose of LVL-RecAP did not restore acellular cementum layer to the root surface, the PDL space associated with the molar molar and the alveolar bone border was better preserved (Figure 11i).

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The 800916-1/1 group was compared to WT at Ρ53-50 to determine effects on mature tooth structure and function. Radiographic imaging 401 demonstrated reduced mineralization of alveolar and confluent bone around the molar tooth in the lower jaw of Alpl-/- mice, compared with controls (Figure 11j-m). The formation of molar tooth and dentin appears largely normal in LVL-RecAP16 mice, 5 and this was confirmed by CT analysis of the first molar (Table). Enamel in the LVL group.

RecAP16 was not different from WT in BV/TV or TMD. Molar crowns and roots showed a moderate but significant reduction of 4-10% in BV/TV and TMD compared to 771 indicating mineralization of the dentin in which was not completely salvaged, and a 15% reduction in root thickness. Alveolar bone mineralization remained severely defective in the LVL-RecAP16 group, with 87/17 decreased by 27% 10 and TMD reduced by 13% compared to WT. The incisor teeth in treated Alpl −/− mice also remained severely affected (Figure 11k-m).

By histological analysis, 080016-/1 mice present a mixture of mineralized alveolar bone, osteoclasts, and a reduced but present PDL space (Figure 11np). We did not observe tooth loss in the 5804016-/1 group by p53, although gingival attachment remained impaired as evidenced by the lack of cementum layer, PDL disorganization, detachment from the root surface, and epithelial ingrowth.

Conductivity down. However, small areas of PDL connection to the tooth were observed in association with organized PDL fibers (Figure 11S F) suggesting the presence of some compromised connection that may act to trap the molars.

Table 016 07 Analyzes of dental alveolar tissue. Mandibular first molars 20 and their associated alveolar bone were compared at Ρ53-Ρ50 in n) WT=5) and Alpl-/- mice treated with

2580016 (n 4). Values are reported as averages ± 5.

<tr><td><p dir="rtl">Fish</p><p dir="rtl">(micro mola</p></td><td><p>g) TMD</p><p dir="rtl">Decisive)</p></td><td><p>(%) 8//1/</p></td><td><p dir="rtl">BV(mm3)</p></td><td><p dir="rtl">TV(mm3)</p></td><td></td></tr>

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<tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">Enamel</p></td></tr><tr><td><p dir="rtl">٧٠,٢ ±</p></td><td><p dir="rtl">١,٦٩ ±</p></td><td><p dir="rtl">٩٨,٧٧ ±</p></td><td><p dir="rtl">٠,٢٤ ±</p></td><td><p dir="rtl">٠,٢٥ ±</p></td><td><p>WT</p></td></tr><tr><td><p dir="rtl">٠,٥</p></td><td><p dir="rtl">٠,٠٥</p></td><td><p dir="rtl">٠,٥٢</p></td><td><p dir="rtl">٠,٠٢</p></td><td><p dir="rtl">٠,٠٣</p></td><td></td></tr><tr><td><p dir="rtl">٦٣,٠ ±</p></td><td><p dir="rtl">١,٦٨ ±</p></td><td><p dir="rtl">٩٨,٩٧ ±</p></td><td><p dir="rtl">٠,٢٠ ±</p></td><td><p dir="rtl">٠,٢٠ ±</p></td><td><p>LVL-</p></td></tr><tr><td><p dir="rtl">٠,٥</p></td><td><p dir="rtl">٠,٠٣</p></td><td><p dir="rtl">٠,٢٧</p></td><td><p dir="rtl">٠,٠٢ *</p></td><td><p dir="rtl">٠,٠٢ *</p></td><td><p>RecAP16</p></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">the crown</p></td></tr><tr><td><p>N.D</p></td><td><p>N.D</p></td><td><p dir="rtl">٩١,٠٦ ±</p></td><td><p dir="rtl">٠,٥٦ ±</p></td><td><p dir="rtl">٠,٦١ ±</p></td><td><p>WT</p></td></tr><tr><td></td><td></td><td><p dir="rtl">٠,٦٤</p></td><td><p dir="rtl">٠,٠٣</p></td><td><p dir="rtl">٠,٠٤</p></td><td></td></tr><tr><td><p>N.D</p></td><td><p>N.D</p></td><td><p dir="rtl">٨٦,٩٢ ±</p></td><td><p dir="rtl">٠,٤٦ ±</p></td><td><p dir="rtl">٠,٥٣ ±</p></td><td><p>\\،\-</p></td></tr><tr><td></td><td></td><td><p dir="rtl">٠,٦٩ *</p></td><td><p dir="rtl">٠,٠٣ *</p></td><td><p dir="rtl">٠,٠٣ *</p></td><td><p>RecAP16</p></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">the roots</p></td></tr><tr><td><p dir="rtl">١٤٠,٤</p></td><td><p dir="rtl">١,٠٧ ±</p></td><td><p dir="rtl">٨٥,٨٤ ±</p></td><td><p dir="rtl">٠,٥٦ ±</p></td><td><p dir="rtl">٠,٦٥ ±</p></td><td><p>WT</p></td></tr><tr><td><p dir="rtl">± ٨,٤</p></td><td><p dir="rtl">٠,٠١</p></td><td><p dir="rtl">٠,٧٤</p></td><td><p dir="rtl">٠,٠٥</p></td><td><p dir="rtl">٠,٠٥</p></td><td></td></tr><tr><td><p dir="rtl">١٢١,٥</p></td><td><p dir="rtl">١,٠٢ ±</p></td><td><p dir="rtl">٧٧,٢٧ ±</p></td><td><p dir="rtl">٠,٤٤ ±</p></td><td><p dir="rtl">٠,٥٦ ±</p></td><td><p dir="rtl">-0l1</p></td></tr><tr><td><p dir="rtl">± ١,٧</p></td><td><p dir="rtl">٠,٠١ *</p></td><td><p dir="rtl">٢,٣٧ *</p></td><td><p dir="rtl">٠,٠٤ *</p></td><td><p dir="rtl">٠,٠٦</p></td><td><p>RecAP16</p></td></tr><tr><td><p>*</p></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td><p dir="rtl">Alveolar bone</p></td></tr><tr><td><p>N.D</p></td><td><p dir="rtl">١,٠١ ±</p></td><td><p dir="rtl">٦٥,٧٢ ±</p></td><td><p dir="rtl">٠,٢٩ ±</p></td><td><p dir="rtl">٠,٤٤ ±</p></td><td><p>WT</p></td></tr><tr><td></td><td><p dir="rtl">٠,٠٢</p></td><td><p dir="rtl">٥,٠٨</p></td><td><p dir="rtl">٠,٠٥</p></td><td><p dir="rtl">٠,٠٤</p></td><td></td></tr><tr><td><p>N.D</p></td><td><p dir="rtl">٠,٨٨ ±</p></td><td><p dir="rtl">٤٧,٩١ ±</p></td><td><p dir="rtl">٠,١٥ ±</p></td><td><p dir="rtl">٠,٣٢ ±</p></td><td><p>LVL-</p></td></tr><tr><td></td><td><p dir="rtl">٠,٠٢ *</p></td><td><p dir="rtl">٤,٢٥ *</p></td><td><p dir="rtl">٠,٠٢ *</p></td><td><p dir="rtl">٠,٠٥ *</p></td><td><p>RecAP16</p></td></tr>

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* p &gt; 0.05 by independent samples t-test

ND = not specified

Example references 8

Anderson HC, Sipe JB, Hessle Dhanyamraju R., Atti Camacho

NP, Millan JL Impaired calcification around matrix vesicles of growth 5 plate and bone in alkaline phosphatase-deficient mice. Am. J. Pathol.

2004,164(3):841-847.

Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ and MOller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. Bone Miner Res 2010:25(7):1468-86. 10

Fedde KN, Blair L, Silverstein, J, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa 8, Millan, JL, MacGregor GR, Whyte MP, Alkaline phosphatase knockout mice recapitulate the metabolic and skeletal defects

of infantile hypophosphatasia. J Bone Miner Res 1999:14:2015-2026.

Foster BL, Nagatomo KJ, Nociti FH, Fong H, Dunn D, Tran AB, Wang W, 15

Narisawa S, Millan JL, Somerman MJ 2012 Central role of pyrophosphate in acellular cementum formation. PLoS One 7(6):638393.

Foster 8.., Nagatomo KJ, Tso HW, Tran AB, Nociti FH, Jr., Narisawa S., Yadav MC, McKee MD, Millan A.L. Somerman MJ Tooth root dentin mineralization defects in a mouse model of hypophosphatasia. 20

J Bone Miner Res. 2013,28(2):271-82.

Foster BL, Nociti FH, Jr., Somerman MJ (20148). The rachitic tooth. Endocr Rev 35(1):1-34.

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Foster BL, Ramnitz MS, Gafni RI, Burke AB, Boyce AM, Lee JS et al. (20140). Rare Bone Diseases and Their Dental, Oral, and Craniofacial Manifestations. J Dent Res 93(7 500):75-195.

Hessle L., Johnson KA, Anderson HC, Narisawa S., Sali A., Goding JW, Terkeltaub R., Millan JL Tissue-nonspecific alkaline phosphatase 5 and plasma cell membrane glycoprotein-l are central antagonistic regulators of bone mineralization. Proc. Natl. Acad. Sci. USA

2002:99(14):9445-9449.

Huesa C., Yadav MC, Finnila MA, Goodyear SR, Robins SP, Tanner

KE, Aspden RM, Millan JL, Farquharson C. PHOSPHol is essential 10 for mechanically competent mineralization and the avoidance of spontaneous fractures. Bone. 2011,48(5):1066-1074.

Liu J, Nam HK, Campbell C, Gasque KC, Millan JL, Hatch NE. Tissue-nonspecific alkaline phosphatase deficiency causes abnormal craniofacial bone development in the 0(-/-) mouse model of infantile 15 hypophosphatasia. Bone 2014,67:81-94.

McKee MD, Nakano., Masica DL, Gray JJ, Lemire I., Heft R., Whyte MP, Crine P., Millan JL Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia. J.Dent. Res. 2011,90(4):470-

٢٠ .476

Meganck JA, Kozloff KM, Thornton MM, Broski SM and Goldstein SA. Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD. Bone 2009,45(6):1104-1116.

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Millan and Narisawa S, Lemire I, Loisel TP, Boileau G, Leonard P, Gramatikova S, Terkeltaub R, Pleshko Camacho N, McKee MD, Crine P and Whyte MP, Enzyme replacement therapy for murine 10000050115. Bone Miner Res 2008; 23: 777-787.

Narisawa S, Wennberg C. Millan JL, Abnormal vitamin 86 metabolism in 5 alkaline phosphatase knock-out mice causes multiple abnormalities, but not the impaired bone mineralization. Pathol 2001; 193: 125-133.

Narisawa S, Frohlander N, Millan L, Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn 1997; 208: 432-446. 10

Umoh Lal, Sampaio AV, Welch I, Pitelka V, Goldberg HA, Underhill TM et al. In vivo micro-CT analysis of bone remodeling in a rat calvarial defect model. Phys Med Biol 2009,54(7):2147-61.

Yadav MC, Lemire I., Leonard P., Boileau G., Blond L., Beliveau M., Cory E., Sah RL, Whyte MP, Crine P., Millan A. to. Dose response of bone- 15 targeted enzyme replacement for murine hypophosphatasia. Bone. 2011,49(2):250-256.

Yadav MC, de Oliveira RC, Foster BL, Fong H., Cory E., Narisawa 8., Sah RL, Somerman M., Whyte MP, Millan A. to. Enzyme replacement prevents enamel defects in hypophosphatasia mice. Bone Miner. Res. 20 2012,27(8):1722-1734.

Yadav, M. C., Huesa, C., Narisawa, S., Hoylaerts, M. F., Moreau, A., Farquharson, C. and Millan, L. L. Ablation of osteopontin improves the

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skeletal phenotype of Phosphol-/- mice. J. Bone Miner. Res. InPress (2014).

Example 9

Alkaline phosphatase enzyme protects against kidney inflammation

These methods:

Cell Culture

Routinely, ciPTEC were cultured on 33 March 2010 (Wilmer, 2010). Cells were transfected with Simian virus T40 antigen and the essential catalytic subunit of human telomerase 10, and allowed to replicate stably. Cells were cultured in

Supplemented with (Gibco, Paisly, United Kingdom) 0/1005 phenol red-free F-12

ITS (µg/ml insulin, µg/ml transferrin, ng/ml

36 nanograms (Sigma-Aldrich, 20006001, The Netherlands; selenium)

Hydrocortisone (Sigma-Aldrich), 10 ng/ml Epidermal Growth Factor 15 (Sigma-Aldrich), 40 pg/ml Tri-Iodine

Greiner Bio-One, ) Thyronine 000100008-10 and 10% fetal bovine serum.

KremsmOnster, Aus-tria. Before the experiment, the cells were planted in a dish with wells (0.4840 cells/cm2) and incubated for one day at 33°C, followed by a maturation period of 7 days at 37°C. On the day of the experiment, the cells were incubated for two hours with LVL. -RecAP (1 e or 10 units/ml taste of

Followed by (Kiffer-Moreira, 2014) (AM-Pharma, Bunnik, The Nether-lands 20 p.). coli 0127:98, Sigma-Aldrich) LPS incubation for 24 hours with 10 μg/ml

Dissolved in 10 mM HEPES HBSS, pH 7.4 (HEPES: Roche

Alternatively, 10 were given.) Diagnostics, Mannheim, Germany; HBSS: Gibco

units/ml LVL-RecAP (-17 μg/ml) to cells incubated in LPS concomitantly or

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after two hours. Control cells were incubated with culture medium only. E. coli (DLPS 055:857 Sigma-Aldrich 10 µg/ml) and inactive LVL-RecAP (17 µg/ml Good Taste from AM-Pharma) were used as negative controls. In the different experimental arms

Ebioscience, Vienna, ) Human TNF-α has been replaced by the recombination protein LPS

• Austria(, or PBMCs supernatant solution, prestimulated for 24 h with or without LPS (1 ng/ml). All experiments (n = 5) were performed in duplicate.

Isolation of peripheral blood mononuclear cells

PBMCs were isolated from villous envelopes obtained from healthy blood donors (blood n, bank Nijmegen = 5) by differential centrifugation over GE (Ficoll-Pague Plus).

RPMI-1640 in PBMCs was resuspended (Healthcare, Diegem, Belgium 10).

(Gibco) enriched with 0.5 mg/ml gentamicin (Sigma-Aldrich), 1 mM pyruvate (Gibco) and 2 mM glutamax. Cells were cultured in 96-well plates at a density of 0. 5

15 hours. All experiments were performed in duplicate.

ATP measurement and cell vitality experiment

Supernatant solution was collected 30 minutes after LPS administration, with or without pretreatment with LVL RecAP, followed by direct measurement of ATP production using the ATP CLS II Bioluminescence Experiment Kit.

(Roche Diagnostics) according to the manufacturer's protocol. Cell viability was assessed after 24 h of 20 LPS incubation by performing an MTT assay. Briefly, the medium was replaced with 100 μl solution

Prewarming MTT (Sigma-Aldrich; 0.5 mg/ml in culture medium) and incubating for 3 h at 037°C. This is followed by adding 200 microliters of DMSO to dissolve the formazan crystals deposited inside the cells. Dye fading was measured at 570 nM using a 0.67 nM wavelength correction.

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Animal model

Animal experiments were performed in accordance with National Institutes of Health guidelines, and protocols were approved by the Institutional Review Board for Animal Experiments. Sprague's rats were divided

to three Dawley pathogen-free (RjHanxSD; Janvier, France) males

5 groups: placebo (n=6) LPS=6) or 0500-1/1+105 (n=). A basal value plasma sample (lithium-heparin 00000-11000 blood) was collected 7 days before the experiment from

Sarstedt, Etten-Leur, the ) Multivette during a cut in the tail vein using

Netherlands). 3 days before the experiment, the basal value of renal function was assessed as FITC-Sinestrin (Schock-Kusch, 2011) (t1/2). At t= 0 hours, placebo (0.9%) was given

10 NaCl, saline) or 0.3 mg/kg E. coli 0127:38 (BW LPS, dissolved in saline) as an intravenous bolus to induce LPS-induced renal failure. At t= 1.5 hours, it was completed

Obtain plasma as previously described. At t=2 h, the rat received an intravenous bolus of placebo or LVL-RecAP (1000 U/kg BW, diluted in saline) followed by a second measurement of renal function. At t = 1 hour, all animals received 15 ml of saline solution (sc) to prevent dehydration, followed by a 16-hour duration of urine collection. At t = 21.5 hours, the third cross-section measurement was performed

Dermis. At t = 24 hours, the rats were anesthetized (0i0p, 3 mg/kg BW xylazine and 80 mg/kg BW ketamine 10%), a retrobulbar blood sample was drawn lithium-heparin to obtain plasma, and the full infusion into the body began (6 minutes saline solution). + 0 IU/ml heparin, 210 mbar; 3 minutes 4% paraformaldehyde (PFA);

20 210 ml bar). After saline infusion, the right kidney was carefully removed, dried abruptly, and kept at -080°C until treatment. The left kidney was removed after PFA infusion, and was preserved in 4% PFA at 4°C until processing for histological analysis and immunohistochemistry. One animal from the 880-/+105 group and one urine sample from the placebo group were excluded because of injection and collection difficulties, respectively.

25 Measurements of kidney function

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Renal function was assessed in awake, freely moving rats by clearance kinetics measured transcutaneously free

Commercially available numbered sinistrin (Fresenius Kabi, Linz, Austria).

Secret S

GFR, by using the new measuring device according to previously published (Schock-Kusch, 2011

2009 Schock-Kusch). Briefly, rats were anesthetized by isoflurane inhalation (5% induction e 1.5-2% follow-up, Abbott Laboratories, Illinois, USA) and the back hair was shaved. The visible part of the device is fixed to the hairless area using a specially designed double-sided adhesive

While the electronic part of the device (Lohmann GmbH, Neuwied, Germany) after determining the baseline signal (Lohmann Biomedical, Malone, USA) was integrated into the rodent's jacket.

FITC—sinistrin (mg per 100 g BW, diluted in buffered saline) 10 was injected into the tail vein. The animals were then allowed to recover from anesthesia while the measurement continued for approximately 120 minutes

minutes after injection. 11/2 was calculated by a one-component model based on the measured removal kinetics of transdermal sinistrin (Schock-Kusch, 2009). In addition, kidney function variables were determined in plasma and urine samples using the Hitachi 704 automated analyzer (Boehringer

Urea molecular excretion and endogenous clearance were calculated (Mannheim, Mannheim, Germany

15 of creatinine using average plasma values of t=1.5 and t=24.

Histology and immunohistochemistry analysis

After fixation for at least 24 hours, the tissue was processed, embedded in Paraplast, and sectioned thickly

3 micromolar. For routine histological analysis, HE staining was performed on kidney tissue. Kidney injury was assessed using a grading system on a scale from 0 to 4 (0 = no change: 4 = severe damage e.g. 20 observed microtubule cell changes). 1-KIM was detected by a goat anti-mouse primary antibody

and secondary antibody KIM-1 (AF3689, R&amp;D Systems, Abingdon, UK 1:50).

Rabbit anti-goat IgG (1:200; P0449, DAKO, Heverlee, Belgium). Done

View immunostaining using Vector system reactants) /01451 Eline ABC

Diaminobenzidine and 3'-diaminobenzidine (Labs, Amsterdam, Netherlands

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(DAB, Sigma-Aldrich), followed by haematoxyline staining. All scoring was conducted anonymously.

Cytokines and markers of kidney injury

5 R&amp;D Systems (Human ELISA) kits were used to determine IL-6, TNF-α, and IL-8 in supernatant solution according to the manufacturer's instructions. Plasma cytokine levels (IL- and IL-1e) were determined

6, 10-INF-γ, TNF-α, IL) by Luminex test according to the manufacturer's instructions

(R&amp;D Systems) ELISA by NGAL and KIM-1 (Millipore, Cork, Ireland).

According to the manufacturer's instructions.

Tissue homogenization 10 Tissue homogenization

Qiagen, Venlo, The ) TissueLyser LT suddenly dehydrated kidneys were homogenized by

H

Netherlands) according to the manufacturer's instructions in the tissue protein extraction reactant (-T

Supplementing with EDTA-free protease inhibitor combination tablets (PER; Thermo Scientific, Rockford, USA) specifies complete EDTA-free (Roche Applied Science, Almere, The Netherlands).

15 Total protein content using the bicinchonicic acid protein experiment kit

(Thermo Scientific) and the samples were stored at -80°C until the experiment was conducted.

Real-time PCR analyses

RNA was extracted from frozen cell pellet or kidney powder (2000

0-15010000[U, Sartorius Stedim Biotech, Aubagne Cedex, 30 seconds.

Cr

20 France) with the reactant Trizol. The RNA was reverse transcribed into cDNA using the Moloney Murine Leukemia Virus (MLV) enzyme.

Reverse Transcriptase (Invitrogen, Breda, The Netherlands).

Real-time quantitative PCR (RQ-PCR) was performed using Applied® Taqman

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GAPDH genes were amplified and normalized for expression (Biosystems, Carlsbad, USA

(ciPTEC: Ct: 18.9+0.1 Renal tissue Ct:renal tissue: 24.8+0.2). The PCR reaction began with a 2-minute incubation step at 5°C followed by an initial denaturation step of 10 minutes at 95°C and 40 cycles of 15 s at 95°C and 1 minute at 6°C. Differences between groups were calculated by the comparative ΔΔCt method. Primers/probe combinations are summarized in Table 17.

Urinary purine content in urine

The content of adenosine in urine (ATP, ADP, AMP, urinary adenosine and cAMP) is determined by HPLC. Briefly, 4 volumes of urine were mixed with 1 volume of chloroacetaldehyde (6-fold diluted in 1 M Sigma-Aldrich pH 44.5 acetate buffer solution), followed by derivatization (6 min 70°C, 5 rpm). Centrifugation (3 minutes, room temperature, 13,400 rpm) and then the supernatant solution was transferred to an HPLC vial and injected. Purines were separated by a Thermo HPLC Scientific system using a Polaris C18-A column (15 x 4.6 mm) with sequential gradient filtration.

15 Using elution product A (1, “M K2HPO4, 1 mM TBAHS (pH 6.5), 2% MeOH) and elution product B (H2O: ACN: THF; 5:49:1). Retention times were 7.1 (adenosine), 8.4 (AMP), 12.5 (ADP), 16.2 (ATP) and 1.48 minutes (cAMP). Quantification was based on peak areas of samples and reference standards measured by fluorescence (excitation: 280 nM: emission: 42 nM).

»2 Statistical analysis

Data were expressed as mean ± SEM or median [25th percentile 75th percentile]. Normality of data was assessed by the Kolmogorov-Smirnov test. Statistical differences between groups were estimated by ANOVA with post hoc comparisons using Bonferroni's multiple comparison test or by the Kruskal-Wallis test using

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Dunn's test. A two-sided p value of less than 0.05 is considered statistically significant. All done

Graphpad Programs Inc. Tests using Windows for Graphpad Prism 5.00 (San Diego, CA, USA).

LVL-RecAP attenuates the LPS-stimulated inflammatory response in vitro

Human ciPTEC 5 pre-treatment LVL-RecAP dose-dependently (Wilmer, 2010).

It attenuates LPS-stimulated cytokine production of IL-6, TNF-α, and IL-8 at the gene and protein levels (Figure 12-). Detoxified LPS (dLPS) was used as a negative comparator and had no effect. Similar protective results at the protein level were obtained when LVL-RecAP was administered concomitantly with LPS or 2 h after exposure to LPS (Figure 12c). has been done

10 A comparative experiment to investigate whether LVL-RecAP dephosphorylates cytokines secreted into the medium, which is not the case (Figure 17). To confirm that the LVL-RecAP-induced decrease in cytokine production was due to the dephosphorylation nature of the enzyme, the effect of inactive LVL-RecAP lacking hydrolytic properties was investigated. Inactive LVL-RecAP did not attenuate the LPS-stimulated inflammatory response in ciPTEC (Figure 12d).

15 The in vitro effects of LVL‐RecAP are kidney‐specific and not restricted to LPS‐stimulated inflammation

To further investigate the nephroprotective mechanism of LVL-RecAP, ciPTEC were incubated with a proinflammatory cytokine TNF-α, which is unable to be dephosphorylated by bovine IAP (Chen, 2010). TNF-α-stimulated cytokine production of IL-6 and IL-8 was also attenuated by pretreatment with LVL-RecAP, while inactive LVL-RecAP had no effect (Figure 13a).

20 In the etiology of sepsis-associated AKI LPS stimulates the local inflammatory response through binding to TLR4 expressed on ciPTEC (Ct) PTEC: 30.5+3.9 • p e). Another hallmark of the disease is a systemic inflammatory response affecting both renal epithelial and endothelial cells resulting in the development of AKI (Peters, 2014). To mimic this endotoxin-induced renal inflammation, ciPTEC were incubated with a supernatant solution of peripheral blood mononuclear cells.

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Simulation with PBMCs (LPS, 1 ng/ml LPS). This stimulates the production of IL-6 and 8-^, which were reduced when ciPTEC were pretreated with 804-/1 (Figure 13, -TNF 0 was not observed). In addition to the finding of attenuation of TNF-α-mediated inflammatory responses by LVL-RecAP treatment, this suggests the presence of another mediator targeted by LVL-RecAP. In contrast

5 Pretreatment of PBMCs with LVL-RecAP did not affect the LPS-stimulated inflammatory response in these cells (Figure 13c), suggesting that the effects of LVL-RecAP are kidney-specific.

LVL-RecAP can provide nephroprotective effects in vitro through the ATP/adidecine pathway

adenosine

A second potential target of LVL-RecAP is ATP, which is released during LVL-induced cell stress

10 For example, inflammation and hypoxia (Eltzschig, 2012). Extracellular ATP has serious effects and can even be converted by ectonucleotidase enzymes (eg AP) into AMP, ADP and ultimately into adenosine, exerting an anti-inflammatory and tissue-protective effect.

Bauerle, 2011, A3, A2B, and A2A by binding to one of the adenosine receptors

2008 ,Di Sole). Surprisingly, expression of adenosine receptors was not affected

15 81 A2B and A3 in ciPTEC by LPS incubation (data not shown), expression of A2A was upregulated once LPS was mimicked (2-fold increase: 4.1+40.4 • h 0.001 compared to placebo). This effect was attenuated by LVL-RecAP combination treatment (two-fold increase: 2.9±0.2; h 0.001 compared to placebo: p&lt;0.05 compared to LPS), suggesting a role for the adenosine pathway in the protective effect. For LVL-RecAP. In addition, we observed an increase in extracellular ATP concentrations

20 after LPS incubation, which was more evident with the higher LPS concentration but was reversed by LVL-RecAP preincubation (Figure 14). LPS did not affect cell viability up to 24 h (data not shown). This supports the assumption that LVL-RecAP may exert its nephroprotective effect through the ATP/adidecine pathway.

LVL-RecAP treatment during LPS-stimulated AKI in rats attenuates renal function failure

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To confirm the beneficial effects of LVL-RecAP in vivo, AKI was induced in rats by 0.3 mg LPS (mg/kg BW). Renal function was assessed by transdermal measurement of sinestrin kinetics.

Decoded fluorescein isothiocyanate (FITC) according to previously reported (Schock-Kusch, 2011). Sinistrin is excreted by the kidneys

5 Filtration only and its disappearance of the plasma component through the dermis can be measured in real time) - Schock

In a more AKI way this allows checking the development of AKI (Kusch, 2011, Schock-Kusch, 2009

Accuracy compared to the commonly used creatinine. Before LPS injection, a baseline blood sample was drawn to determine clinical variables and plasma cytokines and the baseline FITC-sinestrin half-life (t1/2) was determined from the measured kinetics to ensure homogeneity between groups.

10 (data not shown). After 1.5 hours, treatment with LPS resulted in increased levels of cytokines in plasma, abnormalities in multiple plasma variables (Table 18), erection of hair, diarrhea, and decreased spontaneous activity, confirming the presence of systemic inflammation. Two hours after LPS administration, rats were treated with LVL-RecAP (1000 U/kg BW) or placebo (saline) immediately followed by transdermal renal function measurements. LPS significantly prolongs the half-life of FITC—

15 sinestrin, which reveals a significant decrease in kidney function. This trend was attenuated by LVL-RecAP treatment (Figure E1A). In all groups kidney function was fully restored within 24 hours (Figure 15B). Plasma urea levels were also significantly lower in animals treated with LVL-RecAP compared to animals that received LPS without LVL-RecAP (Table). LVL-RecAP treatment also prevents the LPS-stimulated increase in molecular urea secretion

20 (Figure 15c) and the LPS-stimulated decrease in endogenous creatinine clearance (Figure 15d). The biological activity of LVL-RecAP was confirmed in plasma and showed an 8-fold increase 2 hours after injection (Placebo: 293±12 U/mL: LPS: 13+260 U/mL? LPS+AP: 602150 U/mL: • 0 h ,0001).

LVL-RecAP prevents kidney injury during LPS-stimulated AKI in vivo

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The nephroprotective effect of LVL-RecAP on LPS-induced AKI was further investigated by evaluating renal histological analysis and specific tubular injury markers. No differences were found on histological analysis between treatment groups with changes ranging from no damage (“”) to secondary degenerating changes such as a foamy appearance and micro-swelling of proximal tubular cells (1) and a moderate appearance of foam and swelling as well as a few instances of cell fading (2 ) (Placebo: 1 [0.75-2] LPS: 1.5 [0-2 -LPS+ LVL

RecAP: 1 [.-,]). Treatment with LPS resulted in a significant increase in renal expression levels of IL-1

<p dir="rtl">6, while other cytokines and markers of injury (MPO, myeloperoxidase BAX; myeloperoxidase, iNOS-associated protein 8012, inducible nitric oxide synthase) were not affected (Table 20). LVL-RecAP could not be downgraded</p>

<p dir="rtl">10 decreased the expression levels of IL-6 in the kidney but did not improve the renal expression of anti-inflammatory cytokine 1-10 (Table 20). In addition, LPS administration resulted in a significant increase in urinary excretion of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, which was accompanied by elevated renal gene expression levels. This effect was avoided by co-administration of -LVL</p>

<p dir="rtl">15 RecAP (Figure 16a-b Table 20). Similar effects of LVL-RecAP were observed on plasma NGAL levels (Figure 16C) and on renal protein levels of 1-KIM (Figure 16D), which is primarily located at the apical surface of proximal tubular epithelial cells (Figure 16E).</p>

Reduced urinary adenosine excretion during LPS-stimulated AKI in vivo

<p dir="rtl">20 In order to further elucidate the nephroprotective mechanism of LVL-RecAP, we investigated the role of the ATP-adenosine pathway. LPS treatment tended to reduce gene expression levels in the kidney for all four adenosine receptors, with only the A3 adenosine receptor reaching statistical significance (Table 20). Surprisingly, treatment with LPS significantly reduced urinary excretion of adenosine (placebo: +0 pg adenosine/10 mcg creatinine: LPS: 19.4±6.3 pg).</p>

<p dir="rtl">25 grams of adenosine / 10 micrograms of creatinine: p&gt;0.001), without altering the secretion of ADP, ATP, and cAMP.</p>

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and AMP (data not shown). This may suggest that the kidney benefits from adenosine during LPS-stimulated AKI. Treatment with LVL-RecAP had no effect on adenosine receptor gene expression (Table 20) or on urinary adenosine excretion (5000-+5: 16.7+6.8 pg adenosine/10 mcg creatinine: 20 0, 001 compared to placebo) compared to LPS alone.

5 tables

Table 17. Primer/probe specifications

<tr><td><p dir="rtl">Experience identity</p></td><td><p dir="rtl">Genetic name</p></td><td><p dir="rtl">Genetic code</p></td><td></td></tr><tr><td></td><td></td><td></td><td><p>ciPTEC</p></td></tr><tr><td><p>150275899191</p></td><td><p dir="rtl">Glyceraldehyde-3-phosphate dehydrogenase</p><p>glyceraldehyde-3-phosphate</p><p>dehydrogenase</p></td><td><p>GAPDH</p></td><td></td></tr><tr><td><p>150111362491</p></td><td><p dir="rtl">Tumor necrosis factor</p><p>tumor necrosis</p><p>factor. factor</p></td><td><p>TNF-α</p></td><td></td></tr><tr><td><p>HsOO985639_m1</p></td><td><p>interleukin interleukin</p><p dir="rtl">٦</p></td><td><p>IL-6</p></td><td></td></tr><tr><td><p>Hs00174103_m1</p></td><td><p dir="rtl">Interleukin 8</p></td><td><p>IL-8</p></td><td></td></tr><tr><td><p>HsOO152939_m1</p></td><td><p dir="rtl">Future similar to 4 toll</p></td><td><p>TLR4</p></td><td></td></tr>

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<tr><td><p>HsOO379752_m1</p></td><td><p dir="rtl">Adenosine Al receptor</p><p>adenosine Al</p><p>receptor</p></td><td><p>ADORA1</p></td><td></td></tr><tr><td><p>HsOO169123_m1</p></td><td><p dir="rtl">Adenosine A2a receptor</p></td><td><p>00542</p></td><td></td></tr><tr><td><p>HsOO386497_m1</p></td><td><p dir="rtl">Adenosine 20 receptor</p></td><td><p>ADORA2B</p></td><td></td></tr><tr><td><p>HsO156O269_m1</p></td><td><p dir="rtl">Adenosine A3 receptor</p></td><td><p>ADORA3</p></td><td></td></tr><tr><td></td><td></td><td></td><td><p dir="rtl">Rat kidney</p></td></tr><tr><td><p>RnO1775763 gl</p></td><td><p dir="rtl">Glyceraldehyde-3-</p><p dir="rtl">Phosphate dehydrogenase</p></td><td><p>GAPDH</p></td><td></td></tr><tr><td><p>RnOO58O432_m1</p></td><td><p dir="rtl">Interleukin 1 beta</p></td><td><p>IL-1e</p></td><td></td></tr><tr><td><p>RnO141O33O_m1</p></td><td><p dir="rtl">Interleukin 6</p></td><td><p>II -6</p></td><td></td></tr><tr><td><p>RnOO5634O9_m1</p></td><td><p dir="rtl">Interleukin 10</p></td><td><p>,1-10</p></td><td></td></tr><tr><td><p>Rn99999017_m1</p></td><td><p dir="rtl">Tumor necrosis factor</p></td><td><p>TNF-α</p></td><td></td></tr><tr><td><p>RnOO594O78_m1</p></td><td><p dir="rtl">Interferon gamma</p><p>interferon gamma</p></td><td><p>IFN-γ</p></td><td></td></tr><tr><td><p>RnOO5977O3_m1</p></td><td><p dir="rtl">Cellular receptor for hepatitis C virus</p><p dir="rtl">١</p></td><td><p>HAVCRl</p></td><td></td></tr>

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<tr><td><p>RnOO59O612_m1</p></td><td><p dir="rtl">lipocalin</p><p dir="rtl">٢</p></td><td><p>LCN2</p></td><td></td></tr><tr><td><p>RnO146O2O4_m1</p></td><td><p dir="rtl">Myeloperoxidase</p><p>myeloperoxidase</p></td><td><p>MPO</p></td><td></td></tr><tr><td><p>0500253208291</p></td><td><p dir="rtl">X-linked protein</p><p>Bcl2</p></td><td><p>BAX</p></td><td></td></tr><tr><td><p>RnOO561646_m1</p></td><td><p dir="rtl">Nitric oxide synthase</p><p>nitric oxide</p><p>synthase</p><p dir="rtl">2, can be stimulated</p></td><td><p>NOS2</p></td><td></td></tr><tr><td><p>0500056766801</p></td><td><p dir="rtl">Adenosine A1 receptor</p></td><td><p>ADORA1</p></td><td></td></tr><tr><td><p>RnOO583935_m1</p></td><td><p dir="rtl">Adenosine receptor 22</p></td><td><p>ADORA2A</p></td><td></td></tr><tr><td><p>RnOO567697_m1</p></td><td><p dir="rtl">Adenosine 20 receptor</p></td><td><p>ADORA2B</p></td><td></td></tr><tr><td><p>Rn00563680_m1</p></td><td><p dir="rtl">Adenosine A3 receptor</p></td><td><p>ADORA3</p></td><td></td></tr>

Table 18. Plasma cytokines and experimental variables

<tr><td><p>LPS + recAP</p></td><td><p>LPS</p></td><td><p dir="rtl">Placebo</p></td><td></td></tr>

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<tr><td></td><td></td><td></td><td><p dir="rtl">Cytokines</p></td></tr><tr><td><p dir="rtl">٥٧٦٤+١٢٥٩*</p></td><td><p dir="rtl">٦٩١٣+١٣٦٢*</p></td><td><p dir="rtl">٩+٩</p></td><td><p dir="rtl">13-AA (Pico</p><p dir="rtl">g/ml)</p></td></tr><tr><td><p dir="rtl">٧٠٢٤٧±١٣٨١٢#</p></td><td><p dir="rtl">٧٤٢٩٤+١١٢٤٠*</p></td><td><p>N.D</p></td><td><p dir="rtl">IL - 6(pg/ml)</p></td></tr><tr><td></td><td><p dir="rtl">١٠٠٥٤±٢٠١٧#</p></td><td><p dir="rtl">١٧ +١٧</p></td><td><p dir="rtl">IL - 10(pg/ml)</p></td></tr><tr><td><p dir="rtl">١٠٥١٤±٨٨٩# *</p></td><td><p dir="rtl">٢١٠٧١+٣٣٧٥*</p></td><td><p dir="rtl">١±١</p></td><td><p dir="rtl">L0-3No1(Pico</p><p dir="rtl">g/ml)</p></td></tr><tr><td><p>N.D</p></td><td><p>N.D</p></td><td><p>N.D</p></td><td><p dir="rtl">7-3 No (Biko</p><p dir="rtl">g/ml)</p></td></tr><tr><td></td><td></td><td></td><td><p dir="rtl">Variables in</p><p dir="rtl">Plasma</p></td></tr><tr><td><p dir="rtl">٠,٢٥ +٠,٠٤</p></td><td><p dir="rtl">٠,٠٥٦٠,٣٠</p></td><td><p dir="rtl">٠,١٦+٠,٠١</p></td><td><p dir="rtl">Creatinine</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">٤٨+٣ *</p></td><td><p dir="rtl">٣٩±٢#</p></td><td><p dir="rtl">٢٨+٢</p></td><td><p dir="rtl">Urea</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">٣٣+٣</p></td><td><p dir="rtl">٣٤+٢</p></td><td><p dir="rtl">٢٤+٥</p></td><td><p dir="rtl">Laknat</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">٢١٦±١٣#</p></td><td><p dir="rtl">٢٦٠±٢٥#</p></td><td><p dir="rtl">١٤٣+٣</p></td><td><p dir="rtl">glucose</p><p dir="rtl">(mg/dL)</p></td></tr>

٦٣١٣

-١٠٢-

<tr><td><p dir="rtl">٥٦ [٥٦-٥٨]</p></td><td><p dir="rtl">٥٨ [٤٨-٦١]</p></td><td><p dir="rtl">٥٨ [٥٦-٥٨]</p></td><td><p dir="rtl">Safe mg</p></td><td><p dir="rtl">Protein (liter)</p></td></tr><tr><td><p dir="rtl">٢,٤١±٦.,.#</p></td><td><p># , 3.2,43</p></td><td><p dir="rtl">٥٩,٠,٠٣±2</p></td><td><p dir="rtl">(Ml</p></td><td><p dir="rtl">Like sodom</p><p dir="rtl">mol/L)</p></td></tr><tr><td><p dir="rtl">٢,٨١+٠,١٠</p></td><td><p dir="rtl">٢,٧٦+٠,٠٩</p></td><td><p dir="rtl">٠١,±3»٠,١</p></td><td><p dir="rtl">Other (ml</p></td><td><p dir="rtl">phosphor</p><p dir="rtl">Organic mol/L)</p></td></tr><tr><td><p dir="rtl">٣١٤٦</p></td><td><p dir="rtl">2±١٤٨</p></td><td><p dir="rtl">2±١٤٩</p></td><td><p dir="rtl">(Ml</p></td><td><p dir="rtl">Molar sodium</p></td></tr><tr><td><p dir="rtl">٥,٠٨ [٤,٧٦-</p><p dir="rtl">٥,١٨]</p></td><td><p dir="rtl">٤,٨٧ [٤,٦٦-</p><p dir="rtl">٥,٢٣]</p></td><td><p dir="rtl">٥,٨٩ [٥,٦٦-</p><p dir="rtl">٦,٢١]</p></td><td><p dir="rtl">(Ml</p></td><td><p dir="rtl">Molalra potassium</p></td></tr>

Plasma parameters were determined 1.5 hours after LPS administration. Data were expressed as mean +8 and median [25th percentile, 75th percentile] based on the distribution of each variable. Differences in the distribution of plasma variables compared to t=24 are likely related to sample size. Significant differences were estimated using one-way ANOVA and Bonferroni post hoc test or Kruskal-Wallis test with Dunns post hoc test. Placebo LPS 06 05+80 n= e. #p&lt;05, compared to placebo. * p&lt;5,,0 compared to LPS.LPS, lipopolysaccharide? recAP, gene recombination alkaline phosphatase; ND: Not specified.

Table 19. Plasma and urine variables

<tr><td><p>LPS+</p><p>recAP</p></td><td><p>LPS</p></td><td><p dir="rtl">Placebo</p></td><td></td></tr>

٦٣١٣

-١٠٣-

<tr><td></td><td></td><td></td><td><p dir="rtl">Plasma variables</p></td></tr><tr><td><p dir="rtl">٠,٢٠+٠,٠١</p></td><td><p dir="rtl">٠,٠٢٦٠,٢٤</p></td><td><p dir="rtl">٠,٠١٠,٢٠</p></td><td><p dir="rtl">Creatinine</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">٣٦+٢*</p></td><td><p dir="rtl">٤٨±٤#</p></td><td><p dir="rtl">٢٨±٢</p></td><td><p>Urea</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">١٢,٠ [١٠-</p><p dir="rtl">١٤]</p></td><td><p dir="rtl">١٤ [١٢-٢١]</p></td><td><p dir="rtl">١٠ [٨-٣١]</p></td><td><p dir="rtl">Lactate</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">١٦١+٣</p></td><td><p dir="rtl">١٣٢+١١</p></td><td><p dir="rtl">١٤٤±٧</p></td><td><p dir="rtl">glucose</p><p dir="rtl">(mg/dL)</p></td></tr><tr><td><p dir="rtl">٥٤٦٠٤-٦٥٨</p></td><td><p dir="rtl">٥٦ [٥٢-٥٧]</p></td><td><p dir="rtl">٥٦ [٥٤-٥٧]</p></td><td><p dir="rtl">protein</p><p dir="rtl">(mg/ml liter)</p></td></tr><tr><td><p dir="rtl">٢,٧٠ [٢,٦-</p><p dir="rtl">٢,٨]</p></td><td><p dir="rtl">٢,٦٥ [٢,٦-</p><p dir="rtl">٢,٧]</p></td><td><p dir="rtl">٢,٨٤ [٢,٧-</p><p dir="rtl">٣,١]</p></td><td><p dir="rtl">Calcium (Mmol.r.a.)</p></td></tr><tr><td><p dir="rtl">2.95 EGP 0.09</p></td><td><p dir="rtl">٠٦,٠,١٠±3</p></td><td><p dir="rtl">٢,٩٥+٠,٠٩</p></td><td><p dir="rtl">Inorganic phosphorus (mmol/L)</p></td></tr><tr><td><p dir="rtl">١٥٠ [١٤١-</p><p dir="rtl">١٥٤]</p></td><td><p dir="rtl">١٥٤ [١٤٧-</p><p dir="rtl">١٥٥]</p></td><td><p dir="rtl">١٥٠ [١٤٨-</p><p dir="rtl">١٥٧]</p></td><td><p dir="rtl">Sodium (mmol/L)</p></td></tr><tr><td><p dir="rtl">٤,٤٢+٠,٢٤</p></td><td><p dir="rtl">3٢,٤±٠,١٠</p></td><td><p dir="rtl">٤,٢٤±٠,٠٤</p></td><td><p dir="rtl">Potassium (mmol/L)</p></td></tr>

٦٣١٣

-١٠٤-

<tr><td></td><td></td><td></td><td><p dir="rtl">Urine variables</p></td></tr><tr><td><p dir="rtl">٥,٧+٠,٦</p></td><td><p dir="rtl">٦,١±٠,٣</p></td><td><p dir="rtl">٥,٤±٠,٣</p></td><td><p dir="rtl">Creatinine (mg)</p></td></tr><tr><td><p dir="rtl">٤٤٠+١٩*</p></td><td><p dir="rtl">٤٨٢+٢١*</p></td><td><p dir="rtl">٢٦٧±٢٦</p></td><td><p dir="rtl">Urea (mg)</p></td></tr><tr><td><p dir="rtl">٣١٠ [٠-٤١٩]</p></td><td><p dir="rtl">٠ [٠-٦٦٣]</p></td><td><p dir="rtl">٠ [٠-٥٤]</p></td><td><p dir="rtl">Albumin (µg)</p></td></tr><tr><td><p dir="rtl">٠,٩٩±٠,٢٧</p></td><td><p dir="rtl">١,٧٤+٠,٢٨</p></td><td><p dir="rtl">١,٥٩+٠,٢٥</p></td><td><p dir="rtl">Glucose (mg)</p></td></tr><tr><td><p dir="rtl">٦١+٩</p></td><td><p dir="rtl">٦٧±٩</p></td><td><p dir="rtl">٣8±٦</p></td><td><p dir="rtl">Protein (micrograms)</p></td></tr><tr><td><p dir="rtl">١,٧٦٧,٤</p></td><td><p dir="rtl">٧,٢+٠,٩</p></td><td><p dir="rtl">٤,١-٠,٧</p></td><td><p dir="rtl">Calcium (micromol)</p></td></tr><tr><td><p dir="rtl">٠,٦٤ [٠,٦-</p><p dir="rtl">٠,٨]</p></td><td><p dir="rtl">٠,٦٣ [٠,٦-</p><p dir="rtl">١,٠]</p></td><td><p dir="rtl">٠,٥٠ [٠,٣-</p><p dir="rtl">٠,٩]</p></td><td><p dir="rtl">Inorganic phosphorus (mmol)</p></td></tr><tr><td><p dir="rtl">١,٥ [١,٤-</p><p dir="rtl">٢,٤]</p></td><td><p dir="rtl">١,٥ [١,٣-</p><p dir="rtl">١,٧]</p></td><td><p dir="rtl">١,٦ [١,٣-</p><p dir="rtl">٢,٠]</p></td><td><p dir="rtl">Sodium (mmol)</p></td></tr><tr><td><p dir="rtl">١,٩±٠,١#</p></td><td><p dir="rtl">٢١٣±٠١٤#</p></td><td><p dir="rtl">١,٦±٠,٣</p></td><td><p dir="rtl">Potassium (mmol)</p></td></tr>

Plasma parameters were determined 24 hours after LPS administration. Urine variables were determined between 1 and 21 h after LPS administration. Data were expressed as mean +5 and median [25th percentile 75th percentile] based on the distribution of each variable. Significant differences were estimated using the Kruskal-Wallis test with Dunns' post hoc test or one-way ANOVA with onferroni's post hoc test. Placebo LPS [= 06 n LPS+recAP=

٦٣١٣

-١٠٥-

H; Urine variables: placebo n— 05 # p&lt;5 0, 0 compared to placebo. * p&lt;5”, compared to LPS. LPS, lipopolysaccharide: recAP, gene recombination alkaline phosphatase.

Table 20. Renal gene expression levels

<tr><td></td><td colspan="3"><p dir="rtl">Ct values</p></td><td colspan="3"><p dir="rtl">Double increase (AAct82)</p></td></tr><tr><td></td><td><p dir="rtl">Placebo</p></td><td><p>LPS</p></td><td><p>LPS+</p><p>recAP</p></td><td><p dir="rtl">Placebo</p></td><td><p>LPS</p></td><td><p>LPS+</p><p>recAP</p></td></tr><tr><td><p dir="rtl">Cytokines</p></td><td></td><td colspan="2"></td><td></td><td></td><td></td></tr><tr><td></td><td><p dir="rtl">٢٧,٧±٠,٥</p></td><td><p dir="rtl">٢٦,٦±</p><p dir="rtl">٠,٢</p></td><td><p dir="rtl">٢٦,٩±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">١,١±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">١,٦±,٠</p><p dir="rtl">٥</p></td><td><p dir="rtl">7,١±٠,٥</p></td></tr><tr><td><p>IL-6</p></td><td><p dir="rtl">٣٧,٤</p><p dir="rtl">[٣٦,٣-</p><p dir="rtl">٣٨,٠]</p></td><td><p dir="rtl">٣٣,٥</p><p dir="rtl">[٣٣,٣-</p><p dir="rtl">٣٤,٨]</p></td><td><p dir="rtl">٣٤,٧</p><p dir="rtl">[٣٤,٤-</p><p dir="rtl">٣٦,٤[</p></td><td><p dir="rtl">١,٢±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">٠,٤,٩</p><p dir="rtl">٥#</p></td><td><p dir="rtl">٥,٢±١,٢#</p></td></tr><tr><td><p>IL-10</p></td><td><p dir="rtl">٣٤,٨±٠,٤</p></td><td><p dir="rtl">٣٢,٥±</p><p dir="rtl">٠,١</p></td><td><p dir="rtl">٣٢,٩±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">١,١±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">٣,٣±,٠</p><p dir="rtl">٩</p></td><td><p dir="rtl">٥,٥±١,٢#</p></td></tr><tr><td><p>TNF-</p><p>α</p></td><td><p dir="rtl">٣٦,٧±٠,٢</p></td><td><p dir="rtl">8,٣٥±</p><p dir="rtl">٠,٤</p></td><td><p dir="rtl">٢,٣7±,٠</p><p dir="rtl">٥</p></td><td><p dir="rtl">١,١±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">١,٠±,٠</p><p dir="rtl">١</p></td><td><p dir="rtl">١,٠±٠,٣</p></td></tr><tr><td><p>INF-γ</p></td><td><p dir="rtl">٣٦,٣± ٠,٣</p></td><td><p dir="rtl">٣٥,٣±</p><p dir="rtl">٠,٤</p></td><td><p dir="rtl">٣٥,٦±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">١,١±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">١,٣±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">١,٧±٠,٢</p></td></tr><tr><td><p dir="rtl">Numbers</p><p dir="rtl">injury</p></td><td></td><td></td><td></td><td></td><td></td><td></td></tr>

٦٣١٣

-١٠٦-

<tr><td><p>KIM-1</p></td><td><p dir="rtl">٣١,٧±٠,٤</p></td><td><p dir="rtl">٢٢,٩±</p><p dir="rtl">٠,٧</p></td><td><p dir="rtl">٢٥,٣±,٠</p><p dir="rtl">٦</p></td><td><p dir="rtl">٠,٨</p><p dir="rtl">[٠,٦-</p><p dir="rtl">١,٩]</p></td><td><p dir="rtl">٤٣٠</p><p dir="rtl">[١٩٥-</p><p dir="rtl">٥٣٠]#</p></td><td><p dir="rtl">١١٣ [٤٣-</p><p dir="rtl">٣٣٦]</p></td></tr><tr><td><p>NGAL</p></td><td><p dir="rtl">٢٩,١±٢,»</p></td><td><p dir="rtl">١,٤</p></td><td><p dir="rtl">٢٥,٥±,١</p><p dir="rtl">٦</p></td><td><p dir="rtl">١,٢±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">٣٣±٩#</p></td><td><p dir="rtl">٨٢٨</p></td></tr><tr><td><p>MPO</p></td><td><p dir="rtl">٣٢,٤+٠,٣</p></td><td><p dir="rtl">٠,٧</p></td><td><p dir="rtl">٣٣,٠±,١</p><p dir="rtl">٠</p></td><td><p dir="rtl">١,٠</p><p dir="rtl">[٠,٤-</p><p dir="rtl">٢,٩]</p></td><td><p dir="rtl">١,٤</p><p dir="rtl">[٠,٤-</p><p dir="rtl">٣,٩]</p></td><td><p dir="rtl">٠,٦ [٠,٣-</p><p dir="rtl">٣,٢]</p></td></tr><tr><td><p>BAX</p></td><td></td><td><p dir="rtl">٠,١</p></td><td><p dir="rtl">٢٥,١±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">٠,٩</p><p dir="rtl">[٠,٧-</p><p dir="rtl">١,٥]</p></td><td><p dir="rtl">٠,٧</p><p dir="rtl">[٠,٣-</p><p dir="rtl">٢,١]</p></td><td><p dir="rtl">١,٠ [٠,٥-</p><p dir="rtl">١,٣]</p></td></tr><tr><td><p>iNOS</p></td><td><p dir="rtl">٣٦,٢±٠,٥</p></td><td><p dir="rtl">٠,٢</p></td><td><p dir="rtl">٣٤,٨+,٠</p><p dir="rtl">٨</p></td><td><p dir="rtl">١,١±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">١,٨±,٠</p><p dir="rtl">٥</p></td><td><p dir="rtl">٢,٦±٠,٨</p></td></tr><tr><td><p dir="rtl">Adenosine receptors</p></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><p>Al</p></td><td><p dir="rtl">٢٨,٦±٠,٣</p></td><td><p dir="rtl">٢٧,٩±</p><p dir="rtl">٠,٣</p></td><td><p dir="rtl">٢٩,٣±,٠</p><p dir="rtl">٦</p></td><td><p dir="rtl">١,٣</p><p dir="rtl">[٠,٤-</p><p dir="rtl">٢,٣]</p></td><td><p dir="rtl">٠,٧</p><p dir="rtl">[٠,٥-</p><p dir="rtl">١,٤]</p></td><td><p dir="rtl">٠,٨ [٠,٣-</p><p dir="rtl">١,٥]</p></td></tr><tr><td><p>A2A</p></td><td><p dir="rtl">٢٧,٠±٠,١</p></td><td><p dir="rtl">٢٦,٤±</p><p dir="rtl">٠,٢</p></td><td><p dir="rtl">٢٧,٣±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">١,٣±,٠</p><p dir="rtl">٤</p></td><td><p dir="rtl">٠,٨±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">٠,٩±٠,٢</p></td></tr>

٦٣١٣

-١٠٧-

<tr><td><p>A2B</p></td><td><p dir="rtl">٢٩,٠</p><p dir="rtl">[٢٨,٩-</p><p dir="rtl">٢٩,٣]</p></td><td><p dir="rtl">٢٨,٤</p><p dir="rtl">[٢٨,١-</p><p dir="rtl">٢٩,٠]</p></td><td><p dir="rtl">٢٩,٠</p><p dir="rtl">[٢٨,٩-</p><p dir="rtl">٣٠,٢]</p></td><td><p dir="rtl">١,٢±,٠</p><p dir="rtl">٣</p></td><td><p dir="rtl">٠,٨±,٠</p><p dir="rtl">٢</p></td><td><p dir="rtl">٠,٨±٠,١</p></td></tr><tr><td><p>A3</p></td><td><p dir="rtl">٣٤,٣+٠,٣</p></td><td><p dir="rtl">٣٤,٢±</p><p dir="rtl">٠,٤</p></td><td><p dir="rtl">±3٥,٠,٠</p><p dir="rtl">٦</p></td><td><p dir="rtl">١,٠±,٠</p><p dir="rtl">١</p></td><td><p dir="rtl">٠,٥±,٠</p><p dir="rtl">١#</p></td><td><p dir="rtl">٠,٧+٠,١</p></td></tr>

Data were expressed as mean ± SEM, median [25th percentile

75th percentile] based on the distribution of each variable. Significant differences for twofold increase were estimated using the Kruskal-Wallis test with Dunns' post hoc test or one-way ANOVA with Bonferroni post hoc test. Placebo LPS 6; nLPS+recAP= e. # E •0.05 compared to placebo. * •0.05 compared to LPS. LPS, lipopolysaccharide? recAP, gene recombination alkaline phosphatase; MPO, myeloperoxidase BAX; myeloperoxidase, iNOS X-linked protein; Bcl2, inducible nitric oxide synthase.

References example 9

Bauerle, J.D., Grenz, Kim, A. L., Lee, H0T., and Eltzschig, 1.. 2011. 10

Adenosine generation and signaling during acute kidney injury. J Am Soc

Nephrol 22:14-20.

Chen, K.T., Malo, M.S., Moss, A.K., Zeller, 5., Johnson, P., Ebrahimi, F., Mostafa, G., Alam, S.N., Ramasamy, S., Warren, H.S., et al. 2010.

Identification of specific targets for the gut mucosal defense factor intestinal 15 alkaline phosphatase. Am for Physiol Gastrointest liver Physiol 299:(467-

475.

Di Sole, F. 2008. Adenosine and renal tubular function. Curr Opin Nephrol

Hypertens 17:399-407.

٦٣١٣

-١٠٨-

Eltzschig, H.K., Sitkovsky, ./., and Robson, 8.0. 2012. Purinergic signaling during inflammation. N Engl J Med 367:2322-2333.

Kiffer-Moreira, T., Sheen, C.R., Gasque, K.C., Bolean, Ciancaglini, P., van Elsas, A., Hoylaerts, M.F., and Millan, J.L. 2014. Catalytic identification of a heat-stable, chimeric human alkaline phosphatase with therapeutic 5 potential. PLoS One 9:689374.

Peters, E., Heemskerk, S., Masereeuw, R., and Pickkers, P. 2014. Alkaline Phosphatase: A Possible Treatment for Sepsis-Associated Acute Kidney Injury in Critically Ill Patients. Am J Kidney Dis. 63:1038-48

Schock-Kusch, D., Sadick, M., Henninger, N., Kraenzlin, 8., Claus, G., 10

Kloetzer, H.M., Weiss, C., Pill, W.L. and Gretz, 2009. No. Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats. Nephrol Dial Transplant 24:2997-3001.

Schock-Kusch, D., 2011. 15

Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves. Kidney Int 79:1254-1258.

Wilmer, M.J., Saleem, M.A., Masereeuw, R., Zella, L., van der Velden, 1. Russel, F.G., Mathieson, P.W., Monnens, L.A., van den Heuvel, LP, and 20. Levtchenko, EN 2010. Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters. Cell Tissue Res 339:449-457

٦٣١٣

-١٠٩-

Example 10

Comparison of LVL-RecAP with RecAP under different temperature conditions

»01 Materials

1 06 01 Reference standards

LVL-RecAP e

Batch No.: NB1963P1

PRA ID: 14-049

Protein content: 9.9 mg/ml (009280)

Activity: 6537 IU/mL (660 IU/mg)

“1 Storage conditions: nominal at —”7 m

Expiration date: January 8, 2015

RecAP

Batch number: 2013-052 62 lot

PRA ID: 14-311

15 Protein content: 13.3 mg/ml (00280)

Activity: 1 987 units/ml (742 units/mg)

Storage conditions: at 2-8°C

Expiration date: June 6, 2016

»01 06 2 is an empty array

٦٣١٣

-١١٠-

The following biological matrix was used to prepare sample solutions.

Matrix: serum

Species: human

Supplier: Sera Laboratories International, Haywards Heath, UK

E. Storage conditions: At nominal storage temperature — 20°C

PRA IDs: 14-0624 14-0647 and 14-0652

Expiration date: May 2, 2016 (14-0624),

May 6, 2016 (14-0647 and 14-0652)

10. 07 Roads

10 010 07 1 Preparation of solutions

<p dir="rtl">010 07 01 1 2 molar sodium hydroxide</p>

A 2 M NaOH solution was prepared by dissolving 8 g NaOH in approximately 90 mL Milli-Q water and after cooling to room temperature the volume was adjusted to 100 mL. The solution was stored at room temperature for a maximum of one month.

15 010 07 01 2 1 molar Magnesium chloride

A 1 M magnesium chloride solution was prepared by dissolving 4.06 g magnesium chloride hexahydrate.

magnesium chloride hexahydrate in approximately 16 ml of 0Milli-Q water. After dissolving, the volume was adjusted to 20 ml of liter. The solution was stored at a nominal temperature of +4°C for a maximum of one month.

20 010 07 01 3 0.1 molar Zinc chloride

٦٣١٣

-١١١-

A solution of 0.1 M zinc chloride was prepared by dissolving 272.5 mg zinc chloride in approximately 16 ml of Milli-Q water. After dissolving, the volume was adjusted to 20 ml.

The solution was stored at a nominal temperature of +4°C for a maximum of one month.

<p dir="rtl">10. 07 1. 4 Method 0.025 M glycine solution 9 pH 9.6 at 25°C method</p>

A 0.025 M glycine solution, pH 9.6, was prepared by dissolving 3.76 g of PL.

Glycine in approximately 1800 ml of Milli-Q water. The solution was warmed to 25 °C and adjusted to pH 9.6 using 2 M NaOH (see section »01 07 01 1). The volume was brought to 2000 mL and the pH was rechecked. The pH “1” should be pH 9.6 at 25°C. The solution was stored at a nominal temperature of +4°C for a maximum of one week.

<p dir="rtl">» 01 07 01 AH Method of buffer solution of an enzyme diluent at 25°C</p>

A buffer solution of an enzyme diluent was prepared by mixing 5.5 ml L 1 M magnesium chloride (see Section 01 07 01 2) with 5 ml L 1 M zinc chloride (see Section 01 07 01 15 3) and »»5 ml L 25»,»Molar glycine pH 9.6 solution (see section »01 07 01

<p dir="rtl">4). To this solution, 5 g mannitol and 25 g bovine serum albumin were added and dissolved under stirring. The pH was checked and if necessary adjusted to pH 9.6 at 25°C using 2 M NaOH. A buffer solution of the enzyme diluent is prepared fresh every day.</p>

<p dir="rtl">»2 »01 07 01 6 Method 0.0103 M p-nitrophenyl phosphate pH 9.6 at 25 degrees</p>

percentage

0.0103 molar p-Nitrophenyl phosphate was prepared. Secret number

pH 9.6 by dissolving 1528 mg p-nitrophenyl phosphate in approximately 36 mL solution.

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<p dir="rtl">0.025 M Glycine pH 9.6 (see Section 10.7.01 4). The pH was checked and if necessary adjusted to pH 9.6 at 25°C using 2 M NaOH (see Section 1 01 07 010). After checking the pH, the volume was adjusted to 0 0 4 mL using a solution of 0.025 M glycine, pH 09.6 5. The solution was kept at a nominal value of +4 C for a maximum of 5 days.</p>

<p dir="rtl">0 10 07 01 7 Substrate method Operating at 25°C</p>

The working substrate was prepared by mixing 120 ml L 0.0103 M p-Nitrophenyl phosphate solution pH 9.6 (see Section 010 07 01 6) with 1.25 ml L 1 M magnesium chloride solution (see Section 010 07 01). To this secret solution

Approximately 10 mL of 0.025 M glycine solution pH 9.6 was added (see section 010 07 01 4) and the pH was checked and, if necessary, adjusted to pH 9.6 at 25°C using 2 M hydroxide. Sodium (see section 1 01 07 010). The volume was adjusted to 5 4 1 mL using 0.025 M glycine solution pH 09.6 The working substrate was prepared fresh every day.

15 010 07 01 8 Method 0.025 M glycine solution pH 9.6 at 37°C

A 0.025 M glycine solution, pH 9.6, was prepared by dissolving 3.76 g of glycine.

Glycine in approximately 1800 mL of 0Milli-Q water. The solution was warmed to 25°C and adjusted to pH 9.6 using 2 M NaOH (see Section 101.07.010). The volume was brought to 2000 mL and the pH was rechecked. The pH should be 20. The pH should be 9.6 at 37°C. The solution was kept at a nominal value of +4 C for a maximum of one week

<p dir="rtl">0 10 07 01 9 Method of buffer solution of an enzyme diluent at 37°C</p>

A buffer solution of an enzyme diluent was prepared by mixing 0.5 ml L 1 M magnesium chloride (see Section 2 01 07 010) with 0.5 mL 0.1 M Zinc chloride (see Section 1 01 07 010

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<p dir="rtl">3) and 500 mL of 0.025 M glycine solution, pH 6.9 (see section 01.071.</p>

<p dir="rtl">8). To this solution, 5.00 g mannitol and H2, 5 g bovine serum albumin were added and dissolved under stirring. The pH was checked and if necessary adjusted to pH 9.6 at 37°C using 2 M NaOH. A buffer solution of a new enzyme diluent was prepared each day.</p>

<p dir="rtl">»01 07 01 10 Method 0.0103 M •-nitrophenyl phosphate pH 9.6 at 37°C</p>

0.0103 M p-diphenyl phosphate, pH 9.6, was prepared by dissolving 1528 p.m.

mg p—nitrophenyl phosphate in approximately »36 ml L 0.025 M glycine solution pH »1 9.6 (see section »01 07 01 8). The pH was checked and, if necessary, adjusted

to pH 9.6 at 37°C using 2 M NaOH (see section »01 07 01 1). After checking the pH the volume was adjusted to 400 mL using 0.025 M glycine pH 9.6 solution. The solution was kept at a nominal value of 4 C for a maximum of a few days.

15 »01 07 01 11 Operating substrate method at 37°C

The working substrate was prepared by mixing 12 ml L 0.0103 M p-nitrophenyl phosphate solution pH 9.6 (see section 01 07 01 1) with 10 H2 ml L 1 M chloride solution.

Magnesium (see section “01 07 01”). To this solution, approximately 1 mL/L of 0.025 M glycine solution, pH 9.6, was added (see section “01 07 01 4”) and the pH “2” was checked and, if necessary, adjusted to pH 9.6 at 37 degrees. Celsius using

2 M sodium hydroxide (see section “01 07 01 1(0) The volume was adjusted to 145 mL using H2”, “M glycine solution pH 09.6 Working substrate was prepared fresh every day0

» 01 07 2 LVL-RecAP Spike Solution (500 mcg/ml)

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The recAP spike solution was prepared by diluting 252.5 μL LVL-RecAP (Section 10.6.1) to 5.00 mL using a buffer solution of enzyme diluent (Section 10.7.1.5). The final concentration of the spike solution is 500 μg/ml This solution was used to prepare recAP spiked human serum samples (Section 10.07 4).

3 07 010 5 RecAP Spike Solution (500 µg m/mL) RecAP spike solution was prepared by diluting 188.0 µL RecAP (Section 1 06 010) to 5.00 mL with an enzyme diluent buffer solution (Section 07 010 01 5). The final concentration of the spike solution was 50 µg/ml. This solution was used to prepare RecAP spiked human serum samples (Section 010 07).

10 010 07 4 Preparation of human serum samples LVL-recAP Spiked Serum samples were prepared.

By LVL-RecAP dates into three complete individual batches of serum using the following concentration:

<tr><td><p dir="rtl">Total volume (mL)</p></td><td><p dir="rtl">Dates volume (μl)</p></td><td><p dir="rtl">Calculated activity (U/L)</p></td><td><p dir="rtl">Concentration (µg/ml)</p></td><td><p dir="rtl">Serum samples</p><p>LVL-</p><p>RecAP</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">١٠٠</p></td><td><p dir="rtl">٦٦٠٣</p></td><td><p dir="rtl">٠.١٠</p></td><td><p dir="rtl">١</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">٠٠٨٠</p></td><td><p dir="rtl">٥٢٨٢</p></td><td><p dir="rtl">٠٠.٨</p></td><td><p dir="rtl">٢</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">٠٦٠</p></td><td><p dir="rtl">٣٩٦٢</p></td><td><p dir="rtl">٠٠.٦</p></td><td><p dir="rtl">٣</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">٠٠٤٠</p></td><td><p dir="rtl">٢٦٤١</p></td><td><p dir="rtl">٤-٠٠</p></td><td><p dir="rtl">٤</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">٠.٢٠</p></td><td><p dir="rtl">١٣٢١</p></td><td><p dir="rtl">٢-٠٠</p></td><td><p dir="rtl">٥</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">٠.١٠</p></td><td><p dir="rtl">٦٦٠</p></td><td><p dir="rtl">١-٠٠</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">٠٠.٥</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">Endogenous</p></td><td><p dir="rtl">Endogenous</p></td><td><p dir="rtl">٧</p></td></tr>

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Serum samples were kept at a nominal value of -70°C until analysis.

5 07 010 Preparation of human serum samples RecAP Spiked

Serum samples were prepared by RecAP dates into three complete individual batches of serum using...

Next focus:

<tr><td><p dir="rtl">Total</p><p dir="rtl">Volume (mL)</p></td><td><p dir="rtl">Date size</p><p dir="rtl">(µL)</p></td><td><p dir="rtl">Calculated activity (U/L)</p></td><td><p dir="rtl">Concentration (µg/ml)</p></td><td><p dir="rtl">Serum samples</p><p>RecAP</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٠.٩٠</p></td><td><p dir="rtl">٦٦٨٠</p></td><td><p dir="rtl">٠٠٠٩</p></td><td><p dir="rtl">١</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٠.٧٢</p></td><td><p dir="rtl">٥٣٤٤</p></td><td><p dir="rtl">٢٠٠٧</p></td><td><p dir="rtl">٢</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٠.٥٤</p></td><td><p dir="rtl">٤٠٨٨</p></td><td><p dir="rtl">٤٠.٥</p></td><td><p dir="rtl">٣</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٠.٣٦</p></td><td><p dir="rtl">٢٦٧٢</p></td><td><p dir="rtl">٦٠.٣</p></td><td><p dir="rtl">٤</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٠.١٨</p></td><td><p dir="rtl">١٣٣٦</p></td><td><p dir="rtl">٨٠.١</p></td><td><p dir="rtl">H</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٩..٠</p></td><td><p dir="rtl">٦٦٨</p></td><td><p dir="rtl">٩٠٠٠٠</p></td><td><p dir="rtl">٦</p></td></tr><tr><td><p dir="rtl">E.00</p></td><td><p dir="rtl">٠</p></td><td><p dir="rtl">Endogenous</p></td><td><p dir="rtl">Endogenous</p></td><td><p dir="rtl">٧</p></td></tr>

5 Serum samples were stored at a nominal value of -70°C until analysis.

6 07 010 Preparation of sample solutions for enzyme activity 560-471 and RecAP

Sample solutions for enzyme activity 90-471 and RecAP were prepared by diluting the 05000-1/0 product sample or the RecAP product sample with a buffer solution of the enzyme diluent (section 01.07.010E in the 25°C method or section 9.01.07.010 in the 37°C method). ).

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Sample 1 of sample 5000-1 and/or RecAP was diluted by taking 125 µl of the LVL-RecAP and/or RecAP serum sample and diluting it to 2.50 ml with enzyme diluent buffer solution to prepare the final sample solution for LVL-RecAP enzyme activity or RecAP.

Sample 2 of LVL-RecAP and/or RecAP was diluted by taking 125 µL of LVL-RecAP and/or RecAP serum sample 5 and diluting it to 2.00 mL with enzyme diluent buffer solution to prepare the final sample solution for LVL-RecAP enzyme activity or RecAP.

Sample 3 of the LVL-RecAP and/or RecAP sample was diluted by taking 167 µL of the LVL-RecAP and/or RecAP serum sample and diluting it to 2.00 mL with enzyme diluent buffer solution to prepare the final sample solution for LVL-RecAP enzyme activity or RecAP.

10 Sample 4 of eye LVL-RecAP and/or RecAP was diluted by taking 250 µl of the LVL-RecAP and/or RecAP serum sample and diluting it to 2.00 ml with enzyme diluent buffer solution to prepare the final sample solution for LVL-RecAP enzyme activity. Or RecAP.

• The sample was diluted from the LVL-RecAP and/or RecAP sample by taking 5 0 0 µL of the LVL-RecAP and/or RecAP serum sample and diluting it to 2.00 mL with enzyme diluent 15 buffer solution to prepare the final sample solution for LVL enzyme activity -RecAP and/or RecAP.

Sample 6 of the LVL-RecAP and/or RecAP sample was diluted by taking 1,000 μL of the LVL-RecAP and/or RecAP serum sample and diluting it to 2.00 mL with enzyme diluent buffer solution to prepare the final sample solution for LVL-RecAP enzyme activity and /or RecAP.

The endogenous sample (sample 7) was diluted from the LVL-RecAP and/or RecAP sample by taking 20 1000 µL of the LVL-RecAP and/or RecAP serum sample and diluting it to 2.00 mL

With a buffer solution of enzyme diluent to prepare the final sample solution for LVL-RecAP and/or RecAP enzyme activity.

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10. 8. Implementation results and discussions

010 8. 1 Equipment and settings

The following method and settings were used:

Spectrophotometer: Thermo Fisher Evolution 300 UV/VIS with single-cell 5 Peltier heater array at 25°C (3-18-AN) or 37°C (4-18-ΑΝ) with

Magnetic stirring device

Wavelength: 5 0 4 nanomolar

Measurement: for 3 minutes, measuring every 15 seconds. The first minute is not taken into account in the calculations.

Cuvette type: glass

10 The following solutions were pipetted into a glass cuvette. The temperature of the solutions was 25°C ±

<p dir="rtl">00.5°C for 3-18-AN or 37°C ± 00.5°C for 4-18-AN.</p><table border="1"><tbody><tr><td><p dir="rtl">Empty</p></td><td><p dir="rtl">Sample solution</p></td><td><p dir="rtl">Reactive substance</p></td></tr><tr><td><p dir="rtl">1450 microliter</p></td><td><p dir="rtl">1450 microliter</p></td><td><p dir="rtl">How to operate</p></td></tr><tr><td><p dir="rtl">50.0 microliter</p></td><td></td><td><p dir="rtl">Enzyme diluent</p></td></tr><tr><td></td><td><p dir="rtl">50.0 microliter</p></td><td><p dir="rtl">Damn</p></td></tr><tr><td><p dir="rtl">1500 microliter</p></td><td><p dir="rtl">1500 microliter</p></td><td><p dir="rtl">My aesthetic size</p></td></tr></tbody></table>

،

The working substrate and enzyme diluent were first mixed before sample addition. The solution was mixed and the cuvette was immediately placed in the spectrophotometer and the increase in absorbance was measured from 1 to 3 min in steps of 15 s to obtain at least two data points. At 3 minutes (last) data point

15 The optical density was &lt;1. 5. Moreover, sin was acceptable: it had to be a relationship

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The correlation coefficient (r) for each transcript is » 00.99. All sample results with a correlation coefficient (r) &gt; 0.990 were taken into account during the evaluation. Sample results with a correlation coefficient (r) &lt; 0.990 are recorded for information only. Enzyme activity was performed in duplicate, one test per dilution.

<p dir="rtl">»01 8. 2 LVL-RecAP and RecAP enzyme activity</p>

e despite the description in the study plan that the difference in two independent results (enzyme activity) should be h e. »% To accept two individual results for dilution, all correlation coefficient (r) results for each measurement &gt; 0.990 were used in the evaluation. This is due to the method of enzyme activity of LVL-RecAP drug product samples being verified at 2°C ± 0.5°C and not to the origin of the alkaline phosphatase enzyme and/or other conditions.

»1 Figure 19 displays the relationship between the enzyme activities of RecAP in human serum at 2 °C and at 37 °C. Figure 2 shows the relationship between the enzyme activities of LVL-RecAP in human serum at 2°C and 37°C.

Figure 2 displays activity/μg protein at 2°C and 37°C for RecAP and 0LVL-RecAP. The values represent the average activity between 2°C and 37°C 15 for the given protein concentration.

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