CN116761620A

CN116761620A - Recombinant alkaline phosphatase for the treatment of acute respiratory distress syndrome

Info

Publication number: CN116761620A
Application number: CN202180061670.2A
Authority: CN
Inventors: 杰伦·埃拉萨斯沙普; 埃瑞克·简·范德伯格
Original assignee: AM Pharma BV
Current assignee: AM Pharma BV
Priority date: 2020-09-09
Filing date: 2021-09-09
Publication date: 2023-09-15

Abstract

The present disclosure relates to alkaline phosphatase, and in particular to the use of improved alkaline phosphatase, such as RecAP, for preventing, treating, curing or alleviating the symptoms of Acute Respiratory Distress Syndrome (ARDS). The present application relates to methods of preserving lung function, shortening the duration of mechanical ventilation therapy, and increasing the P/F ratio.

Description

Recombinant alkaline phosphatase for the treatment of acute respiratory distress syndrome

The application relates to the field of medicine. In particular, the present application relates to alkaline phosphatase and in particular to the use of modified alkaline phosphatase (such as RecAP) for the prevention, treatment, cure or alleviation of symptoms of Acute Respiratory Distress Syndrome (ARDS). The application also relates to methods of preserving lung function, shortening the duration of mechanical ventilation therapy, increasing the P/F ratio.

Background

Acute Respiratory Distress Syndrome (ARDS) is a pulmonary respiratory dysfunction characterized by extensive inflammation of the lungs. Symptoms include shortness of breath, and skin blushing (Fan, E; brodie, D; slutsky, AS (20.2.20.2018), "Acute Respiratory Distress Syndrome: advances in Diagnosis and Treatment". JAMA.319 (7): 698-710). ARDS has an overall poor prognosis with mortality of about 40% (Lewis, sharon R.; pratcard, michael W.; thomas, carmel M.; smith, andrew F. (7.23.2019), "Pharmacological agents for adults with acute respiratory distress syndrome". The Cochrane Database of Systematic reviews.7:CD 004477). The quality of life decline is common to those who survive ARDS (Matthay, MA; zemans, RL; zimmerman, GA; arabic, YM; beitler, JR; mercat, A; herridge, M; randolph, AG; calfee, CS (14 days 3 months 2019), "Acute respiratory distress syndrome". Nature reviews. Diseases primers.5 (1): 18).

Worldwide, ARDS affects more than 300 tens of thousands of people per year (Fan E et al (2018)). Although The term "adult respiratory distress syndrome" is sometimes used to distinguish ARDS from "infant respiratory distress syndrome" in newborns, the international consensus is that "acute respiratory distress syndrome" is The best term, as ARDS affects people of all ages (Bernard G, artigas A, brigham K, carlet J, falke K, hudson L, lamy M, legall J, morris A, spragg R (1994), "The American-European Consensus Conference on ARDS. Definitions, mechanics, relevant outcomes, and clinical trial coordination". Am J Respir Crit Care Med.149 (3 Pt 1): 818-24). There are separate diagnostic criteria for children and children in areas of less world resources (Matthay MA et al (2019)).

The annual incidence of ARDS is typically 13-23 per 100,000 of the general population. ARDS is more common in mechanically ventilated people with acute lung injury (Lewandowski K, lewandowski M (2006), "Epidemiology of ARDS". Miniva anestesiol.72 (6): 473-7). ARDS incidence increases due to COVID-19 (Guo, YR; cao, QD; hong, ZS; tan, YY; chen, SD; jin, HJ; tan, KS; wang, DY; yan, Y (13. 2020), "The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak-an update on The status". Military Medical research.7 (1): 11; solaimanzadeh, I (20. 2020), "Acetazolamide, nifedipine and Phosphodiesterase Inhibitors: rationale for Their Utilization as Adjunctive Countermeasures in The Treatment of Coronavirus Disease 2019 (COVID-19)," Cureus.12 (3): e 7343).

On a global scale, and in the absence of viral pandemics (such as covd-19), acute sepsis is the most common trigger leading to ARDS (Goldman, lee (2011). Goldman's Cecil Medicine (24 th edition). Philiadelphia: elsevier samenders. 635 page ISBN 978-1437727883). Other triggers include mechanical ventilation, pneumonia, gilles disease, drowning, circulatory shock, aspiration, trauma-especially pulmonary contusion-major surgery, massive blood transfusion (Laar, alexander p.j.; binnnekade, jan m.; prins, david, van Stein, danielle, hofsta, jorrit j.; schultz, marcus j.; juffermans, nicole p.; month 3. Risk factors and outcome of transfusion-related acute lung injury in the critically ill: a new case-control study; "Critical Care medicine 38 (3): 771-778), smoke inhalation, drug reaction or excess, fat embolism, and pulmonary edema reperfusion after pulmonary embolism or pulmonary embolism.

In addition to treatment of underlying causes (e.g., antibiotics or antiviral or anti-inflammatory drugs), ARDS is often treated in the Intensive Care Unit (ICU) with mechanical ventilation. Mechanical ventilation is typically delivered through a hard tube (endotracheal tube) that enters the oral cavity and is secured in the airway, or through a tracheostomy when prolonged ventilation (2 weeks or more) is required. The effect of non-invasive ventilation is limited to the very early stages of the disease, or to the prevention of exacerbation of respiratory distress in atypical pneumonia, individuals with bruising of the lungs or patients with major surgery at risk of developing ARDS. No particular ventilator pattern is known to improve the mortality of ARDS, which is between 35% -50% (Fan E et al (2018)). Thus, there is a need for methods of treating ARDS.

SUMMARY

The present disclosure provides a method of treating Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof comprising administering to the subject an effective amount of Alkaline Phosphatase (AP). In a preferred embodiment, the AP is administered in at least one dose of 500U/kg to 2,000U/kg.

The severity of ARDS is generally classified as "mild", "moderate" and "severe" ARDS. Mild, moderate and severe ARDS are defined herein by the following criteria:

the following PaO ₂ /FiO ₂ Ratio reduction (PaO) ₂ /FiO ₂ The ratio is the arterial oxygen partial pressure (PaO) ₂ Expressed in mmHg) and the Fraction of Inhaled Oxygen (FiO) ₂ Expressed as a fraction, rather than a percentage), and also referred to as a P/F ratio):

- >200 and ≡300mmHg (> 26.66kPa and ≡40.00 kPa): mild ARDS;

- >100 and ≡200mmHg (> 13.33 and ≡26.66 kPa): moderate ARDS;

-.ltoreq.100 mmHg (. Ltoreq.13.33 kPa): severe ARDS;

wherein the P/F ratio is 5cmH ₂ A minimum Positive End Expiratory Pressure (PEEP) measurement of O.

In some aspects, the subject suffers from severe or moderate ARDS according to the definition above (i.e., using 5 cmH) ₂ The P/F ratio of the minimum PEEP of O is less than or equal to 200 mmHg). In some aspects, the subject suffers from severe ARDS (i.e., using 5 cmH) ₂ The P/F ratio of the minimum PEEP of O is less than or equal to 100 mmHg). In some aspects, the subject suffers from moderate ARDS (i.e., using 5cmH prior to treatment with AP ₂ The P/F ratio of the minimum PEEP of O is 100-200 mmHg). In some aspects, ARDS is associated with and/or caused by sepsis. In other aspects, the ARDS is associated with and/or infected by a viral infection, preferably a coronavirus infection, more preferably a Severe Acute Respiratory Syndrome (SARS) associated coronavirus infectionRespiratory Syndrome (SARS) associated coronavirus infection. In some aspects, the AP is a human AP. In some aspects, the AP is a recombinant AP. In some aspects, the recombinant AP is chimeric. In some aspects, the chimeric AP has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to RecAP (SEQ ID NO: 1).

In some aspects, the increase in respiratory function comprises an increase in the P/F ratio of the subject relative to the P/F ratio of the subject without treatment. In some aspects, sepsis in the subject is detected less than 96 hours prior to AP administration. In some aspects, sepsis is detected less than 72 hours prior to ARDS detection.

In some aspects, the treatment according to the invention is initiated within 24 hours after detecting sepsis in the subject. In some aspects, treatment according to the invention is initiated within 24 hours after detection of ARDS in the subject. In some aspects, the AP is administered once daily. In some aspects, the AP is administered intravenously. In some aspects, P is administered a at a dose of three times per day. In some aspects, the dosage of the AP is 0.8mg/kg or 1.6mg/kg RecAP (e.g., clinical grade RecAP for use in the present disclosure). In some aspects, the dosage of the AP is 500U/kg or 1000U/kg RecAP (e.g., clinical grade RecAP for use in the present disclosure).

In some aspects, the administration of at least one dose of AP shortens or stops the duration of mechanical ventilation therapy in a subject undergoing mechanical ventilation therapy. In some aspects, at least one dose of AP administration results in a maintenance or increase in the P/F ratio of the subject.

Brief Description of Drawings

Fig. 1 is a table showing the proposed RecAP dose group.

FIG. 2 shows the amino acid sequence of RecAP (SEQ ID NO: 1).

FIG. 3 shows a timeline of STOP-AKI studies, which includes determining AUC _1-7 RRT incidence and mortality, and dose regimen and time frame of MAKE.

Figure 4 is a flow chart showing patient entry, randomization and follow-up in the trial. Abbreviations: AKI (acute kidney injury), AUC (area under time corrected curve), ECC (endogenous creatinine clearance), ICF (informed consent), ITT (intent to treat), IV (intravenous), MAKE (major renal adverse events), NS (unspecified), recAP (human recombinant alkaline phosphatase with sequence SEQ ID NO: 1), RRT (renal replacement therapy), sA-AKI (sepsis-related acute kidney injury), SCr (serum creatinine).

Fig. 5 is a table showing the demographics and baseline characteristics of all groups in the trial, indicating that there were no significant differences in any baseline characteristics between groups. For variables lacking data, the data is summarized based on the adjusted numbers. Body Mass Index (BMI) is the weight in kilograms divided by the square of the height in meters. Acute physiological and chronic health assessment II (APACHE II) scores ranged from 0 to 71, with higher scores indicating higher disease severity. Sequential Organ Failure Assessment (SOFA) scores ranged from 0-24, with higher scores indicating more severe dysfunction. The Simplified Acute Physiology Score (SAPS) ranged from 0 to 163, with higher overall scores indicating higher disease severity. The estimated glomerular filtration rate (eGFR) was calculated according to the equation of CKD-EPI (chronic kidney disease epidemiological co-organization): 141 x min (Scr/κ, 1) α x max (Scr/κ, 1) -1.209 x 0.993 age 1.018[ if female ] x1.159 [ if black). Sca is serum creatinine (mg/dL), k is 0.7 in women and 0.9 in men, α is-0.329 in women and-0.111 in men, min represents the minimum or 1 of Sca/k, and max represents the maximum or 1 of Sca/k. (www.kidney.org/content/ckd-epi-creatine-evaluation-2009). Acute Kidney Injury (AKI) phase is graded according to the AKI network definition (www.akinet.org/akinstruments. Php). Median and quartile values, or numbers and percentages, are depicted.

Fig. 6 is a graph showing AUC versus dose for a linear increase in RecAP concentration. Patient exposure in STOP-AKI clinical trials was slightly higher than in healthy subjects. Dose linearity and proportionality were observed over the dose range (0.4-1.6 mg/kg).

FIG. 7 is a table showing primary and secondary endpoints (placebo and RecAP 0.6mg/kg group). AUC (AUC) _1–7 ECC curves from day 1 to day 7 are shownThe lower area is divided by 7 to provide time corrected clearance in ml/min. Abbreviations: CI (confidence interval), CKD-EPI (chronic kidney disease epidemiological co-organization), ECC (endogenous creatinine clearance), eGFR (estimated glomerular filtration rate), ICU (intensive care unit), IQR (quartile range), MAKE (major renal adverse events), RRT (renal replacement therapy), SOFA (sequential organ failure assessment).

RRT occurrence represents the percentage of patients requiring RRT after randomization.

And ii with the advance of the last observation.

* MAKE 28: RRT was received before or at day 28, or die before or at day 28. A ratio meeting at least one criterion.

MAKE 60: on day 60, eGFR was estimated using CKD-EPI equation based on serum creatinine<A chronic RRT is required at 60ml/min, either before or at day 60, or die before or at day 60. A ratio meeting at least one criterion.

MAKE 90:60: on day 90, eGFR was estimated using CKD-EPI equation based on serum creatinine<A chronic RRT is required at 60ml/min, or before or at day 90, or hospitalized for new AKI before or at day 90, or die before or at day 90. A ratio meeting at least one criterion.

And.

Representing the odds ratio (for the classification variable).

Representing the hazard ratio (for the event).

Fig. 8 shows the primary and secondary endpoints for all treatment groups (RecAP at 0.4.mg/kg, 0.8mg/kg or 1.6mg/kg dose, or placebo).

Fig. 9A shows quality of life (QoL) assessment measured by EuroQoL (EQ 5D) score at ICU (left panel) and day 90 (right panel).

FIG. 9B shows lung function parameters (P/F ratio, tidal volume, and PEEP) in the 1.6mg/kg RecAP dose group and placebo group, expressed as median. The values are displayed below the graph.

FIG. 9C shows liver function parameters (alanine aminotransferase and aspartate aminotransferase activity), expressed as median, for the 1.6mg/kg RecAP dose group and placebo group. The values are displayed below the graph.

Fig. 10 shows the results of the post hoc multivariate analysis.

Fig. 11 shows the observed treatment emergency adverse events during STOP-AKI studies.

FIG. 12 shows the P/F ratio of STOP-AKI in a population of subjects with measurable screening or day 1 values, randomized to placebo or high dose recAP, up to and including day 29. The numbers represent the number of patients per treatment group at that particular time point. The average is shown with a 95-% confidence interval (CL) of bootstrap. The time of each treatment group was slightly shifted to improve the discrimination of CL.

FIG. 13 shows the fold change in P/F ratio of STOP-AKI in a population of subjects with measurable screening or day 1 values, randomized into placebo or high dose recAP, up to and including day 29. The numbers represent the number of patients per treatment group at that particular time point. The average is shown with the 95-% confidence interval of bootstrap. The time of each treatment group was slightly shifted to improve the discrimination of CL.

FIG. 14 shows the fold change in P/F ratio of STOP-AKI in a population of subjects with measurable screening or day 1 values, randomized into placebo or high dose recAP, up to and including day 29. The numbers represent the number of patients per treatment group at that particular time point. The average is shown with the 95-% confidence interval of bootstrap. The time of each treatment group was slightly shifted to improve the discrimination of CL. The mean basis value data according to the packet ARDS standard are shown in the different figures.

FIG. 15 shows survival of STOP-AKI patients randomized to placebo or high dose recAP up to and including day 29. The percentage of surviving patients in the placebo or high dose AP group for severe to moderate ARDS patients at baseline relative to day 0 is depicted. On day 0, the number of patients in the placebo group was 40 and the high dose recap group was 42.

Detailed description of the preferred embodiments

It is an object of the present disclosure to provide methods for preserving or improving respiratory function in a subject, especially in cases where the subject has or is at risk of acute respiratory syndrome (ARDS). It is another object of the present disclosure to prevent or shorten the duration of mechanical ventilation therapy to maintain or increase the P/F ratio of a subject, and/or to increase survival.

Accordingly, the present disclosure relates to the use of Alkaline Phosphatase (AP) (e.g., recAP) to preserve or improve respiratory function in subjects suffering from or at risk of ARDS. The present disclosure provides, for example, methods for treating a subject with severe or moderate ARDS, comprising administering an AP (e.g., recAP) to the subject. In some aspects, each dose of AP administered to a subject comprises at least 500U/kg (0.8 mg/kg in the case of clinical grade RecAP). In some particular aspects, the dosage of AP per dose is at least 1,000U/g (1.6 mg/kg in the case of clinical grade RecAP). In some aspects, the ARDS is sepsis-related ARDS. In other aspects, the ARDS is caused or exacerbated by a viral infection, preferably by a coronavirus or an orthomyxovirus infection, more preferably by a Severe Acute Respiratory Syndrome (SARS) -associated coronavirus infection such as, for example, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, which is a strain of coronavirus (covd-19) causing a respiratory disease that is a pandemic in year 2020 covd-19), in the case of an orthomyxovirus infection, the infection is preferably caused by an influenza virus, such as influenza a, b, c or d, more preferably influenza a or b.

RecAP is a chimeric AP that combines the properties of two human isozymes, namely intestinal and placental AP (Kiffer-Moreira et al, PLoS One 2014; 9:e89374). Substitution of the coronary domain of intestinal AP (the most biologically active isoenzyme of both) with the coronary domain of placental AP (which has the longest half-life) resulted in a highly stable biologically active enzyme (Kiffer-Moreira et al, PLoS One 2014; 9:e89374). See, for example, U.S. patent application publications US20170009216 and US20160250299, the disclosures of which are incorporated herein by reference in their entirety.

In some aspects, if an ARDS is present and the ARDS has been determined to be moderate or severe, an AP (e.g., recAP) is administered to the subject. Thus, in some aspects of the disclosure, the methods disclosed herein include measuring the severity of ARDS (e.g., by determining the P/F ratio) prior to treatment with an AP to determine whether a subject has moderate ARDS or severe ARDS.

Diagnostic criteria for ARDS are constantly changing as the understanding of pathophysiology is enhanced. The international consensus standard for ARDS was recently updated in 2012 and is referred to AS "2012 Berlin definition (2012 Berlin definition)" (rannieri VM, rubenfeld GD, thompson BT, ferguson ND, calwell E, fan E, camporota L, sletsky AS (6. 2012), "Acute respiratory distress syndrome: the Berlin definition. ARDS Definition Task Force". Jama.307 (23): 2526-33;Ferguson ND,Fan E,Camporota L,Antonelli M,Anzueto A,Beale R,Brochard L,Brower R,Esteban A, et al, (10. 2012), "The Berlin definition of ARDS: an expanded rationale, just identification, and supplementary material". Interactive Care med.38 (10): 1573-82). In addition to The overall enlarged diagnostic threshold, other significant changes compared to The previous 1994 consensus standard (Bernard G, artigas A, brigham K, carlet J, falke K, hudson L, lamy M, legall J, morris A, spragg R (1994), "The American-European Consensus Conference on ARDS. Definitions, mechanics, relevant outcomes, and clinical trial coordination', am J Respir Crit Care Med.149 (3 Pt 1): 818-24) include discouraging use of The term" acute lung injury "and define ARDS severity levels as a function of The reduction in oxygen content in blood.

According to the definition of berlin in 2012, the characteristics of adult ARDS are as follows:

acute onset lung injury, within 1 week of overt clinical injury, accompanied by progression of respiratory symptoms;

bilateral shadows on chest images (chest radiographs or CT) cannot be explained by other lung pathologies (such as effusion, lobe/lung collapse or nodules);

respiratory failure cannot be explained by heart failure or excessive volume;

-PaO ₂ /FiO ₂ ratio reduction (PaO) ₂ /FiO ₂ Ratio reduction indicates reduced arterial oxygenation from available inhaled gas):

mild ARDS: 200 and 300mmHg (> 26.66kPa and 40.00 kPa);

moderate ARDS: 100 and 200mmHg or less (> 13.33 and 26.66kPa or less);

severe ARDS: less than or equal to 100mmHg (less than or equal to 13.33 kPa);

considering PaO ₂ /FiO ₂ Ratio, berlin definition requires 5cmH ₂ Minimum Positive End Expiratory Pressure (PEEP) of O. This degree of PEEP can be delivered non-invasively using Continuous Positive Airway Pressure (CPAP) to diagnose mild ARDS.

In this regard, it is noted that ARDS and Acute Lung Injury (ALI) as used herein may be used interchangeably within the limits defined above, i.e., using other criteria defined by P/F limits and 2012 Berlin to distinguish between mild, moderate and severe disease, as known in the art (Hernu, R., wall, F., thiollire, F., et al, an attempt to validate the modification of the American-European consensus definition of acute lung injury/acute respiratory distress syndrome by the Berlin definition in a university home Care Med39,2161-2170 (2013)).

In some aspects of the disclosure, ARDS is classified as severe if the baseline P/F ratio is less than or equal to 100 mmHg. In some aspects, ARDS is classified as moderate if the baseline P/F ratio is >100 and 200 mmHg. In other aspects, other diagnostic measurements may be used to determine the severity of ARDS. In some aspects of the disclosure, if ARDS is determined to be mild or absent (e.g., if the baseline P/F ratio >200 mmHg), no AP is administered to the subject.

In some aspects, the AP (e.g., recAP) is administered prophylactically. In some aspects, the methods disclosed herein comprise administering at least one dose of AP (e.g., recAP) to a subject, wherein each dose is at least 500U/kg (0.8 mg/kg for the clinical grade RecAP used) or 1000U/kg (1.6 mg/kg for the clinical grade RecAP used). In some specific aspects, the dosage regimen comprises administering from 0.5mg/kg to 2mg/kg of AP (e.g., recAP) at a daily dose by intravenous infusion for at least three days. In some specific aspects, the dosage regimen comprises administering from 0.5mg/kg to 2mg/kg of AP (e.g., recAP) at a daily dose by intravenous infusion for at least three days.

For easier understanding of the present disclosure, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

I. Definition of the definition

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "a/an" and the terms "one or more" and "at least one" are used interchangeably herein.

Furthermore, as used herein, "and/or" is to be taken as a specific disclosure of each of two particular features or components, whether or not the other features or components are present. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include "a and B", "a or B", (only) "a", and (only) "B". Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; (only) A; (only) B; and (only) C.

Where aspects are described herein using the language "comprising," other similar aspects described in terms of "consisting of … (comprising of)", and/or "consisting essentially of … (consisting essentially of)" are also provided.

The term "about" as used in connection with a numerical value throughout the specification and claims means an interval of accuracy familiar to and acceptable to those skilled in the art. Typically, this accuracy interval is + -15%.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Units, prefixes, and symbols are expressed in terms of their international system of units (SI) acceptance.

Numerical ranges include numbers defining the range. Where a range of values is recited, it is understood that each intermediate integer value and each fraction thereof between the recited upper and lower limits of the range, and each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may independently be included in or excluded from the range, and each range where either, neither, or both limits are included in the invention is also encompassed within the invention. If the lower limit value is preceded by ">", the lower limit of the range will be excluded from the range. If the upper limit value is preceded by a "<", the upper limit of the range will be excluded from the range. If the lower limit value is preceded by ". Gtoreq," the lower limit of the range is included in the range. If the upper limit value is preceded by "+.ltoreq", then the upper limit of the range is included in the range. If a value is explicitly recited, it is to be understood that values in approximately the same amount or quantity as the recited value are also within the scope of the present invention. If a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the invention. Conversely, if a different element or group of elements is disclosed separately, then a combination thereof is also disclosed. Where any element of the present invention is disclosed as having multiple alternatives, examples of the invention are also disclosed herein, wherein each alternative is excluded alone or in any combination with the other alternatives; more than one element of the present invention may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

As used herein, the term "treatment" or "treatment of …" refers to (i) reducing the potential or risk of a disease or disorder (e.g., ARDS), (ii) reducing the occurrence of a disease or disorder (e.g., ARDS), (iii) reducing the severity of a disease or disorder (e.g., ARDS) (e.g., alleviating symptoms), or (iv) combinations thereof.

For example, treatment may refer to the ability of a therapy to prevent or reduce the risk of occurrence of lung injury caused by or associated with ARDS, and/or to cure or alleviate symptoms, signs, or etiologies of lung injury (e.g., ARDS) when administered to a subject. Treatment also refers to alleviation or diminishment of at least one clinical symptom and/or inhibition or delay of progression of the condition, and/or prevention or delay of onset of the disease or condition, when compared to untreated (or placebo-treated) groups. Thus, the term "treatment" or "treatment of …" (or grammatically equivalent terms) refers to a therapeutic treatment regimen of ARDS that may prevent (further) pulmonary damage caused by or associated with ARDS. In the case of respiratory disease, it is preferred that the subject has an improved or reduced respiratory function after treatment with AP by the methods disclosed herein relative to a subject not treated with the AP.

As used herein, the term "preserving" includes preventing, slowing, stopping and/or at least partially reversing the reduction of lung function. The term "increase" is not necessarily limited to increasing the respiratory function to a value equal to or higher than before the treatment occurs. It includes partial restoration of respiratory function.

The term "subject" or "patient" as used herein refers to any subject, particularly a mammalian subject, in need of treatment or prognosis of lung injury (e.g., ARDS). As used herein, the term "subject" or "patient" includes any human or non-human animal. As used herein, phrases such as "patient with ARDS" or "patient with sepsis" include subjects (e.g., mammalian subjects) that would benefit from administration of AP therapy as disclosed herein.

In some aspects of the disclosure, the object is an object that has not been experienced. An unresubject refers to a subject that is not administered a therapy (e.g., a therapeutic agent). In some aspects, the subject who has not experienced is not treated with the therapeutic agent until it is diagnosed with a lung injury (e.g., ARDS) or a disease or condition that may cause lung injury (e.g., sepsis).

In another aspect, the subject is treated and/or one or more doses of the therapeutic agent prior to being diagnosed with a lung injury (e.g., ARDS) or a disease or condition that may cause lung injury and/or ARDS (e.g., sepsis).

In some aspects, if the baseline P/F ratio of the subject is below a predetermined P/F ratio threshold level, or if the baseline P/F ratio is within a predetermined range, at least one therapeutically effective dose of AP (e.g., recAP) may be administered to the subject.

The terms "therapeutic agent" and "drug" as used herein also refer to any therapeutically active substance administered to a subject suffering from a disease or disorder, such as a lung injury, e.g., ARDS, or a disease or condition that may cause lung injury, e.g., sepsis, to produce a desired, generally beneficial effect. The therapeutic agent may also be a prodrug that is metabolized to the desired therapeutically active substance when administered to a subject. In some aspects, the therapeutic agent is a prophylactic agent. In addition, the therapeutic agent may be formulated pharmaceutically. The therapeutic agent may also be or comprise a radioisotope or agent that is activated by some other form of energy (e.g., light or ultrasonic energy), or by other systemically administrable circulating molecules.

In some aspects of the disclosure, a therapeutic agent for treating, preventing, or alleviating symptoms of a disease or condition (e.g., sepsis) that is or may cause lung injury (e.g., ARDS) may comprise AP (e.g., recAP); alone or in combination with one or more standard therapeutic agents commonly used to treat lung injury (e.g., ARDS) or diseases or conditions that may cause lung injury (e.g., sepsis).

As used herein, a "therapeutically effective" amount is an amount of a therapeutic agent that provides some improvement or benefit to a subject suffering from a disease or disorder, such as a lung injury, e.g., ARDS, or a disease or condition that may cause lung injury, e.g., sepsis. Thus, a "therapeutically effective" amount refers to an amount that provides some alleviation, alleviation and/or reduction of at least one clinical symptom of a disease or disorder, such as a lung injury, e.g., ARDS, or a disease or condition that may cause a lung injury, e.g., sepsis.

Clinical symptoms associated with lung injury (e.g., ARDS) or diseases or conditions that may cause lung injury (e.g., sepsis) that may be treated by the compositions, methods, specific dosage regimens of the present disclosure are well known to those of skill in the art. Furthermore, those skilled in the art will recognize that the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject. In some aspects, the term "therapeutically effective" refers to an amount of a therapeutic agent that is capable of altering the level of a biomarker (e.g., the P/F ratio in a patient in need thereof).

As used herein, a "sufficient amount" or "sufficient amount" to achieve a particular result in a patient suffering from a disease or disorder (e.g., a lung injury, such as ARDS, or a disease or condition that may cause a lung injury (e.g., sepsis)) refers to an amount of a therapeutic agent (e.g., an AP, such as RecAP) effective to produce a desired effect, which is optionally a therapeutic effect (i.e., by administering a therapeutically effective amount). In some aspects, such a particular outcome is an improvement in lung function.

As used herein, the term "healthcare provider" refers to a person or institution that directly interacts with and manages living subjects (e.g., human patients). Non-limiting examples of healthcare providers include doctors, nurses, technicians, therapists, pharmacists, consultants, alternative medical practitioners, medical facilities, doctor offices, hospitals, emergency rooms, clinics, emergency care centers, alternative medical clinics/facilities, and any other entity that provides general and/or specialized treatments, evaluations, maintenance, therapies, medication and/or advice associated with all or any portion of a patient's health condition, including but not limited to general medical, specialty medical, surgical, and/or any other type of treatment, evaluation, maintenance, therapy, medication, and/or advice.

As used herein, the term "clinical laboratory" refers to a facility for examining or processing substances derived from living subjects (e.g., humans). Non-limiting examples of treatment include biological, biochemical, serological, chemical, immunohematological, hematological, biophysical, cytological, pathological, genetic, or other examination of substances derived from the human body for the purpose of providing information, for example, for diagnosis, prevention or treatment of any disease or injury, or for health assessment of a living subject (e.g., a human). These examinations may also include procedures to collect or otherwise obtain a sample, prepare, determine, measure, or otherwise describe the presence or absence of various substances in a body of a living subject (e.g., a human), or a sample obtained from the body of a living subject (e.g., a human).

As used herein, the term "healthcare benefit provider" includes an individual party, organization, or community that provides, presents, gives, pays all or part of a fee, or is otherwise associated with a patient obtaining one or more healthcare benefits, benefit plans, medical insurance, and/or healthcare fee account plans.

In some aspects, a healthcare provider may administer a treatment to prevent, treat, or alleviate symptoms of a disease or disorder (e.g., a lung injury, such as ARDS, or a disease or condition that may cause lung injury (e.g., sepsis)) or instruct another healthcare provider. The healthcare provider may perform or instruct other healthcare providers or patients to perform the following actions: the method includes the steps of obtaining a sample, processing the sample, submitting the sample, receiving the sample, transferring the sample, analyzing or measuring the sample, quantifying the sample, providing a result obtained after analyzing/measuring/quantifying the sample, receiving a result obtained after analyzing/measuring/quantifying the sample, comparing/scoring a result obtained after analyzing/measuring/quantifying one or more samples, providing a comparison/score from one or more samples, obtaining a comparison/score from one or more samples, administering a treatment (e.g., AP, such as RecAP), starting administration of the treatment, stopping administration of the treatment, continuing administration of the treatment, temporarily interrupting administration of the treatment, increasing the amount of the therapeutic agent administered, decreasing the amount of the therapeutic agent administered, continuing administration of an amount of the therapeutic agent, increasing the frequency of administration of the therapeutic agent, decreasing the frequency of administration of the therapeutic agent, maintaining the same dosing frequency of the therapeutic agent, replacing the therapy or therapeutic agent by at least one other therapy or therapeutic agent, combining the therapy or therapeutic agent with at least one other therapeutic method or additional therapeutic agent.

In some aspects, the healthcare benefit provider may authorize or reject, for example, collect a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide a result obtained after analyzing/measuring/quantifying a sample, transfer a result obtained after analyzing/measuring/quantifying a sample, compare/score a result obtained after analyzing/measuring/quantifying one or more samples, transfer a comparison/score from one or more samples, administer a treatment or therapeutic agent, begin administration of a treatment or therapeutic agent, stop administration of a treatment or therapeutic agent, continue administration of a treatment or therapeutic agent, temporarily discontinue administration of a treatment or therapeutic agent, increase the amount of a therapeutic agent administered, decrease the amount of a therapeutic agent administered, continue administration of a certain amount of a therapeutic agent, increase the frequency of administration of a therapeutic agent, decrease the frequency of administration of a therapeutic agent, maintain the same frequency of administration of a therapeutic agent, replace a treatment or therapeutic agent by at least one other treatment or therapeutic agent, or combine a treatment or therapeutic agent with at least one other treatment or additional therapeutic agent.

In addition, the healthcare benefit provider may, for example, authorize or reject a therapy prescription, authorize or reject therapy coverage, authorize or reject therapy fee reimbursement, determine or reject therapy eligibility, and the like.

In some aspects, a clinical laboratory may, for example, collect or obtain a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide a result obtained after analyzing/measuring/quantifying a sample, receive a result obtained after analyzing/measuring/quantifying a sample, compare/score a result obtained after analyzing/measuring/quantifying one or more samples, provide a comparison/score from one or more samples, obtain a comparison/score from one or more samples, or other related activities.

Treatment of ARDS with AP

In certain aspects, the disclosure relates to methods for preserving or improving lung function in a population of subjects (particularly ARDS patients) that have been determined to respond particularly well to AP therapy.

Analysis of data from the STOP-AKI study recently described in Pickkers et al (Pickkers P, mehta RL, murray PT, et al, effect of Human Recombinant Alkaline Phosphatase on 7-Day Creatinine Clearance in Patients With Sepsis-Associated Acute Kidney Injury: ARandomized Clinical Trial. JAMA.2018;320 (19): 1998-2009) has established a statistically significant correlation between thresholds corresponding to ARDS severity and pulmonary function improvement (as evidenced by improvement in P/F ratio relative to placebo). In STOP-AKI clinical trials, patients were graded according to markers, e.g., lung function, and in particular P/F ratios, surprisingly identified the specific effect of AP administration on a particular subset. One or more parameters defining each of these subgroups (e.g., a series of thresholds, such as P/F ratio thresholds) may be used, for example, to personalize AP therapy to a particular subgroup, to select patients for treatment, to make decisions related to AP treatment (e.g., modify dosing or dosage plans), or to evaluate the likelihood of a positive outcome.

Thus, in some aspects, the methods disclosed herein relate to administering an AP (e.g., recAP) to a subject determined to have a moderate or severe ARDS, including administering an AP (e.g., recAP) to the subject. The skilled person is familiar with how to classify the ARDS severity of a subject as moderate or severe. As described above, berlin 2012 defines the classification of moderate ARDS as having>P/F of 100 and 200mmHg or less, and severe ARDS are classified as having P/F of 100mmHg or less (13.33 kPa or less). Thus, persons with moderate or severe ARDS have a P/F ratio of 200mmHg or less, and the Berlin definition also requires a minimum Positive End Expiratory Pressure (PEEP) of 5cmH when the P/F ratio is considered ₂ O. In a preferred embodiment, therefore, the present invention provides Alkaline Phosphatase (AP) for use in a method of treating Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof, wherein the subject has moderate or severe ARDS, if 5cmH is used ₂ The P/F ratio is measured by the minimum Positive End Expiratory Pressure (PEEP) of O, and the P/F ratio is less than or equal to 200mmHg. When administered to subjects suffering from moderate to severe ARDS at a dose of at least 500U/kg,AP administration is particularly effective. AP is particularly effective when administered at a dose of at least 1,000U/kg.

In some aspects, the AP (e.g., recAP) is administered at the following doses: at least about 500U/kg, at least about 600U/kg, at least about 700U/kg, at least about 800U/kg, at least about 900U/kg, at least about 1000U/kg, at least about 1100U/kg, at least about 1200U/kg, at least about 1300U/kg, at least about 1400U/kg, at least about 1500U/kg, at least about 1600U/kg, at least about 1700U/kg, at least about 1800U/kg, at least about 1900U/kg, or at least about 2000U/kg per dose. In some aspects, the AP (e.g., recAP) is administered at a dose in excess of 2000U/kg per dose. In some aspects, the AP (e.g., recAP) is administered at a dose of less than 500U/kg per dose.

In some aspects, the AP (e.g., recAP) is administered at the following doses: about 500U/kg to about 1500U/kg, about 600U/kg to about 1400U/kg, about 700U/kg to about 1300U/kg, about 800U/kg to about 1200U/kg, or about 900U/kg to about 1100U/kg. In some specific aspects, the AP is administered at a dose of about 1000U/kg.

In some aspects, the AP is a human AP. In some aspects, the AP is a recombinant AP. In some aspects, the AP is a chimeric AP. In a particular aspect, the chimeric AP is RecAP (SEQ ID NO: 1). In some aspects, an AP disclosed herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO. 1. In some aspects, an AP is a functional fragment (i.e., a fragment of an AP, e.g., an AP that retains at least about 10%, at least about 20%, at least about 30%, at least 40%, at least about 50%, at least about 60%, at least 70%, at least about 80%, or at least about 90% of the AP activity of the corresponding full-length AP). In some aspects, the AP is a variant or derivative of the AP disclosed herein. Other APs that may be used as disclosed herein are discussed in detail below.

In some aspects, the AP is a RecAP (e.g., clinical grade RecAP as used in the present disclosure), and it is administered at the following doses: at least about 0.1mg/kg, at least about 0.2mg/kg, at least about 0.3mg/kg, at least about 0.4mg/kg, at least about 0.5mg/kg, at least about 0.6mg/kg, at least about 0.7mg/kg, at least about 0.8mg/kg, at least about 0.9mg/kg, at least about 1mg/kg, at least about 1.1mg/kg, at least about 1.3mg/kg, at least about 1.4mg/kg, at least about 1.5mg/kg, at least about 1.6mg/kg, at least about 1.7mg/kg, at least about 1.8mg/kg, at least about 1.9mg/kg, at least about 2mg/kg, at least about 2.1mg/kg, at least about 2.2mg/kg, at least about 2.3mg/kg, or at least about 2.4mg/kg per dose. In some aspects, the AP is administered at a dose in excess of 2.4mg/kg per dose. In some aspects, the AP is a RecAP (e.g., clinical grade RecAP as used in the present disclosure), and it is administered at the following doses: at least about 100U/kg, at least about 200U/kg, at least about 300U/kg, at least about 400U/kg, at least about 500U/kg, at least about 600U/kg, at least about 700U/kg, at least about 800U/kg, at least about 900U/kg, at least about 1000U/kg, at least about 1100U/kg, at least about 1200U/kg, at least about 1300U/kg, at least about 1400U/kg, at least about 1500U/kg, at least about 1600U/kg, at least about 1700U/kg, at least about 1800U/kg, at least about 1900U/kg, or at least about 2000U/kg. AP is RecAP and it is administered at a dose below 100U/kg. AP is RecAP and it is administered at a dose exceeding 2000U/kg.

In some aspects, the AP is a RecAP (e.g., clinical grade RecAP as used in the present disclosure), and it is administered at the following doses: about 0.8mg/kg to about 2.4mg/kg, about 0.9mg/kg to about 2.3mg/kg, about 1mg/kg to about 2.2mg/kg, about 1.1mg/kg to about 2.1mg/kg, about 1.2mg/kg to about 2mg/kg, about 1.3mg/kg to about 1.9mg/kg, about 1.4mg/kg to about 1.8mg/kg, or about 1.5mg/kg to about 1.7mg/kg. In some specific aspects, the AP is administered at a dose of about 1.6 mg/kg.

In some aspects, the AP is a RecAP (e.g., a clinical grade RecAP as used in the present disclosure), and it has the following specific activities: at least about 100U/mg, at least about 200U/mg, at least about 300U/mg, at least about 400U/mg, at least about 500U/mg, at least about 600U/mg, at least about 700U/mg, at least about 800U/mg, at least about 900U/mg, at least about 1000U/mg, at least about 1100U/mg, at least about 1200U/mg, at least about 1300U/mg, at least about 1400U/mg, at least about 1500U/mg, at least about 1600U/mg, at least about 1700U/mg, at least about 1800U/mg, at least about 1900U/mg, or at least about 2000U/mg.

In some aspects, the AP is a RecAP (e.g., a clinical grade RecAP as used in the present disclosure), and it has a specific activity of about 1000u:1.6 mg. In some aspects, the AP is RecAP, and it has the following specific activities: about 600U/mg to about 700U/mg, or about 500U/mg to about 800U/mg, or about 400U/mg to about 900U/mg, or about 300U/mg to about 1000U/mg, or about 200U/mg to about 1100U/mg, or 100U/mg to about 1200U/mg. In some aspects, the AP is RecAP and has a specific activity of less than 100U/mg. In some aspects, the AP is RecAP and has a specific activity of greater than 1200U/mg.

In some aspects, only one dose of AP (e.g., recAP) is administered per treatment (e.g., one dose per day for 1-7 days). In other aspects, more than one dose of AP is administered. In some aspects, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 doses of AP are administered (e.g., at least two doses per day for 1-7 days).

In some aspects, the AP dose is administered daily. In other aspects, the AP dose is administered every 2, 3, 4, 5, 6, or 7 days.

In some aspects, a single dose is administered daily. In some aspects, 2, 3 or more doses are administered daily.

In some aspects, treatment with AP is for less than about 4 days. In some aspects, the treatment with AP is for less than 3 days, less than 2 days, or less than 1 day.

In a particular aspect, the AP is administered at an administration dose of about 1000U/kg per day over 3 consecutive days. In some particular aspects, when the AP is RecAP, the AP is administered at an administration dose of 1.6mg/kg per day over 3 consecutive days. In some aspects, the dosage of each dose of AP (e.g., recAP) (e.g., clinical grade RecAP as used in this disclosure) is from about 0.10mg/kg to about 3mg/kg, or from about 0.20mg/kg to about 2.9mg/kg, or from about 0.3mg/kg to about 2.8mg/kg, or from about 0.4mg/kg to about 2.7mg/kg, or from about 0.5mg/kg to about 2.6mg/kg, or from about 0.6mg/kg to about 2.5mg/kg, or from about 0.7mg/kg to about 2.4mg/kg, or from about 0.8mg/kg to about 2.3mg/kg, or from about 0.9mg/kg to about 2.2mg/kg, or from about 1mg/kg to about 2.1mg/kg, or from about 1.2mg/kg to about 1.9mg/kg, or from about 1.3mg/kg to about 1.8mg/kg, or from about 1.7mg/kg to about 1.4 mg/kg. In some aspects, the dosage of each dose of AP (e.g., recAP) comprises at least about 0.1mg AP/kg, at least about 0.2mg AP/kg, at least about 0.3mg AP/kg, at least about 0.4mg AP/kg, at least about 0.5mg AP/kg, at least about 0.6mg AP/kg, at least about 0.7mg AP/kg, at least about 0.8mg AP/kg, at least about 0.9mg AP/kg, at least about 1mg AP/kg, at least about 1.1mg AP/kg, at least about 1.2mg AP/kg, at least about 1.3mg AP/kg, at least about 1.4mg AP/kg, at least about 1.5mg AP/kg, at least about 1.6mg AP/kg, at least about 1.7mg AP/kg, at least about 1.8mg AP/kg, at least about 1.9mg AP/kg, at least about 2.1mg AP/kg, at least about 2.2mg AP/kg, at least about 3.3 mg AP/kg, at least about 2.4mg AP/kg, at least about 2.5mg AP/kg, at least about 2.6mg AP/kg.

AP may be administered via different routes, such as intravenous, rectal, bronchial or oral. In some specific aspects, the AP is administered intravenously, e.g., via intravenous infusion. In some aspects, the AP is administered intravenously via continuous infusion.

Although short-term preservation of lung function may immediately have life-saving consequences, it is preferred that the effect of the AP on lung function is durable.

The P/F ratio may be determined by methods known in the art. Arterial pO as measured by Arterial Blood Gas (ABG) ₂ Is a preferred method for calculating the P/F ratio. However, as shown in the following table, when pO is caused by the unavailability of ABG ₂ Unknown, spO measured by pulse oximeter may be used ₂ To approximate pO ₂ . It is important to note that when SpO ₂ At 98% -100%, from SpO ₂ Estimating pO ₂ And becomes unreliable.

SpO ₂ (％) 86 87 88 89 90 91 92 93 94 95 96 97

PO ₂ (mmHg) 51 52 54 56 58 60 64 68 73 80 90 110

Examples: assume a patient with 40% oxygen content has pulse oximetry SpO ₂ 95%. Referring to the above table, 95% SpO ₂ pO equal to 80mmHg ₂ . P/F ratio=80 divided by 0.40=200. Patients may be stable when receiving 40% oxygen, but still suffer from acute respiratory failure. If oxygen is pumped out, the patient breathes the indoor air, pO ₂ Will be only 40mmHg (well below the acute respiratory failure threshold of 60mmHg in room air).

Oxygen supplementation may be administered through a mask or nasal cannula ("NC"). Venturi masks (Venti masks) are designed with a specific fixed concentration (FIO) ₂ ) Delivering a controlled flow of oxygen: 24%, 28%, 31%, 35%, 40% or 50%. Non-rebreathing ("NRB") masks are designed to deliver approximately 100% oxygen. Providing 40% or more supplemental oxygen means that the physician is treating acute respiratory failure, as only acute respiratory failure patients need as much oxygen. The nasal cannula provides oxygen in liters of oxygen per minute (L/min or "LPM") at an adjustable flow rate. Actual FIO delivered through nasal cannula ₂ (percent oxygen) is somewhat unstable and less reliable than a mask, but can be estimated as shown in the table below. FIO derived from nasal cannula flow rate ₂ And can then be used to calculate the P/F ratio.

Flow Rate (L/min) 12 3 4 5 6

FIO ₂ (％) 24 28 32 36 40 44

Examples: the patient had pO on the ABG while receiving 5L/min of oxygen ₂ 85mmHg. Due to 5L/min of oxygen (FIO) equal to 40% ₂ 0.40), the P/F ratio=85 divided by 0.40=212.5.

In some aspects, administration of AP to a subject as disclosed herein can improve a number of respiratory function related parameters, such as the P/F ratio of the subject, oxygen saturation in arterial blood (pO ₂ ) Pulse oxygen saturation (SpO) ₂ ) An oxygenation index and/or a respiration index.

In some aspects, administration of an AP (e.g., recAP) to a subject results in an increase in the P/F ratio relative to the P/F ratio of a subject without treatment. In some aspects, the P/F ratio is increased by at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200mmHg relative to the P/F ratio of the subject without treatment.

In some aspects, administration of an AP (e.g., recAP) to a subject increases the P/F ratio of the subject relative to a baseline P/F ratio. In some aspects, the P/F ratio of the subject is increased by at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200mmHg relative to the baseline P/F ratio. In some aspects, administration of AP (e.g., recAP) to an ARDS patient results in a decrease in severity of ARDS from "severe" to "moderate", or from "moderate" to "mild", from "severe" to "mild", or from "moderate" or "severe" to "no ARDS", i.e., P/F >300mmHg.

In this context, the baseline P/F ratio is defined as the P/F ratio before or after the start of treatment. The baseline P/F ratio is preferably determined on day-2, day-1, day 0 or day 1, i.e. preferably within 48 hours before and up to 24 hours after the start of AP treatment. More preferably, the baseline P/F ratio is determined within 24 hours before and up to 12 hours after the start of AP treatment, more preferably between 12 hours before and up to 6 hours after the start of AP treatment, more preferably between 12 hours before and up to the start of AP treatment, and most preferably between 6 hours before and up to the start of AP treatment. AP treatment is preferably continued until the P/F ratio is at least 200 or higher, more preferably at least 300 or higher, most preferably until the subject is considered no longer suffering from ARDS.

In some aspects, an increase in lung function (e.g., an increase in P/F ratio) is observed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after AP administration.

In some aspects of the disclosure, the ARDS of the subject is associated with or due to sepsis or viral infection. In some aspects, ARDS in the subject is associated with or due to sepsis or viral infection, and AP (e.g., recAP) is administered to the subject only when sepsis or viral infection is determined less than 96 hours prior to deciding to begin treatment. In other aspects, the ARDS of the subject is accompanied or due to sepsis or viral infection, and the AP (e.g., recAP) is administered to the subject only when sepsis or viral infection is determined less than 72 hours prior to ARDS detection.

In some aspects of the disclosure, ARDS of the subject is associated with or due to sepsis or viral infection, and treatment of the subject with AP (e.g., recAP) is initiated within 24 hours after the sepsis or viral infection is determined. The presence of sepsis may be detected, for example, by using criteria developed for determining sepsis (e.g., SIRS or SOFA). Since the first definition of "sepsis" in 1992, its definition was re-evaluated a number of times, with emphasis on the prevailing opinion at that time, sepsis was caused by the Systemic Inflammatory Response Syndrome (SIRS) of the host to infection (Bone RC, balk RA, cerra FB, et al American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepis. Crit Care Med 1992;20 (6): 864-874). In 2001, the list of diagnostic criteria was expanded, but no alternative criteria were provided (Levy MM, fink MP, marshall JC, et al, international Sepsis Definitions conference.2001SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions conference. Intercept Care Med.2003;29 (4): 530-538). In 2016, the "sepsis-3" standard, which is now widely used, was recommended, which no longer uses the SIRS standard. In contrast, sequential [ Sepsis related ] organ failure assessment (SOFA) was used to diagnose Sepsis (Singer M, deutschman CS, seymour CW et al The Third International Consensus Definitions for Sepsis and Septic Shock (sepss-3) JAMA.2016;315 (8): 801-810.Doi: 10.1001/jama.2016.0287). STOP-AKI studies described in the examples were performed between 2014 and 2017 and used SIRS and SOFA scores to identify and/or rank sepsis patients. For definition and determination of "Sepsis" as used herein, the Sepsis-3 standard is preferably used, which is based on SOFA scores and is widely described in Singer et al (Singer M, deutschman CS, seymour CW et al, the Third International Consensus Definitions for Sepsis and Septic Shock (sepss-3). JAMA.2016;315 (8): 801-810.Doi: 10.1001/jama.2016.0287).

In some aspects of the disclosure, AP is administered to a subject at risk of sepsis to prevent a decrease in lung function.

In some aspects, the AP treatment of the subject is initiated within 48 hours after detection of ARDS. In a preferred embodiment, the AP treatment of the subject is initiated within 48 hours, preferably within 24 hours, more preferably within 12 hours, most preferably within 6 hours after detection of the moderate or severe ARDS.

In some aspects of the methods disclosed herein, the administration of at least one dose of AP shortens or stops the duration of mechanical ventilation therapy in a subject undergoing mechanical ventilation therapy.

In some aspects of the methods disclosed herein, the AP administration increases lung function or prevents lung function from decreasing below a critical threshold. Thus, in some aspects, AP administration can prevent lung function from dropping below a critical threshold. Thus, in some aspects, to determine the risk of the human's lung function falling below a certain threshold level, an indicator of lung function (e.g., a P/F ratio) is determined prior to administration of the AP to preserve lung function.

In some aspects, the methods disclosed herein comprise detecting a change in the level of one, two, three, or more biomarkers (e.g., a change in the P/F ratio) alone or in combination to detect a marker of lung function.

In some aspects, the methods disclosed herein include predicting an increased clinical response to an AP (e.g., recAP) treatment based on a detected lung function parameter (e.g., baseline P/F ratio). In some aspects, the methods of the present disclosure include assessing whether a lung function parameter (e.g., baseline P/F ratio) falls within a certain range, or whether it is above or below a certain threshold (e.g., the P/F ratio threshold for ARDS severity). Thus, if, for example, a lung function parameter (e.g., baseline P/F ratio) alone or in combination with other biomarkers indicates that the patient would benefit from AP treatment, treatment (e.g., increasing or decreasing dose, or increasing or decreasing dose frequency) may be initiated or maintained or modified.

Conversely, if, for example, a lung function parameter (e.g., baseline P/F ratio) alone or in combination with other biomarkers indicates that the patient will not benefit from AP therapy, therapy may be discontinued, temporarily suspended, modified (e.g., increased or decreased dose or increased or decreased frequency of administration), etc.

In other words, specific levels of a lung function parameter (e.g., baseline P/F ratio), alone or in combination with other molecules or clinical biomarkers, are correlated with the clinical efficacy of AP treatment and can be used to predict clinical outcome in a specific population of patients with sepsis and/or ARDS.

For some jurisdictions, the present invention provides Alkaline Phosphatase (AP) for use in a method of treating Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof, wherein the subject has a P/F ratio of less than or equal to 200mmHg. Also provided is the use of Alkaline Phosphatase (AP) for the manufacture of a medicament for treating Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof, wherein the subject has a P/F ratio of less than or equal to 200mmHg.

The invention also provides Alkaline Phosphatase (AP) for use in a method of treating acute respiratory syndrome (ARDS) in a subject in need thereof, the method comprising administering to the subject an effective amount of Alkaline Phosphatase (AP), wherein

(i) The subject suffers from moderate or severe ARDS prior to treatment with AP, and

(ii) The AP is administered at a dose of at least one dose of 300U/kg to 2,000U/kg.

In moderate or severe ARDS, 5cmH is used according to the definition of berlin 2012 ₂ The P/F ratio is less than or equal to 200mmHg when measured by the minimum Positive End Expiratory Pressure (PEEP).

In a preferred embodiment, the subject has a moderate ARDS with a P/F ratio of >100 and ∈200mmHg prior to treatment with AP. In another preferred embodiment, the subject has severe ARDS with a P/F ratio of <100mmHg prior to treatment with AP.

In some preferred embodiments, there is provided an AP for use according to the invention or a use according to the invention, wherein the AP is a human AP.

In some preferred embodiments, the AP is a recombinant AP, preferably a chimeric AP, more preferably an AP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of RecAP (SEQ ID NO: 1). In some preferred embodiments, the AP is a recombinant AP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of RecAP (SEQ ID NO: 1), provided that amino acid 279 is leucine (L), amino acid 328 is valine (V), and amino acid 478 is leucine (L).

In a preferred embodiment, an AP for use according to the invention or a use according to the invention is provided, wherein administration of the AP results in an increase in pulmonary function in a subject. The increase in pulmonary function preferably comprises an increase in the P/F ratio of the subject relative to the P/F ratio of a subject without treatment.

In some preferred embodiments, there is provided an AP for use according to the invention or a use according to the invention, wherein the ARDS is associated with or caused by sepsis. Preferably sepsis is detected less than 96 hours prior to AP administration.

In a more preferred embodiment, there is provided an AP for use according to the invention or a use according to the invention, wherein the ARDS is associated with or caused by sepsis and sepsis is detected less than 72 hours prior to ARDS detection. Preferably, the AP treatment is initiated within 24 hours after detection of sepsis and/or after detection of ARDS.

In other preferred embodiments, there is provided an AP for use according to the invention or a use according to the invention, wherein the ARDS is associated with or caused by a viral infection. The viral infection preferably comprises a coronavirus infection, more preferably a Severe Acute Respiratory Syndrome (SARS) -associated coronavirus infection. In some preferred embodiments, the coronavirus is SARS-CoV-2.

In a preferred embodiment, an AP for use according to the invention or a use according to the invention is provided, wherein the AP is administered once daily. In a preferred embodiment, the AP is administered intravenously. In a preferred embodiment, the AP is administered at a dose of three times per day.

In some preferred embodiments, there is provided an AP for use according to the invention or a use according to the invention, wherein the AP is RecAP and the dose is 0.6mg/kg (or 375U/kg) to 3.2mg/kg (or 2,000U/kg) of RecAP, preferably 0.8mg/kg (or 500U/kg) to 2.0mg/kg (or 1250U/kg), more preferably about 1.6mg/kg (or 1000U/kg).

Some preferred embodiments provide an AP for use according to the invention or a use according to the invention, wherein the AP is administered in a dose of at least one dose of 500U/kg to 2,000U/kg. Preferably, the AP dose is 500U/kg or 1000U/kg RecAP. In some preferred embodiments, the AP dose is 0.8mg/kg or 1.6mg/kg RecAP.

In a preferred embodiment, there is provided an AP for use according to the invention or a use according to the invention, wherein administration of at least one dose of AP results in maintenance or increase of the P/F ratio in a subject. Preferably, the administration of the at least one dose of AP is such that the duration of mechanical ventilation therapy in the subject undergoing mechanical ventilation therapy is shortened or stopped.

The term "biomarker" as used herein refers to a factor that is a unique indicator of a biological process, biological event, and/or pathological condition, e.g., a predictor of clinical response to treatment with AP (e.g., recAP). As used herein, the term biomarker includes clinical markers and molecular biomarkers (biomarkers). Thus, in the context of the present disclosure, the term "biomarker" includes, for example, "a biological biomarker" or "a molecular biomarker". In some aspects, biological or molecular biomarkers for assessing lung function include inflammatory markers, such as C-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6), or interleukin 8 (IL-8). More specific biomarkers associated with diagnosis and prognosis of ARDS are advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surface active protein D (SP-D), endothelin-8, fas and Fas ligand, procollagen peptides of type I and III (PCP), octane, acetaldehyde and 3-methylheptane. In general, these are cell-specific for epithelial or endothelial lesions or are involved in inflammatory or infectious responses (GarcIia-Larden MI, lorente JA, flares C, slotsky AS, villar J.biomarks for the acute respiratory distress syndrome: how to make the diagnosis more prese. Ann Transl Med.2017;5 (14): 283).

As disclosed above, the term "biomarker" also includes "clinical biomarkers," also referred to as "clinical status markers," that can predict a response to a biological therapy, e.g., gender, age, concomitant medication, smoking status, body Mass Index (BMI), and the like.

As discussed above, the critical approach based on baseline P/F values is applied to classify ARDS as either "severe ARDS" or "moderate ARDS". For example, the difference in P/F levels observed in subjects with "severe ARDS", "moderate to severe ARDS" or "mild ARDS" can be used to predict clinical outcome when treating patients with AP (e.g., recAP). Thus, if the P/F ratio of a subject is below a certain threshold (e.g., 200 mmHg), the subject will be a candidate for treatment with a certain AP therapy, e.g., treatment with a certain AP regimen comprising one or more doses of RecAP.

In some aspects, merely determining that the P/F ratio is below a predetermined threshold level is sufficient to identify the subject as a candidate for treatment with a certain AP therapy (e.g., with RecAP therapy). Thus, in some aspects of the methods disclosed herein, the P/F ratio may be used alone. However, in other aspects, the P/F ratio may be combined with other measurements of lung function (e.g., using spirometry) and/or molecular or clinical biomarkers, such as, for example, markers selected from the group consisting of: c-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6) or interleukin 8 (IL-8), advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surfactant protein D (SP-D), endothelin-8, fas and Fas ligands, procollagen peptides of types I and III (PCP), octane, acetaldehyde, and 3-methyl heptane.

These findings may be applied, for example, to methods of designing new defined therapies (e.g., by selecting a patient as a candidate for a certain AP therapy), methods of treating decreased lung function, preventing decreased lung function, increasing lung function or preserving lung function (e.g., to treat ARDS), methods of monitoring the efficacy of AP therapy, or methods of adjusting formulations, dosage regimens, or routes of administration.

The methods disclosed herein include prescribing, initiating and/or altering prophylaxis and/or treatment based on the severity of ARDS as generally classified based on the P/F ratio of the subject (as described above), alone or in combination with one or more additional biomarkers.

The present disclosure provides a method of determining whether to treat a patient suffering from ARDS with a treatment regimen comprising administration of AP, wherein the method comprises: (a) Determining the P/F ratio (or another lung function parameter), and optionally measuring or instructing a clinical laboratory to measure the level of an additional biomarker, such as, for example, a C-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6) or interleukin 8 (IL-8), advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surface active protein D (SP-D), interleukin-8, fas and Fas ligand, procollagen type I and III peptide (PCP), octane, acetaldehyde and/or 3-methylheptane, in a sample taken from the patient, and (b) if the patient is determined to have a higher or lower P/F ratio (or another lung function parameter) than the predetermined threshold level of each biomarker, or a higher or lower level of a biomarker, such as, for example, C-reactive protein (CRP), fas-6, IL-2, fas end product (SP-8), interleukin-2) or interleukin (IL-8), in a sample than the predetermined threshold level of each biomarker, or a higher or lower level of each biomarker than the predetermined threshold level of one or more of the biomarkers, and (b) if the patient is determined to have a higher or lower P/F ratio (or another lung function parameter than the predetermined threshold level of each biomarker, type I and III procollagen peptides (PCP), octane, acetaldehyde and/or 3-methyl heptane), then the treatment or instruct the healthcare provider to treat the patient with a treatment regimen comprising administration of an AP (e.g., recAP), or to pause the treatment, not start the treatment, reject the treatment, or instruct the healthcare provider to pause, not start or reject the treatment.

In one aspect, the present disclosure provides a method of determining whether to treat a patient suffering from ARDS with a treatment regimen comprising administration of AP, wherein the method comprises: (a) Determining a P/F ratio (or another lung function parameter), and optionally measuring or instructing a clinical laboratory to measure the level of an additional biomarker in a sample taken from the patient, such as, for example, C-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6) or interleukin 8 (IL-8), advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surface active protein D (SP-D), interleukin-8, fas and Fas ligand, procollagen type I and III peptide (PCP), octane, acetaldehyde and/or 3-methyl heptane, and (b) providing a healthcare regimen comprising the administration of AP to the patient if the patient is determined to have a lower or decreased P/F ratio (or another lung function parameter) as compared to a predetermined threshold level, and optionally as compared to one or more threshold levels of the predetermined biomarker, or as compared to one or more levels of the biomarker in one or more controls.

In one aspect, the present disclosure provides a method of determining whether to treat a patient suffering from ARDS with a treatment regimen comprising administration of AP, wherein the method comprises: (a) Determining a P/F ratio (or another lung function parameter), and optionally measuring or instructing a clinical laboratory to measure the level of an additional biomarker, such as, for example, C-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6) or interleukin 8 (IL-8), advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surface active protein D (SP-D), interleukin-8, fas and Fas ligand, procollagen peptides type I and III (PCP), octane, acetaldehyde and/or 3-methyl heptane, in a sample taken from the patient, and (b) if the patient is determined to have a higher or increased P/F ratio (or another lung function parameter) compared to a predetermined threshold level, and optionally a higher or lower level of at least one optional biomarker in the sample compared to one or more threshold levels of the biomarker in one or more controls, or a patient rejection of a therapeutic regimen, including, for example, disabling therapy, or otherwise providing therapy to the patient (AP) including, for example, disabling therapy, or not providing therapy to the patient (AP) if the patient is determined to have a higher or lower than a predetermined threshold level of P/F ratio (CRP-C).

Also provided is a method of selecting a patient diagnosed with ARDS as a candidate for treatment with an AP comprising: (a) Determining a P/F ratio (or another lung function parameter), and optionally measuring or instructing a clinical laboratory to measure the level of an additional biomarker in a sample taken from the patient, such as, for example, C-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6) or interleukin 8 (IL-8), advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surface active protein D (SP-D), interleukin-8, fas and Fas ligand, procollagen peptides type I and III (PCP), octane, acetaldehyde and/or 3-methyl heptane, and (b) providing a healthcare therapy to the patient if the patient is determined to have a lower or decreased P/F ratio (or another lung function parameter) compared to a predetermined threshold level, and optionally a higher or lower level of at least one optional additional biomarker in the sample compared to one or more levels of the biomarker in one or more controls.

Also provided is a method of selecting a patient diagnosed with ARDS as a candidate for treatment with an AP comprising: (a) Determining a P/F ratio (or another lung function parameter), and optionally measuring or instructing a clinical laboratory to measure the level of an additional biomarker in a sample taken from the patient, such as, for example, C-reactive protein (CRP), fibrinogen, interleukin 6 (IL-6) or interleukin 8 (IL-8), advanced glycation end product Receptor (RAGE), angiopoietin-2 (Ang-2), surface active protein D (SP-D), interleukin-8, fas and Fas ligand, procollagen peptides type I and III (PCP), octane, acetaldehyde and/or 3-methyl heptane, and (b) if the patient is determined to have a higher or increased P/F ratio (or another lung function parameter) compared to a predetermined threshold level, and optionally a lower or reduced level of at least one optional additional biomarker in the sample compared to a predetermined threshold level or levels of the biomarker in one or more controls, refusing (e.g., not providing therapy to the patient, for example, withholding therapy to the patient, or not providing therapy to the patient (RecAP), for example, pausing therapy to the patient).

In some aspects, the disclosed methods may require ordering and/or performing one or more additional assays. For example, the P/F ratio (or another lung function parameter) determination assay may be repeated to exclude false negative results, and/or one or more additional P/F ratio (or another lung function parameter) determination assays may be performed to monitor the status of the subject. Conversely, it may be desirable to repeat the P/F ratio (or another lung function parameter) determination to exclude false positive results.

In some aspects, the presence of a P/F ratio (or another lung function parameter) above or below a predetermined threshold level in a patient with ARDS may be used in combination with one or more clinical or molecular biomarkers specific for sepsis or to cause a specific infection with sepsis in the ARDS patient with or caused by sepsis.

Those of skill in the art will appreciate that the P/F ratio (or another lung function parameter) may be used in accordance with the methods disclosed herein, including but not limited to therapeutic, diagnostic, and monitoring methods, as a positive selector, i.e., if the P/F ratio (or another lung function parameter) of a patient is below or above a predetermined P/F ratio (or another lung function parameter) threshold level, or if the P/F ratio (or another lung function parameter) increases or decreases relative to the P/F ratio (or another lung function parameter) in one or more controls, a particular action (e.g., treating the patient) will be taken.

Those of skill in the art will appreciate that the P/F ratio (or another lung function parameter) may be used in accordance with the methods disclosed herein, including but not limited to therapeutic, diagnostic, and monitoring methods, as a negative selector, i.e., if the P/F ratio (or another lung function parameter) of a patient is below or above a predetermined P/F ratio (or another lung function parameter) threshold level, or if the P/F ratio (or another lung function parameter) increases or decreases relative to the P/F ratio (or another lung function parameter) in one or more controls, then a particular action (e.g., treating the patient) will be taken.

In one aspect, the disclosure includes a method of facilitating a healthcare provider, a healthcare welfare provider, or a clinical laboratory to determine whether a patient will benefit from AP therapy.

In one aspect, the methods disclosed herein include diagnosing based at least in part on the patient's P/F ratio (or another lung function parameter), which may be a differential diagnosis. In some aspects, the methods disclosed herein include informing a subject of the outcome of the determination of the P/F ratio (or another lung function parameter) and/or diagnosis based at least in part on the P/F ratio (or another lung function parameter). The patient may be notified orally, in writing, and/or electronically. The diagnosis may also be recorded in a patient medical record.

The term "medical record" or "patient medical record" refers to an account of patient examination and/or treatment that generally includes one or more of the following: medical history and complaints of patients, physical examination results of doctors, results of diagnostic tests and procedures, and patient medications and treatment procedures. Medical records are typically made by one or more doctors and/or doctor assistants, and are written, transcribed, or otherwise recorded records and/or histories of various diseases or injuries that require medical care and/or vaccination, and/or allergy, and/or treatment, and/or prognosis, and/or often, health information about parents, siblings, and/or professions. The doctor can view the record when diagnosing the condition.

The medical records may be in paper form and/or may be stored in a computer readable medium. The medical records may be maintained by a laboratory, doctor's office, hospital, medical care maintenance organization, insurance company, and/or personal medical records website. In some aspects, a diagnosis based at least in part on the determined P/F ratio is recorded on or in a medical alert article such as a card, a wearable article, and/or a Radio Frequency Identification (RFID) tag. As used herein, the term "article of wear" refers to any article that is wearable on the body of a subject, including but not limited to a tag, bracelet, necklace, arm band, or headband.

As used herein, the term "diagnosis" means detecting a disease or determining the stage or extent of a disease. Typically, diagnosis of a disease is based on an assessment of one or more factors and/or symptoms indicative of the disease. That is, diagnosis may be based on the presence, absence, or amount of a factor indicative of the presence or absence of a disease or disorder. Each factor or symptom considered to be indicative of a particular disease diagnosis need not be uniquely related to the particular disease, e.g., there may be a differential diagnosis that can be inferred from the diagnostic factor or symptom. Also, there may be cases where there are factors or symptoms indicative of a particular disease in an individual who does not have the particular disease.

The term "diagnosis" also includes determining the therapeutic effect of a drug therapy (e.g., AP therapy), or predicting the response pattern to a drug therapy. The diagnostic methods may be used alone or in combination with other diagnostic and/or staging methods known in the medical arts for a particular disease.

As used herein, the term "differential diagnosis" refers to determining which of two or more diseases with similar symptoms is likely to cause symptoms in a subject based on clinical data analysis. The term is also used to refer to determining whether a patient is susceptible to AP therapy, depending on whether the patient's determined P/F ratio (or another lung function parameter) is above or below a predetermined threshold level, or is elevated or lowered relative to the level of one or more controls.

The term "prognosis" as used herein refers to the prediction of the likely course and outcome of a clinical condition or disease (e.g., sepsis or ARDS). Prognosis is typically made by assessing factors or symptoms of a disease that indicate an advantageous or adverse course or outcome of the disease. The phrase "determining prognosis" as used herein refers to a process by which one of skill in the art is able to predict the course or outcome of a patient condition. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Conversely, the skilled artisan will appreciate that the term "prognosis" refers to an increase in the probability that a process or result will occur; that is, a process or outcome is more likely to occur in patients exhibiting a given condition when compared to those individuals not exhibiting the condition.

The terms "good prognosis" and "positive prognosis" or "poor prognosis" and "negative prognosis" as used herein are relative terms used to predict the likely course and/or likely outcome of a condition or disease (e.g., sepsis or ARDS). A good or positive prognosis predicts a better outcome for the condition than a poor or negative prognosis. In a general sense, a "good prognosis" is a result that is relatively better than many other possible prognosis that may be associated with a particular condition, while a result of a poor prognosis prediction is relatively worse than many other possible prognosis that may be associated with a particular condition. Typical examples of good or positive prognosis include increased lung function, preservation of lung function, increased P/F ratio (or another lung function parameter), etc.

The present disclosure includes methods of treating ARDS in a subject, or an AP in a method of treating ARDS in a subject, based on a change in P/F ratio (or another pulmonary function parameter) expression. The present disclosure provides a method of treating a patient suffering from ARDS, or an AP for use in a method of treating ARDS in a patient, wherein the method comprises: if the patient is determined to have a lower or decreased P/F ratio (or another lung function parameter) than a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to the P/F ratio (or another lung function parameter) in one or more controls, then the AP is administered to the patient.

The present disclosure also provides a method of treating a patient suffering from ARDS, or an AP for use in a method of treating ARDS in a patient, wherein the method comprises: (a) MeasuringQuantity pO ₂ And FiO ₂ (or deriving pO from another measurable lung function parameter) ₂ And FiO ₂ ) And determining a P/F ratio (or another lung function parameter), and (b) administering the AP to the patient if the patient has a lower or reduced P/F ratio (or another lung function parameter) compared to a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to the level of the P/F ratio (or another lung function parameter) in one or more controls.

Also provided are methods of treating a patient suffering from ARDS, or an AP for use in a method of treating ARDS in a patient, wherein the method comprises: (a) Determining a P/F ratio (or another lung function parameter) in the patient, and (b) suspending or not initiating administration of an AP (e.g., recAP) to the patient if the patient has a higher or increased P/F ratio (or another lung function parameter) compared to a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to a level of the P/F ratio (or another lung function parameter) in one or more controls.

The present disclosure also provides a method of treating a patient suffering from ARDS, or an AP for use in a method of treating ARDS in a patient, wherein the method comprises: (a) Measuring pO of patient ₂ And FiO ₂ (or deriving pO from another measurable lung function parameter) ₂ And FiO ₂ ) And determining a P/F ratio (or another lung function parameter), and (b) determining whether the P/F ratio (or another lung function parameter) is higher or increased, or lower or decreased, than a predetermined P/F ratio (or another lung function parameter) threshold level. In some aspects, the method further comprises administering the AP (e.g., recAP) to the patient or suggesting that the healthcare provider administer the AP (e.g., recAP) to the patient if the patient is determined to have a lower or reduced P/F ratio (or another lung function parameter) compared to a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to a P/F ratio (or another lung function parameter) level in one or more controls; or if the patient is determined to have a higher or increased P/F ratio (or another lung function parameter) than a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to the P/F ratio (or another lung function parameter) level in one or more controls, halting or rejecting AP was applied.

Also provided are methods of treating a patient suffering from ARDS, or an AP for use in a method of treating ARDS in a patient, wherein the method comprises: (a) Measuring pO of patient ₂ And FiO ₂ (or deriving pO from another measurable lung function parameter) ₂ And FiO ₂ ) And determining a P/F ratio (or another lung function parameter), and (b) administering the AP to the patient if the patient is determined to have a lower or decreased P/F ratio (or another lung function parameter) compared to a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to a P/F ratio (or another lung function parameter) level in one or more controls; or if the patient is determined to have a higher or increased P/F ratio (or another lung function parameter) than a predetermined P/F ratio (or another lung function parameter) threshold level, or compared to a P/F ratio (or another lung function parameter) level in one or more controls, halting, not starting or refusing administration of AP to the patient.

The present disclosure also provides a method of measuring AP efficacy or pharmacodynamics in a patient diagnosed with ARDS, comprising: (a) Making a first determination of the patient's P/F ratio (or another pulmonary function parameter); (b) administering an AP (e.g., recAP); and (c) making a second determination of the P/F ratio (or another lung function parameter) of the patient, wherein an increase in the P/F ratio (or another lung function parameter) in the second determination as compared to the P/F ratio (or another lung function parameter) of the patient in the first determination indicates that the patient is responsive to treatment with the AP (e.g., recAP).

The present disclosure also provides a method of measuring AP efficacy or pharmacodynamics in a patient diagnosed with ARDS, comprising: (a) Making a first determination of the patient's P/F ratio (or another pulmonary function parameter); (b) administering an AP (e.g., recAP); and (c) making a second determination of the P/F ratio (or another lung function parameter) of the patient, wherein a decrease in the P/F ratio (or another lung function parameter) in the second determination as compared to the P/F ratio (or another lung function parameter) of the patient in the first determination indicates that the patient is not responding to treatment with the AP (e.g., recAP).

In some aspects, the second determination is made 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after administration of the AP (e.g., recAP), or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks, or at a time in between.

In certain aspects, in all of the therapeutic methods disclosed herein, a "loading" dose of AP is administered to achieve a desired level of lung function in the patient. If the AP loading dose does not significantly affect the patient's lung function, it may be decided to stop the treatment-e.g., to alter alternative therapies.

If loading the dose increases patient lung function, it may be decided to reduce the AP dose size or frequency to a "maintenance" dose. It is important to note that the method provided herein is a guideline for the healthcare provider to administer the treatment, and that the final treatment decision will be based on the healthcare provider's rational judgment.

Formulations, dosage regimens, and routes of administration of an AP (e.g., recAP) can be adjusted according to the methods disclosed herein to provide an effective amount for optimal therapeutic response. With respect to administration of AP, AP may be administered by any suitable means, composition and route known in the art. With respect to bolus administration, a single bolus may be administered, several administrations may be divided over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment regimen.

Alkaline Phosphatase (AP)

Alkaline phosphatase (AP; EC 3.1.3.1 according to IUBMB enzyme nomenclature) is an enzyme that catalyzes the reaction of phosphatase monoester and H2O to alcohol and phosphate. Other names of APs are alkaline phosphomonoesterases; a phosphomonoesterase; a glycerophosphate enzyme; alkaline phosphate hydrolase; alkaline phenylphosphatase; orthophosphoric monoester phosphohydrolase (alkaline optimum). The systematic name of AP is phosphomonoesters phosphohydrolases (alkaline optimum).

AP is a widely specific enzyme that also catalyzes transphosphorylation. In humans and other mammals, at least four different but related APs are known. They are intestinal, placenta-like and liver/bone/kidney (or tissue non-specific) APs. The first three are located together on chromosome 2, while the tissue-nonspecific form is located on chromosome 1.

The term "AP of the present disclosure" refers to an isolated alkaline phosphatase, which includes splice variants, isoforms and polymorphisms thereof. Recombinant and chimeric APs are also included. In a specific aspect, the AP is RecAP. The amino acid sequence of RecAP is shown in fig. 2. In some aspects, an AP as used herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 1. In some aspects, an AP is a functional fragment (i.e., a fragment of an AP, e.g., an AP that retains at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least 70%, at least about 80%, or at least about 90% of the AP activity of the corresponding full-length AP). In some aspects, the AP is a variant or derivative of the AP disclosed herein.

The AP for use according to the present disclosure may be a commercial AP enzyme, or any composition comprising an AP enzyme and any means capable of producing a functional AP enzyme in the context of the present invention, such as a DNA or RNA nucleic acid encoding an AP protein.

The nucleic acid encoding the AP may be embedded in a suitable vector, such as a plasmid, phagemid, phage, (retrovirus), transposon, gene therapy vector and other vectors capable of inducing or conferring production of AP. In the context of the present disclosure, natural or recombinant microorganisms (such as bacteria, fungi, protozoa, and yeast) may also be used as a source of AP.

The AP-containing compositions for use according to the present disclosure may comprise eukaryotic APs (e.g., mammalian APs), which may be tissue-nonspecific AP types (e.g., liver-bone or kidney types), or tissue-specific APs (e.g., placental AP, intestinal AP, and placenta-like AP). The latter, also known as germ cell AP, localizes to testis, thymus and certain germ cell tumors and is closely related to both placental and intestinal forms of AP.

In some aspects, the mammalian AP is human or bovine AP. Non-limiting examples of human AP sequences can be found in the NCBI (Genpept) collection and include: np_001622 (intestinal AP), np_001623 (placental AP), np_112603 (placental-like AP) or np_000469 (tissue non-specific AP). In some aspects, the AP comprises a polymorphism. In some aspects, the AP is placental AP, placenta-like AP, intestinal AP, liver/bone/kidney AP, or a combination thereof. In some aspects, the AP is a recombinant AP.

From a conformational point of view, AP is roughly composed of two domains: a coronal domain and an active site domain. The active site domain can be divided into different parts such as catalytic residues and three metal ion sites (Zn 1, zn2 and Mg 3). From a primary structural point of view, the coronal domain is flanked by amino acids that form the active site domain. The amino acid sequence of AP and the relative positions of the catalytic domain and the coronal domain are known to the skilled person.

In some aspects of the disclosure, the AP is an isolated or recombinant AP comprising a coronary domain and a catalytic domain, wherein the coronary domain and the catalytic domain are obtained from different APs, and wherein at least one of the different phosphatases is a human phosphatase. In some aspects, the AP is, for example, ECAP (Escherichia coli) AP or one of seven known BIAPs (Niu Changdao AP).

In some aspects, the AP is an isolated or recombinant AP comprising a crown domain and a catalytic domain, wherein the crown domain and the catalytic domain are obtained from different APs, and wherein the different APs are human APs. This is particularly useful if the modified phosphatase is subsequently used in human therapy. The AP used in the disclosed methods may be modified, e.g., a genetically modified human-derived AP that is not immunogenic or only weakly immunogenic.

The modified APs disclosed herein may be used, for example, for "in vitro" or "ex vivo" diagnosis or treatment. Such modified phosphatases may include, for example, human and E.coli AP, or may consist of bovine and E.coli AP.

In some aspects of the disclosure, the AP is an isolated or recombinant AP comprising a coronary domain and a catalytic domain, wherein the coronary domain and the catalytic domain are obtained from different APs, and wherein the coronary domain is a coronary domain of placental AP (ALPP), and wherein the catalytic domain is a catalytic domain of intestinal AP (ALPI). In some aspects, at least one of the different APs is a human phosphatase. In other aspects, both different APs are human phosphatases.

Domain exchange mutants suitable for use in the human AP-based methods disclosed herein are listed in table 1.

Table 1 domain exchanged alkaline phosphatase. ALPI is intestinal AP, ALPP is placental AP, GCAP is placenta-like (or germ cell) AP, and TNAP is tissue-nonspecific AP.

Catalytic domain	Coronary domains	Called as
			ALPI	GCAP	catALPI/coronal GCAP
	TNAP	catALPI/coronary TNAP
				ALPP	catALPI/crown ALPP
ALPP	GCAP	catALPP/coronal GCAP
				TNAP	catALPP/coronary TNAP
	ALPI	catALPP/crown ALPI
			GCAP	ALPI	catGCAP/crown ALPI
	ALPP	catGCAP/crown ALPP
				TNAP	catGCAP/coronary TNAP
TNAP	ALPI	catTNAP/crown ALPI
				ALPP	catTNAP/crown ALPP
	GCAP	catTNAP/coronal GCAP

In some aspects, the AP is a combination between the catalytic domain of ECAP or any human form (ALPI, ALPP, GCAP or TNAP) and the coronal domain of BIAP. In addition, combinations of the catalytic domain of BIAP with any human form of the coronal domain can also be generated.

In some aspects, the modified AP is an AP that is linked to the cell membrane under natural conditions via a Glycosyl Phosphatidylinositol (GPI) anchor, but is modified such that it is no longer linked to the cell membrane. All isozymes are functionally active in the cell membrane and defective forms of GPI anchors do not occur naturally at detectable levels. Although serum AP activity has been demonstrated, it is generally believed that the enzyme is still present in the shed membrane fraction or membrane vesicles. AP activity in milk is also present in the fraction containing membrane vesicles. The GPI anchor is stored in the cell as a precursor molecule, where it is linked to the attachment site by a transamidase. The backbone of the GPI anchor is identical in mammals, but cell type dependent modifications are known.

In some aspects, the AP is human for treatment of a human subject. This is preferred due to the fact that AP forms obtained from other species may be immunogenic in human subjects and that treatment may elicit immune responses and pathological side effects. In some subjects, even fatal side effects (i.e., anaphylactic shock) may occur, and thus, it is preferable to minimize the risk of immune side effects by using the human AP form.

Since isolation of AP from humans is not feasible, human recombinant forms of AP protein can be routinely produced in different recombinant expression platforms. However, GPI modification and expression and purification of membrane anchored proteins are notoriously difficult; GPI proteins are difficult to separate from membranes and difficult to isolate and purify. Thus, in some aspects, the recombinant Ap comprises a modification in the GPI signal sequence, wherein the modification results in a secreted Ap, i.e., the Ap is not linked to a cell membrane.

No generic sequence is responsible for the GPI anchor linkage, but there are some specific common features:

(i) Hydrophobic extension of the amino acid at the C-terminus (at least 11 amino acids, but preferably more than 11 amino acids);

(ii) Upstream of the hydrophobic region, a spacer of hydrophilic amino acids (5-12 amino acids);

(iii) GPI is linked to a small amino acid: glycine, aspartic acid, asparagine, alanine, serine or cysteine; and

(iv) The 2 subsequent amino acids downstream of the GPI attachment site must be small amino acids and in most cases they are selected from the group consisting of: glycine, aspartic acid, asparagine, alanine, serine or cysteine.

In some aspects, the recombinant AP comprises a modification in the GPI signal sequence, wherein the modification results in a secreted AP that is biologically active, i.e., that exhibits activity on a biologically relevant substrate. In some aspects, the secreted AP is human AP. In some aspects, the secreted human AP is human liver-kidney-bone phosphatase, human intestinal AP, or human placenta-like alkaline phosphatase.

Based on the above common features, the skilled person may introduce modifications, e.g. by inserting one or more amino acids, which would disrupt the common part and would render the AP incapable of linking to the GPI anchor. Thus, in some aspects, a recombinant AP comprises a modification in a GPI signal sequence that results in a secreted AP, wherein the modification comprises a mutation or deletion of at least one amino acid in the sequence comprising the consensus GPI signal sequence.

In some aspects, the AP is an AP disclosed in U.S. patent No. 8,557,545. In some aspects, the AP is a chimeric AP or chimeric AP-like protein, such as those described in US2017/0009216 and US 2014/0193388. Preferably, AP is a recombinant alkaline phosphatase comprising a catalytic domain of ALPI (intestinal alkaline phosphatase) and a coronal domain of ALPP (placental alkaline phosphatase). More preferably, the AP is RecAP (corresponding to SEQ ID NO:1 and described in U.S. patent application publication No. US 2017/0009216).

In some aspects, the APs of the present disclosure comprise (i) a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% sequence identity to the coronal domain of human ALPI, and (ii) a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% sequence identity to the catalytic domain of human ALPI.

In some aspects, the sequence having the sequence identity to the crown domain of ALPP is located at approximately the same position in a protein according to the invention as the crown domain of ALPP in a native ALPP protein.

The percent identity, or the term "% sequence identity", of an amino acid or nucleic acid sequence is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that are identical to residues in a reference sequence after aligning the residues in the candidate amino acid or nucleic acid sequence to residues in the reference sequence and introducing gaps, if necessary, to achieve the maximum percent identity. In a preferred embodiment, the calculation of the percentage of sequence identity is performed without introducing gaps. Methods and computer programs for alignment are well known in the art, such as "Align 2" or BLAST services of the national center for Biotechnology information (the National Center for Biotechnology Information, NCBI).

In some embodiments, the invention provides Alkaline Phosphatase (AP) for use in a method of treating Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof, the method comprising administering to the subject an effective amount of Alkaline Phosphatase (AP), wherein the subject has moderate or severe ARDS prior to treatment with AP and the AP administration results in an increase in respiratory function. In a preferred embodiment, the subject has severe ARDS prior to treatment with AP. In another preferred embodiment, the subject has a moderate ARDS prior to treatment with AP.

In some preferred embodiments, the AP is administered in a dose of at least one dose of 500U/kg to 2,000U/kg. Preferably, ARDS is associated with or caused by sepsis, or ARDS is associated with or caused by a viral infection. In the case where ARDS is associated with or caused by sepsis, sepsis is preferably detected within less than 96 hours prior to AP administration, more preferably within less than 72 hours prior to ARDS detection. Preferably, the treatment is initiated within 24 hours after sepsis detection.

In some preferred embodiments, the AP is a human AP, and/or the AP is a recombinant AP. If the AP is recombinant, it is preferably a chimeric AP. Preferably, the chimeric AP has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of RecAP (SEQ ID NO: 1).

In some preferred embodiments, there is provided an AP for use according to the present invention, wherein the increase in respiratory function comprises an increase in P/F ratio relative to the P/F ratio without treatment with the AP.

In some preferred embodiments, there is provided an AP for use according to the invention, wherein the treatment is started within 24 hours after detection of ARDS.

In some preferred embodiments, the AP is administered once daily and/or intravenously. Preferably, the AP is administered in three doses per day. Preferably, the AP dose is about 0.8mg/kg or about 1.6mg/kg of RecAP per dose, and/or the AP dose is about 500U/kg or about 1000U/kg of RecAP. In this context, "about" means +/-20%, preferably +/-10%, most preferably +/-5% of the indicated dose. For average individuals (e.g., individuals weighing about 60-80kg, each dose will be about 30,000U to about 160,000U, preferably about 30,000U to about 80,000U, more preferably about 60,000U to about 80,000U, the dose expressed in mg will be equivalent to about 48mg to about 256mg, preferably 48mg to about 128mg, more preferably 96-128mg, thus, there is also provided an AP for use according to the present invention, wherein the dose is about 30,000U to about 160,000U, preferably about 30,000U to about 80,000U, more preferably about 60,000U to about 80,000U, there is also provided an AP for use according to the present invention, wherein the dose is about 48mg to about 256mg, preferably 48mg to about 128mg, more preferably 96-128mg.

In some preferred embodiments, there is provided an AP for use according to the present invention, wherein at least one dose of AP is administered such that the duration of mechanical ventilation therapy of a subject undergoing mechanical ventilation therapy is shortened or stopped.

Preferably, at least one dose of AP is administered such that the P/F ratio in the subject is maintained or increased. More specifically, the AP for use according to the invention allows a reduced risk of mortality in the subject. Treatment of one or more subjects as part of a group of subjects suffering from moderate or acute respiratory distress syndrome will result in a reduction in mortality in the group of subjects.

Although a feature may appear to be described in the present application as part of the same embodiment or as part of a separate embodiment, the scope of the invention also includes embodiments that include any combination of all or some of the features described herein.

The invention is further illustrated in the following examples. These examples are not intended to limit the scope of the invention but are merely illustrative of the invention.

Examples

Example 1

Human recombinant alkaline phosphatase for acute respiratory distress syndrome

Acute Respiratory Distress Syndrome (ARDS) adversely affects long-term lung outcome and survival. In preclinical studies, administration of the detoxification enzyme alkaline phosphatase may improve lung inflammation parameters. We studied the efficacy and safety of human recombinant alkaline phosphatase (RecAP) (FIG. 2) in ARDS patients.

In part 1 of the adaptation phase 2a/2b STOP-AKI trial, patients received RecAP 0.4, 0.8 or 1.6mg/kg or placebo randomly once a day for 3 days to determine the optimal dose.

In part 2, the dose was compared to placebo. The primary endpoint was the time-corrected area under the curve (AUC) of endogenous creatinine clearance on days 1-7 _1-7 ECC), renal Replacement Therapy (RRT) as a key secondary endpoint. Pulmonary function was also analyzed. A total of 301 patients were enrolled. No difference in (severe) adverse events between RecAP and placebo was observed between the groups. RecAP appears to be safe and well tolerated.

Method

STOP-AKI assay design and participants

The STOP-AKI test is an international randomized, double-blind, placebo-controlled, four-group, parallel-group, dose-finding adaptation phase 2a/2b test performed in critically ill adults with sepsis-related acute kidney injury. The protocols and main results of STOP-AKI studies have been previously published (Pickkers P, mehta RL, murray PT et al Effect of Human Recombinant Alkaline Phosphatase on 7-Day Creatinine Clearance in Patients With Sepsis-Associated Acute Kidney Injury: A Randomized Clinical Trial. JAMA.2018;320 (19): 1998-2009).

ICU patients aged 18 years or older and diagnosed with sepsis (Levy et al, crit Care Med2003; 31:1250-6) and first diagnosed with AKI (Mehta et al, crit Care 2007; 11:R31) were eligible for study. Inclusion and exclusion criteria and primary results are described in Pickkers et al (JAMA 2018).

Results measurement

The main objective of this study was to study the optimal therapeutic dose of RecAP and evaluate its effect on renal function. The effect on other organs (e.g. lung) was also investigated.

The effect of safety, tolerability, pharmacokinetics (in the first 120 patients), immunogenicity, systemic and urinary biomarkers, and RecAP on the quality of life and other non-renal parameters of the SA-AKI patients were also studied. Pulmonary function analysis is described in detail below.

By partial pressure of arterial oxygen in mechanically ventilated patients (PaO ₂ ) Score of inhaled oxygen (FiO) ₂ ) (P/F ratio) Carrico index, positive End Expiratory Pressure (PEEP), and tidal volume assessed lung function.

Number of days without mechanical ventilation: days from randomization to day 28 (including #), days with and without mechanical ventilation. The day of no ventilation is defined as the day when the patient is not using ventilation (invasive or non-invasive mechanical ventilation).

Days of ventilation: for those patients who used mechanical ventilation at the beginning of this period, days from the start of the first administration of study drug to the cessation of mechanical ventilation (from day 1 to day 28, inclusive).

Statistical analysis

Data analysis was performed on patients who obtained informed consent and were randomly assigned to treatment groups according to the intent-to-treat principle. Details related to the sample size are as follows.

Calculation of sample size

In section 1, the sample size for each treatment group plan is n ₁ =30 patients, in part 2, an additional n ₂ =85 patients were enrolled into the optimal RecAP dose group and placebo-treated group (total sample size n=290 patients). Custom programming simulations were performed using SAS software v.9.2 to determine the efficacy and type I error rate of selected sample amounts and design in many different dose-response scenarios. The standard deviation of the primary endpoint was assumed to be 49ml/min for each scenario, the placebo group assumed to respond to 60ml/min, and the RecAP dose group was between 60ml/min (no treatment effect) and 79ml/min (strong treatment effect).

Fifty thousand simulations were performed to show a single-sided type I error rate of 2.4% (and thus well controlled at a single-sided level of 2.5% significance). Efficacy is defined as the probability of rejecting null hypothesis (no difference between treatment groups) when one or more RecAP dose groups have a therapeutic effect, defined as a response of 69.5 ml/min. This was studied in seven scenarios, each of which was simulated 10 000 times. The selected design achieves 79% -86% efficacy for scenes with strong therapeutic effects for medium and high RecAP dose groups; there was a different response to the low dose group and only between 66% and 67% when the high dose group had a strong therapeutic effect. Since the determination of sample size is based on the number of patients required for the intent-to-treat (ITT) analysis, patients that were randomly assigned and subsequently withdrawn prior to study completion were not replaced.

According to the study protocol, for patients receiving study drug, the study protocol analysis compares the intervention group to the placebo group, and on days 1 through 7, there are no more than two ECC value deletions, detailed in the statistical analysis program.

For descriptive statistics, continuous variables are expressed as mean values with standard deviation or median values with quartile range, depending on their distribution. Comparing the normal distribution variables using student t-test; the Mann-Whitney U test is used to compare non-normally distributed variables. The classification (and binary) variables are expressed as a percentage as numbers and are analyzed using the chi-square test. Survival analysis of Kaplan-Meier curves was used for graphical display. Cox proportional risk regression analysis was used to evaluate the survival rate with RecAP versus placebo and the risk ratio for RRT free, shock free and mechanical ventilation days during days 1-28 of the study.

The primary efficacy endpoint was analyzed by analysis of variance. All analysis of secondary endpoints is for exploratory purposes only and therefore does not require multiple adjustments. Post hoc multivariate analysis was performed to determine the robustness of RecAP effects. All statistical tests (SAS software version 9.4; SAS Institute inc., cary, NC, u.s.a.) on the intended treatment population were bilateral, with a 5% level of significance.

Results

Participants (participants)

Of the 326 patients that passed the initial screening, 301 patients were enrolled in 53 sites in 11 countries in the european union and north america (fig. 4). Patients received RecAP 0.4mg/kg (n=30), 0.8mg/kg (n=32), 1.6mg/kg (n=29 in part 1 and n=82 in part 2) or placebo (n=86 in part 1, n=30 and part 2). In addition to slight baseline differences in renal function between groups (fig. 5), randomization resulted in a good balance of demographics and patient characteristics.

Safety of

43% of patients receiving RecAP 1.6mg/kg and 50% of patients receiving placebo reported serious adverse events triggered by treatment. See fig. 11. No RecAP dose dependence of incidence and nature of Adverse Events (AE) was observed. The anti-drug antibody titres for 9 patients treated with RecAP were slightly above the limit of detection.

Discussion of the invention

In this multinational double-blind, randomized controlled trial involving SA-AKI patients, we observed that addition of RecAP in standard care had a great effect on survival, with improved and sustained recovery of renal function and long-term clinical outcomeFruit (including composite MAKE endpoint). Because this is a phase 2 trial demonstrated by the principle of dose discovery, it was designed to include clinical results related to renal dysfunction (e.g., short term ECC) and long term, more patient-centric (e.g., MAKE) _{Day 60-90} Survival) associated endpoint.

In summary, recAP treatment is considered safe and well tolerated. In sepsis patients with AKI, recAP therapy was observed to provide long-term renal function, MAKE _60-90 And a significant improvement in survival.

Example 2

Determination of RecAP enzyme Activity and protein concentration

Activity measurement:

the recAP enzyme activity was determined based on the conversion (hydrolysis) of 4-nitrophenol phosphate to yellow 4-nitrophenol. The change in optical density at 405nm per unit time is a measure of alkaline phosphatase activity. The assay buffer consisted of 0.25M glycine buffer at 25℃pH9.6 with 2mM MgCl ₂ And 0.1mM ZnCl ₂ And 8.5mM 4-nitrophenol phosphate.

The unit (U) of recAP, expressed as U/mL, is the amount of enzyme that hydrolyzes 1. Mu. Mol of 4-nitrophenol phosphate per minute at pH9.6 and 25 ℃.

Protein concentration:

determination of total protein concentration in RecAP drug substance and drug product was performed by UV/Vis analysis. RecAP solution was analyzed at 280nm and absorbance was a measure of protein content (mg/mL) using the following formula:

concentration (mg/mL) = [ a/(a×b) ]×df where a=a280; b = path length; a=1.01 mL mg-1cm-1 mass extinction coefficient; DF is the dilution factor.

Example 3

Exploring P/F ratio

In preparing a study of the potential beneficial effects in covd-19 treatment, there is interest in exploring the effect of recAP (recombinant alkaline phosphatase) on related biomarkers or endpoints observed in STOP-AKI studies, as described in example 1 and Pickkers et al (JAMA 2018). One of the relevant endpoints is the ratio of arterial oxygen partial pressure to inhaled oxygen fraction, i.e. the P/F ratio. Thus, the purpose of this study was: the potential effect of recAP on the P/F ratio observed in the STOP-AKI study was explored.

The relevant tables of the analytical data model (ADaM) dataset, in particular the adverse drug reaction (ADRe) dataset, provided by AM-Pharma, are read into statistical analysis program R (R-project. Org; version 3.4.4 (2018-03-15)). A patient population with P/F ratio measurements was included that received either placebo or 1.6mg/kg recAP treatment at random. As described in the definition of Berlin 2012, PEEP is at least 5cm H for all patients with P/F ratios measured as 300 or less ₂ O. Averaging the baseline of the P/F ratio determined in the screened samples and the samples collected on day 1 to increase the stability of the values and reduce the occurrence of missing values, and labeled mean base (MeanBase); patients lacking mean basal values were excluded from analysis. Absolute and normalized values are plotted as a function of time after the initial dose. After the sub-setting (sub-setting) is a three-digit number of the mean base value, the graph exploration is also repeated. The average is plotted with bootstrap confidence intervals. The trends observed in these plots were checked using linear regression and non-parametric tests at relevant time points.

Figures 12-14 provide the progression over time of the ratio of arterial oxygen partial pressure to fraction of inhaled oxygen (i.e., the P/F ratio) after initiation of treatment. When considering the complete data set, the absolute P/F ratio does not change in a specific way over time and there is no significant difference between placebo and treatment groups, see fig. 12. As the study was discontinued due to death or for other reasons, the number of patients decreased over the study time and the width of the bootstrap confidence interval increased accordingly. However, if the P/F ratio of each patient was determined to be a "fold change" from the baseline value, the patient administered recAP showed an increase over time compared to placebo-treated patients (fig. 13).

As can be seen in fig. 14, this difference between recAP-treated patients and placebo-treated patients was largely due to those patients with P/F ratios below 200mmHg at baseline (i.e., moderate to severe ARDS patients), whereas only slight, transient differences were seen in the mild ARDS group (200 mmHg < P/F ratio <300 mmHg) and the patient group showing no ARDS at baseline (P/F ratio >300 mmHg). In summary, those patients who developed moderate to severe ARDS (P/F ratio <200 mmHg) at baseline most benefited from recAP treatment.

As can be seen in fig. 15, alkaline phosphatase treatment resulted in a significant reduction in mortality in a group of patients with moderate to severe ARDS (P/F ratio <200 mmHG) at baseline when compared to placebo-treated patients with P/F ratio <200 mmHG. Thus, in an embodiment, there is provided an AP for use according to the invention, wherein administration of at least one dose of AP results in a reduced risk of mortality in a subject. In a preferred embodiment, the reduction is a reduction in risk of total mortality. Preferably, the risk of mortality in the subject is reduced relative to when the subject is not undergoing AP treatment. Also provided is an AP for use according to the invention, wherein administration of at least one dose of AP results in a reduction in mortality in a group of said subjects. In a preferred embodiment, the reduction is a reduction in total mortality. Preferably, mortality in the group of subjects treated for AP is reduced relative to the group of subjects with severe to moderate ARDS not treated for AP.

Mortality, risk of mortality, total cause mortality, and total cause risk of mortality have their usual meanings in the context of the present invention. Mortality or total cause mortality in this context means the rate of death from all causes of death from a population over a given period of time. In this context, risk of death or risk of total cause of death means risk of death from all causes of an individual over a given period of time.

It should be understood that the detailed description section, rather than the summary and abstract sections, is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more, but not all exemplary embodiments of the invention as contemplated by the inventors, and thus are not intended to limit the invention and the appended claims in any way.

The invention has been described above with the aid of functional building blocks illustrating certain functions and relationships thereof. For ease of description, the boundaries of these functional building blocks are arbitrarily defined herein. Alternate boundaries may be defined so long as the specified functions and relationships thereof are appropriately performed.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments without undue experimentation without departing from the generic concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present application should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All publications, patents, patent applications, and/or other documents cited in this disclosure are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Claims

1. Alkaline Phosphatase (AP) for use in a method of treating Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof, wherein the subject has moderate or severe ARDS.

2. The AP for use of claim 1, wherein the subject suffers from moderate or severe ARDS when 5cmH is used ₂ The minimum Positive End Expiratory Pressure (PEEP) of O has a P/F ratio of 200mmHg or less when measured.

3. The AP for use according to claim 2, wherein the subject has severe ARDS and has a P/F ratio of less than or equal to 100mmHg prior to treatment with AP.

4. The AP for use according to claim 2, wherein the subject has a moderate ARDS, prior to treatment with AP, with a P/F ratio of >100 and ∈200 mmHg.

5. The AP for use of any one of claims 1-4, wherein the AP is administered at a dose of at least one dose of 500U/kg to 2,000U/kg.

6. The AP for use of any one of claims 1-5, wherein the ARDS is associated with or caused by sepsis.

7. The AP for use of any one of claims 1-6, wherein the ARDS is associated with or caused by a viral infection.

8. The AP for use of any one of claims 1-7, wherein the AP is a human AP.

9. The AP for use of any one of claims 1-8, wherein the AP is a recombinant AP, preferably wherein the recombinant AP is chimeric.

10. The AP for use of any one of claims 1-9, wherein the AP has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of RecAP (SEQ ID NO: 1).

11. The AP for use according to any one of claims 1-10, wherein administration of the AP results in an increase in respiratory function, preferably wherein the increase in respiratory function comprises an increase in the P/F ratio of the subject relative to the P/F ratio of the subject not treated with the AP.

12. The AP for use of any one of claims 1-11, wherein the AP treatment begins within 48 hours after detection of ARDS.

13. The AP for use of any one of claims 1-12, wherein the at least one dose of AP administration shortens or stops the duration of mechanical ventilation therapy in a subject undergoing mechanical ventilation therapy.

14. The AP for use of any one of claims 1-13, wherein the AP is administered once daily.

15. The AP for use of any one of claims 1-14, wherein the AP is administered intravenously.

16. The AP for use of any one of claims 1-15, wherein the AP is administered at a dose of three times per day.

17. The AP for use of any one of claims 1-16, wherein the dosage of AP is 0.8mg/kg or 1.6mg/kg RecAP.

18. The AP for use of any one of claims 1-17, wherein the dosage of AP is 500U/kg or 1000U/kg RecAP.

19. The AP for use of any one of claims 1-18, wherein the at least one dose of AP administration maintains or increases the P/F ratio of the subject.

20. The AP for use of any one of claims 1-19, wherein the at least one dose of AP administration results in a reduced risk of mortality for the subject.

21. The AP for use of any one of claims 1-19, wherein the at least one dose of AP administration results in a reduction in mortality in a group of subjects including the subject.