AF-16 or AF-17 for acute treatment of brain oedema in patients with traumatic brain injury

Abstract

The current invention relates to a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the measurement of ICP to at least 18 mmHg. The herein disclosed use of antisecretory peptides in acute brain oedema reduces mortality risk in patients with acute brain oedema due to a TBI by at the least 20%.

Description

[0001] Acute Treatment of Brain Oedema in Patients with Traumatic Brain Injury

[0002] Field of invention

[0003] Traumatic brain injury (TBI) constitutes a global epidemic. Overall outcome is poor with mortality ranging from 10-70 % and significant long-term impairment and morbidity. Several experimental reports have claimed effect on traumatic brain oedema, but all clinical trials have failed.

[0004] The current invention relates to a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the measurement of ICP to at least 18, such as at least 20 mmHg. The herein disclosed very early administration of antisecretory peptides to patients suffering from acute brain oedema due to TBI potently reduces mortality risk in these patients by at least 20%.

[0005] Background of the invention

[0006] The causes of traumatic brain injury (TBI) are very heterogenous, but the common denominator is an external force to the head resulting in a brain injury. The nature of this primary injury is multi-faceted as it depends on e.g. velocity, force, severity, and haemorrhage pattern. A common denominator is, however, that the primary injury cannot be reversed. Secondary injury in TBI develops as a diverse array of pathophysiologic phenomena, including hypoperfusion, mitochondrial dysfunction, oxidative injury, as well as further disruption to the blood-brain-barrier (BBB) (Prakash and Carmichael 2015, Winkler et al., 2016).

[0007] The clinical severity of TBI is commonly based on the Glasgow Coma Scale (GCS) scoring from 3 (worst) to 15 (no impairment) (Teasdale and Jennett, 1974). The scoring is summed to classify TBI as mild (GCS 13-15), moderate (GCS 9-12) or severe (GCS 3-8). Around 90% of all TBIs are considered mild (Maas et aL, 2017, Tagliaferri et aL, 2006) and those patients generally, but not universally, make a good functional recovery. Subjects with GCS scores in the moderate-severe range have a substantially higher risk of mortality (Godoy et aL, 2016; Sandhaug et aL, 2015).

[0008] Not all TBI result in brain oedema, but brain oedema in patients with TBI is a key secondary contributor to post-traumatic morbidity and mortality (Shah and Kimberley, 2016). Although Starling forces are physical drivers of oedema based on differences in intravascular vs extracellular hydrostatic and oncotic pressures, the molecular pathophysiology underlying cerebral oedema is complex and remains incompletely understood. Cerebral oedema is commonly seen in a variety of brain injuries including ischemic stroke, subarachnoid haemorrhage, traumatic brain injury, subdural, epidural, or intracerebral hematoma, hydrocephalus, brain cancer, brain infections, low blood sodium levels, high altitude, and acute liver failure. Diagnosis is based on symptoms and physical examination findings and confirmed by serial neuroimaging. The major types of cerebral oedema include vasogenic, cellular, osmotic, and interstitial. Through these mechanisms, cerebral oedema stems from tumour, trauma, hypoxia, infection, metabolic derangements, or acute hypertension. Causes are widespread and divide into neurological and non- neurological categories.

[0009] The treatment of cerebral oedema depends on the cause and includes monitoring of the person's airway and intracranial pressure, proper positioning, controlled hyperventilation, medications, fluid management, steroids. Extensive cerebral oedema can also be treated surgically with a decompressive craniectomy. Cerebral oedema is a major cause of brain damage and contributes significantly to the mortality of ischemic strokes and traumatic brain injuries.

[0010] There are two main pathophysiological paths in TBI leading to cerebral oedema; cytotoxic oedema leading to cellular swelling, and vasogenic oedema, which is extracellular oedema. Cerebral oedema in patients with TBI is considered a very serious condition since these two pathways can result in disastrous increases in intracranial hypertension (Winkler et aL 2016). Cerebral oedema is reported in any severity of TBI although more frequently in more severe TBL Tucker et aL, (2017) showed that patients with TBI of any severity grade (mild, moderate, severe) with cerebral oedema were eight times (4.6-14; 95% confidence interval [Cl]) more likely to die during their hospital stay compared to patients suffering TBI without oedema. The majority of patients in this study were diagnosed with mild TBI (93%) and did not experience sequalae. However, patients who suffered mild head trauma having evidence of brain oedema were nearly five times (2.03- 11.75 95% Cl) more likely to die than mild head trauma patients without oedema.

[0011] Measurement of cerebral oedema is indirect and generally relies on surrogate markers seen on neuroimaging studies and increased intracranial pressure (ICP). Imaging at admission plays a critical role in the evaluation of patients at risk of developing cerebral oedema after TBI. In the clinic, contrast-enhanced computed tomography (CT) serves as first line of imaging in the acute phase of moderate and severe head trauma due to easy availability and speed and this technique can reliably detect absence of, as well as presence of, cerebral oedema of clinical significance. Magnetic resonance imaging (MRI) can be particularly useful in specific settings e.g. in patients with milder TBI, presenting with Glasgow Coma Score (GCS) 13-15, i.e. responding to verbal stimuli. Follow-up scanning should be performed if there is any clinical deterioration, since evolution of cerebral oedema may occur, thus requiring an alternative treatment approach. Intracranial hypertension, as measured by ICP monitoring, serves as an evaluation and treatment proxy for cerebral oedema and a focus of guideline based TBI care (Carney et al., 2017). Worsening of cerebral oedema should be considered in patients with signs or symptoms of rising ICP, such as decreased level of consciousness, vomiting, headache, gaze deviation and hemiparesis. Of these signs, impaired level of consciousness is the most important clinical indicator of cerebral oedema.

[0012] At present, the goal of oedema management in TBI is to prevent secondary injury by minimizing ICP elevation and maintaining adequate cerebral blood flow which can be estimated and monitored as the cerebral perfusion pressure (CPP). Management of ICP elevation involves a graded algorithmic approach, from general measures (e.g. optimal head and neck positioning, avoidance of dehydration, and maintenance of normothermia) to medical treatment using mannitol, diuretics, corticosteroids or barbiturates (Raslan and Bhardway, 2017). Generally, mannitol or hypertonic saline are used for osmotic therapy in which a concentration gradient is created between fluids at the BBB, favouring the movement of fluid to the intravascular compartment for drainage (Carney et al., 2017). Both treatments are empirical with limited evidence base (Maas and Menon, 2017) and require cautious administration since treatment responses are unpredictable in the individual subject. A recent Cochrane meta-analysis concluded that new multi-centre trials of both hypertonic saline and mannitol are needed to evaluate comparative efficacy and safety in the long-term management of acute TBI (Chen et al., 2020). Regardless of osmotic therapy chosen, patients need to be closely monitored for ongoing fluid balance and to prevent excessive elevation of sodium and chloride levels, and to detect and correct other derangements such as hypokalaemia. Cerebral oedema in TBI with a disrupted BBB may even be worsened by the use of mannitol, due to accumulation of osmotically active molecules leading to further increase of local intracerebral oedema and rebound increases in ICP. In an autopsy study by Hay et al. (2015) including moderate or severe TBI subjects (n=70) neuropathologic evidence of BBB disruption was observed in 88% in at least one brain region and 40% had widespread (two or more regions), diffuse, multifocal BBB disruption. Even after mild TBI, 53% of patients (n=30) the BBB was shown to be affected (Tomkins et al., 2011).

[0013] The Protein Antisecretory Factor (Protein-AF)

[0014] Protein Antisecretory Factor (Protein-AF) is a 41 kDa protein that was originally described to provide protection against diarrhoea diseases and intestinal inflammation (for a review, see Lange and Lbnnroth, 2001 ). The Protein Antisecretory Factor (Protein-AF) has long since been sequenced and its cDNA cloned. The antisecretory activity seems to be mainly exerted by a peptide located between the amino acid positions 35 and 50 on the Protein Antisecretory Factor (Protein-AF) sequence. Immunochemical and immunohistochemical investigations have revealed that the Protein Antisecretory Factor (Protein-AF) is present and may also be synthesized by most tissues and organs in a body. Synthetic peptides, comprising the active sequence, shown in SEQ.ID.NO: 1 (AF-16) or SEQ.ID.NO: 2 (AF- 17) have prior been characterized (WO 97 / 08202; WO 05 / 030246; WO 2007 / 126364; WO 2018 / 015379).

[0015] Antisecretory Factor (AF)-16 has been shown to reduce cerebral oedema in animal brain trauma models that qualitatively replicate several pathological responses and functional deficits exhibited in human TBI (Clausen et al., 2017). These models also result in acute BBB leakage, thereby further representing a clinically relevant feature of brain injury (All uri et al., 2018, Michinaga and Koyama, 2015).

[0016] The effect of AF-16 has been studied in the rat midline fluid percussion injury (mFPI) model, a clinically relevant model of mild to moderate TBI (Clausen et al., 2017). The effect of AF-16 on the reduction of brain water content (BWC) was studied in this model. In addition, cognitive impairment and immunohistology injury markers were evaluated. The mFPI-injury induced oedema in the brain tissue resulted in an increase in BWC at 48 h compared to naive or sham-injured animals. Animals subjected to mFPI-injury and treated with control peptide had significantly higher BWC (p < 0.05) compared to naive and sham-injured animals (no oedema present) treated with control. Treatment with AF-16 reduced the mFPI-induced oedema by approximately 45% (p < 0.05) compared to treatment of oedema with the control peptide. Treatment with AF-16 reduced the BWC to similar levels as the naive and sham-injured animals. With this, it is demonstrated that AF- 16 exerts its effect (i.e. reduction of BWC) only if there is an existent oedema.

[0017] Summary of the present invention

[0018] It has been found that the oedema-reducing effects of AF-16, as analysed by reduction of brain water content (BWC), are present only if there is a resident oedema and not in the unaffected brain. These specific effects of AF-16 on oedema after TBI supports a potential clinical benefit in a justifiable clinical condition; cerebral oedema in patients with TBI.

[0019] Administration of AF-16 is herein demonstrated to be an efficacious treatment of cerebral oedema in TBI patients, and was shown to, if administered in the acute treatment phase, reduce the mortality rate of patients diagnosed with a clinical coma severity of between 3- 12 (Glasgow Coma Scale) GCS, i.e. moderate to severe coma severity, by at least 20%.

[0020] In particular, the current inventors have found that the beneficial effect of the AF-16 peptide on the cerebral oedema in patients with TBI is most prominent, when administered as early as possible after the occurrence of the trauma and / or the development of an elevated ICP, preferably wherein AF-16 is administered intravenously, and treatment is initiated during the initial stage of emergency care and / or hospitalization, even before measuring of the ICP has taken place.

[0021] The current invention therefore relates to the use of a pharmaceutical composition for treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of an elevated intracranial pressure (ICP), such as with an initial increased intracranial pressure (ICP) of at least 12 mmHg.

[0022] In addition, the current invention further relates to the use of a pharmaceutical composition for treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient who had initially been classified with mild coma severity of between 13-15 and who develops an increased intracranial pressure (ICP) of at least 18 mmHg, such as of at least 20 mmHg (unprovoked), which can be measured for at least 60 minutes during monitoring of said patient, within 7 days of arrival to the hospital. The current invention relates to a new medical use and a new administration route of a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity. The new medical use disclosed herein for the first time is for treating acute brain oedema due to a traumatic brain injury (TBI) in a patient, wherein the patient is diagnosed with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization. The acute treatment reduces mortality risk in patients with acute brain oedema due to a traumatic brain injury (TBI) by at least 20%.

[0023] In a preferred embodiment, a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2(AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, is administered no later than 24h after the measurement of ICP to at least 18 mmHg, such as of at least 18 mmHg, 20 mmHg, 21 mmHg, 22 mmHg or higher.

[0024] In a further embodiment, treatment according to the present invention is initialized during the initial stage of emergency care and / or hospitalization, i.e. even before measurement of ICP is done, solely based on the diagnosing of the patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP).

[0025] Since the acute treatment reduces mortality risk in patients with acute brain oedema due to a traumatic brain injury (TBI) by at least 20% and treatment with a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2(AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity has no or at least few documented severe side-effects for the patient, initiating the treatment according to the present invention is considered relatively risk-free. Treatment with a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2(AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity can thus be initiated as a precautionary treatment that can prevent and / or ameliorate the upcoming brain oedema due to a traumatic brain injury (TBI) in a patient if administered as early as possible.

[0026] A pharmaceutical composition for use according to the current invention is intended for treating a patient diagnosed with a clinical coma severity of between 3-12, such as between 3-10 or 4-9 (Glasgow Coma Scale) GCS.

[0027] Typically, a pharmaceutical composition for use according to the current invention comprises at least 7.5 mg / mL of an isolated recombinant and / or synthetically produced peptide as shown in SEQ ID NO:1 (AF-16) and / or I SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity.

[0028] Administering a pharmaceutical composition for use according to the current invention reduces the ICP in the patient at the latest at day 5 after the initializing of the treatment to 8.5 mmHg, 9 mmHg, 9.5 mmHg, 10 mmHg, 10.5 mmHg, 11 mmHg, 11.5 mmHg, 12 mmHg, 12.5 mmHg, or 13 mmHg. Typically, the ICP in said patient is reduced by between 10-25% after 10 min, 15 min, 30 min, 1 h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, or at the most 12h after the first administration.

[0029] A pharmaceutical composition for use according to the current invention can be administered repeatedly, such as during at least 1 , 2, 3, 4, or 5 days of treatment. It can be administered in multiple daily doses, such as administered each 8 hours, such as b.i.d or t.i.d, or be administered continously

[0030] The effect of administering a pharmaceutical composition according to the current invention, can be analysed as reduction in brain oedema and / or assessed as reduction of brain water content (BWC) and / or reduction of the cerebral perfusion pressure (CPP) of said patient.

[0031] In a currently preferred embodiment, the patient to be treated suffers from a blood brain barrier (BBB) disruption and / or an acute BBB leakage.

[0032] In one aspect, a pharmaceutical composition for use according to the current invention is formulated as a solution for intravenous infusion. It typically comprises the active ingredient (peptide AF-16 and / or peptide AF-17) and a pharmaceutically acceptable excipient, such as but not limited to, water, a chelating agent, a tonicity agent and a pH adjusting agent. The pharmaceutical composition is in one embodiment administered at a dosage of 1-50 mg / kg body weight as a 10-60 minute-infusion, such as a 10-40, or 10-30 minute infusion, such as at a dosage of 10 mg / kg body weight as a 10 minute-infusion or at a dosage of 30 mg / kg as a 30 minute-infusion or at a dosage of 50 mg / kg as a 40 minute-infusion or 60 minute-infusion. In a currently preferred embodiment, the pharmaceutical composition is administered each 8 hours at a dosage of 1-50 mg / kg body weight as a 10-60 minute- infusion, such as a 20-40 min infusion, such as a 20, 30 or 40 minute-infusion.

[0033] A pharmaceutical composition for use according to the current invention can be administered in combination with other treatments focused on managing ICP elevation, selected from the group consisting of optimal head and neck positioning, optimized pain relief, optimized sedation, negative fluid balance, controlled ventilation, hypocapnia, avoidance of dehydration, and maintenance of normothermia and / or in combination with other treatments focused on managing ICP elevation comprising a medical treatment using any one selected from the group consisting of mannitol, hypertonic saline solution, diuretics, corticosteroids, barbiturates, and combinations thereof.

[0034] Typically, but not exclusively, the patient is a human patient, such as a human patient with loss of consciousness of more than 6 hours.

[0035] In currently preferred embodiments, the treatment reduces intracranial pressure (ICP) within 30 min-1h after treatment start and leads to signs of clinical benefit, such as but not limited to reducing the need for deep sedation of the patient. Alternatively, the treatment further comprises sedation of the patient without the administration of barbiturates, except during periods of gastroparesis of the patient.

[0036] Administering a pharmaceutical composition for use according to the current invention typically results in a favourable short-term outcome, as well as in a favourable long-term outcome for the patient. In particularly, the treatment typically results in a primary outcome of a decreased 30-day mortality, such as in a primary outcome of a at least 20%, such as at least 25%, such as of at least 50% decreased 30-day mortality.

[0037] In aspects, administering a pharmaceutical composition for use according to the current invention results in a secondary outcome comprising reduction in treatment intensity level, intracranial pressure and / or number of days at the neuro-intensive care unit, a reduction of damage and / or inflammation in the brain of the patient and / or in a lowering of intracranial pressure (ICP) to non-critically elevated levels in 24 hours. In one aspect, the current invention further relates to the use of an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for manufacturing of a pharmaceutical composition for use in treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient a. with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, and b. an initial increased intracranial pressure (ICP) of at least 12 mmHg, wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated no later than 24h after the raise of ICP to at least 18mmHg.

[0038] In another aspect, the current invention relates to a method of treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient a. with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, and b. an initial increased intracranial pressure (ICP) of at least 12 mmHg, comprising administering intravenously a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, wherein the treatment is initiated no later than 24h after the raise of ICP to at least 18 mmHg in said patient.

[0039] Figure legends

[0040] Figure 1 : 1 A Chemical structure of AF-16 and 1B Chemical structure of AF-17 Figure 2: Demography & baseline characteristics of the patients in experiment 2 Figure 3: Max ICP ITTn, all patients & controls

[0041] Figure 4: Average ICP (AUC) ITTn, all patients & controls

[0042] Figure 5: TIL scores ITTn, all patients & controls

[0043] Definitions and abbreviations

[0044] Abbreviations

[0045] AF: antisecretory factor,

[0046] AF-16: a peptide composed of the amino acids VCHSKTRSNPENNVGL (SEQ ID NO:1 ); AF-17: a peptide composed of the amino acids VC(C)HSKTRSNPENNVGL (SEQ ID NO:2)

[0047] BBB: Blood-Brain Barrier b.i.d., bid or bd: (bis in die): twice a day

[0048] CDC: Centers for Disease Control and Prevention

[0049] Cl: Confidence Interval

[0050] CCI: Controlled Cortical Impact

[0051] CPP: Cerebral Perfusion Pressure

[0052] CSF: Cerebrospinal Fluid

[0053] ED: Emergency Department

[0054] EDHD: Emergency department visits, hospitalizations, and deaths

[0055] GBD: Global Burden of Disease

[0056] GHDx: Global Health Data Exchange

[0057] GFAP: Glial Fibrillary Acidic Protein

[0058] GCS: Glasgow Coma Scale

[0059] ICP: Intracranial Pressure

[0060] IV: Intravenous

[0061] ITT: Intent-to-Treat mFPI: Midline Fluid Percussion Injury

[0062] MWM: Morris Water Maze o.d.:"once daily"

[0063] PD: Pharmacodynamic

[0064] PK: Pharmacokinetics

[0065] RTI: Road Traffic Injury

[0066] RTT: Method for measuring a standardized secretion response in rat small intestine, as published in SE 9000028-2 (publication number 466331 ) for measuring content of AF (ASP).

[0067] SC: Subcutaneously

[0068] SD: Standard Deviation

[0069] SPC: Specially Processed Cereals

[0070] TBI: Traumatic Brain Injury tid or t.i.d.: (ter in die): three times a day

[0071] Definitions

[0072] Proteins are biological macromolecules constituted by amino acid residues linked together by peptide bonds. Proteins, as linear polymers of amino acids, are also called polypeptides. Typically, proteins have 50-800 amino acid residues and hence have molecular weights in the range of from about 6,000 to about several hundred thousand Dalton or more. Small proteins are called peptides, polypeptides, or oligopeptides. The terms “protein”, “polypeptide”, “oligopeptide” and “peptide” may be used interchangeably in the present context. Peptides can have very few amino acid residues, such as between 2-50 amino acid residues (aa).

[0073] The term “antisecretory” refers in the present context to inhibiting or decreasing secretion and / or fluid transfer. Hence, the term “Protein Antisecretory Factor (Protein-AF)” refers to a family of proteins capable of inhibiting or decreasing, or otherwise modulating fluid transfer as well as secretion in a body.

[0074] In the present context, the term “equivalent activity” is used interchangeably with “analogous biological activity”.

[0075] In the present context, the terms an “antisecretory factor protein”, “Protein Antisecretory Factor (Protein-AF)”, “AF- protein”, AF, or a homologue, or fragment thereof, may be used interchangeably with the term “antisecretory factors” or “antisecretory factor proteins” as defined in W097 / 08202, and refer to an Protein Antisecretory Factor (Protein-AF) or a peptide or a homologue, and / or a fragment thereof having antisecretory and / or equivalent functional and / or analogue activity, or to a modification thereof not altering the function of the polypeptide. Hence, it is to be understood that an “antisecretory factor”, “antisecretory factor protein”, “antisecretory peptide”, “antisecretory fragment”, or an “Protein Antisecretory Factor (Protein-AF)” in the present context, also can refer to a homologue or fragment thereof. These terms may all be used interchangeably in the context of the present invention. Furthermore, in the present context, the term “antisecretory factor” may be abbreviated “AF”. Protein Antisecretory Factor (Protein-AF) in the present context refers to a protein with antisecretory properties as previously defined in W097 / 08202 and WOOO / 38535. Antisecretory factors have also been disclosed e.g. in W005 / 030246. Also intended by the term antisecretory factor are native antisecretory factors (NASPs) in egg yolk enriched and / or naturally rich in antisecretory factors as disclosed e.g. in SE900028-2 and WOOO / 38535 and in WO2017 / 009004, as further described below. Additionally, alternative names have been used in the literature for the AF protein.

[0076] Also intended by the term antisecretory factor are native antisecretory factors (NASP) which can be provided in egg yolk with a high content of native antisecretory factors (NASP), as e.g. disclosed in SE900028-2 and WOOO / 38535. Detailed description of the invention

[0077] The current invention relates to a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO: 1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient, wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the measurement of an ICP in the patient of at least 18 mmHg.

[0078] Said patient is typically diagnosed with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP).

[0079] In particular, the current invention relates to a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide AF-16 (VCHSKTRSNPENNVGL) and / or AF-17 (VC(C)HSKTRSNPENNVGL) as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of a nonprovoked elevation of ICP above the threshold value of 18 mm Hg during at least 1 hour before the administration of the pharmaceutical composition, wherein said pharmaceutical composition is administered intravenously, wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, wherein the treatment reduces the ICP in said patient with at least 20% and, wherein the treatment therefore reduces the mortality risk in said patient substantially.

[0080] TBI

[0081] Traumatic brain injury (TBI) is a complex injury with a broad spectrum of symptoms and disabilities. Traumatic brain injury (TBI) is also known as intracranial injury, it occurs when an external force traumatically injures the brain. TBI can be classified based on severity, mechanism (closed or penetrating head injury), or other features (e.g., occurring in a specific location or over a widespread area). In the present context, TBI is also meant to include head injury, i.e. it can involve damage to structures other than the brain, such as the scalp and skull.

[0082] TBI is a major cause of death and disability worldwide, especially in children and young adults. Causes include falls, vehicle accidents, and violence. Brain trauma can occur as a consequence of a focal impact upon the head, by a sudden acceleration / deceleration within the cranium or by a complex combination of both movement and sudden impact. In addition to the damage caused at the moment of injury, brain trauma causes secondary injury, a variety of events that take place in the minutes and days following the injury. These processes, which include alterations in cerebral blood flow and the pressure within the skull, contribute substantially to the damage from the initial injury.

[0083] TBI can cause a host of physical, cognitive, social, emotional, and behavioural effects, and outcome can range from complete recovery to permanent disability or death. In the present context, the following terms and definitions refer to the different injuries relating to TBI, all of which are treatable by administering the pharmaceutical composition as described herein to a patient in need thereof: Closed Head Injury, Open Head Injury, Diffuse Axonal Injury, Contusion, Penetrating Trauma, and Secondary Injury as well as vascular dysfunction.

[0084] Oedema due to TBI

[0085] Cerebral oedema in patients with TBI is a minor subset of TBI. The most representative estimate of the proportion of cerebral oedema in patients with TBI is judged to be 1 .7% reported by Tucker et al. (2017). Alternatively, cerebral oedema in patients with TBI can also be considered a different condition than TBI without cerebral oedema (Jha et al., 2019). A TBI-patient with oedema is managed differently to a TBI patient in general (Raslan and Bhardway, 2017; Chen at al. 2020).

[0086] Cerebral oedema is associated with increased ICP (Winkler et al., 2016) and increased mortality (Donkin and Vink, 2010; Akerlund et al, 2020). Cerebral hypertension due to cerebral oedema is closely linked to a poor outcome after TBI (Vik et al., 2008). The risk of suffering cerebral oedema is substantially lower for mild TBI-cases compared to moderate and severe TBI. Neuroimaging techniques, particularly CT and MRI, can be used to differentially diagnose TBI with and without oedema. The causality between cerebral oedema, elevated ICP and severity of morbidity and mortality in TBI is not necessarily linear though. Akerlund et al., 2020, e.g., found that although the adjusted ICP pressure time dose was strongly correlated to mortality, short periods of high ICP appear more confidently related to worse outcome than long periods of moderately high ICP. Additionally, they found that ICP tolerability appears highly dependent on the cerebral autoregulation status where, in the case of impaired cerebrovascular reactivity, no safe ICP levels could be identified, suggesting that safe limits may need to be related to current autoregulatory status in the future.

[0087] The current inventors for the first and surprisingly demonstrate that the presently disclosed use of administration route and dosage form of the antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, is particularly useful for treating and / or preventing mortality in TBI patients with cerebral oedema if administered as soon as possible in the acute medical care after the TBI injury, even before said patient is diagnosed with an elevated ICP. As can be seen in the experimental section, the very early administration of the pharmaceutical composition as described herein effectively reduces the mortality risk of a TBI patient substantially. Without wishing to be limited to a scientific theory, it is considered that the acute treatment of the patient with the herein disclosed pharmaceutical composition prevents and / or ameliorates elevation of ICP in the patient, as well as reduces already elevated levels of ICP, as well as prevents and / or ameliorates the build-up and / or spreading of the cerebral oedema due to the TBI. Lastly, the acute treatment of the patient with the herein disclosed pharmaceutical composition is in addition believed to modulate local and / or systemic inflammatory response(s) in the patient to the cerebral oedema. In summary, the acute treatment of the patient with a pharmaceutical composition described herein during the initial stage of emergency care and / or hospitalization prevents effectively and / or at least drastically reduces and / or ameliorates the pathophysiological process of the TBI in the patient, i.e., abnormal changes in body functions that are the causes, consequences, or concomitants of the TBI process observed in the patient.

[0088] It has in clinically relevant studies by the current inventors been found that the critical parameter for the reduction of mortality in acute TBI patients with cerebral oedema is the immediate administration of the pharmaceutical composition during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the measurement of ICP of at least 12 mmHg.

[0089] In the present invention, the pharmaceutical composition is intended for treating a human patient in the acute stage after the trauma, who typically is diagnosed with an impairment of consciousness level of between 3-12 on a Glasgow Coma Scale (GCS), such as on a level of < 10 on a Glasgow Coma Scale (GCS). In one embodiment, said patient is a patient with loss of consciousness. The treatment of said patient is initiated by administering the pharmaceutical composition during the initial stage of emergency care and / or hospitalization, and / or no later than 24h after the measurement of ICP of at least 12 mmHg.

[0090] The current invention thus relates to the use of an isolated and / or recombinantly produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for treating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the measurement of ICP to at least 18 mmHg.

[0091] A pharmaceutical composition for use according to the current invention typically lowers the ICP in said patient by between 10-25%, such as between 20-25%, such as by at least 20% after 10 min, 15 min, 30 min, 1 h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, or at the most 12h after the first administration.

[0092] Typically, the ICP in said patient is reduced to between 13-8.5 mmHg after at the most 10 min, 15 min, 30 min, 1 h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 12h, 24h, 48h, 72h, 96h, or 130h after the first administration.

[0093] In one embodiment, the pharmaceutical composition for use according to the current invention is administered 3 times daily for 5 days, starting with a first administration no later than 24h after the measurement of ICP to at least 18 mmHg, such as at least 20mmHg, during the initial stage of emergency care and / or hospitalization.

[0094] In one embodiment, the pharmaceutical composition for use according to the current invention is administered 3 times daily for 5 days, starting with a first administration during the initial stage of emergency care and / or hospitalization even before the measurement of ICP.

[0095] In embodiments, the ICP in said patient is analysed as reduction in brain oedema, assessed as reduction of brain water content (BWC). Reduction of mortality

[0096] As seen in the experimental section, acute treatment of brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), with a pharmaceutical composition according to the current invention results in a favourable short-term outcome, as well as in a favourable long-term outcome for the patient.

[0097] In general, acute treatment with a pharmaceutical composition according to the current invention results in a reduction of damage and / or inflammation in the brain of the patient as well as a lowering of intracranial pressure (ICP) to normal levels in 24 hours after the first administration. In particular, a pharmaceutical composition for use according to the current invention reduces the mortality risk in patients with brain oedema due to a traumatic brain injury (TBI). Typically, the risk of mortality in the patient is reduced by at least 20%, such as at least 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35%.

[0098] In one embodiment, acute treatment with a pharmaceutical composition according to the current invention results in a primary outcome of a decreased 30-day mortality, such as in a primary outcome of a at least 20% or 25%, such as of at least 50% decreased 30-day mortality.

[0099] What is more, acute treatment with a pharmaceutical composition according to the current invention results in a secondary outcome comprising reduction in treatment intensity level, intracranial pressure and / or number of days at the neuro-intensive care unit.

[0100] The antisecretory factor

[0101] A pharmaceutical composition according to the current invention comprises an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the measurement of ICP to at least 18, such as at least 20 mmHg. The antisecretory factor (AF) is a class of proteins that occurs naturally in the body. The human antisecretory factor AF protein (Protein-AF) is a 41 kDa protein, comprising 382- 288 amino acids when isolated from the pituitary gland. The active site can be localized to the protein in a region close to the N-terminal parts of the protein, in particular, it can be localized to amino acids 1-163 of SEQ ID NO:3, more specifically to amino acid positions 35 - 50 on the Protein Antisecretory Factor (Protein-AF) sequence.

[0102] Protein-AF is phylogenetically well preserved and is present in most mammalian tissues, including plasma and can appear associated to the 26 S proteasome or free in the cytoplasm as well as in the nucleus (Lange, 1999; Lange and Lbnnroth, 2001 ; Davidson and Hickey, 2004; Kiss, 2005). AF-16 is a synthetic fragment of the endogenous Protein- AF and contains the antisecretory active site of mammalian AF. AF-17 is the predominantly occurring natural metabolite of Protein-AF and AF-16 in human blood.

[0103] AF-16 is an antisecretory peptide that inhibits epithelial transport of fluid and electrolytes. The anti-oedema effect of AF-16 is proposed to be mediated through various mechanisms, including preventing disruption of tight junction proteins. An inhibitory effect on transcellular and paracellular fluid by AF-16 has been demonstrated in a cellular model of the human intestinal epithelial barrier after bacterial toxin-induced fluid transport (Nicolas and Lievin-Le Moal, 2015). It has been shown that AF-16 is taken up by cells through an endocytic mechanism partially dependent on the presence of cell-surface proteoglycans (Matson and Dzebo et al., 2014).

[0104] AF-16 (VCHSKTRSNPENNVGL) is a peptide with the chemical name L-valyl-L-cysteinyl- L-histidyl-L-seryl-L-lysyl-L-threonyl-L-arginyl-L-seryl-L-asparaginyl-L-prolyl-L-glutamyl-L- asparaginyl-L-asparaginyl-L-valyl-L-glycyl-L-leucine.

[0105] AF-17 (VC(C)HSKTRSNPENNVGL) is a peptide with the chemical name L-valyl-L- disulphide-cysteinyl-L-histidyl-L-seryl-L-lysyl-L-threonyl-L-arginyl-L-seryl-L-asparaginyl-L- prolyl-L-glutamyl-L-asparaginyl-L-asparaginyl-L-valyl-L-glycyl-L-leucine.

[0106] Table 1. Properties of AF-16 / AF-17 drug substance

[0107] Figure 1A. Chemical structure of AF-16

[0108] Figure 1B. Chemical structure of AF-17

[0109] The pharmaceutical composition is presented as a solution for IV infusion, comprising in one embodiment 7.5 mg / mL AF-16 and / or AF-17 (as net peptide). It is possible to use as is for infusion or to dilute in NaCI 9 mg / mL solution for infusion before administration.

[0110] The pharmaceutical composition for use according to the current invention is in one embodiment a composition comprising an AF-16 7.5 mg / mL solution for IV infusion as shown in the experimental section:

[0111] Any amino acid sequence being at least 70% identical, such as being at least 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the amino acid sequence of either AF-16 and / or AF-17 peptide is also considered to be inside the scope of the present invention. By a sequence at least, for example 95% identical to a reference amino acid sequence, is intended that the amino acid sequence of e.g. the peptide is identical to the reference sequence, except that the amino acid sequence may include up to 5-point mutations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acids in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.

[0112] In the present invention, a local algorithm program is best suited to determine identity. Local algorithm programs, (such as Smith Waterman) compare a subsequence in one sequence with a subsequence in a second sequence and find the combination of subsequences and the alignment of those sub-sequences, which yields the highest overall similarity score. Internal gaps, if allowed, are penalized. Local algorithms work well for comparing two multi domain proteins, which have a single domain, or just a binding site in common.

[0113] Methods to determine identity and similarity are codified in publicly available programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al (1994)) BLASTP, BLASTN, and FASTA (Altschul, S.F. et al (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S.F. et al, Altschul, S.F. et al (1990)). Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used.

[0114] The isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), for use according to the present invention may further comprise an N-terminal and / or a C-terminal protecting group. One example of an N-terminal protecting group includes acetyl. One example of a C-terminal protecting group includes amide. As specified in the accompanying sequence listing, some of the amino acids in the herein- specified sequences may be replaced by other amino acids. In the following in this paragraph, the position of a particular amino acid in a particular amino acid sequence is calculated from the left, denoting the most N-terminal amino acid as being in position 1 in that particular sequence. Any amino acid substitution(s) as specified below may be performed independently of any other amino acid substitution(s) in that sequence. In SEQ ID NO 1 ; the C in position 2 may be replaced by S, In SEQ ID NO:1 or 2; the H in position 3 may be replaced with R or K, S in position 4 may be replaced with L, and / or T in position 6 may be replaced with A.

[0115] Pharmaceutical composition

[0116] The current invention in one embodiment relates to the use of a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, which is formulated as a solution for intravenous infusion.

[0117] Such as pharmaceutical composition can further comprise a pharmaceutically acceptable excipient, such as, but not limited to, water, a chelating agent, a tonicity agent and a pH adjusting agent.

[0118] A “pharmaceutical composition”, in the present context, refers to a composition comprising a therapeutically active amount of an antisecretory peptide as shown in SEQ ID NO:1 (AF- 16) and / or SEQ ID NO:2 (AF-17), or a homologue and / or fragment thereof which may be any salt derived therefrom, optionally in combination with a pharmaceutically active excipient, such as a carrier or a vehicle. Said pharmaceutical composition is formulated for the appropriate route of administration, which is intravenous administration. The pharmaceutical composition upon administration presents the active substance to the body of a human or an animal. Said pharmaceutical composition is in the form of a liquid solution.

[0119] The term “pharmaceutically active salt”, in the current context refers to a salt of an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a homologue and / or fragment thereof which may be any salt derived therefrom, based on so-called Hofmeister series. Other examples of pharmaceutically active salts comprise trifluoroacetate, acetate and lysine chloride, the invention is not limited thereto. In one embodiment of the present invention, the pharmaceutical composition according to the invention further comprises a pharmaceutically acceptable excipient. The choice of pharmaceutically acceptable excipient and their optimum concentration for use according to the present invention can readily be determined by the skilled person by experimentation. Pharmaceutically acceptable excipients for use according to the present invention include solvents, buffering agents, preservatives, chelating agents, antioxidants, and stabilizers, emulsifying agents, suspending agents and / or diluents. The pharmaceutical compositions of the invention may be formulated according to conventional pharmaceutical practice, e.g. according to “Remington: The science and practice of pharmacy”, 21st edition, ISBN 0-7817-4673-6 or “Encyclopedia of pharmaceutical technology”, 2nd edition, ed. Swarbrick J., ISBN: 0-8247-2152-7.

[0120] In a currently preferred embodiment, the pharmaceutical excipient is selected from the group consisting of water, a chelating agent, a tonicity agent and a pH adjusting agent.

[0121] A pharmaceutically acceptable excipient is a substance that is substantially harmless to the individual to which the composition is to be administered. Such an excipient normally fulfills the requirements given by the national health authorities. Official pharmacopoeias such as e.g. the British Pharmacopoeia, the United States of America Pharmacopoeia and The European Pharmacopoeia set standards for pharmaceutically acceptable excipients.

[0122] The pharmaceutically acceptable excipients may include solvents, buffering agents, preservatives, chelating agents, antioxidants, and stabilizers, emulsifying agents, suspending agents and / or diluents. Examples of the different agents are given bellow.

[0123] Example of various agents:

[0124] Examples of solvents include but are not limited to water, alcohols, blood, plasma, cerebrospinal fluid, ascites fluid and lymph fluid.

[0125] Examples of buffering agents include but are not limited to citric acid, acetic acid, tartaric acid, lactic acid, hydrogen phosphoric acid, bicarbonates, phosphates, diethylamide, etc.

[0126] Examples of chelating agents include but are not limited to EDTA, di-sodium EDTA dihydrate and citric acid. Examples of antioxidants include but are not limited to butylated hydroxyl anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof.

[0127] Examples of diluents and disintegrating agents include but are not limited to lactose, saccharose, glucose, emdex, calcium phosphates, calcium carbonate, calcium sulphate, mannitol, starches and microcrystalline cellulose.

[0128] Examples of binding agents include but are not limited to saccharose, sorbitol, gum acacia, sodium alginate, gelatine, chitosan, starches, cellulose, carboxymethylcellulose, methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone and polyetyleneglycol.

[0129] In one embodiment of the invention, said pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Such an excipient may be any preferable excipient chosen to be appropriate for the specific purpose. Examples of excipients are disclosed herein.

[0130] Typically, the pharmaceutical composition comprises an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity (the active ingredient), water, a chelating agent, a tonicity agent and a pH adjusting agent.

[0131] The patient

[0132] The current invention relates to a pharmaceutical composition for treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of an elevated intracranial pressure (ICP), such as with an initial increased intracranial pressure (ICP) of at least 12 mmHg,

[0133] A patient in the current context is a human patient.

[0134] A patient to be treated according to the current invention suffers from an acute cerebral oedema due to a TBI.

[0135] A patient to be treated according to the current invention is typically diagnosed with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), a blood brain barrier (BBB) disruption and / or an acute BBB leakage.

[0136] In some embodiments, the patient has a measured ICP of at least 12, 13, 14, 15, 16, 17, or 18mmHg, 20mmHg, such as at least 21 mmHg, 22mmHg or higher, such as between 13-16mmHg, 12-18mmHg or 14-15mmHg, wherein this ICP level is induced by a brain oedema.

[0137] In one embodiment, the patient to be treated suffers from a blood brain barrier (BBB) disruption and / or an acute BBB leakage.

[0138] In one aspect, the patient to be treated suffers from a loss of consciousness, such as a loss of consciousness of > 6 hours.

[0139] In one aspect, administering a pharmaceutical composition for use according to the current invention reduces the cerebral perfusion pressure (CPP) of said patient.

[0140] Typically, a patient to be treated by administering a pharmaceutical composition for use according to the current invention is admitted to hospital due to TBI and displays a closed head injury, preferably signs of cerebral oedema on brain CT or MRI as determined by the investigator, and is diagnosed with a clinical coma on the Glasgow Coma Scale (GCS) of 3-12, such as of 4 to 9 and that requires ICP monitoring.

[0141] After intraparenchymal ICP, an ICP >20 mmHg (unprovoked), such as an ICP >18 mmHg (unprovoked) is preferably measured for a duration of >60 minutes, within 7 days of arrival to hospital.

[0142] Preferably again, the typical patient displays a systolic blood pressure >100 mmHg at enrolment.

[0143] In one currently preferred aspect, a patient to be treated is diagnosed with a Glasgow Coma Score (GCS) of 4-9 (inclusive) due to TBI and has a documented unprovoked ICP of 18 mmHg or higher for at least one hour and signs of brain oedema on a CT or MRI scan. These criteria are allowed to develop during and after admission to hospital ICU. GCS

[0144] The current invention relates to a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient, wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, wherein said patient is diagnosed with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP).

[0145] In one aspect, said patient has a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, such as between 3-8, such as between 3-7, 3-6, 3-5, 3-4, such as between 4-9, or such as between 9-10, 9-11 or 9-12 (Glasgow Coma Scale) GCS. Typically, said patient has a clinical coma severity of at the most 12, such as at the most 11 , 10, 9, 8, 7, 6, 5, 4 or 3 (Glasgow Coma Scale) GCS.

[0146] ICP

[0147] ICP in the current context means intracranial pressure (ICP). Measuring and / or monitoring ICP via an intraventricular catheter one of the two established and accurate monitoring methods. To insert an intraventricular catheter, a hole is drilled through the skull. The catheter is inserted through the brain into the lateral ventricle. This area of the brain contains cerebrospinal fluid (CSF). The other methods for measuring ICP is via a pressure sensor that is inserted through the skull bone through a drilled hole, and then placed between the meningeal sheaths. The evaluation of increased ICP should include detailed history taking, physical examination, and ancillary studies. A funduscopic exam can reveal papilledema which is a tell-tale sign of raised ICP as the cerebrospinal fluid is in continuity with the fluid around the optic nerve.

[0148] The current invention relates to a pharmaceutical composition for treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of an elevated intracranial pressure (ICP), such as with an initial increased intracranial pressure (ICP) of at least 12 mmHg.

[0149] In the current context, suspicion of and / or imminent danger of elevated intracranial pressure (ICP) means that the patient is suspected to have an ICP of at least 12, 13, 14, 15, 16, 17, or 18mmHg, 20mmHg, such as at least 21mmHg, 22mmHg or higher, such as between 13-16mmHg, 12-18mmHg or 14-15mmHg, wherein this ICP level is induced by a brain oedema.

[0150] Administering a pharmaceutical composition for use according to the current invention reduces the ICP in said patient to between 13-8.5mmHg after at the most 10min, 15 min, 30min, 1 h, 1.5hs, 2hs, 2.5hs, 3hs, 4hs, 5hs, 6hs, 12hs, 24hs, 48hs, 72hs, 96hs, or 130hs after the first administration.

[0151] Acute Treatment

[0152] The current invention relates to a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient, wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization.

[0153] The initial stage of emergency care and / or hospitalization in the current context relates to the time period following immediately after when the patient has been diagnosed with a clinical coma severity of between 3-12, such as between 4-9 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), as judged by a healthcare professional, either in prehospital care, after admittance to an emergency care unit, after admittance to an intensive care unit, and any time in between these localizations, and lasts until the patient after regained consciousness (GCS 13-15) has permanently left the Intensive care unit.

[0154] It has been by the current inventors for the first time shown to be imminent to the favourable outcome of the treatment that the treatment is initiated as soon as possible after the initial trauma, since a longer time with a cerebral oedema with or without an elevated ICP level, such as for a time of more than 15 min, 20 min, or 30, or 60 min after prehospital care, after admittance to an emergency care unit, after admittance to an intensive care unit, and any time in between these localizations, exponentially increases the mortality risk and the risk of other unfavourable outcomes, such as lack of recovery or severe disability. Early treatment with the pharmaceutical composition of the current invention leading to a prevented, ameliorated and / or reduced cerebral oedema, inflammation and / or ICP, thereby also lowering the risk of the need for more advanced treatment options such as craniectomy and / or controlled hypothermia and / or barbiturate coma, which in themselves infer a high risk of severe complications such as fatal infections and sepsis.

[0155] As can be seen in the experimental section, acute treatment according to the invention described herein of TBI patients with an acute cerebral oedema, diagnosed with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), leads to a severely reduced mortality risk of no less than 20%.

[0156] Reduction of ICP

[0157] Typically, the treatment with the pharmaceutical composition described herein reduces intracranial pressure (ICP) within 30minutes-1 h after treatment start. In one embodiment, the treatment reduces intracranial pressure (ICP) to below 20mmHg, such as to below 18 mmHg. In one embodiment, the ICP is reduced within 12h after treatment start.

[0158] In one embodiment of the current invention, the ICP is reduced to between 13-8.5mmHg at the latest at day 5 after the initializing of the treatment, such as to 8.5mmHg, 9mmHg, 9.5mmHg, lOmmHg, 10.5mmHg, 11mmHg, 11.5mmHg, 12 mmHg, 12.5mmHg, or 13mmHg. In one embodiment of the current invention, the ICP is reduced to between 13- 8.5mmHg at the latest at day 5 after the initializing of the treatment, such as to between 8.5-12 mmHg, 9-13mmHg, 9.5-12 mmHg, 10-13mmHg, 8.5 -10.5mmHg, 11-13mmHg, 11.5mmHg-12 mmHg, 10-12 mmHg, 9-12.5mmHg, or 12-13mmHg.

[0159] ICP will be measured continuously, and is e.g., measured invasively, using an intraventricular catheter and / or a pressure sensitive probe placed on the meninges, and / or noninvasively, e.g., using transcranial Doppler pulsatile index technique. Obtained measurement values are sampled in a computer system, and data points are then computer derived into average levels over variable time windows, which can be done by presetting a surveillance monitor, with time windows varying from seconds to minutes. Later data derivation to average values can then be done post hoc, to obtain average ICP level values over hourly or multi-hourly intervals.

[0160] In one embodiment, the ICP in said patient is reduced by between 10-25% after 10min, 15 min, 30min, 1 h, 1 ,5hs, 2hs, 2.5hs, 3hs, 4hs, 5hs, 6hs, or at the most 12hs after the initialization of the treatment. In one embodiment, the ICP in said patient is reduced by between 10-25% after at the most12hs, such as at the most 24hs, 48hs, 72hs, 96hs, or 130hs after the initialization of the treatment.

[0161] Reduction of ICP can be measured in mmHg and evaluated as a comparison to a normal range, as a nominal reduction from the initial (baseline) measurement in mmHg, or as a percentage reduction from baseline levels, or as a nominal difference from an upper level of normal.

[0162] Method of treatment

[0163] The current invention relates to a pharmaceutical composition for use in treating and / or ameliorating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), wherein the said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization.

[0164] The present invention relates to methods of treating and / or preventing the development of acute oedema in TBI in a patient in need thereof, by administering a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, to said patient at the latest 24h after the measurement of ICP to at least 18 mmHg.

[0165] Said pharmaceutical composition is administered intravenously.

[0166] A treatment according to the present invention typically leads to signs of clinical benefit and / or reduces the need for deep sedation of the patient.

[0167] In one embodiment, treatment according to the present invention further comprises sedation of the patient without the administration of barbiturates, except during periods of gastroparesis of the patient.

[0168] A treatment according to the present invention typically results in a favourable short-term outcome, as well as in a favourable long-term outcome, such as in a primary outcome of a decreased 30-day mortality. In one embodiment, the treatment results in a primary outcome of a at least 20%, such as of at least 25%, such as of at least 50% decreased 30-day mortality. A treatment according to the present invention further results in a secondary outcome comprising reduction in treatment intensity level, intracranial pressure and / or number of days at the neuro-intensive care unit.

[0169] A treatment according to the present invention further in particular results in a reduction of damage and inflammation in the patient’s brain due to the cerebral and / or brain oedema resulting from the TBL

[0170] A treatment according to the present invention results in a lowering of intracranial pressure (ICP) to normal / non-critically elevated levels in at the latest 24 hours, such as lowering of intracranial pressure (ICP) to of at the most 20 mmHg in 24 hours after the initialization of the treatment. Normal / non-critical levels here being determined in comparison to a normal range, as a nominal reduction from the initial (baseline) measurement in mmHg, or as a percentage reduction from baseline levels, or as a nominal difference from an upper level of normal.

[0171] In one aspect, treatment with a pharmaceutical composition according to the current invention, is combined with other treatments focused on managing ICP elevation, selected from the group consisting of optimal head and neck positioning, optimized pain relief, optimized sedation, negative fluid balance, controlled ventilation, hypocapnia, avoidance of dehydration, and maintenance of normothermia.

[0172] In one aspect, treatment with a pharmaceutical composition according to the current invention, is combined with other treatments focused on managing ICP elevation comprising a medical treatment using anyone selected from the group consisting of mannitol, hypertonic saline solution, diuretics, corticosteroids, barbiturates, and combinations thereof.

[0173] In one aspect, a pharmaceutical composition according to the current invention, is administered to a human patient with loss of consciousness, such as with loss of consciousness of more than 6 hours.

[0174] Typically, treatment with a pharmaceutical composition for use according to the current invention reduces intracranial pressure (ICP) within 10 min - 1 h after intravenous administration of said pharmaceutical composition, such as within 30 min - 1 h after intravenous administration pharmaceutical composition. Treatment with a pharmaceutical composition for use according to the current invention leads to signs of clinical benefit, such as reducing the need for deep sedation of the patient. Alternatively, treatment with a pharmaceutical composition according to the current invention can further comprise sedation of the patient without the administration of barbiturates, except during periods of gastroparesis of the patient.

[0175] One aspect of the current invention relates to a use of an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for manufacturing of a pharmaceutical composition for use in treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, and an initial increased intracranial pressure (ICP) of at least 12 mmHg, wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated no later than 24h after the raise of ICP to at least 18 mmHg.

[0176] One aspect of the current invention relates to a method of treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, and an initial increased intracranial pressure (ICP) of at least 12 mmHg, comprising administering intravenously a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, wherein the treatment is initiated no later than 24h after the raise of ICP to at least 18 mmHg in said patient.

[0177] Administration route

[0178] The pharmaceutical composition for use according to the invention is in the present context administrated intravenously, preferably through a drip and / or a syringe.

[0179] A pharmaceutical composition for use according to the current invention is administered intravenously as an infusion.

[0180] In a currently preferred aspect, patients are administered AF-16 and / or AF-17 as a 40- minute intravenous infusion every 8 hours for a total of 5 days. Patients will be managed according to local standard care and will receive standard of care pharmacological and non-pharmacological treatment as clinically indicated. (Veerappan et al, 2022; Neurotrauma Society of India, 2022; Carney et al, 2017). Intraparenchymal ICP monitoring is in place for all patients at the start of the treatment.

[0181] Dosage / Intervals

[0182] The current invention relates to a pharmaceutical composition for use in treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of an elevated intracranial pressure (ICP), such as with an initial increased intracranial pressure (ICP) of at least 12 mmHg, comprising administering intravenously a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the raise of ICP to at least 18 mmHg in said patient, and wherein the composition comprises at least 7.5 mg / mL of said isolated recombinant and / or synthetically produced peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity.

[0183] In one embodiment, the treatment with the pharmaceutical composition for use according to the current invention is initiated during the initial stage of emergency care and / or hospitalization even before the measurement of ICP.

[0184] In one embodiment, the pharmaceutical composition is administered to a patient when said patient has a measured ICP of at least 12, 13, 14, 15, 16, 17, or 18 mmHg, 20 mmHg, such as at least 21 mmHg, 22 mmHg or higher.

[0185] In one embodiment, the pharmaceutical composition is administered once.

[0186] In one embodiment, the pharmaceutical composition is administered repeatedly, such as twice, three times or more daily.

[0187] In one embodiment, the pharmaceutical composition is administered once daily, or repeatedly daily during at least 1 , 2, 3, 4, or 5 days of treatment. In a preferred embodiment, a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide as shown in SEQ ID NO:1 (AF-16) and / or SEQ ID NO:2 (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, is administered no later than 24h after the measurement of ICP to at least 18 mmHg, such as of at least 18 mmHg, 20 mmHg, 21 mmHg, 22 mmHg or higher.

[0188] The pharmaceutical composition is in one embodiment administered at a dosage of 1-50 mg / kg body weight as a 10-60 minute-infusion, such as a 10-40 minute infusion, such as a 10-30 minute infusion, such as at a dosage of 10 mg / kg body weight as a 10 minute- infusion or at a dosage of 30 mg / kg body weight as a 30 minute-infusion or at a dosage of 50 mg / kg body weight as a 40 minute-infusion.

[0189] In one embodiment, the pharmaceutical composition is administered at a dosage of 1- 50mg / kg as a 10-40 minute-infusion three times daily, such as at a dosage of at least 30mg / kg as a 40 minute-infusion. In a currently preferred embodiment, the pharmaceutical composition is administered at a dosage of 50mg / kg as a 40 minute-infusion three times daily.

[0190] In one aspect, a pharmaceutical composition for use according to the current invention is administered at a dosage of 1-50 mg / kg body weight as a 10-60 minute-infusion, such as a 40 minute-infusion three (3) times daily (tid).

[0191] In one aspect, a pharmaceutical composition for use according to the current invention is administered at a dosage of 1-50 mg / kg body weight.

[0192] In one aspect, a pharmaceutical composition for use according to the current invention is administered as a 10-60 minute-infusion, such as a 40 minute-infusion.

[0193] In one aspect, a pharmaceutical composition for use according to the current invention is administered at a dosage of 10 mg / kg body weight as a 10 minute-infusion.

[0194] In one aspect, a pharmaceutical composition for use according to the current invention is administered at a dosage of 30 mg / kg body weight as a 30 minute-infusion.

[0195] In one embodiment, the pharmaceutical composition is administered at a dosage of 50 mg / kg body weight as a 40 minute-infusion. In one embodiment, the pharmaceutical composition for use according to the current invention is administered 3 times daily for 5 days, starting with a first administration no later than 24h after the measurement of ICP to at least 18 mmHg, during the initial stage of emergency care and / or hospitalization. Such as at a dosage of 50 mg / kg body weight as a 40 minute-infusion which is administered 3 times daily for 5 days.

[0196] In one embodiment, the pharmaceutical composition for use according to the current invention is administered 3 times daily for 5 days, starting with a first administration during the initial stage of emergency care and / or hospitalization even before the measurement of ICP.

[0197] In a currently preferred aspect, AF-16 and / or AF-17 will be given as an intravenous infusion over 40 minutes in a separate intravenous line 3 times daily, every 8 hours, for 5 days.

[0198] AF-16: 450 mg of AF-16 (as net peptide) in a 10 mL vial (7.5 mg / mL solution for intravenous infusion), i.e. for one dose, six vials are used.

[0199] The IMP vials are stored at -20oC. The medication vials is added to a NaCI infusion bag (for example Baxter Viaflo 100 mL).

[0200] The duration of the treatment is preferably three times daily intravenous administrations of AF-16 and / or AF-17 for 5 consecutive days.

[0201] Experimental section

[0202] Experiment 1

[0203] Safety, tolerability, and pharmacokinetics of AF-16 evaluated in an adaptive, randomised, double-blind, single-centre, placebo-controlled phase I, First-In-Human (FIH) study.

[0204] The completed First-in-Human clinical study, combines single and multiple ascending doses of AF-16 intravenous infusion administered. A total of 73 healthy adult volunteers (60 females, 13 males) were exposed to AF-16 as single or multiple doses up to OD for 5 days or TID for 2 days. The study was divided in 2 parts. Part I, a Single Ascending Dose (SAD) in 48 healthy female subjects (36 subjects received a 40-min intravenous infusion of AF-16). Part II, a Multiple Ascending Dose (MAD) in 49 healthy female and male subjects. MAD cohorts 1 and 2 comprised 2 sequential ascending dose cohorts, in which AF-16 was administered once daily (OD) for 5 consecutive days to 12 female subjects. MAD cohorts 3, 4 and 5 comprised 3 sequential ascending dose cohorts, in which AF-16 was administered 3 times daily (TID) for 2 consecutive days. From MAD cohort 3 and onwards, male subjects were also included (cohort 3: 6 females and 6 males; cohort 4: 4 females and 3 males; cohort 5: 3 females and 3 males). In summary, AF-16 administered intravenously in single ascending doses (3 mg to 1350 mg) and in multiple ascending doses (75 mg to 675 mg once daily for 5 days, 225 mg to 675 mg 3 times daily for 2 days) to healthy volunteers was safe and well tolerated as assessed by AEs, ECGs, vital signs, safety laboratory parameters and physical examinations. There were no serious adverse effects, or any adverse effects assessed as related to AF-16 study medication. AF-16 rapidly reached a maximum blood concentration, and the blood concentration decreased rapidly after end of infusion. The mean CL was high. The apparent terminal half-life was estimated to approximately 26 to 101 minutes, with a considerably shorter initial half-life. Dose proportionality analysis suggests a proportional to somewhat supra-proportional increase in exposure relative to dose, with large variations between dose groups and dose occasions. Similar patterns were seen for AF-17.

[0205] Generally, doses and concentrations are expressed as net peptide content, i.e. 7.5 mg AF-16 corresponds to approximately 10 mg AF-16 TFA as the net peptide content in the drug substance batches is approximately 75%. However, in most safety pharmacology and all toxicity studies the doses and concentrations are expressed as AF-16 TFA, i.e. 10 mg AF-16 TFA corresponds to approximately 7.5 mg AF-16. Table 1 is a help to ’translate’ doses depending on how they are expressed.

[0206] Table 1 : AF-16 doses expressed as AF-16 TFA and AF-16 and concentrations of AF-16 in pM and ng / mL

[0207] Composition

[0208] The composition is an AF-16 7.5 mg / mL solution for intravenous infusion in water: No novel excipients or excipients of human / animal origin are used in the formulation.

[0209] AF-16 powder for solution for infusion contains a synthetic fragment of the endogenous Antisecretory Factor (AF) protein. AF-16 consists of 16 natural amino acids and is isolated as a lyophilized powder where trifluoroacetic acid (TFA) acts as a counter ion.

[0210] For phase 1 (FIH): A frozen solution, AF-16 7.5 mg / mL solution for intravenous infusion, that will be thawed at room temperature and diluted to suitable volumes, using NaCI 9 mg / mL solution for infusion from Baxter Viaflo at the CTU prior to dosing will be used. The solution is isotonic.

[0211] AF-16 7.5 mg / mL solution for intravenous infusion is stored frozen at -20°C. It should be thawed in room temperature prior to administration. In the phase 2 study it will be diluted with NaCI 9 mg / mL solution for infusion to 250 ml. The diluted solution is stable at room temperature for 24 hours. A tabular overview of the clinical trials conducted with AF-16 following a 40 min intravenous infusion is given in the Table 2 below.

[0212] Table 2: Summary of completed studies with AF-16

[0213] Pharmacokinetics and Drug Metabolism (AF-16)

[0214] Single Intravenous Infusion

[0215] The PK profile of AF-16 in healthy female volunteers was investigated following administration of AF-16 TFA as an intravenous infusion over 40-min. The maximum mean blood concentration (Cmax) was reached quickly after start of infusion of AF-16 and the mean blood concentrations declined rapidly after the end of infusion (40 minutes).

[0216] The AF-16 exposure, both in terms of Cmax and AUCo-iast, increased with dose in the range 3 mg to 1350 mg. The variability of the exposure parameters was rather high; the calculated coefficients of variation (CV%) for Cmax and AUCo-iast ranged from of 31 .3 to 60.5% and from 24.6 to 39.5%, respectively. The median time to Cmax (Tmax) per dose group for AF-16 varied between 20 to 30 minutes.

[0217] AUCo -inf, Lambda_z, ti / 2, CL, Vzor Vssfor AF-16 were not possible to determine for dose groups 1 to 3 (3 to 75 mg). In addition, some calculated AUCo-inf values did not fulfil all the Lambda_z acceptance criteria. Because of these reservations associated with AUCo-inf determination, the parameter AUCo-iast was used for the dose proportionality analysis. Lambda_z was calculated with the best fit option with start not before 0.91 hour.

[0218] The blood CL for AF-16 was high. The mean CL for dose groups 4 (225 mg), 5 (675 mg) and 6 (1350 mg) was 1319, 1421 and 915.7 L / h, respectively. For dose groups 4 (225 mg), 5 (675 mg) and 6 (1350 mg), the mean distribution volume associated with the terminal elimination phase (Vz) and mean the distribution volume at steady state (Vss) ranged between 628.0 and 1019 L and between 90.58 and 127.8 L, respectively. The apparent mean AF-16 terminal half-life (ti / 2(z>) for dose groups 4 (225 mg), 5 (675 mg) and 6 (1350 mg) ranged between 26 to 33 minutes. Rough estimates, based on the period immediately after end of infusion, suggests a considerably shorter initial half-life (approximately 1.5 minutes).

[0219] Dose proportionality (single dose)

[0220] Dose proportionality analyses of all individual AUCo-iast and Cmax values for AF-16 within the 6 dose groups revealed dose proportionality constants of 1 .22 for AUCo-iast and 1 .23 for Cmax, which suggest a somewhat supra-proportional increase in exposure relative to dose within the 3-1350 mg range.

[0221] Multiple Intravenous Infusions

[0222] The PK profile of AF-16 in healthy female and male volunteers was investigated following administration of AF-16 TFA as an intravenous infusion over 40-min for up to 5 days once daily (OD) (75 mg and 675 mg) or three time a day (TID) for 2 days (225 mg, 450 mg, and 675 mg). In line with SAD part of the study, the maximum mean blood concentration of AF-16 was reached quickly after start of infusion for both the first and the last dose, and the mean blood concentrations declined rapidly after the end of infusion (40 minutes).

[0223] The exposure, both in terms of Cmax and AUCo-iast, increased with dose in the range 75 mg OD to 675 mg TID. There were no obvious differences between males and females regarding exposure.

[0224] As calculated from the last dose in each group, the blood CLSSfor AF-16 was high. The mean CL for dose groups 2 (675 mg OD), 3 (225 mg TID), 4 (450 mg TID) and 5 (675 mg TID) was 1640, 1395, 1295 and 1861 L / h, respectively. For dose groups 2 (675 mg OD), 3 (225 mg TID), 4 (450 mg TID) and 5 (675 mg TID), the mean Vssranged between 83.90 and 382.2 L.

[0225] The mean AF-16 ti / 2(z) for dose groups 2 (675 mg OD), 3 (225 mg TID), 4 (450 mg TID) and 5 (675 mg TID) was 34, 38, 62 and 101 minutes, respectively. The ti / 2(z) increase with dose, which is likely due to later timepoints above LLOQ at higher doses. Rough estimates, based on the period immediately after end of infusion, suggests a considerably shorter initial ti / 2 (approximately 1.5 minutes).

[0226] Based on AUCo-iast and Cmax, an apparent accumulation is indicated for all dose groups, up to an almost 2-fold higher mean exposure in dose group 3 (225 mg TID) after the last dose compared to the first dose (mean accumulation ratio: 1 .975 for Cmax and 1.834 for AUCo-iast). The apparent accumulation cannot be explained from residual pre-dose concentrations of AF-16.

[0227] Dose proportionality (repeated dosing)

[0228] After the first dose, dose proportionality analyses of all individual AUCo-iast and Cmax values for AF-16 within the 5 dose groups (4 dose levels) revealed dose proportionality constants of 1 .19 for AUCo-iast and 1 .13 for Cmax. The corresponding values after the last dose were 1 .16 for AUCo-iast and 1 .06 for Cmax. Similarly to what was seen in the SAD part, this suggests a somewhat supra-proportional increase in exposure relative to dose within the 75 to 675 mg range after multiple dosing. However, dose-normalised PK parameters for AUCo-iast and Cmax showed high variability between dose groups and dose occasions.

[0229] Pharmacokinetics and Drug Metabolism (AF-17, metabolite of AF-16)

[0230] Single Intravenous Infusion

[0231] AF-16 is rapidly metabolized to AF-17 (cysteine adduct) in blood. The maximum mean blood concentration (Cmax) was reached quickly after start of infusion of AF-16 and the mean blood concentrations declined rapidly after the end of infusion (40 minutes). Similar patterns were seen for AF-17 as for AF-16, although the higher LLOQ for AF-17 likely explains the differences in the shapes of the blood-concentration-time curves. Blood concentrations of AF-17 was not determined for dose group 1 (AF-16, 3 mg) and in dose group 2 (AF-16, 15 mg), there were too few blood concentrations above the LLOQ to allow calculation of PK parameters.

[0232] The AF-17 exposure, both in terms of Cmax and AUCo-iast, increased with dose in the range 75 mg to 1350 mg. In general, the blood exposure to AF-17 was higher than for AF-16. Tmax for AF-17 was approximately 25 minutes for all 4 dose groups. Rough estimates, based on the period immediately after end of infusion, suggests a short initial half-life for AF-17 (approximately 2 minutes).

[0233] Lambdazwas calculated with the best fit option with start not before 0.91 hour and was not possible to be determined for any AF-17 PK profiles. Hence, it was not possible to determine AUCo-int and ti / 2(z) for AF-17.

[0234] Dose proportionality (single dose) For AF-17, dose proportionality analysis of individual AUCo-iastand Cmax values for dose groups 3 to 6 revealed dose proportionality constants of 0.98 for AUCo-iast and 0.86 for Cmax, which suggests a proportional increase in AF-17 exposure relative to dose of AF-16 in the 75 to 1350 mg range.

[0235] Multiple Intravenous Infusions

[0236] Mean blood concentrations of AF-17 were analysed after the first and last dose following the infusion of AF-16 TFA. Similar patterns were seen for AF-17 as for AF-16, although the higher LLOQ for AF-17 likely explains the differences in the shapes of the blood concentration-time curves. No obvious trend of increased concentrations with every dose was seen, however with daily variations.

[0237] As calculated from the last dose in each group, the AF-17 exposure, both in terms of Cmax and AUCo -last, increased with dose in the range 75 mg OD to 675 mg TID, with the highest exposure seen in dose group 2 (675 mg OD). There were no obvious differences in the formation of AF-17 between males and females. Based on Cmax and AUCo-iast for AF-17, no accumulation was indicated between the first and last dose. The accumulation ratio for Cmax and AUCo-iast ranged from 1 .087 to 1 .270 and 1 .027 to 1 .259, respectively.

[0238] Dose proportionality (repeated dosing)

[0239] After the first dose, dose proportionality analyses of all individual AUCo-iast and Cmax values for AF-17 within the 5 dose groups of AF-16 (4 dose levels) revealed dose proportionality constants of 1 .14 for AUCo-iast and 1 .05 for Cmax. The corresponding values after the last dose were 1.12 for AUCo-iast and 1.02 for Cmax. Similar to what was seen in the SAD part, this suggests a fairly proportional increase in AF-17 exposure relative to dose of AF-16 in the 75 to 675 mg range, which is also what is suggested from dose-normalised PK parameters.

[0240] Dosage and administration

[0241] AF-16 is presented as a sterile, isotonic solution for intravenous infusion, delivered in frozen condition. The drug product was thawed in room temperature and diluted in NaCI 9 mg / mL according to a separate handling instruction. A dose of 450 mg was given as intravenous infusion for 40 minutes administered in a peripheral vein, with a dosing interval of 8 hours. Co-administration with other intravenous treatments than normal saline solution has not been studied and is therefore not allowed in the study.

[0242] Pharmacokinetics Single Intravenous Infusion (SAD)

[0243] The PK profile of AF-16 in healthy female volunteers was investigated following single dose administration of AF-16 TFA as an intravenous infusion over 40-min. The maximum mean blood concentration (Cmax) was reached quickly after start of infusion of AF-16 and the mean blood concentrations declined rapidly after the end of infusion.

[0244] The AF-16 exposure, both in terms of Cmax and AUCo-iast, increased with dose in the range 3 mg to 1350 mg, with a rather high variability of the exposure. The median time to Cmax (tmax) per dose group for AF-16 varied between 20 to 30 minutes.

[0245] The blood CL for AF-16 was high. The mean CL for dose groups 4 (225 mg), 5 (675 mg) and 6 (1350 mg) was 1319, 1421 and 915.7 L / h, respectively. For dose groups 4 (225 mg), 5 (675 mg) and 6 (1350 mg), the mean distribution volume associated with the terminal elimination phase (Vz) and mean the distribution volume at steady state (Vss) ranged between 628.0 and 1019 L and between 90.58 and 127.8 L, respectively.

[0246] The apparent mean AF-16 terminal half-life (ti / 2(z>) for dose groups 4 (225 mg), 5 (675 mg) and 6 (1350 mg) ranged between 26 to 33 minutes. Rough estimates, based on the period immediately after end of infusion, suggests a considerably shorter initial half-life of approximately 1.5 minutes.

[0247] Dose proportionality analyses suggest a somewhat supra-proportional increase in exposure relative to dose within the 3-1350 mg range.

[0248] Multiple Intravenous Infusions (MAD)

[0249] The PK profile of AF-16 in healthy female and male volunteers was investigated following administration of AF-16 TFA as an intravenous infusion over 40-min for up to 5 days once daily (OD) (75 mg and 675 mg) or three time a day (TID) for 2 days (225 mg, 450 mg, and 675 mg). In line with SAD part of the study, the maximum mean blood concentration of AF-16 was reached quickly after start of infusion for both the first and the last dose, and the mean blood concentrations declined rapidly after the end of infusion (40 minutes), The exposure, both in terms of Cmax and AUCo-iast, increased with dose in the range 75 mg OD to 675 mg TID. There were no obvious differences between males and females regarding exposure.

[0250] As calculated from the last dose in each group, the blood CLSSfor AF-16 was high. The mean CL for dose groups 2 (675 mg OD), 3 (225 mg TID), 4 (450 mg TID) and 5 (675 mg TID) was 1640, 1395, 1295 and 1861 L / h, respectively. For dose groups 2 (675 mg OD), 3 (225 mg TID), 4 (450 mg TID) and 5 (675 mg TID), the mean Vssranged between 83.90 and 382.2 L.

[0251] The mean AF-16 ti / 2(z) for dose groups 2 (675 mg OD), 3 (225 mg TID), 4 (450 mg TID) and 5 (675 mg TID) was 34, 38, 62 and 101 minutes, respectively. The ti / 2(z) increase with dose, which is likely due to later timepoints above LLOQ at higher doses. Rough estimates, based on the period immediately after end of infusion, suggests a considerably shorter initial ti / 2 of approximately 1.5 minutes.

[0252] Based on AUCo-iast and Cmax, an apparent accumulation is indicated for all dose groups, up to an almost 2-fold higher mean exposure in dose group 3 (225 mg TID) after the last dose compared to the first dose (mean accumulation ratio: 1 .975 for Cmax and 1 .834 for AUCo-iast). The apparent accumulation cannot be explained from residual pre-dose concentrations of AF-16.

[0253] After the first dose, dose proportionality analyses of all individual AUCo-iast and Cmax values for AF-16 within the 5 dose groups (4 dose levels) revealed dose proportionality constants of 1 .19 for AUCo-iast and 1 .13 for Cmax. The corresponding values after the last dose were 1 .16 for AUCo-iast and 1 .06 for Cmax. Similar to what was seen in the SAD part, this suggests a somewhat supra-proportional increase in exposure relative to dose within the 75 to 675 mg range after multiple dosing. However, dose-normalised PK parameters for AUCo-iastand Cmax showed high variability between dose groups and dose occasions.

[0254] Safety Evaluation

[0255] A total of 73 healthy adult volunteers (60 females, 13 males) were exposed to AF-16 as single or multiple doses up to OD for 5 days or TID for 2 days. There were no deaths, other SAEs or withdrawals due to AEs in the study.

[0256] In the completed study:

[0257] • One subject in the MAD part chose to stop further treatment during Dose 6, due to experiencing AE (anxiety, assessed as unlikely related to the IMP treatment). The subject completed the follow up visits and hence the study.

[0258] • A vast majority of the AEs were mild in intensity. The most common AE in both SAD and MAD parts was headache, which was reported in similar proportion by subjects receiving AF-16 or placebo. In the SAD part, more AEs were reported in the higher dose groups. In the MAD part, with the exception of infusion site reactions, there was no obvious trend in terms of AE reporting frequency with increasing dose of AF-16. Mild infusion site reactions (mainly infusion site pain) were more common in dose groups with 3 times daily dosing and more common for subjects receiving active treatment.

[0259] • There were no clinically significant changes from baseline in mean ECG, vital signs, clinical chemistry, haematology, coagulation or urinalysis parameters or physical examination findings over time and no clinically significant patterns in any parameter associated with increasing dose of AF-16.

[0260] • In the MAD part, 1 subject had elevated ALT and AST values assessed as abnormal clinically significant at 6 days after last dose of AF-16. The subject had low or normal ALT / AST values at screening and the values then increased slightly over time throughout the study. At 48 hours after start of dosing, the subject was confirmed to have a Cytomegalovirus infection, believed to be the cause of the increasing transaminases. ALT / AST values had returned to normal at 3 weeks after dosing.

[0261] • In the MAD part, there was no indication of AF-16 anti-drug antibody development at 25 days after the first IMP treatment.

[0262] In conclusion, in the First in Human, combined SAD and MAD, study, AF-16 demonstrated an acceptable safety and tolerability in healthy Caucasian male and females when administered as single and multiple doses of up to 1350 mg as single dose and in doses up to 2025 mg daily as multiple doses, given as intravenous infusion over 40 minutes. There were no deaths, other SAEs or withdrawals due to AEs. All AEs were mild or moderate.

[0263] Safety margins and precautions

[0264] Based on the obtained non-clinical exposure, the safety margin towards the intended clinical dose of 450 mg i.v. TID, there are indications of different safety margins for men and women. For male subjects there is a 6-fold safety margin compared to rat, where there NOAEL was based on findings in Sertoli cells, and 38-fold in minipig, where there were no histopathological findings. For female subjects there is a 70-fold compared to rat and a 60-fold safety margin compared to minipig.

[0265] Safety data from the First in Human study shows no safety concerns. There are no antidotes to AF-16. Should the investigator note any possible treatment related adverse effects of clinical significance during an ongoing i.v. infusion of AF-16, the infusion should be terminated. Any adverse effects of clinical significance should be treated symptomatically. Due to the short half-life of AF-16 of 1 .5 minutes, any treatment related adverse effects would be expected to spontaneously wane within a few minutes.

[0266] In conclusion, AF-16 administered intravenously in single ascending doses (3 mg to 1350 mg) and in multiple ascending doses (75 mg to 675 mg once daily for 5 days, 225 mg to 675 mg 3 times daily for 2 days) to healthy volunteers was safe and well tolerated as assessed by AEs, ECGs, vital signs, safety laboratory parameters and physical examinations.

[0267] Experiment 2

[0268] TBI patients with acute brain oedema:

[0269] This Phase 2a exploratory study was a multicenter, open-label, historical cohort control study to evaluate the safety and tolerability of AF-16 in patients with cerebral oedema (brain swelling) caused by TBI. The study was conducted during 2023 and involved 5 trial sites located in Europe.

[0270] Study Objectives:

[0271] Primary Objective:

[0272] • To evaluate the safety and tolerability of 5 days of antisecretory factor AF-16 treatment in patients with cerebral oedema (brain swelling) because of TBI.

[0273] Secondary Objectives:

[0274] • To explore the efficacy of 5 days of AF-16 treatment compared with historical controls as measured by intracranial pressure (ICP) in patients with cerebral oedema (brain swelling) because of TBI.

[0275] • To explore the use of concomitant therapies (therapy intensity level [TIL]) after 5 days of AF-16 treatment compared with historical controls in patients with cerebral oedema (brain swelling) because of TBI.

[0276] Exploratory Objectives

[0277] • To determine exposure of AF-16 and its metabolite AF-17.

[0278] • To explore the effect of 5 days of treatment with AF-16 on the Glasgow Outcome Scale- Extended (GOS-E) on Day 14 and Day 30 compared with historical controls. • To explore the effect on cerebral oedema (brain swelling) as assessed by computed tomography (CT) after 5 days of treatment with AF-16 (if feasible).

[0279] • To explore the efficacy of 5 days of AF-16 treatment compared with historical controls as measured by the use of concomitant therapies (TIL).

[0280] • To explore the effect of AF-16 treatment on blood biomarkers of neuronal damage.

[0281] These endpoints were assessed as:

[0282] Primary endpoint assessment:

[0283] • Frequency and severity of adverse events (AEs) in patients treated with AF-16

[0284] Assessment of secondary endpoints:

[0285] • Changes in vital signs, physical examination results, electrocardiogram, clinical chemistry, hematology, coagulation, and hormonal values during the study period

[0286] Secondary endpoint assessments:

[0287] ICP was measured continuously during the time spent in the ICU. Collected endpoints included daily maximum ICP and average daily ICP.

[0288] TIL area under the curve (AUC) from Day 1 through Day 5 or earlier in the case of early discontinuation

[0289] • Total time spent with insults of ICP >20 mmHg lasting longer than 5 minutes from the start of AF-16 treatment to the end of AF-16 treatment (i.e., Day 5 or earlier in the case of early discontinuation)

[0290] Results:

[0291] Demographics:

[0292] The control group consists of 22 historical controls with prospectively collected data from the CENTER-TBI study, matched for age, sex, type of injury and severity of injury (CT scan classification, Marshall Score (a scoring tool for categorizing CT findings related to TBI), and Glasgow Coma Scale [GCS]).

[0293] Six patients were recruited to the trial. One was excluded due to having a penetrating injury (exclusion criterion). The safety analysis included five patients (i.e., all those having received at least one dose of AF-16) and the efficacy analysis included 4 patients (i.e., all those having received > 24 hours of treatment with AF-16). The historical cohort patients included in the study (N-22) were selected from the CENTER-TBI study via PSM. All five patients in the AF-16 / safety population suffered a closed TBI caused by accidental fall (three patients; 60.0%), traffic accident either as a pedestrian (one patient; 20%) or a car driver / passenger (one patient; 20%). In the historical cohort, all 22 (100.0%) patients suffered a closed TBI caused by road traffic incident (12 patients; 54.5%), incidental fall (8 patients; 36.4%), other causes (one patient; 4.5%) or from an unknown reason (one patient; 4.5%).

[0294] Intracranial haemorrhage was present in all five patients (100.0%) in both the AF-16 and historical control groups. The haemorrhage was untreated in one patient in the AF- 16 / safety group, whereas in the historical control 8 (36.4%) were untreated. In the AF-16 group, two patients (40.0%) were categorized as having a Class II in Marshall CT score, one patient (20.0%) Class IV and another two patients (40.0%) Class V. In the historical controls, nine patients (40.9%) had a Class II Marshall CT score, six patients (27.3%) Class III and seven patients (31 .8%) Class VI.

[0295] Safety results:

[0296] All five patients (100.0%) in the AF-16 / safety group experienced at least one TEAE and at least one treatment-emergent SAE occurred in three patients (60.0%); all were considered unrelated to the study treatment.

[0297] The most frequently reported TEAEs with an incidence of 40% were Pneumonia (two patients) and constipation (two patients). Three TEAEs of ‘severe’ intensity were observed in three patients (60.0%) in the AF-16 / safety group including, Intracranial pressure increased (SOC: Nervous system disorders).

[0298] Haemoglobin decreased (SOC: Investigations), and Brain death (SOC: General disorders and administration site conditions). None of these events were deemed related to AF-16 by investigators.

[0299] Three deaths occurred during the study, and all were deemed unrelated to AF-16 by investigators. The two deaths were fatal outcomes following AEs. The third death occurred in a patient who received only a single dose of AF-16 and was therefore included in the safety population.

[0300] A total of five treatment-emergent SAEs occurred in three patients (60.0%) in the AF- 16 / safety group. The most commonly reported SAEs were Pneumonia aspiration and Brain death. TEAEs that led to the study treatment discontinuation occurred in two (40.0%) patients in the AF-16 / safety group. Two such events (Intracranial pressure increased and Brain death) were ‘severe’ in intensity, thus deemed serious. One event (Brain death) resulted in patient death.

[0301] Overall, there were no laboratory changes related to IP, and all out-of-range values, except for the anaemia related to the SAE anaemia, were, as expected, the result of the TBI as such or due to the pulmonary AEs. Similarly, there were no changes in ECG, vital signs, physical findings, or other observations related to safety that were not considered as expected as a result of a the TBI and the standard of care treatment of the TBI.

[0302] Efficacy results:

[0303] The baseline mean maximum ICP in the AF-16 efficacy analysis group was higher than historical control. It was 23.25 (±4.031 ), and 14.31 (±8.911) and 11.00 (±6.164) in the AF- 16, and historical cohorts respectively. Despite showing lower value at baseline, the mean maximum ICP numerically increased after baseline, although SD values were high across all groups. These findings were also reflected in the change from baseline where a negative change (decline) was observed in the AF-16 group and the control group on any of the days (Day 1 to Day 7) of assessment.

[0304] Similar results were also seen when examining average ICP, in absolute terms. When changes from baseline were considered, in the AF-16 group and in alignment with the results observed with maximum ICP, the average ICP remained below baseline for all 7 days of observation, with corresponding negative values for the change from baseline, except on Day 2. The results for the change from baseline were more inconsistent for the control group, though an increase from baseline was seen on most of the seven days of observation.

[0305] The mean 24-hour AUC ICP for the AF-16 group was higher than for the control group, on each day assessed. There was an increase (from 15.75±2.021 to 16.63±8.845) on Day 2 followed by a decline on Day 3 and Day 4 and it remained approximately the same until Day 5. Similar patterns were observed in the control group, although with numerically slightly lower mean AUC values. In line with the 24-hour AUC results, the historical control group showed lower mean ICP Day 1 to Day 5 AUC values than the AF-16 group, demonstrating that the patients in the AF-16 group had more severe level of injury than the patients in the control group.

[0306] In terms of absolute 24-hour TIL score however, although the absolute value of the mean TIL score was numerically higher in the AF-16 group, it declined more consistently in comparison to historical controls by Day 7.

[0307] MMRM analysis of the ICP and TIL AUC data revealed no significant differences for the 24-hour AUC values between the AF-16 and historical controls, on any of the 5 days of assessment. The same was also found for overall, Day 1 to Day 5 AUCs.

[0308] Results

[0309] Table 3: Summary

[0310] All AF-16 Patients survival data

[0311] Table 4: Patient 1 Table 7: Patient 4

[0312] Table 8: Overall survival comparison

[0313] Table 9: Granular comparison of 30-day mortality in relation to expected risk Conclusions:

[0314] Delays in the study recruitment and consequently product stability complications, prohibited enrollment of the originally planned 12 patients to allow for a more comprehensive dataset and statistical analyses. Nevertheless, the differences observed between the AF-16 group and the PSM-selected control group, namely a decline in the maximum and mean ICP values over the 7-day observation period, as well as in the TIL scores are, and to some extent in the ICP and TIL AUC data are encouraging, especially considering this higher ICP, higher risk TBI population. These effects are likely to underscore the fact that the death rate seen following treatment with AF-16 is lower than what would have been expected and that it is extremely important to start the AF-16 treatment as soon as possible, i.e., that the treatment is initiated during the initial stage of emergency care and / or hospitalization.

[0315] References

[0316] • Prakash and Carmichael 2015

[0317] • Winkler et al., 2016

[0318] • Teasdale and Jennett, 1974

[0319] • Maas AIR, Menon DK, Adelson PD, Andelic N, Bell MJ, Belli A, et al. Trauma=c brain injury: integrated approaches to improve preven=on, clinical care, and research. Lancet Neurol. (2017) 16:987-1048. doi: 10.1016 / S1474- 4422(17)30371 -X

[0320] • Tagliaferri F1 , Compagnone C, Korsic M, Servadei F, Kraus J.

[0321] • A systematic review of brain injury epidemiology in Europe. Acta Neurochir 148:255- 68, 2006.

[0322] • Godoy et al. , 2016;

[0323] • Sandhaug et al., 2015

[0324] • Shah and Kimberley, 2016

[0325] • Tucker, B., Aston, J., Dines, M., Caraman, E., Yacyshyn, M., McCarthy, M., et al., 2017.

[0326] • Early brain oedema is a predictor of in-hospital mortality in traumal c brain injury. J. Emerg. Med. 53, 18-29.

[0327] • Carney N et al. Guidelines for the management of severe traumatic brain injury 4th edition. Brain trauma foundation. Braintrauma.org 2016.

[0328] • Carney N, Token AM, O'Reilly C, et al. Guidelines for the Management of Severe Traumalc Brain Injury, Fourth Edilon. Neurosurgery 2017;80(1 ):6-15. DOI: 10.1227 / NEU.0000000000001432.

[0329] • Raslan and Bhardway, 201

[0330] • Maas, A.LR., Menon, D.K., Adelson, P.D., Andelic, N., Bell, M.J., Belli, A., et al., 2017. Traumalc brain injury: integrated approaches to improve prevenion, clinical care, and research. Lancet Neurol. 16, 987-1048. • Chen et al., 2020

[0331] • Hay et al. (2015)

[0332] • Lange S, Lonnroth I. The Antisecretory Factor: synthesis, anatomical and cellular distribution and biological action in experimental and clinical studies. Int Rev Cyt 210:39-75, 2001.

[0333] • WO 97 / 08202;

[0334] • WO 05 / 030246;

[0335] • WO 2007 / 126364;

[0336] • WO 2018 / 015379

[0337] • Clausen F, Hansson HA, Raud J, Marklund N. Intranasal Administration of the Antisecretory Peptide AF-16 Reduces Edema and Improves Cognitive Function Following Diffuse Traumatic Brain Injury in the Rat. Front Neurol 14(8):39, 2017.

[0338] • Alluri et al., 2018,

[0339] • Michinaga S, Koyama YA-O. Pathophysiological Responses and Roles of Astrocytes in Traumatic Brain Injury. LID - 10.3390 / ijms22126418 [doi] LID - 6418. (14220067 (Electronic)) (In eng).

[0340] • SE 9000028-2

[0341] • WOOO / 38535

[0342] • WO 2017 / 009004

[0343] • Jha, R.M., P.M. Kochanek, and J.M. Simard, Pathophysiology and treatment of cerebral oedema in trauma:c brain injury. Neuropharmacology, 2019. 145(Pt B): p. 230-246.

[0344] • Vik et al., 2008

[0345] • Lange S, Jennische E, Johansson E, Lonnroth I. The Antisecretory Factor — synthesis and intracellular localization in porcine tissues. Cell Tissue Res 296:607-17, 1999.

[0346] • Davidson TS, Hickey WF. Distribution and immunoregulatory properties of antisecretory factor. Lab Invest 84:307-19, 2004a.

[0347] • Kiss P, Szabo A, Hunyadi-Gulyas E, Medzihradszky KF, Lipinszki Z, Pal M, et al. Zn2+-induced reversible dissociation of subunit Rpn10 / p54 of the Drosophila 26 S proteasome. Biochem J 391 :301-10, 2005.

[0348] • Nicolas V, Lievin-Le Moal V. Antisecretory factor peptide AF-16 inhibits the secreted autotransporter toxin-stimulated transcellular and paracellular passages of fluid in cultured human enterocyte-like cells. Infect Immun 83:907-22, 2015.

[0349] • Matson Dzebo M, Reymer A, Fant K, Lincoln P, Norden B, Rocha S. Enhanced cellular uptake of antisecretory peptide AF-16 through proteoglycan binding. Biochemistry 53:6566-73, 2014. Remington: The science and practice of pharmacy”, 21st edition, ISBN 0-7817-4673-6 or “Encyclopedia of pharmaceutical technology”, 2nd edition, ed. Swarbrick J., ISBN: 0-8247-2152-7

[0350] • Akerlund CAI, Holst A, Stocchetti N, et al. Clustering identifies endotypes of traumatic brain injury in an intensive care cohort: a CENTER-TBI study. Critical Care 2022;26(1):1-15. DOI: 10.1186 / s13054-022-04079-w.

[0351] • Akerlund CAI, et al., Impact of duration and magnitude of raised intracranial pressure on outcome after severe traumatic brain injury: A CENTER-TBI high- resolution group studyPLOS ONE | https: / / doi.org / 10.1371 / journal.pone.0243427 December 14, 2020

Claims

53Claims1 . A pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide consisting of the amino acid sequence VCHSKTRSNPENNVGL (AF-16 ) and / or consisting of the amino acid sequence VC(C)HSKTRSNPENNVGL (AF-17), or a pharmaceutically active salt thereof, having equivalent functional activity, for use in treating acute brain oedema due to a traumatic brain injury (TBI) in a patient with a. a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of b. a non-provoked elevation of ICP above the threshold value of 18 mm Hg during at least 1 hour before the administration of the pharmaceutical composition, wherein said pharmaceutical composition is administered intravenously, wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, wherein the treatment reduces the ICP in said patient with at least 20% and, wherein the treatment reduces the mortality risk in said patient.

2. A pharmaceutical composition for use according to claim 1 , wherein said patient is diagnosed with a clinical coma severity of between 4-9 (Glasgow Coma Scale) GCS.

3. A pharmaceutical composition for use according to claim 1 or 2, wherein said patient is diagnosed with a non-provoked elevation of ICP above the threshold value of 18 ± 4 mm Hg during at least 1 hour before the administration of the pharmaceutical composition, such as with an ICP >20 mmHg.

4. A pharmaceutical composition for use according to any of the preceding claims, wherein the treatment reduces the ICP in said patient with at least 20-25%.

5. A pharmaceutical composition for use according to any of the preceding claims, wherein the treatment reduces the mortality risk of said patient by at least 20%.

6. A pharmaceutical composition for use according to any of the preceding claims, wherein said pharmaceutical composition is administered no later than 24h after54 the measurement of a non-provoked elevation of ICP above the threshold value of 18 ± 4 mm Hg.

7. A pharmaceutical composition for use according to any of the preceding claims, wherein said pharmaceutical composition is administered as an intravenous infusion three times per day (tid).

8. A pharmaceutical composition for use according to any of the preceding claims, wherein said pharmaceutical composition is administered as a 40-minute intravenous infusion every 8 hours for at least 5 consecutive days.

9. A pharmaceutical composition for use according to any of the preceding claims, wherein the patient has a documented unprovoked ICP of 18 mmHg or higher for at least one hour and signs of brain oedema on a CT or MRI scan.

10. A pharmaceutical composition for use according to any of the preceding claims, wherein the patient has a documented unprovoked ICP of 18 mmHg or higher for at least one hour within 7 days of arrival to hospital.

11. A pharmaceutical composition for use according to any of the preceding claims, wherein the patient has a closed head injury.

12. A pharmaceutical composition for use according to any of preceding claims, wherein the composition comprises at least 7.5 mg / mL of said isolated recombinant and / or synthetically produced peptide AF-16 and / or AF-17, or a pharmaceutically active salt thereof, having equivalent functional activity.

13. A pharmaceutical composition for use according to any one of the preceding claims, wherein the administration of said pharmaceutical composition reduces the ICP in said patient at the latest at day 5 after the initializing of the treatment.

14. A pharmaceutical composition for use according to claim 6, wherein the ICP is reduced to between 13-8.5 mmHg at the latest at day 5 after the initializing of the treatment, such as to 8.5 mmHg, 9 mmHg, 9.5 mmHg, 10 mmHg, 10.5 mmHg, 11 mmHg, 11.5 mmHg, 12 mmHg, 12.5 mmHg, or 13 mmHg.5515. A pharmaceutical composition for use according to any one of the preceding claims, wherein the ICP in said patient is reduced by between 10-25% after 10 min, 15 min, 30 min, 1 h, 1 ,5h, 2h, 2.5h, 3h, 4h, 5h, 6h, or at the most 12h after initializing of the treatment.

16. A pharmaceutical composition for use according to any one of the preceding claims, wherein the ICP in said patient is reduced by between 10-25% after at the most 12h, 24h, 48h, 72h, 96h, or 130h after initializing of the treatment.

17. A pharmaceutical composition for use according to any one of the preceding claims, wherein the pharmaceutical composition is administered repeatedly, such as each 8 hours.

18. A pharmaceutical composition for use according to any one of the preceding claims, wherein the pharmaceutical composition is administered during at least 5 days of treatment.

19. A pharmaceutical composition for use according to any one of the preceding claims, wherein the ICP in said patient is reduced by between 10-25% after 10 min, 15 min, 30 min, 1 h, 1 ,5h, 2h, 2.5h, 3h, 4h, 5h, 6h, or at the most 12h after each administration.

20. A pharmaceutical composition for use according any one of the preceding claims, wherein the ICP in said patient is reduced to between 13-8.5 mmHg after at the most 10 min, 15 min, 30 min, 1 h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 12h, 24h, 48h, 72h, 96h, or 130h after each administration.

21. A pharmaceutical composition for use according to any one of the preceding claims, wherein the ICP in said patient is analysed as reduction in brain oedema, assessed as reduction of brain water content (BWC).

22. A pharmaceutical composition for use according to any one of the preceding claims, wherein the administration of said pharmaceutical composition to said patient reduces the cerebral perfusion pressure (CPP) of said patient.5623. A pharmaceutical composition for use according to any one of the preceding claims, wherein the patient suffers from a blood-brain barrier (BBB) disruption and / or an acute BBB leakage.

24. A pharmaceutical composition for use according to any one of the preceding claims, formulated as a solution for intravenous infusion.

25. A pharmaceutical composition for use according to any one of the preceding claims, further comprising a pharmaceutically acceptable excipient.

26. A pharmaceutical composition according to any one of the preceding claims, comprising the active ingredient A-16 and / or AF-17 and / or a pharmaceutically active salt thereof, water, a chelating agent and a tonicity agent and a pH adjusting agent.

27. A pharmaceutical composition for use according to any one of the preceding claims, which is administered at a dosage of 1-50mg / kg as a 10-40 minute- infusion, such as a 40 minute-infusion.

28. A pharmaceutical composition for use according to any one of the preceding claims, in combination with other treatments focused on managing ICP elevation, selected from the group consisting of optimal head and neck positioning, optimized pain relief, optimized sedation, negative fluid balance, controlled ventilation, hypocapnia, avoidance of dehydration, and maintenance of normothermia.

29. A pharmaceutical composition for use according to any one of the preceding claims, in combination with other treatments focused on managing ICP elevation comprising a medical treatment using anyone selected from the group consisting of mannitol, hypertonic saline solution, diuretics, corticosteroids, barbiturates, and combinations thereof.

30. A pharmaceutical composition for use according to any one of the preceding claims, wherein the patient is a human patient.

31. A pharmaceutical composition for use according to any one of the preceding claims, for use in treating a human patient with loss of consciousness.

32. A pharmaceutical composition for use according to any one of the preceding claims, for use in treating a human patient with loss of consciousness of more than 6 hours.

33. A pharmaceutical composition for use according to any one of the preceding claims, wherein the treatment leads to signs of clinical benefit.

34. A pharmaceutical composition for use according to any one of the preceding claims, wherein the treatment reduces the need for deep sedation of the patient.

35. A pharmaceutical composition for use according to any one of the preceding claims, wherein the treatment further comprises sedation of the patient without the administration of barbiturates, except during periods of gastroparesis of the patient.

36. Use of an isolated recombinant and / or synthetically produced antisecretory peptide AF-16 and / or AF-17, or a pharmaceutically active salt thereof, having equivalent functional activity, for manufacturing of a pharmaceutical composition for treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), such as an initial increased intracranial pressure (ICP) of at least 12 mmHg, wherein said pharmaceutical composition is administered intravenously and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the raise of ICP to at least 18 mmHg.

37. A method of treating acute cerebral oedema due to a traumatic brain injury (TBI) in a patient with a clinical coma severity of between 3-12 (Glasgow Coma Scale) GCS, leading to suspicion of and / or imminent danger of elevated intracranial pressure (ICP), such as an initial increased intracranial pressure (ICP) of at least 12 mmHg, comprising administering intravenously a pharmaceutical composition comprising an isolated recombinant and / or synthetically produced antisecretory peptide AF-16 and / or AF-17, or a pharmaceutically active salt thereof, having equivalent functional activity, and wherein the treatment is initiated during the initial stage of emergency care and / or hospitalization, such as no later than 24h after the raise of ICP to at least 18 mmHg.

Images