Phosphoinositide 3 kinase (PI3k) inhibitor for use in the treatment of neurological diseases

Abstract

The invention refers to the field of neurodegenerative diseases including Alzheimer's Disease and a treatment thereof with a Benzoxazepine compound. Benzoxazepine compounds are e.g. known in the treatment of breast cancer and according to the use of the invention are a first therapeutic and prophylactic treatment for neurodegenerative diseases, including Alzheimer's Disease. The invention further relates to test systems for identifying, mapping, elaborating and evaluating said and further therapeutic and prophylactic uses of compounds of interest and / or other pharmaceutical compositions for the treatment of neurodegenerative diseases, including Alzheimer's Disease. (AD)

Description

[0001] PHOSPHOINOSITIDE 3 KINASE (PI3K) INHIBITOR FOR USE IN THE TREATMENT OF NEUROLOGICAL DISEASES

[0002] The invention refers to the field of Alzheimer’s Disease (AD) and further neurological disorders, and further provides a new treatment approach for such neurological disorders and AD. For the treatment of neurological disorders is thus provided PI3K inhibitors and particularly provided are PI3K-i n hi bitors identifiable as Benzoxazepine or compositions thereof. Benzoxazepine compounds are known and approved for use in the treatment of certain types of breast cancer.

[0003] Additionally, the invention is based on a brain-specific test systems capable of simulating physiological and pathological conditions of the human brain. This system has been used fortesting, identifying, mapping, elaborating and evaluating substances for their effects on the brain.

[0004] As one result, identified with the test system, PI3K inhibitors were identified for their therapeutic and / or prophylactic use in the treatment of neurological disorders, beside others for dementia, Alzheimer’s Disease (AD), vascular Dementia, Down syndrome (DS), glioblastoma, brain metastases or diabetic polyneuropathy.

[0005] Alzheimer’s disease will be one of the major challenges of the century. Presently every third person above the age of 90 is affected, every seventh person above the age of 80 is affected. The global situation shows an incidence, that has increased from 1990 to 2019 by around 145%, resulting in more than 7 million cases recorded worldwide in 2019. Considering the predictable demographic development this sums up to more than 150 million Alzheimer patients in 2050. As there is no treatment, not only the families that have the challenge of caretaking but also society will dramatically be affected. So far, medicine and research are hampered as only unsuitable test systems or animal models seems available or feasible for scientific analysis of early stages of Alzheimer.

[0006] Presently, there is not yet a reliable test system that allows the testing of drugs or medicaments suitable for the treatment of neurological disorders and in particular of Alzheimer, neither is there any transferability of results from any such systems to humans and the pathology of Alzheimer’s disease, which can be extracted from the high failure rate of preclinical promising results of AD drugs in clinical trials in humans.

[0007] Moreover, it is ethically impermissible and scientifically unsound to expose neurologically healthy human subjects to investigational compounds for the assessment of therapeutic efficacy against neurological disorders such as Alzheimer’s disease. Accordingly, because first-in-human administration of novel molecular entities is proscribed— and Phase I clinical studies necessarily entail human dosingwhile subsequent safety and pharmacodynamic evaluations require testing in two distinct non-rodent species, the absence of predictive and reliable in-vitro assays or representative animal models constitutes a fundamental need to develop new test systems for the subsequent testing of new substances in the treatments of neurological disorders, and in particular Alzheimer’s disease.

[0008] Another pressing challenge in Alzheimer’s disease research is that the typical time progression to develop the disease takes several decades. A lack of suitable test systems, e.g., biomarkers, to identify the Alzheimer’s disease patient cohort at least 5 to 10 years prior to the initial, symptomatic onset of Alzheimer’s disease state, which is described as “mild cognitive impairment”, makes it even more challenging to identify or test high potential drug candidates that can treat the disease early on.

[0009] Notwithstanding the desirability of evaluating candidate therapeutics for Alzheimer’s disease, conventional clinical trial paradigms are rendered infeasible by the protracted natural history of the disorder, which precludes meaningful data collection within practicable timeframes. Even where high-risk subjects receive investigational compounds, assessment of whether disease onset is prevented, delayed, or attenuated to a less severe phenotype necessitates longitudinal follow-up of at least five to ten years. Furthermore, establishing a causal relationship between therapeutic intervention and neuropathological outcome is rendered impracticable by the requirement for brain tissue analysis years after treatment initiation, often only post-mortem.

[0010] It is to the credits of the inventor that there is now a research tool suitable to address this need and to simulate the physiological and pathological alterations for human brain cells. With the new test system, it is now possible to analyze, identify, classify, quantify, observe and evaluate the effect of pharmaceutical drugs or other blood-brain-barrier transmittable components on neurons, glia cells, e.g., microglia and astrocytes and all the other cellular, extracellular or intracellular functions in the brain, which are involved in the development and / or progression of neurological diseases such as Alzheimer’s disease. As an example with this research tool the inventor identified a drug, which is capable to prevent and reduce pathological alterations for human brain cells typical for AD.

[0011] Accordingly, the invention refers to the treatment of neurodegenerative diseases including Alzheimer’s Disease and a treatment thereof with a Benzoxazepine compound. Benzoxazepine compounds are known to be used for the treatment of breast cancer and according to the use of the invention are a first therapeutic and prophylactic treatment approach for neurological and neurodegenerative diseases, such as Alzheimer’s disease.

[0012] The invention further illustrates how the new test systems will be used to analyze, identify, map, elaborate and evaluate further therapeutic and prophylactic uses of compounds of interest and / or other pharmaceutical compositions for and in the treatment of neurological and neurodegenerative diseases, including Alzheimer’s Disease (AD). Though, in the following sections the treatment of AD is used as an example, however other neurological or neurodegenerative diseases and disorders as well as other suitable drugs to treat them can be identified with this invention.

[0013] Neurodegenerative diseases and disorders suitable for such treatment approaches include e.g.

[0014] Frontotemporal dementia, Dementia with Lewy bodies, Parkinson’s disease, Huntington’s disease, Progressive supranuclear palsy, Corticobasal degeneration, Multisystem atrophy and Progressive myoclonic epilepsy’s and many more as later explicitly mentioned. For example, Alzheimer’s disease (AD) is a major cause of suffering and death globally, and with 60-70% of all 9.9 million new estimated Dementia cases each year the largest part among the humans directly suffering from Dementia and accompanying complications. AD is a slowly progressive neurodegenerative disease, which goes along with dementia, loss of memory, loss of decision making, loss of orientation, significant morbidity, multiple comorbidities and consequentially leading to a major loss of quality of life for the patient but also her / his family or responsible care takers (Barker et al., (2020)).

[0015] In the next decade AD will rapidly become an increasingly severe problem for societies all over the world. A prediction states, that in 2030, the number of Americans diagnosed with AD will have experienced an increase of 35%.

[0016] Scientific reviews such as Barker eto / ., (2020) summarize the clinical picture includingthe symptoms and progression of AD development, which typically starts with a loss of short-term memory, later follows cognitive failure and confusion, and finally an inability to carry out tasks required for successful daily living.

[0017] Despite immense efforts for better understanding AD, it must be accepted that the cause of AD onset is still unclear. There are several hypothesizes proposed to explain the pathophysiology of AD. One established hypothesis considers the development of plaques due to the accumulation of A-beta- amyloid-peptides (A|3) within the extracellular matrix of brain- and CNS-tissue as a hallmark or even potential root cause of AD. Another hypothesis speculates about the hyperphosphorylation of Tau protein (pTau), being intracellular neurofibrillary tangles, as possibly driving pathomechanism underlying AD. It is further believed that these two driving forces are interconnected, and that Azaccumulation increases intracellular hyperphosphorylation of pTau within neurons and / or within the brain- and CNS-tissue. According to Barkers etal., the accumulation and hyperphosphorylation are causally correlated with the death of neurons and consequentially the AD symptoms.

[0018] Additionally, and confirmingthe existence of a genetic predisposition for AD, which triggers an even earlier onset of AD, comparative studies between healthy patients and AD patients have helped to identify AD risk genes, of which it is assumed that their expression is correlated with or maybe even causes the onset of AD. Presently known examples thereof are glia risk genes, e.g.: BLNK, ADAM10, ADAM17, TREM2, RhoH, and SIGLEC11. This list is however not to be understood as a complete list, and more recent studies also identify the PI3K cascade and genes participating in this cascade as possibly being involved in the pathology of AD. For example, Barker etal. (2020) speculates on the PI3K pathway as one explanation for the pathophysiology of AD. However, the paper focuses on the participation PI3K pathway in cancer, particularly breast cancer. Disturbingly, it seems that PI3K involvement in AD and cancer is quite contradictory as it seems to trigger very distinct and unequal effects of dysregulation. Barker etal. summarizes that most cancers, including breast cancer, involve or are caused by unrestrained cell proliferation. For breast cancer, it was found that such cell proliferation often coexists with a PI3K mutation. In those so-called PI3Kinase-dependent or -associated forms of breast cancer there is found a hyperactivity of the PI3K / Akt signaling pathway, which then leads to the cancerogenic dysregulation and cell proliferation.

[0019] The situation within the brain or brain cells, such as neurons and glia, e.g., microglia or astrocytes, the PI3K activity and / or regulation seems to be different. For example, for neurons it is believed that they remain in a metabolic stable status, meaning neither do they proliferate anymore, nor do they grow any further. Thus, it can be said under healthy conditions, neurons remain in stable state, and even though they may express receptors, which in many other cells would lead to cell growth, they will not actively proliferate. Neurons in the brain, are terminally differentiated post-mitotic cells, which if forced into a renewed cycle usually die.

[0020] It is exactly such a hypothesis, which nicely illustrates the actual problem of AD research. As there is no suitable animal model for studying AD and as it is unacceptable to randomly treat patients of which one only assumes that they might - one day - develop AD, any such speculation regarding a potential involvement of e.g. the PI3K pathway in AD cannot properly be tested.

[0021] It is due to the efforts and development of the inventorthat a novel method and system for testing the effect of high potential drug candidates and potentially interesting substances or drugs for treatment of neurons, microglia and astrocytes were established. With this method and system, the inventor was capable to newly identify suitable drugs that lead to a significant reduction of protein concentrations of e.g., Af?> and pTau by interfering with and / or inhibiting the PI3K pathway.

[0022] For the first time it is possible on the basis of this system to not only observe the development of the pathophysiology of AD (Klostermeier et al., - in press) but - even more interesting - to identify substances and / or compositions, which interfere, also reduce and delay or even may revert the pathophysiology of AD.

[0023] The present invention thus overcomes two longstanding obstacles that have impeded both fundamental research into neurodegenerative disorders and the identification of effective therapies therefor. First, the inventor has formulated a novel and previously unrecognized mechanistic hypothesis governing the initiation and progression of Alzheimer’s disease. Second, the inventor has devised a groundbreaking paradigm delineating the requisite mode of action of a substance or composition capable of preventing, mitigating or even reversing Alzheimer’s pathology.

[0024] The invention thus provides a 3D cell culture system for co-culturing and real-time monitoring of multiple neural cell types under both physiological and pathophysiological conditions. By enabling controlled simulation of neuronal, glial and other brain-cell interactions, this system permits rigorous evaluation of existing and newly proposed hypotheses concerning the onset, development and treatment of neurodegenerative diseases, and in a particular example of Alzheimer’s disease.

[0025] Solving the above-mentioned problems, the inventor provides a system that allows to simulate and to observe the interaction between different brain cells not only in physiological, but also under pathophysiological conditions. With this system it is possible to test and thus better understand previously suggested hypothesis regarding the onset or development of various neu regenerative diseases and also AD.

[0026] The discovery of the inventor that an essential involvement in the development of AD or more general in the development of neurological and neurodegenerative diseases is caused by changes of tissue’s mechanobiology in the brain (in press - Klostermeier et. al.,) is used in the herein provided system to firstly show the pathological development and thereafter, to use the system to identify substances which can stop and / or reduce such pathological development.

[0027] The herein presented teaching of the inventor for the treatment of AD in the future allows a treatment regime, which is capable to effectively reduce the protein concentrations of AB and pTau firstly in the system but correspondingly also in the human brain. The inventor shows for the first time that by inhibiting the PI3K pathway the A and pTau protein accumulation can be reduced. By effectively reducing such toxic proteins in the brain and, thus, by reducing an accumulation and the formation of AB plaques as well as reducing hyperphosphorylated pTau in the brain an unexpected improvement of the pathological brain alternations is obtainable and was obtained in the inventive 3D cell culture system.

[0028] For the first time, the invention delivers a dual-target therapeutic modality that concurrently attenuates both A|3 and phosphorylated tau species and, furthermore, intercepts the pathogenic cascade at an upstream juncture, in contrast to existing antibody therapies which are confined to removal of established A3 plaques. As a result, this innovative intervention is anticipated to exert a markedly enhanced effect on the initiation and progression of Alzheimer’s disease, holding strong potential to open new avenues of treatment for patients, allowing new hope for their families and caregivers, and thereby to alleviate the broader societal burden.

[0029] Accordingly, the inventive treatment approach focuses in its most general embodiment on the functional inhibition of the PI3K pathway. Additionally, as one result of employing the new test system the inventor could identify a PI3K inhibitor as a drug suitable to be used in the treatment of Alzheimer’s disease. Thus, as a result of employing the new system and method the inventor could identify a drug suitable to be used in the treatment of neurological disorders, particularly AD.

[0030] The invention thus provides the class of PI3K-i n hi bitors for use in the treatment of neurological disorders. In the context of the invention the term “neurological disorder” refers to or identifies pathophysiological condition concerning the brain and primarily cells, such as neurons and glia cells in the brain, but also pathophysiological condition of e.g. neurons in the periphery. Furthermore, the term neurological disorder includes neurodegenerative diseases, neuropathological disorders and polyneuropathies.

[0031] Examples of “neurodegenerative diseases” include, but are not limited to, Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis, Dementia, Vascular dementia, Frontotemporal dementia, Dementia with Lewy bodies, Parkinson’s disease, Huntington’s disease, Progressive supranuclear palsy, Corticobasal degeneration and Multisystem atrophy.

[0032] The term “neuropathological disorder” refers to or describes diseases and / or disorders of the central nervous system and / or the peripheral nervous system, optionally, but not necessarily, including the muscles. Examples of neuropathological disorders include, but are not limited to genetic diseases, amongst others Down Syndrome, migraine, inflammatory diseases, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, meningitis, encephalitis, Creutzfeldt-Jakob disease, neuroborreliosis, multiple sclerosis, neuromyelitis optica, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, epilepsies and seizure disorders, progressive myoclonic epilepsy, traumatic brain injuries, spinal cord injuries, nervous system tumors, brain tumors, spinal cord tumors, peripheral nerve tumors, glioma, glioblastoma, brain metastasis, neuromuscular disorders such as myasthenia gravis, muscular dystrophies and spinal muscular atrophy.

[0033] The term “polyneuropathy” refers to or describes an impairment of all parts of the nervous system. An example of polyneuropathies includes, but is not limited to diabetic polyneuropathy.

[0034] In the context of the invention the terms “treatment” and “treat” refer to or describe both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired neurological disorder, such as neurodegeneration. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “T reatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0035] In the context of the invention the term “PI3K” refers to or describes a group of enzymes known as “PI3- Kinases”, also referred to as Phosphatidylinositol 3-kinases. Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases capable of phosphorylating the 3’-hydroxyl group of the inositol ring of phosphatidylinositol. PI3Ks integrate signals from growth factors, cytokines and other extracellular stimuli into intracellular signals to regulate cell growth, proliferation, survival, motility and metabolism.

[0036] Furthermore, the PI3K pathway is an extracellularly started intracellularly proceeding signaling cascade. Under physiological conditions it starts with the binding of an activating ligand on the transmembrane PI3K-Receptor (PI3K), leading also to the change of the intracellular part of PI3K pathway and - it is believed - eventually to the accumulation of hyperphosphorylated pTau in neurons.

[0037] In the context of the invention the term “PI3K-i n hi bitor” refers to or describes substances, compounds or compositions, which themselves and / or in their activated form inhibit PI3K, i.e. slow down, prevent or stop in a concentration-dependent manner the enzymatic activity of PI3K.

[0038] By providing therapeutically active concentrations of the PI3K inhibitor to a patient the PI3K-cascade can be slowed down, interrupted or even stopped. Thus, a treatment with a PI3K-i n hi bitor advantageously reduces the negative effects transmitted by an activated PI3K-cascade on brain cells or other neuronal cells and supports a physiological neuroplastic restructuring. Therefore, this invention provides a substance suitable as first in class for use in the treatment of neurological disorders.

[0039] According to the invention herewith provided is a substance or composition of Formula (I) for the use in the treatment of a neurological disorder, neu regenerative disease or disorder, and among others Alzheimer’s Disease.

[0040] Formula (I), which also can be identified as Benzoxazepine.

[0041] Such Benzoxazepine are known in the art and particularly from W02011036280 or WO2013182668 where they are used in the treatment of cancer, in particularly in a specific breast cancer having mutations in the PI3K pathway, which cause tumor growth and metastasis.

[0042] According to one embodiment Formula (I) is characterized by a first residue R1and the second residue A, the first residue Rxand the second residue A selected from a group of substituents as identified below and thus, leading to the known compounds GDC-0032 and GDC-0077.

[0043] In one preferred embodiment of the substance or composition of Formula (I) for the use according to the invention is defined by the residue R1being a lH-pyrazol-l-yl-2-methylpropanamide, as shown and the second residue A being a l-isopropyl-3-methyl-lH-l,2,4-triazol-5-yl, as shown leadingto an Benzoxazepine compound of Formula (II), namely

[0044] The Benzoxazepine compound of Formula (II), and it’s usability for the treatment of cancer in humans is known from EP2858666B1. The Benzoxazepine compound of Formula II is also known as GDC-0032, 2- (4-(2-(l-isopropyl-3-methyl-lH-l,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[l,2-d][l,4]oxazepin-9-yl)- lH-pyrazol-l-yl)-2-methylpropanamide orTaselisib (INN).

[0045] The Benzoxazepine of Formula (II), is believed to be an unspecific PI3K-i nhibitor, i.e. binding the various PI3Ks. The provision of an unspecific PI3K-inhibitor, namely the Benzoxazepine compound of Formula (II), is particularly advantageous as a first in class substance for the treatment in neurological disorders and thus, is a good starting point for further comparison with other compounds regarding their effect, efficiency in reducing the expression of target genes or the reestablishing of physiological conditions in brain cells.

[0046] In a second preferred embodiment of the substance or composition of Formula (I) for the use according to the invention is defined by the residue R1being a (S)-2-(2-amino)propenamide, as shown and the second residue A being a (S)-4-(difluoromethyl)-2-oxooxazolidin as shown leadingto an Benzoxazepine compound of Formula (III)

[0047] The Benzoxazepine compound of Formula (III), and it’s usability for the treatment of cancer in humans is known from WO 2020023297A1. The Benzoxazepine compound of Formula (III) is also known as GDC- 0077, (S)-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[l,2- d][l,4]oxazepin-9-yl)amino)propenamide or as Inavolisib (=INN).

[0048] What is remarkable about this specific Benzoxazepine compound of Formula (III) is, it’s selectivity in inhibiting the PI3Ka isoform with higher affinity as the PI3K|3, PI3K6, and Pi3Ky isoforms.

[0049] Furthermore, according to the invention also stereoisomer, constitutional isomer, geometric isomer, tautomer or pharmaceutically acceptable salt of the Formula (I), (II) or (III) are provided for the treatment of neurological disorder, neu regenerative disease or disorder and Alzheimer’s Disease.

[0050] In the context of the invention the term “pharmaceutically acceptable salt”, refers to or describes pharmaceutically acceptable organic or inorganic salts of a compound of the invention. They must be compatible chemically and / or toxicologically, with the other ingredients comprising a substance or composition. Exemplary pharmaceutically acceptable salt include, but are not limited to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ions. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0051] According to the invention the substance or composition is used in the treatment of the neurological disorder selected from the group of neurological disorders comprising neurodegenerative diseases and neuropathological disorder, including but not limited to Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), Dementia, Vascular dementia, Frontotemporal dementia, Dementia with Lewy bodies, Parkinson’s disease, Huntington’s disease, progressive supranuclear palsy, corticobasal degeneration, migraine, multisystem atrophy, Down syndrome (DS), inflammatory bowel diseases, Crohn's disease, ulcerative colitis, meningitis and encephalitis, migraine, Creutzfeldt-Jakob disease and neuroborreliosis (Lyme disease), multiple sclerosis, neuromyelitis optica, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, epilepsies or seizure disorders, progressive myoclonic epilepsy, traumatic brain injuries, spinal cord injuries, glioblastoma, brain metastasis, neuromuscular disorders, myasthenia gravis, muscular dystrophies, spinal muscular atrophy, polyneuropathies and diabetic polyneuropathy.

[0052] The substance or composition according to the invention is particularly advantageous for the use in those diseases, caused by, related to, accompanied with and / or following a neuroplasticity rearrangement, disorders or trauma. Genetic predisposition can trigger or increase the risk for a neurological or neuropathological disorder.

[0053] In another certain embodiment the substance or composition according to the invention is administered oral, intravenous, subcutaneous, intrathecal, sublingual, transbuccal, transdermal, nasal, pulmonal, rectal, as Antibody-Drug-complex and / or as nanoparticles.

[0054] According to this further embodiment the galenic formulations of the substance or composition of the invention is relevant, particularly when treating disorders of the brain and in the brain. The brain, as separated from the peripheral immune system by the blood-brain-barrier (BBB) relies also on the innate immune system for defense. It is believed that part of this innate immune response includes production of A[3-peptide and activation of the resident macrophages, glia, resulting in neuroinflammation, neuronal loss and ultimately death. Unless constantly cleared from the extracellular brain space such A|3 formed plaques are toxic, insoluble oligomeric forms, which may cause neuroinflammation, trigger phagocytic activity of microglia cells and contribute to neuronal death.

[0055] For a transfer through the BBB the skilled practitioner may provide suitable pharmaceutical formulations. Alternatively, the skilled practitioner may use complexes of the substance of the invention with BBB crossing antibodies, likely single chain antibodies, forming so-called Antibody-Drug- Complexes (ADC), which are known to transport cargo molecules into the brain. In another embodiment the substance or composition according to the invention, is administrable as a retard composition. The term “retard composition” refers to or describes a formulation of the compound or composition designed for a slow or delayed release.

[0056] Interestingly, it is shown in the examples, that not only the dose, which is used in the treatment of breast cancer but also a substantially lower dose is significant and effectively modifying the metabolism of and gene expression in brain cells. Experimental data show, that already a dose of 10 nM (in the Figure the abbreviation nm was used for nanomolar) is sufficient for a drastic reduction of the protein concentration and thus a potentially curing effect.

[0057] The term “therapeutically effective amount” refers to or describes a selectable concentration of a pharmacologically active substance of Formula (I), (II), (III) and / or a pharmacologically active metabolite of said substances respective to time and body weight.

[0058] In another embodiment the substance or composition according to the invention is used as a prophylactic or post-expositiona I treatment. In the context if the invention the term “prophylactic” describes or relates to an administration of the substance of the invention after a first notice of a risk factor for developing e.g. neurological disorder. Alternatively, a prophylactic use also is indicated where there is a genetic predisposition for the development of a neurological disorder or any of the above- mentioned specific diseases. The term “post-expositional” describes or relates to an immediate administration after exposure to an acute risk-factor for developing the neurological disorder, which can be a trauma e.g. induce by an internal bleeding, concussion, injury, the application of brute force on the head or similar incidences.

[0059] Such prophylactic or post-expositional treatment with the substance or composition of the invention, which stabilized the metabolism of the affected brain cells, is expected to be particularly useful for avoiding long-term brain damage.

[0060] The substance of the present invention has been identified in a 3D cell culture system for identifying and testing substances and / or compositions of interest for their effect on brain cells, wherein the 3D cell culture system is suitable for the cultivation of one or more species of neuronal cells or brain cells, wherein the 3D cell culture system comprises at least two different cell cultivation hydrogels made from Alginate-dialdehyde (ADA) and collagens.

[0061] The at least two hydrogels, i.e. the at least first and at least second hydrogel are made according to the Examples of ADA and collagen, for the present testing ADA PH124 and PH176 (VIVAPHARM JRS Pharma, Ulm, Germany) were used for the Synthesis as described in Example 2. These ADAs, namely PH124 und PH176 lead to the differences in mechano-stability and viscoelasticity of the resulting hydrogels that resembles the human healthy and pathological brain. Thus, the 3D cell culture system and test system according to the invention is characterized by the use of hydrogels with such individually distinct biomechanics, which are adjustable while preparing the hydrogels. Explicitly, and for testing brain specific drug candidates the at least first and at least second hydrogel have defined and distinct stiffness and elasticity. Together the stiffness and elasticity of the individual hydrogel can be described and is defined by its viscoelasticity.

[0062] In the context of the present invention the term “viscoelasticity” describes the property of materials that exhibit both viscous and elastic characteristics when undergoing deformation. Viscoelastic materials have time-dependent strain, meaning they exhibit both immediate elastic response and delayed viscous response under stress.

[0063] Further in this context the term “viscous” or “viscosity” refers to a measure of a fluid's resistance to flow. It describes the internal friction within the fluid that arises when adjacent layers move at different speeds. For biomaterials, viscosity can describe how the material resists deformation under shear stress. Still further in this context the term “elastic” or “elasticity” refers to the property of a material to return to its original shape after the removal of a force that caused deformation. In biomaterials, elasticity is critical in understanding how tissues and other biological materials respond to mechanical forces. This viscoelasticity of the individual hydrogels is individually designed depending on the test situation and purpose of the 3D cell culture system.

[0064] One way to design the individual hydrogels of the 3D cell culture system is to mimic the specific viscoelasticity in the at least first hydrogel with the physiological conditions of a certain organ, e.g. of healthy brain tissue. In the corresponding at least second hydrogel would then be mimicked a viscoelasticity of altered conditions for said organ, and in the example with the brain such at least second hydrogel has then the viscoelasticity of pathological brain condition.

[0065] Such corresponding hydrogels with distinct and different viscoelasticity are then populated with the test cells, which subsequently can be exposed to a substance of interest for testing the effects on these cells under distinct, namely e.g. physiological and pathological brain conditions. In such a 3D cell culture system it is now possible to compare and evaluate the effects of a substance of interest on the different neuronal cells or brain cells. It was previously shown that such brain cells without the exposure to any drug do show a response to the viscoelasticity of the hydrogel (Klostermeier et al., in press], which can be measured e.g. as potential gene activation and overexpression followed by an protein accumulation.

[0066] The response to differences in the viscoelasticity of the hydrogel was shown to resemble the situation in the human brain, where in e.g. injured brain the stiffness and elasticity of the extracellular matrix and / or tissue are amended by the influence of cytokines, infiltrating blood or other inflammatory factors. This influence leads to a more “stiff” tissue condition, which is imitated in the ‘stiff test gels’ of the present invention.

[0067] Similarly, it was shown by the inventorthat within the 3D cell culture system by comparing the 3D development of brain cells in the corresponding hydrogels with different viscoelasticity the development and metabolism of said brain cells depends on the viscoelasticity. In other words, in a healthy brain, which has a viscoelasticity with a stiffness of around 1 kPa and shear modulus around of 0.4-1.4 kPa, as it is also provided in the “soft” hydrogel of the presently described 3D cell culture system, the cultivated brain cells develop in a physiological manner. If tested for e.g. the expression of certain genes, and in particular for so-called AD risk genes, or alternatively, if tested for the accumulation of AB or pTau, it was shown that in soft brain tissue and soft hydrogels no risk genes overexpression and / or protein accumulation was found.

[0068] However, in the hydrogel with the pathological viscoelasticity, which is throughout the application defined as stiff, and wherein the findings imitate and resemble tissue conditions of an AD affected brain, and wherein the cultivated cells behave differently and resemble morphological the pathological findings in AD, the results are different. It was found that in such stiff hydrogels the tested brain cells show clear overexpression of AD risk genes and / do accumulate A or pTau.

[0069] On the bases of these findings, it is now possible to test substances of interest on their effects on cultivated cells e.g. brain cells under different viscoelasticity conditions. For the AD research this allows for the first time to test substances of interest on their effect on brain cells under the pathophysiological condition of an AD brain and compare them to physiological conditions of the human brain.

[0070] It is particularly, the comparison between the at least first and at least second hydrogel of the inventive test system, wherein each hydrogel has a distinct and different viscoelasticity allowing deep insights into the development, the physiology and the pathology of different cells and tissues. In case that such cells are exposed to a substance of interest it also allows to observe and evaluate the specific influence of said substance on the cells of interest.

[0071] Accordingly, the herein described 3D cell culture system is suitable to compare, illustrate and / or measure the effect of a substance of interest on the cultured cells in a pharmacological, toxicological, metabolic and / or dose-dependent manner. Such testing in the 3D cell culture system as described herein can be made for individual cell types or collective cell agglomerates as found in tissue probes or primary tissues extracted in a biopsy.

[0072] Particularly, interesting for the present application is the selection of brain cells including neurons, glia cells, e.g. astrocytes, microglia cells and combinations thereof. Additionally, also suitable is the 3D cell culture system for the testing of cell lines derived from brain cells or stem cells differentiated into brain cells.

[0073] All such cells can be tested in the 3D cell culture system on the effect of a substance of interest on such brain cells by employing a method for testing substances and / or compositions which comprises the steps of firstly preparing of the 3D cell culture systems as described above with at least a first and a second hydrogel having selected but distinct viscoelasticity. In a second step the hydrogels are populated with cells to be tested e.g. from the group of brain cells comprising neurons, glia cells, microglia cells and combinations thereof. Alternatively, also cells selected from cell lines derived from brain cells, tissue samples or biopsies derived from patient brains or stem cells differentiated into brain cells have been selected for this method.

[0074] For this, the selected cells will be counted and then mixed into the pre-pared - still liquid - hydrogel solution by gently pipetting the cell-hydrogel mixture up and down until a homogeneous mixture of cel Is- hydrogel solution is created. By slowly increasing the temperature to conditions suitable for the cells to be tested, namely 32°C to 37°C, the hydrogel solution of the present invention starts polymerizing and starts to build up the viscoelasticity predefined by the selection of the polymer preparation, e.g. alginate preparation.

[0075] The cells can then be directly tested after mixing them into the hydrogel or optionally can be cultivated for a defined period to establish e.g. 3D structures, which might be of interest for the testing. The culturing of the brain cells or any other cell types in the at least first and the at least second hydrogel is performed under suitable conditions depending on the cell types. For comparing the results between the hydrogels with different viscoelasticity the cell culture conditions, besides the gel’s biomechanics, are to be identical.

[0076] Consequently, after the culture conditions have been established in the next step of the method the cultivated cells in the at least first and the at least second hydrogel get exposed to at least one compound of interest. The exposure can be a one-time exposure or a repetitive exposure. Furthermore, the exposure can be performed by mixing the substance of interest into the hydrogel e.g. while preparing the gels, drippling the diluted substance to the hydrogel, by soaking the hydrogel or by flooding the hydrogel after the establishment of the test cells in the hydrogel.

[0077] Either right from the beginning of the exposure or after a defined time the cells are analyzed for differences regarding physiological and / or pathological changes. Such analysis cover for example - but is not limited to - tests for metabolic regulation, tests for morphology development, analysis of gene expression or RNA expression, as well as protein expression and / or protein accumulation. Other molecular biological relevant tests or analysis for identifying different reactions are used as well e.g. tests for monitoring protein degradation.

[0078] The data collected from such analysis, or the various tests are, optionally, extracted and prepared for external evaluation or Al-driven evaluation. Alternatively, the collected data are analyzed by comparing the data of a first hydrogel with the data of a second hydrogel, which is typically a negative (untreated) control, or alternatively a hydrogel with a different viscoelasticity.

[0079] Such comparison then allows the identification of substances that cause physiological and / or pathophysiological differences in the cells exposed to said substance. Such comparison further allows to identify substances which are capable of reversing on a cellular level a pathological condition into a normal or physiological condition.

[0080] With this new method and 3D cell culture system for the first time it is possible to not only observe the development of the pathophysiology of e.g. AD but - even more interesting - to identify substances and / or compositions, which interfere, also reduce and delay or even may revert the pathophysiology of AD. Such observations are e.g. intensified by testing whereby the cells of interest are exposed in one or several hydrogels of controlled or standardized stiffness (e.g. still hydrogels) to the substance of interest in comparison to a negative (untreated by the substance of interest) control or a physiological control being a hydrogel with physiological viscoelasticity and treated with the substance of interest.

[0081] Advantageously, by testing further substances more knowledge about the so far still not well understood pathomechanism of neurological disorders including the pathology of e.g. AD may be collected.

[0082] The main characteristic of the method and the 3D cell culture system herein described is that the 3D cell culture system comprises two or more hydrogels, which are made in a highly reproducible manner from primarily - but not limited to - size-defined ADA and collagen, whereby each hydrogel can be reproducible prepared in a distinct stiffness and elasticity and thus, viscoelasticity. While the viscoelasticity can be matched with a tissue specific viscoelasticity, and in this context also with the physiological and pathophysiological viscoelasticity specific for a defined tissue.

[0083] Alternatively, the individual hydrogels can also have gradually or stepwise increasing or decreasing viscoelasticity, in other words having matching viscoelasticity.

[0084] Another characteristic the 3D cell culture system herein described is that the two or more hydrogels are free from any cytokines or free from any other cell stimulating or modulating substances, such as cytokines, growth factors or other inhibiting and / or activating factors. Accordingly, the method herein described and the cells growing in the 3D cell culture system are not affected by any such stimulating, inhibiting and / or activating factors.

[0085] The method herein described is particularly suitable for testing any compound of interest for identifying effects of such compounds regarding its pharmaceutical activity or its metabolical effects on the cells or tissues tested.

[0086] This is particularly helpful for testing already established pharmaceutical drugs and their potential of repurposing them for a treatment of a new therapeutical indication. Particularly interesting in this regard is the testing of pharmaceutical drugs selected from small molecules, antibodies, receptor ligands, biologies and / or biosimilars.

[0087] Further interesting is the testing of pharmaceutical drugs in comparison to the same drug in combination of a pharmaceutical composition comprising adjuvants, pharmaceutical acceptable carrier, diluents and / or excipients. Such comparison allows to characterize and distinguish the pharmaceutical effect form possible side effects caused by additional compounds in a pharmaceutical composition.

[0088] Even more interesting is the testing of a compound of interest on a target tissue when administered in combination with additional and further drugs a patient is taking or might have to take for other unrelated treatments. With such experimental setup, it is for the first time possible to effectively evaluate side effects of combination therapy on the healthy brain but also on already pathologically modified brain tissue. It is thus, further possible to identify also unwanted effects or side effects of drug combination while being used in parallel or in combination therapies, which would otherwise not be predictable or only show due to their side effects.

[0089] It goes without saying that the method and the 3D test system herein described has a tremendous potential for identifying not only side effects of pharmacological drugs or different combinations of pharmacological drugs, but interestingly, for the first time it became feasible to also analyze and test for side effects on the brain tissue and the brain cells of various drugs or combination thereof with or without normal nutrients (e.g. ethanol).

[0090] The method and the 3D cell culture test system herein described has thus the potential not only to replace many in vitro testing but also to reduce or replace preclinical animal testing.

[0091] Particularly in the context of brain research, where suitable animal models are rare, a method as presented herein capable of simulating brain tissue and simulatingthe physiological but also the pathological condition of the human brain is allowing a much quicker and more reliable testing of suitable substances or drugs for the treatment of neurological disorders.

[0092] With this system, which simulates in vivo conditions for the brain, it will also be possible to test for suitable amounts of a substance of interest regarding its therapeutically effective doses or even suitable or necessary doses regimes.

[0093] The results of such doses regime testing also allow to identify a dose, which is potentially lower than the effective treatment dose, but still shows a curing or stabilizing effect and thus can lead to the development of the long-term, low-level treatments for neurological disorders and neurodegenerative disorders.

[0094] Such long-term and / or low-level treatment is advantageously administrable as a prophylactic treatment avoiding the development of neurological disorders and diseases or reducing the risk of developing such neurological disorders and diseases, as it might exist e.g. for patients with a genetic predisposition e.g. for AD.

[0095] Additionally, it will become feasible to prophy lactica Uy treat neurological disorders which dependent on or are caused by changes of the extracellular viscoelasticity of the CNS. One example for a risk that will become prophylactically treatable are brain injuries such as concussion, trauma or encephalitis. The invention further provides a 3D cell culture system, which due to the composition and preparation of the hydrogels provided resemble the stiffness and elasticity, wherein stiffness and elasticity together form a definable viscoelasticity of various human organs. The present application primarily demonstrates the suitability of the 3D cell culture system on the example and for the viscoelasticity of the human brain. This however is only the most complex human organ and is understood as the most complex example. For other human tissues or organs, which have also a characteristic or defined viscoelasticity, the 3D cell culture system of the invention works correspondingly.

[0096] This 3D cell culture system is particularly suitable for identifying and testing substances, e.g. toxic substances, pharmacological substances, but also regular micronutrient or food ingrediencies on their effect on human cells or organs. While it is typically suitable or sufficient to compare the results from 2 matching hydrogels, the system can also comprise several, such matching hydrogels, such as 3, 4, 5, 6, hydrogels. The number of hydrogels included in the system depends on the hypothesis to be tested and the viscoelasticity to be established in the test. The term viscoelasticity is defined for each hydrogel by the parameters for the biomechanical characteristics, namely stiffness and elasticity of the hydrogel.

[0097] According to the invention the 3D cell culture system comprises at least a first and at least a second 3- dimensional (3D) hydrogel made from ADA and collagen and which due to different percentages in the composition of ADA and collagen and / or different ways of polymerization will be adjustable in their biomechanic characteristics. In a preferred embodiment the biomechanics of the at least first and the at least second hydrogel have distinct but matching stiffness and elasticity.

[0098] The at least first and the at least second hydrogel used in the 3D cell culture system have distinct characteristics to allow a comparison and the evaluation of the different parameters on the effect to be tested, namely the effect ofthe testing substance on the human cells or organ.

[0099] At the same time the invention claims that the hydrogels in the 3D cell culture system should be matching. In the context of the invention matching depend on the question asked or hypothesis tested and refers to a distinction, which is reasonable. If e.g. the effect of a substance on a human organ in comparison to a tumor of the same organ is to be tested the matching hydrogels need to resemble on the one hand the normal human organ viscoelasticity and on the other hand the viscoelasticity of the tumor of this organ. For each hypothesis tested the matching will likely be different but the hydrogels prepared for the test need to be “matching”.

[0100] According to further embodiments the 3D cell culture system is suitable to identify and test by comparison effects that would be considered e.g. pharmacological, toxicological or metabolic in nature. Additional test regarding the nature of the effects are generally known to the practitioner and could be e.g. a cytological analysis, a DNA / RNA analysis or e.g. an analysis of distinct (viscoelasticity dependent) gene expression.

[0101] According to one embodiment the 3D cell culture system is particularly suitable to test brain cells. Typically, brain cells are most complicated to grow or keep in cell culture in general and even more complicated to culture under conditions, which allow the morphology in the 3D cell culture to be similar if not identical to normal in situ data of human brain. Even more complicated and thus unreliable is the culturing of mixed cultures of human brain cells. With the 3D cell culture system of the present invention, it is successfully shown for the first time, that mixed cultures of human brain cells will form 3 dimensional structures that resemble the morphology of human brain structures.

[0102] According to further embodiments the human brain cells suitable to be cultures in the 3D system of the invention are selected from the group comprising neurons, glia cells, astrocytes, microglia cells and combinations thereof, which may optionally derive from cell lines, biopsies of patients and / or stem cells. Alternatively, also other human cells, which typically are found in complex organ organizations are selectable for culturing and testing the 3D cell culture system of the invention. Organs with a high degree in complexity regarding their viscoelasticity are e.g. liver, kidney, intestines, bones, cartilage, skin, lung, or even the eye. Thus, the 3D cell culture system of the invention can be adjusted to resemble the viscoelasticity of all these organs and thus can be used to test the various cells of these organs regarding the effect of testing substances.

[0103] According to further embodiments the present application provides also a method for testing substances and / or compositions comprising a compound of interest on their effect on human cells, which comprises the subsequently described steps.

[0104] For the testing depending on the hypothesis to be answered, firstly the at least two hydrogels with different viscoelasticity will be prepared according to prepublished techniques or as described under Example 1. These hydrogels are prepared in a manner that they match the tissue viscoelasticity of the cells to be tested. Alternatively, at least one gel will match the healthy organ viscoelasticity while the at least second gel will be prepared with a diverging or pathological viscoelasticity.

[0105] For populating the at least first and the at least second hydrogel the cells to be tested, preferably human cells, will be diluted in suitable culture medium and mixed with the hydrogel stock solution. The ratio of cells in culture media to hydrogel stock solution regulates the viscoelasticity.

[0106] According to one preferred embodiment human brain cells selected from neurons, glia cells, astrocytes, microglia cells and combinations thereof, or alternatively, cell lines derived from brain cells, or cells from biopsies of patients and / or stem cells differentiated into brain cells can be used in the different methods of testing for e.g. effective pharmaceutical drugs or for side effects of pharmaceutical drugs according to the invention.

[0107] It goes without saying that the culture conditions of the cells to be tested depend on the cell type and need to be adjusted for each cell type or hypothesis to be tested. Consequently, the culture conditions of the cells in the at least first and the at least second hydrogel are to be adjusted to the cell type and then are kept under suitable but identical cell culture conditions to allow comparison of the results. The cells cultivated in the at least first and the at least second hydrogel are then exposed to at least one substance of interest or to be tested. This exposure can occur by adding to the gel, flooding the gel or by soaking the gel with a solution containing the substance of interest. This exposure can be continuously or punctual, depending on the hypothesis to be tested. Further depending on the hypothesis, the exposure can contain different concentrations of the substance to be tested.

[0108] Tests and technologies to analyze the cells cultivated in the hydrogel after the exposure are known to the practitioner and can differences regarding e.g. physiological or / and pathological changes particularly differences in metabolic regulation or morphology, differences in gene expression, differences in RNA expression or differences in protein expression and protein degradation.

[0109] The data from such analysis can be directly compared and illustrate the differences caused by the variations in viscoelasticity of the gels.

[0110] In other embodiments not only the differences in viscoelasticity may be relevant, but it may be relevant to set up the 3D cell culture test system with multiple gels having a viscoelasticity that resembles a specific organ and is suitable for the cells to be tested. In such settings it is testable whether the substance of interest may cause pathological developments, cell death, harm to the normal physiology of the cells to be tested, and also what type of effect (e.g. effects on protein degradation or gene expression), a substance of interests causes in the cell. With this analysis also doses dependent effects can easily be identified, collected, extrapolated and compared to data obtained by other test systems.

[0111] In summary, the 3D cell culture system as herein presented and the method to use this cell culture system for identifying compounds that cause physiological and / or pathophysiological differences in the cells exposed to said compound can not only lead to the identification of new effective drugs, or the concrete doses need for the drug to be effective, but can also evaluate and compare the effect of different drugs in comparable setting to identical cells. This flexibility makes the 3D cell culture test system the ideal tool for pharmacological and / or toxicological testing and will help to replace many animal models, also for cosmetic purposes.

[0112] In the context of the herein described 3D cell culture system orthe method forthe cultivation of one or more species of cells comprises two or more hydrogels made from the polymer ADA (alginate dialdehyde) collagen and optionally laminin, it is known to the practitioner where to get the starting materials such as suitable ADA and collagen, additionally the method to prepare such hydrogels is described in W02020 / 245302 Al. In one example the hydrogels having each a distinct stiffness, elasticity or viscoelasticity have been prepared by usingtwo different alginates (PH124 and PH176, VIVAPHARM JRS Pharma, Ulm, Germany) and / or different concentrations of these alginates. Suitable ranges for the testing are between 0.01 and 10 Pa and depend on the hypothesis tested in the method. If the effect of the tissue viscoelasticity is of interest the test gels involved show preferably a stepwise increase, further preferably increases in regular steps and further preferably increases in e.g. 1 Pa steps. The 3D cell culture system for the cultivation of one or more species of cells is particularly interesting in comparison to presently known hydrogels, particularly the known Matrigel, as the hydrogels of the invention are free from any cytokines or other cell stimulating and / or activating factors.

[0113] With the method of the invention, it is possible to screen and / or test one or multiple compounds of interest to which the cells cultivated in the 3D cell culture system will be exposed for identifying their potential as pharmaceutically-active compound, metabolically-active compound. Compounds that seem of interest and that have an effect of the test cells are then additionally analyzed. With this screening technique the method of the present invention allows the quick identification of potentially interesting compound with an organ specific focus for further pharmaceutical approaches and medical studies. Particularly interesting is the method also for screening and / or testing of well characterized or known pharmaceutical drugs with the intention to identify repurposing opportunities. It is e.g. possible to identify additional, previously unknown, further medical uses - sometimes - with a different organ specificity or suitable for different medical indications. Such repurposing activities with the method of the present invention have identified e.g. the pharmaceutical effects on brain cells which led to the present application. Similar repurposing tests are possible for many or most known drugs but also small molecules, receptor ligands, biologicals, biosimilars or combinations thereof.

[0114] The method according to the invention can also be useful in non-medical approaches, where the effects of regular, non-pharmaceutically active compound, non-toxic compounds and / or nutrients and combination of several nutrients can be tested on the cells cultivated in the 3D cell culture system. It is particularly interesting to test in such contexts the effects of combination of compounds and / or complex combination of several compounds together with other non-pharmaceutical compounds or also pharmaceutical compounds. Such settings allow to analyze patient specific risks or advantages but also can identify the risk and the occurrence of side effects caused by such complex combinations.

[0115] In any such approaches the gel is adapted to provide enhanced predictability of both the expected therapeutic efficacy and potential adverse-effect profile as subsequently observed in patients. In this context it is also feasible to identify with the disclosed method the risks and the occurrence of side effects on cell species and / or organs caused by combinations or synergistic effects of drugs with selected pharmaceutical carrier, diluents or excipients.

[0116] The method as disclosed herein is thus particularly suitable to avoid and / or reduce animal experiments and / or animal models for testing. One advantage is that the disclosed method using one or several hydrogels with defined biomechanical characteristics can be standardized and reproduced in a highly reliable manner. Already for this reason it advantageously for a II kinds of testing and the effects of test substance on human cells in general or from very specific organs.

[0117] It is thus to understand that the method disclosed herein is also particularly useful for screening and testing for side effects, cytotoxic effects and / or effects of cosmetically employed substances on specific cell types e.g. of the skin, mucosal or the eye. Accordingly, the term “cosmetically employed substances” refers to non-pharmaceutical active compounds or combinations of such compounds, which are used or to be used in cosmetic, disinfecting, washing compositions but also compounds or combinations of such compounds used in food or as additions to food preparations.

[0118] Still the most striking and highly fascinating use of the method of the present invention is the use of the 3D cell culture system for the testing of effects on various combinations of human brain cells. Presently, there are no animal models or any other systems that allow to test and / or analyze the effect of one or several substances, compound or combination thereof on the human brain or individualized brain cells. This lack of test systems hampers the development of medication for neurodegenerative diseases dramatically. The more recent problems or stagnation in the research area of Alzheimer treatment is only one example.

[0119] The 3D cell culture system and the method of testing as disclosed herein provide a long-needed model system for the research on neurodegenerative disorders and / or brain diseases, as well as the development or analysis of potentially suitable therapies for neurodegenerative diseases and / or disorders. The 3D cell culture system and the method of testing as disclosed herein also allows a highly reproducible test system to identify suitable dilutions or concentrations that show the desired effects. Further it allows also to identify the cut-off level, where no effect can be seen any longer in the cells to be tested and / or also an analysis of the metabolic effects on the cells to be tested in a dosis regime approach with stepwise changing doses applied.

[0120] The 3D cell culture system for the method oftesting as disclosed herein can be provided in various settings, where e.g. the at least first and the at least second hydrogel are placed in separate containers, seeded with the same cells and then treated by e.g. administering or flooding the test-substance in identical ways but in separate containers. Alternatively, and even more interesting is a setting where the at least first and the at least second hydrogel are placed and seeded in separate containers, which have a permeable or semi-permeable e.g. wall, top or bottom through which the test-substances are soaked, added and / or diffused into the hydrogels.

[0121] It is particularly advantageous that the step of identifying compounds which cause physiological and / or pathophysiological differences in the organ specific cells exposed to said compound, identifies compounds that are suitable for treating disorders and diseases. Additionally, in case of neurological diseases or disorders the method and 3D cell culture system as described herein allows also to identify substances which are useful for the prophylactic administration of such substances.

[0122] In this case the use of the method as described herein is not only a speculation that a certain substance could help or built on a long-term evaluation, but the method as described can in vitro reliably test and observe (even long-term) the reaction of human brain cells (or other organ specific cells) on the effects of the test substance. Thus, for the first time a method and test system are provided which allows to evaluate in vitro the long-term effect of substances on human brain cells (or other organ specific cells). It is, however, not only the fact that a reliable long-term observation is possible with the method described herein, but that also a reliable metabolic / physiological analysis on healthy cells is possible. Thus, the method as described herein helps to identify suitable substances which not only help avoiding but also can counteract the development of neurological disorders and diseases as the metabolic healthy status as well as any deviation or variation thereof is screed and monitored in the cells of interest.

[0123] The method and 3D cell culture test systems allowing the metabolic / physiological monitoring of healthy cells and allow spotting reliable any deviations from a healthy status are not only novel but revolutionary.

[0124] For the area of brain science 3D cell culture system and the method as described herein go one step further as it was for the very first time possible to screen and test human brain cells (e.g. with neurodegenerative markers) in a setting that resembles the extracellular viscoelasticity of the CNS and then identify with the method as described substances that have a curative potential. Such curative potential can be identified as e.g. the downregulation of disease markers as described above and shown in the examples. Any future potential treatment for Alzheimer or any other human neurodegenerative approach should be obliged to undertake such an in vitro testing on its curative potential before starting clinical trials.

[0125] The potential of the method and 3D cell culture test systems herein described is also interesting as it can identify compounds or compositions that show an effect on pathophysiological deviations of brain cells caused by changes of the extracellular viscoelasticity of the central nervous system (CNS). For this the 3D cell couture system is set up with multiple hydrogels that do not only resemble the physiological and / or an age-related viscoelasticity of the brain but also cover all potentially variations of viscoelasticity that the human brain experiences in traumatic accidents or e.g. during and after a stroke. By identifying compounds that are capable of counter acting to the effects of the changes in the viscoelasticity it is - for the first time - possible to find and confirm curative approaches for neurological disorders dependent on or caused by changes of the extracellular viscoelasticity in the brain (concussion).

[0126] The potential of the method and 3D cell culture test systems herein described is not limited to neurodegenerative diseases or disorders. These have been chosen for the present description mainly because it is most obvious that it is an area where in vitro test systems are lacking.

[0127] The method and 3D cell culture test systems herein described are also particularly interesting for personalized medicine approaches to compare the effects and efficacy of a drug, a compound or a combination of substances on the specific cells of an individualized patient in comparison to standardized healthy tissue or even in comparison to standardized tumor cells.

[0128] For this, the method for ex vivo personalized oncology testing, comprises (a) obtaining a cancer cell biopsy from a patient; (b) dissociating the biopsy into multicellular aggregates or single cells;

[0129] (c) suspending the dissociated cells in the hydrogel composition;

[0130] (d) casting the cell-laden hydrogel into a culture well or microfluidic chamber and initiating gelation;

[0131] (e) exposing the hydrogel-embedded cells to one or more candidate agents; and

[0132] (f) evaluating cellular responses.

[0133] This evaluation of cellular responses comprises one or more analysis selected from (i) measuring cell viability via ATP luminescence or live / dead fluorescence assays; (ii) quantifying proliferation via EdU incorporation or Ki-67 immunostaining; (iii) detecting apoptosis via cleaved caspase-3 immunofluorescence; (iv) profiling gene expression by RT-qPCR of sensitivity- or resistance-associated transcripts; and (v) performing time-lapse or high-content imaging to assess morphological changes and invasion.

[0134] The method further comprises analyzing the data obtained in step (f) to determine, for each tested agent, (i) an IC50value or percent inhibition as a measure of therapeutic efficacy, and (ii) off-target cytotoxicity in co-cultured non-tumor cells as a measure of potential adverse effects. The method additionally may comprise integrating the efficacy and toxicity data into a computational decisionsupport framework to generate a personalized treatment recommendation for the patient.

[0135] The method also allows a prognosis wherein the hydrogel-based assay yields a correlation coefficient (R2) greater than 0.8 between the in vitro drug response and clinical outcomes including tumor regression, progression-free survival, or observed toxicities.

[0136] The method and 3D cell culture test systems herein described are furthermore particularly interesting for virological studies or vaccine development as it is possible to compare the effects and efficacy of a vaccine, a drug, a compound or a combination of substances on the virus-infected cells of specific organs in comparison to standardized healthy tissue or cells.

[0137] Description of the Figures

[0138] In the context of the application and in the Figures the abbreviation T-gel stands for “test hydrogel” according to the invention.

[0139] Figure 1 shows the Abeta (hereinafter also identified as Ab, AB or A|3) protein concentration produced by human neuronal stem cells in a 3D in vitro microenvironment. The used test hydrogel (T-gel) “Stiff”, emulating the AD microenvironment, shows A concentration with and without treatment by the test substance according to the invention as described in Example 1 as well as a treatment with different concentrations thereof. Drug concentrations in nanomolar (nm) and protein concentrations in nanogram / milliliter (ng / ml).

[0140] Figure 2 shows the AB protein concentration produced by human neuronal stem cells in a 3D in vitro microenvironment. The used hydrogel T-gel “Soft”, emulating the healthy microenvironment, shows AB protein concentration with and without treatment of the test substance according to the invention as described in Example 1 as well as a treatment with different concentrations thereof.

[0141] Figure 3a und b shows the pTau protein concentration produced by human neuronal stem cells in a 3D in vitro microenvironment. The used hydrogel T-gel “Stiff”, emulating the AD microenvironment, shows pTau protein concentration with and without treatment of the test substance according to the invention as described in Example 1 as well as a treatment with different concentrations thereof.

[0142] Figure 4 shows the pTau protein concentration produced by human neuronal stem cells in a 3D in vitro microenvironment. The used hydrogel T-gel “Soft” shows pTau protein concentration with and without treatment of the test substance according to the invention as described in Example 1 as well as a treatment with different concentrations thereof.

[0143] Figure 5 shows the biomechanical analysis of T-Gels in comparison to Matrigel,

[0144] A. Schematic illustration of the different hydrogel matrices, a 3D network providing structural and biochemical support of the surrounding cells. Graphical illustration of the structural differences of the hydrogels. Matrigel demonstrate no pore size or stiffness adjustment of the hydrogel, while T-Gels offers individually adjustable elastic stiffness and viscosity. B. Biomechanical hydrogel analysis of Matrigel, T-Gels and brain tissue assessed by AFM indentation after 1 day incubation at 37°C. No statistical difference was found between T-Gel soft and mouse brain tissue, whereas Matrigel showed significantly lower storage modulus. T-Gel stiff showed a significantly higher elastic, stiffness compared to T-Gel soft, with a storage modulus of 81.53 Pa. T-Gel stiff, Matrigel and brain tissue showed the same viscosity. T-Gel soft showed a significantly lower viscosity of 36.35 Pa.

[0145] Figure 6 shows the synthetic AB agglomeration in human brain tissue and gels as well as p-Tau and A concentration of AD cells in T-Gels. A - D. Agglomeration of A|3 amyloid in hydrogels and human brain tissue. Immunohistochemical staining of amyloid plaques in human brain using NAB61 antibody and Thioflavin in gels. Synthetic A|3 plaques in Matrigel qualitatively differ and resemble in T-Gels compared to in vivo morphology. E. Staining of AB and p-Tau in T-Gel soft and T-Gel stiff. Immunofluorescent staining of the differentiated AD cells in T-Gel stiff and T-Gel soft after two weeks incubation (green, APP-GFP) with A£ 3D6 (purple) and p-Tau 231 AB9668 (white). Scale bar, 50 pm. F. Boxplot visualizes the results of an ELISA against AB at two timepoints under both soft and stiff gel conditions. Graph shows median and IQR of data points. Significance was tested using a two-way ANOVA (gel: F=11.48, p=0.002, time=145.33, p=2.4e-19; gektime: F=8.61, p=0.0007). Tukeys HSD was then applied to test for differences between gels on individual days (7days p= 0.79; 10 days p=9.2e-5). G. Same as F, visualization of p-Tau abundance. Significance was tested using a two-way ANOVA (gel: F=1.9, p=0.17, time=0.27, p=0.6; gel : time: F=0.003, p=0.96). Tukeys HSD was then applied to test for differences between gels on individual days (7days p= 0.98; 10 days p=2.68e-12) Figure 7 shows the effect of biomechanical matrix changes on microglial gene expression levels with and without synthetic AB. A. Differential expression is reported as direct digital target capture a technique used in genomic studies to capture and enrich specific DNA sequences of interest directly from biological samples for subsequent analysis (delta-delta cycle threshold, ddCT). DdCts of different target genes in T-Gel soft and stiff, with and without synthetic A . The mechanosensitive receptor Piezol shows a significantly decreased expression in T-Gel stiff without AB and a significantly increased expression in T-Gel stiff with AB. Increased gene expression changes were found in ADAMIO in T-Gel stiff with AB but no gene expression changes in T-Gel soft irrespective of AB and stiff without AB. Significantly increased gene expression of ADAM17, TREM2, RHOH and, SIGLEC11 in T-Gel stiff irrespective of AB. Significantly decreased gene expression of BLNK in T-Gel stiff irrespective of AB. B. Activation of Piezol using Yoda showed the same gene expression changes in T-Gel soft with AB as in T- Gel stiff with AB C. Piezol KO clones 1 and 2 showed a rescued gene expression pattern across all genes in T-Gel stiff with AB Box plots with 95% confidence interval and whiskers range, p-values Kruskal-Wallis with Dunn 's post- hoc. Abbreviations: AB = Amyloid beta, soft vs. stiff

[0146] Examples

[0147] Example 1: Substance of interest: GDC-0077

[0148] Tested was a compound of formula III, which was diluted to the various concentrations. The different drug concentrations were added to the cell culture medium which was on top of the stiff and / or soft test gel preparations for efficacy testing.

[0149] Example 2: Cells and cell lines fortesting in the 3D cell culture system

[0150] Human neuronal cell culture with human progenitor Alzheimer’s disease cells

[0151] Expansion and differentiation media for ReNcell VM were prepared following previously established procedures (Nature protocols: A3D human neural cell culture system for modeling Alzheimer's disease DOI https: / / doi.org / 10.1038 / nprot.2015.065 ). ReN FAD (AD cells) (#SCC008FAD2, Alzheimer's In A Dish™ ReN-mGAPIO, Clone D4 NSC Line, Merckmillipore, Germany) were subjected to expansion using ReNcell expansion media (#SCM005, Merckmillipore, Germany), including growth factors 20 ng / ml EGF (#GF144, Sigma, Germany), 20 ng / ml bFGF (#GF003, Sigma, Germany). The removal of growth factors from the ReNcell NCS Maintenance Medium leads to the spontaneous differentiation of ReNcell-immortalized neural progenitor cells. These cells were cultured on T25 or T75 cell culture flasks pre-coated with Matrigel (Merckmillipore, Germany). The cultures were maintained in a controlled environment with 5% CO2at37 °C.

[0152] Example s: Establishing of test hydrogels (T-gel) The test gel (T-gel) is a hydrogel composed essentially of a polymer (ADA), collagen, and laminin as previously described in WO 2020 / 245302 Al. The polymer basis of T-gel is ADA (alginate di-aldehyde) (PMID:32261366).

[0153] To achieve different biomechanical properties, the inventors modified the gel synthesis by using different alginates, in this example PH124 and PH176 (VIVAPHARM JRS Pharma, Ulm, Germany). The ADA synthesis was performed as previously described Sarker eta / ., (J Material Chemistry, Issue 11, 2014) and takes approximately one week. Once synthesized the resulting ADA polymer has a cotton-like structure and can be stored at room temperature in dry conditions.

[0154] For preparing the hydrogels, the selected ADA (here: PH124 or PH176) is dissolved in serum (4xOpti- MEM, ThermoFisher, Germany) followed by adjustment to pH 8.2 usingsodium hydrogen carbonate and filter sterilization using a 0.22 pm filter (Roth, Germany).

[0155] The addition of 7.2 pl ADA solution, 15.8 pl collagen type I (Corning, Germany), 6 pl laminin (1.2 mg, Corning, Germany), 0.4 pl NaHCO3(Roth, Germany), 0.3 pl Pen / Strep (Sigma, Germany), 4.6 pl H2O to ADA at room temperature results in a liquid hydrogel stock solution, which solidifies with a temperature increase to 37 degree Celsius (T-GEL stock). To keep the T-gel stock solution liquid, the compounds are mixed on ice. The T-gel stock is used in a 1:10 solution with a standard culture medium to prepare the test gels.

[0156] Example 4: Preparation of the 3D cell culture test system

[0157] For preparing the “soft” T-gel the stock solution made with ADA PH 123 is diluted 1:10 with cell culture medium and the prewashed test cell as herein described e.g. in Example 2 are mixed into the hydrogel preparation. Correspondingly, is the preparation of the “stiff” T-gel, where the only difference is the selection of ADA being PH176.

[0158] Additionally, the test substance in different concentrations where mixed to the stiff and soft test gels preparation. Then the gel-cell-substance-mixture is then mixed with the suitable test cells and poured into suitable cell culture dishes for incubation in an incubator at 37°C and 5% CO2.

[0159] Example s: Test method for substance of interest

[0160] In a first step one cell culture system is prepared according to Example 4. This cell culture system comprises two hydrogels for the cultivation of suitable cells or cell lines.

[0161] Forthe present testing neurons, capable of expressing Abeta (A ) and pTau are used as described in Example 2. These cells are mixed with the corresponding hydrogel preparation and cultured in the two hydrogels under identical cell culture conditions.

[0162] In a next step the cultivated cells were exposed in the two hydrogels to the substance of interest, herein after substance of Formula III. For exposure the substance of interest is prepared in a suitable diluent with defined concentration, namely 0.0 nm (= untreated), lOnm (nanomolar), lOOnm, 350nm, lpm (micromolar). These preparations are then added onto the gel after the cells are already incubated in the gel. Alternatively, these preparations are mixed into the gel preparation together with the test cells while preparing the T-gels.

[0163] After exposure the test cells are analyzed regarding the physiological and pathological changes in response to the substance of interest. Such analysis covers e.g. test for metabolic regulation and morphology, effects due to the exposure to the substance of interest on gene expression, RNA expression, protein synthesis and protein accumulation. In the present example, the protein concentration for Abeta and pTau produced by ReN cells (Alzheimer’s disease neurons) and the gene expression as RNA expression in microglia was analysed.

[0164] Additionally, the amount of AB protein or pTau produced from the test cells in the different test gels was analyzed and measured in ng / ml or pg / ml. The protein concentration of the untreated cells in comparison to cells treated with the test substance - here Formula III - is illustrated in Figure 1 and 2.

[0165] The comparison between the effects of the substance of interest on the cells in stiff er soft hydrogels as well as in hydrogels exposed or not-exposed to the substance of interest allows to identify substances which are suitable to effectively treat e.g. neurological disorders such as AD.

[0166] Consequently, the data presented, allow the comparison between the microenvironment of the healthy brain versus an AD brain, and additionally prove that the test substance of formula III has particularly in AD brain microenvironment positive effects on (i.e. showing a reduction of) the A and pTau protein accumulation in neurons. Thus, the data included into this application show a substance suitable and promising to reduce the accumulation of A|3 and pTau in neurons and thus, a first drug suitable and promising to treat AD in humans.

[0167] Example 6: Preparation of Aft gels (as control)

[0168] Briefly, lyophilized AJ31-42 (Aggresure™ (AnaSpec) Fremont, USA) was dissolved in 1 ml NT- Gel / HEPES / pH 7.2 solution and plated in the gel preparation made from the 1:10 T-gel stock. The A|3 gels were incubated at 37 °C for 24 h.

[0169] Example 7: AB Detection

[0170] 7,1, Aft analysis - Staining for analyzing the amount in the different test gels

[0171] For A|3 staining, thioflavin S staining 1% ThS solution was added and incubated for 15 min. Followed by a three times wash using PBS. Fluorescence signal (Ex 360 nm; Em 380 nm) was measured using a Axio Observer Z1 (Carl Zeiss).

[0172] 7,2, AB analysis - Immunocytochemistry Cells were treated in the test gels and thereafter isolated from the test gels and washed with PBS. Then the cells were fixed on plates or glass coverslips with 4% paraformaldehyde (Merck Millipore, #1040051000) for 15min at RT. They were washed twice with PBS.

[0173] The fixed cells were stored in PBS at RT or continued with ICC procedure. First, the cells were blocked and permeabilized for 1 hour at 37°C with 3% donkey serum (Pan Biotech, #P30-0101), 0.1% Triton X-

[0174] 100 (Sigma, #T8787) in PBS. Primary antibodies were diluted in blocking solution and the cells were stained for 2h at 37°C. The cells were washed twice with PBS and fluorescently labeled with secondary antibodies (Thermo Fisher Scientific) diluted in blocking solutions for 1 hour at37°C. The cell nuclei were stained for 2min at RT with lpg / ml DAPI (Sigma, #32670). Afterwards, the slides were stored with PBS at RT until microscopy analysis. Images were taken with the Axio Observer Z1 fluorescence microscope (Carl Zeiss) and analyzed with the software ImageJ (version 1.52).

[0175] 7,3, Aft analysis - Gene expression & quantitative PCR

[0176] Primers according to Table 1 for q PCR were purchased from Integrated DNA Technologies (Leuven, Belgium) and are listed afterwards in Table 1 with sequences. In this context the sequence-ID’s refer to a corresponding sequence listing for the present application.

[0177] TABLE 1

[0178] For RNA extraction, cells were plated as describes earlier in T-gel soft and T-gels stiff gels with or without Abeta. RNA was directly isolated accordingto manufacturer’s instructions using the RNeasy Plus Universal Mini Kit (cat. #73404; QIAGEN, Hilden, Germany) and its concentration was determined with a NanoDropTM 2000 spectrophotometer (Thermo Fisher). cDNA was generated directly after RNA isolation accordingto manufacturer’s instructions with High-Capacity cDNA Reverse Transcription Kit (cat. #4368814, Applied Biosystems) and then stored at -20 °C. RNA levels were determined using the Absolute QPCR Mix (SYBR Green, no ROX, cat. #AB1158B, Thermo Scientific).

[0179] Quantitative PCRs were started using the RNA isolated as described above and the different primer pairs as mentioned above to analyze the amount of RNA expressed from the Alzheimer risk genes in response to the treatment with the substance of interest.

[0180] 7,4, Aft analysis - ELISA and p-Tau)

[0181] For A|3 and p-Tau quantification 100000 AD cells were plated in a 1:10 dilution of gekmedium and incubated.

[0182] ELISA analysis was prepared as described in the protocols (A|3, Invitrogen, KHB3491; p-Tau, Invitrogen, KHO0631). In brief, gels were washed with PBS followed by centrifuging at 13000 rounds per minute for 2 minutes. After repeating the last step, the gels were dissolved using 150 pl extraction buffer (Invitrogen, Germany, #FNN0011) including protease phosphatase inhibitor (Thermo Science, Germany, #A32961) vortexed, and incubated on ice for 30 minutes while vortexi ng every 10 minutes. Finally, the supernatant was used for protein quantification in the ELISA.

[0183] Example 8: Neuron and microglia co-culture

[0184] Forthe co-culture experiments cell densities, with 100000 cells per well for AD cell's (#SCC008FAD2, Alzheimer's In A Dish™ ReN-mGAPIO, Clone D4 NSC Line, Merckmillipore, Germany) and ReN G2B2 WT (#SCC008, ReNcell VM humane neural Progenitor cell line, Merckmillipore, Germany) 33 000 cells per well for HMC3 were used.

[0185] T-Gels soft and stiff were used in a 1:10 dilution as described above. AD cells and WT neurons were plated in 1:10 T-Gels and incubated for 7 days at 37 °C, 5% CO2.

[0186] On day 8, 33 000 HMC3 microglia pre-labeled using Cell tracker violet BMQC dye (#C10094, Thermo Fisher scientific, Germany) cells were seeded on top of the T-Gel / neuronal cells. Microglia-neuron interactions were captured using Axio Observer Z1 (Carl Zeiss) for further analysis.

[0187] For testing the substance of interest was soaked into the different test gels or added onto the different test gels in variable concentrations. In this setup not only the direct effect of the therapeutic dose is recognizable, but also the interaction between microglia and neurons in response to the substance of interest is illustrated. The situation is thus even closer to the normal healthy human brain or an AD brain. Analytic testing according to the above-described methods

[0188] Example 9: Characterization of the hydrogel:

[0189] For control purposes also Matrigel* has been used. Matrigel® is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. Briefly, these sarcoma cells are rich in extracellular matrix (ECM) proteins including laminin, collagen IV, heparan sulfate proteoglycans, entactin / nidogen, cytokines and several growth factors. Matrigel is a natural ECM-based hydrogel is a widely used model for 2D and 3D cell culture in vitro. Matrigel is available in multiple formulations and here, we used the standard formulation in 5mL vials (Cat.No. 356234, Corning Life Sciences, Wiesbaden, Germany) used in a 1:10 solution with standard culture medium for each cell type used.

[0190] Both T-gel and Matrigel polymerize quickly at room temperature and for handling before cell culture both hydrogels are kept on ice. All experiments were performed using routine culture medium at routine cell culture conditions (37°C, 5% Oxygen).

[0191] Pore Size Measurements The fiber stucture of T-gels ‘soft’ and ‘stiff’ was imaged using confocal reflection microscopy (Leica SP5, Germany) with a 20x water immersion objective (HCX APO L 20x / 1.00 W, Leica, Germany). Image stacks of the collagen fiber structure were acquired with a volume of 160x160x200pm (voxelsize of 0.31x0.31x0.64pm). From the image stacks, 3D pore diameters were calculated using the covering radius transformation. The mean pore diameters were calculated from three individual samples per condition. Error intervals represent the standard deviation, and the statistical analysis was performed using a two-sided student t-test for p<0.05.

[0192] 3D visualizations of T-gels (volume of 160x160x80pm) were generated using the Python packages

[0193] Pyvista and Saenopy. For better visualization of the collagen fibers, image stacks were Sato filtered. The transparency of the collagen fiber intensity was determined accordingto a sigmoidal transfer function.

[0194] Example 10:

[0195] Cell viability assessment. To compare the effect of the two biomaterials on cell viability and the dynamic of neurite outgrowth in 3D culture, we seeded 150,000 hippocampal neurons / well in 8 replicates for a total of 3 timepoints (7, 10, and 14 days); total of 48 wells. After washing in Hanks balanced salt solution (HBSS, Sigma, Germany), we used the combined viability / cytotoxicity staining kit (LIVE / DEAD™, Lot 2326049, Thermofisher, #L3224). The kit contains 4 pl / ml Calcein-AM and lpl / ml Ethidium homodimer-1 and incubation for 45 min in the dark results in a green cytoplasmic signal (491- 497 nm) when neurons are alive and a red nuclear signal when dead (528-617 nm). After a wash in HBSS we performed fluorescence microscopy (Axio, Zeiss, Germany). For each timepoint and gel, we kept the imaging conditions constant and took 20 dual-color images (LSM file format). For each image and channel, one of the authors (SK) counted the total number of live and dead cells and assigned one of three tiers for neurite density (low, medium, high). We also assessed neurite density by extracting the green channel and quantifying pixels above a uniform threshold using ImageJ / FIJI (Version 2.3.0; http:imagej.net).

[0196] Atomic force microscopy: Briefly, we performed overnight culture of T-Gel and Matrigel on the glass surface of a FD35 dish (World Precision Instruments, #FD35-100). We performed measurements after cooling to room temperature and, to prevent drying, we covered the hydrogels with PBS. For measurements, we glued a 37pm polystyrene beads (microParticles, Germany) onto two cantilevers: Arrow TL1 (NanoWorld, Neuchatel, and SICON (Applied NanoStructures, Inc). We measured each hydrogel at 16 locations using a setpoint of5nN, a Z scanner speed of lOpm / s, and indentation depth of <12pm. We obtained fluidity measurements by holding the cantilever at constant force on the surface for 3 seconds. For plotting of data, we converted the raw hold data into beta values. To plot AFM forcedistance curves, we used the JPK Data Processing software where the Hertz Model was applied to calculate the Young’s Modulus (Poisson’s ratio of 0.5).

[0197] Immunocytochemistry. Cells were fixed on plates or glass coverslips with 4% paraformaldehyde (Merck Millipore, #1040051000) for 15min at RT and washed twice with PBS. The fixed cells were stored in PBS at RT or continued with ICC procedure. First, the cells were blocked and permeabilized for 1 hour at 37°C with 3% donkey serum (Pan Biotech, #P30-0101), 0.1% Triton X-100 (Sigma, #T8787) in PBS. Primary antibodies were diluted in blocking solution, and the cells were stained for 2h at 37°C. The cells were washed twice with PBS and fluorescently labeled with secondary antibodies (Thermo Fisher Scientific) diluted in blocking solutions for 1 hour at 37°C. The cell nuclei were stained for 2min at RT with lpg / ml DAPI (Sigma, #32670). Afterwards, the slides were stored with PBS at RT until microscopy analysis. Images were taken with the Axio Observer Z1 fluorescence microscope (Carl Zeiss) and analyzed with the software ImageJ (version 1.52). Antibodies used IBA1, rabbit, Wako, #019-19741, 1:500, Ab, mouse, Sigma Aldrich, #A8354, 1:100, Donkey anti-rabbit Alexa Fluor 647, Thermo Fisher Scientific, #A31573, 1:750, Donkey anti-mouse Alexa Fluor 546, Thermo Fisher Scientific, #, 1:750, p-Tau, rabbit, Merck, #AB9668, 1:100, Goat anti-rabbit Alexa Fluor405, Merck, #SAB4600461, 1:250. qPCR. For RNA extraction, HMC3 cells were plated as describes earlier in T-gel soft and T-gels stiff gels with or without Ab. RNA was isolated according to manufacturer’s instructions using the RNeasy Plus Universal Mini Kit (QIAGEN, Hilden, #73404) and its concentration was determined with a NanoDrop™ 2000 spectrophotometer (Thermo Fisher). cDNA was generated directly after RNA isolation according to manufacturer’s instructions with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814) and then stored at -20 °C. RNA levels were determined using the ABsolute QPCR Mix (SYBR Green, no ROX, Thermo Scientific, #AB1158B,). Following primers were purchased from Integrated DNATechnologies (Leuven, Belgium):

[0198] Mesoscale Discovery Assay. Chemiluminescence multiplex using the MSD kit enabled the quantification of four distinct cytokines, namely interleukin 1 beta, interleukin 6, interleukin 8, and tumor necrosis factor (IL-lb, IL-6, IL-8, and TNF-a), utilizing the V-PLEX Human Pro Inflammatory Panel II 4-plex kit (MSD, Rockville, #K15053D-l). Pre-plated hydrogels, both with and without synthetic A|3, were prepared. After 48 hours of incubation, 10000 HMC3 cells were seeded per 8-well Ibidi chamber on top of Matrigel, T-Gel soft, and T-Gel stiff substrates. After an additional 24 hours, the positive control was stimulated by adding 100 ng / ml of lipopolysaccharide (LPS, Sigma, #L2630) to the medium, and the medium was not changed. After 24 hours following the LPS stimulation, all supernatants were collected and stored at -80 °C until analysis, following MSD instructions.

[0199] ELISA (AB and p-Tau). For AB and pTau quantification 100000 AD cells were plated in a 1:10 dilution of gekmedium and incubated for 7 to 14 days. ELISA analysis was prepared as described in the protocols (Ab, Invitrogen, #KHB3491; p-Tau, Invitrogen, #KHO0631). In brief, gels were washed with PBS (Gibco, #10010-015) followed by centrifuging at 13000 rounds per minute for 2 minutes. After repeating the last step, the gels were dissolved using 150 pl extraction buffer (Invitrogen, #FNN0011) including protease phosphatase inhibitor (Thermo Science, #A32961) vortexed and incubated on ice for 30 minutes while vortexing every 10 minutes. Finally, the supernatant was used for protein quantification in the ELISA.

[0200] Neuron and microglia co-culture. For the co-culture experiments cells cell densities, with 100.000 cells per well for AD cell's (Alzheimer's In A Dish™ ReN-mGAPIO, Clone D4 NSC Line, Merckmillipore, #SCC008FAD2) and ReN G2B2 WT (ReNcell VM humane neural Progenitor cell line, Merckmillipore, #SCC008) 33000 cells per well for HMC3 were used. T-Gels soft and stiff were used in a 1:10 dilution as described above. AD cells and WT neurons were plated in 1:10 T-Gels and incubated for 7 days at 37 °C, 5% CO2. On day 8, 33000 HMC3 microglia pre-labeled using Cell tracker violet BMQC dye (Thermo Fisher scientific, #C10094) cells were seeded on top of the T-Gel / neuronal cells. Microglia-neuron interactions were captured using Axio Observer Z1 (Carl Zeiss) for further analysis.

[0201] To enable in vitro studies of AD pathology, in which all three hallmarks of the disease are present, we developed an alginate-based matrix with tuneable mechanical properties. These tuneable matrices, which we coined ‘T-gels’, consist of alginate as a backbone and covalently bound collagen I to provide adhesion sites for integrins. Their mechanical properties can be tuned through the usage of different alginates: differences in the sequence of a-L-guluronic acid and [3-D-mannuronic acid result in different elastic and viscous properties. Cross-linking is initiated by heating the gel pre-mix from 0°C to 37°C, similarly, as done with Matrigel, a common gold standard for 3D cell culture in the field.

[0202] For this study, we chose T-gels with elastic storage moduli (a measure of the elastic stiffness of a material) close to those of brain tissue, with one gel mimicking the stiffness of brain tissue and a second one which was about twice as stiff. Both T-gels and brains were significantly stiffer than Matrigel. Loss moduli, which characterize a material’s viscous properties, were similar for brain tissue, stiff T-gels, and Matrigel. Pore sizes of collagen fibres were similar in both T-gel variants, with up to -6% larger pores in stiffer T-gels, corresponding to ~0.4 pm difference, suggesting that cells were not exposed to drastic alterations in the porous structure of their microenvironment.

[0203] To investigate whether T-gels provided a suitable cell environment, we quantified cell survival rates of human neural progenitor cells (ReNcell VM) and primary C57 / B6 mouse hippocampal cells in T-gels and Matrigel using Ethidium homodimer 1 (EthD-1) and Calcein stains. In all tested conditions, cell survival was between 90 and 100 % after 14 days in vitro, indicating that T-gels are suitable for long-term cell culture.

[0204] To test if T-gels facilitated the agglomeration and thus studies of A plaques, as previously been shown in Matrigel, we added monomeric Ali to the gels and assessed plaque formation 24 hours after seeding using immunofluorescence. We found prominent Afi plaques in all matrices. However, in Matrigel, Afi plaques were characterized by sharp edges, and we detected a considerable background signal, likely arisingfrom non-agglomerated monomeric Afi. In contrast, in soft and stiff T-gels, little background signal was detected, and A plaques formed loose fibrillary structures similar to the plaques found in human AD tissue in vivo.

[0205] Familial Alzheimer’s disease (FAD) is an inherited form of AD, characterized by early-onset and is primary liked to mutations in the amyloid precursor protein (APP) and presenilin 1 (PSEN1) genes. These mutations lead to the overproduction of AB and p-Tau, which are critical to the pathogenesis of AD. In order to assess how matrix stiffness may contribute to the development of the three AD hallmarks, we seeded human neural progenitor cells containing two FAD mutations into stiff and soft T- gels. These cells (Alzheimer's In A Dish™ ReN-mGAPIO, Clone D4 NSC line; from here termed ‘AD cells’) overexpress APP and PSEN1. In both gels, AD cells accumulated p-Tau and secreted AB, leading to plaque formation in the matrices as shown by immunofluorescence. Using ELISAs, we quantified the amount of AB and p-Tau in the gels as a function of matrix stiffness and time in culture. AB concentrations were similarly high (-1 ng / ml) in both gels already after 7 days in vitro (DIV). At 10DIV, the AB concentration dropped significantly in the stiff but not in the soft T-gels. After two weeks, however, AB concentrations similarly dropped to about half of the concentrations found at 7 DIV in both gels. In contrast, p-Tau concentrations were similarly low (-0.4 ng / ml) in both gels at 7 DIV and at 14 DIV. The substrate-dependent differences in AB and p-Tau concentrations we identified at 10 DIV suggested that both AD hallmarks are regulated by substrate mechanics.

[0206] Having established that T-gels supported the survival of human-derived neuronal progenitor cells and the accumulation of pTau and AB-plaques in AD cell culture, we next investigated the inflammatory response of human induced microglial cells (hiMGs) to synthetic AB and matrix stiffness. As neuroinflammation is related to microglial morphology, we first quantified the distribution of hiMG cell shapes in soft T-gels. We visualized the cells ’ shapes using immunohistochemistry after 1 DIV and automatically segmented their morphology using the pre-trained deep-learning algorithm ‘cellpose’. We found a wide distribution of cell morphologies, reaching from round shapes (circularity ~1) to heavily elongated ones (circularity ~0). The distribution of the cells’ circularity was heavily left-skewed, with -45% round hiMGs. When lipopolysaccharide (LPS), which induces inflammation in microglia, was added, the fraction of circular hiMGs significantly increased to -60%. In the absence of LPS, the addition of A led to a similar rise in hiMG circularity, indicating that microglial shapes responded similarly to LPS and AB plaques.

[0207] To assess whether this change in cell shape was related to the immune-activation of microglia, we quantified the release of pro-inflammatory cytokines in human microglial cells (HMC3) using Meso Scale Discovery (MSD) assays after 2 DIV. Here, we focused on interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin lb (IL-lb), and tumour necrosis factor a (TNFa), which are all elevated in AD. The application of LPS led to a significant increase in the release of all cytokines by microglia in both soft and stiff T- gels. However, in T-gels containing AB but no LPS, only the release of IL-6 was significantly increased after 2 DIV in both soft and stiff T-gels if compared to control conditions, while that of IL-8, IL-lb, and TNFa was not. These data suggested that the morphology of microglia was not directly related to their immune-activation status, and that AB by itself only caused a mild inflammatory response.

[0208] When testing the effect of 3D substrate stiffness on microglial cytokine expression, levels of IL-6 and IL-8 were significantly increased in stiffer compared to softer substrates, while those of IL-lb and TNFo / p / ?o were largely independent of substrate stiffness. We found a similar trend in cytokine expression in human microglial cells exposed to AB, suggesting that substrate stiffness had a larger effect on microglial immune-activation than AB itself. However, the effect of substrate stiffness on microglial cytokine expression was still considerably weaker than that of LPS. The application of LPS to microglia led to significantly higher cytokine levels in soft substrates if compared to stiff ones. As in the absence of a pro-inflammatory chemical stimulus levels of cytokines were higher in stiffer environments, these data indicated that cytokine release in response to substrate stiffness and LPS was activated by different mechanisms in a non-cooperative manner.

[0209] As substrate stiffness led to an immune activation of HMC3 in T-gels, and microglial activation accompanies AD, we next tested the impact mechanical substrate properties on the expression of several high-risk genes recently implicated in AD progression in microglia. HMC3 cells were cultured in soft and stiff T-gels, either with or without AB, and mRNA levels of the AD risk genes BLNK, ADAM10, ADAM17, TREM2, RhoH, and SIGLEC11 were quantified by qPCR after 1 DIV. Because microglial mechanosensing via mechanosensitive ion channels was recently shown to be potentially involved in AD, we also measured the mRNA expression levels of Piezol, a mechanosensitive ion channel found in microglia.

[0210] The mechanical properties of the T-gels had a strong influence on the expression levels of all tested AD risk genes analysed except for ADAM 10, while the expression levels of Piezol were insensitive to substrate stiffness. The expression levels of BLNK decreased by ~3 fold in stiff T-gels if compared to soft ones. The levels of all other tested genes were significantly increased, between ~5 fold in ADAM 17 and -100 fold in SIGLEC11. In line with the previous set of experiments, the addition of Ali to the T-gels did not alter this microglial response to substrate stiffness. Most of the analysed genes showed the same pattern in response to matrix stiffness as without AB, suggesting that AB is not a critical factor in regulating the expression of AD risk genes in microglial cells. However, substrate stiffness was a strong driver of the expression of these risk genes.

[0211] To test how microglia transduced matrix stiffness into the observed changes in gene expression patterns, we perturbed the activity of the mechanosensitive ion channel, Piezol. Because Piezol activity is increased on stiff substrates, we first tested whether Piezol activation with the specific agonist Yodal would result in a ‘stiff phenotype’ of cells embedded in a soft T-gel in the presence of AB. Indeed, enhanced Piezol activity of human microglia in a soft environment led to enhanced AD risk gene expression as seen in stiff matrices, indicating that the effect of matrix stiffness on microglia is largely mediated by Piezol.

[0212] To corroborate this finding, we tested whether depletion of Piezol in microglia cultured in stiff environments results in a decreased expression of AD risk genes. When CRISPR / Cas9-induced Piezol knock-out HMC3 cells were grown in stiff T-gels, gene expression levels were strongly decreased for all genes investigated if compared to control cells in stiff T-Gels. In fact, RhoH was the only gene whose expression level exceeded that of microglia cultured in soft T-gels. However, it was still a factor of -10 smaller than in microglia grown in stiff T-gels. The expression levels of all other genes except TREM2, whose levels were similar to those found in soft T-gels, were barely detectable and orders of magnitude smaller than in microglia grown in soft T-gels. These data confirmed that the expression of AD risk genes in microglia is regulated by substrate mechanics through the activity of Piezol, with stiffer substrates promoting risk gene expression.

[0213] After having established a system in which all three AD hallmarks can be studied, we next tested if it can be used to study interactions between microglia and neurons expressing A and p-Tau. AD cells and controls (ReNcell VM human neural progenitor cells) were embedded in soft and stiff T-gels and allowed to grow for 7 days. After 7 DIV, HMC3 microglia were added on top of the polymerized T-gels. Cell numbers and spatial relationships were analysed after further 1, 4, and 8 DIV of co-culture.

[0214] At all-time points investigated and in both types of T-gels, the number of microglial cells seen at the level of the neurons was significantly smaller in the cultures containing wild-type neurons than in those with AD neurons. This number remained relatively constant after 1 DIV, suggesting that microglial cells were attracted by the AB plaques and / or pTau deposited by AD cells and migrated through the gels to the neurons within one day, while in the control condition they remained on top of the cultures. Additionally, we observed microglial cells to display a halo-form of clustering around the AD neurons, significantly distinct from random positioning. This is suggestive of active attraction to the neurons.

[0215] In contrast, the number of neuronal cells continuously decreased from 8 DIV in all conditions. At most time points, the number of AD cells was considerably smaller than the number of control neurons despite similar seeding densities, likely because of phagocytosis by microglia cells. Across conditions, the number of neurons was also significantly smaller in stiffer gels, suggesting that microglial cells become activated by increased stiffness of their surroundings, leadingto the observed decrease in neuron number.

[0216] References:

[0217] Barker eto / . (2020) “Mini Review: Opposing Pathologies in Cancer and Alzheimer’s Disease: Does the PI3K / Akt Pathway Provide Clues”, (Frontiers in Endocrinology, June 2020, Vol.11). Sarker et al., (2014), “Fabrication of alginate-gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties" (J Material Chemistry, Issue 11,)

Claims

CLAIMS1. A substance or composition for the use in the treatment of neurological disorders and / or Alzheimer Disease (AD) wherein the substance is a PI3K-i nhibitor.

2. The substance or composition for use in the treatment of neurological disorders according to claim 1, comprising a compound of formula I,wherein the compound of formula I is a Benzoxazepine, a stereoisomer, constitutional isomer, geometric isomer, tautomer or pharmaceutically acceptable salt thereof; and wherein the compound of formula I has a first residue R1and a second residue A.

3. The substance or composition for the use according to claim 1 or 2 wherein the first residue R1is and whereinthe second residue A is or whereinthe first residue R1is and whereinthe second residue A is4. The substance or composition for the use according to any of the claims 1 to 3, wherein the neurological disorder is selected from the group comprising Alzheimer’s Disease, Amyotrophic Lateral Sclerosis (ALS), Dementia, Vascular dementia, Frontotemporal dementia, Dementia with Lewy bodies, Parkinson’s disease, Huntington’s disease, progressive supranuclear palsy, corticobasal degeneration, multisystem atrophy, Down syndrome (DS), inflammatory bowel diseases, Crohn's disease, ulcerative colitis, meningitis and encephalitis, migraine, Creutzfeldt- Jakob disease and neuroborreliosis (Lyme disease), multiple sclerosis, neuromyelitis optica, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, epilepsies or seizure disorders, progressive myoclonic epilepsy, traumatic brain injuries, spinal cord injuries, glioblastoma, brain metastasis, neuromuscular disorders, myasthenia gravis, muscular dystrophies, spinal muscular atrophy, polyneuropathies and diabetic polyneuropathy.

5. The substance or composition for the use according to any of the claims 1 to 4, wherein the substance or composition is administered oral, intravenous, subcutaneous, intrathecal, sublingual, transbuccal, transdermal, nasal, pulmonal, rectal, as Antibody-Drug-complex and / or as nanoparticles.

6. The substance or composition for the use according to any of the previous claims 1 to 5, wherein the substance or composition is administrable as part of a retard composition; and / or wherein the substance or composition comprises the compound of Formula (I) in a therapeutically effective amount.

7. The substance or composition for the use according to any of the previous claims 1 to 6, wherein the treatment is prophylactic or post-expositional prophylactic.

8. A 3D cell culture system for identifying and testing substances and / or compositions to be tested for their effect on human cells comprising an at least first and one at least second hydrogel, wherein the at least first and the at least second hydrogel are essentially made of ADA and collagen,wherein the at least first and the at least second hydrogel are 3-dimensional; and wherein the at least first and the at least second hydrogel have distinct and / or matching stiffness and elasticity, wherein stiffness and elasticity together form a definable viscoelasticity.

9. The 3D cell culture system according to claim 8, wherein the effect is of pharmacological, toxicological, metabolic nature or a combination thereof.

10. The 3D cell culture system according to claim 8 or 9, wherein the brain cells are selected from the group comprising, neurons, glia cells, astrocytes, microglia cells and combinations thereof, which are derived from cell lines, biopsies of patients and / or stem cells differentiated into brain cells.

11. A method for testing substances and / or compositions comprising a compound of interest, for their effect on brain cells, comprising the steps: preparing / providing at least one 3D cell culture system comprising at least two hydrogels according to any of the claims 8 to 10 for the cultivation of one or more species of cells; populating the at least first and the at least second hydrogel with cells selected from the group of brain cells comprising neurons, glia cells, astrocytes, microglia cells and combinations thereof, OR cell lines derived from brain cells, biopsies of patients and / or stem cells differentiated into brain cells. culturing the brain cells in the at least first and the at least second hydrogel under suitable and / or identical cell culture conditions; exposing the cultivated cells in the at least first and the at least second hydrogel to at least one substance of interest; analyzing the cells after exposure for differences regarding physiological or / and pathological changes regarding metabolic regulation or morphology, for differences in gene expression, differences in RNA expression, for differences in protein expression and level, collecting data from the analysis and, optionally, extracting the collected data; comparing the collected data from the at least first and the at least second hydrogel of the 3D cell culture system according to claim x; and identifying compounds that cause physiological and / or pathophysiological differences in the cells exposed to said compound according to the analysis of the data collected in this method.

12. The method according to claim 11, wherein the 3D cell culture system for the cultivation of one or more species of cells comprises two or more hydrogels made essentially from ADA and collagen, which are having each a distinct stiffness, elasticity or viscoelasticity in a rangebetween 0.01 and 10 Pa and being selected for method by matching viscoelasticity which preferably increases stepwise, further preferably increases in regular steps and further preferably increases in 1 Pa steps.

13. The method according to claims 11 to 12, wherein the 3D cell culture system for the cultivation of one or more species of cells comprises two or more hydrogels made from ADA and collagen, which are free from any cytokines or other cell stimulating and / or activating factors.

14. The method according to any of the claims 11 to 13, wherein the compound of interest to which the cells cultivated in the 3D cell culture system are exposed is a pharmaceutically-active compound, a metabolically-active compound, a pharmaceutical drug, a small molecule, a receptor ligand, a biological or biosimilar.

15. The method according to any of the claims 11 to 14, wherein the step of identifying compounds that cause physiological and / or pathophysiological differences in the brain cells exposed to said compound, identifies compounds that are suitable for treating neurological disorders and diseases and / or for the prophylactic administration avoiding the development of neurological disorders and diseases.

16. The method according to any of the claims 11 to 14, wherein the compound of interest to which the cells cultivated in the 3D cell culture system are exposed is a combination of compounds or a combination with other non-pharmaceutical compounds.

17. The method according to any of the claims 11 to 15, wherein the compound of interest to which the cells cultivated in the 3D cell culture system are exposed is in combination with pharmaceutical acceptable carrier, diluents or excipients.

18. The method according to any of the claims 11 to 16, wherein the compound of interest to which the cells cultivated in the 3D cell culture system are exposed is a combination of non- pharmaceutical active compounds.

19. The method according to any of the claims 11 to 17, wherein the compound of interest to which the cells cultivated in the 3D cell culture system are exposed is presented in stepwise dilutions.

20. The method according to any of the claims 11 to 18, wherein the compound of interest to which the cells cultivated in the 3D cell culture system are exposed, is presented onto the various hydrogels of the 3D cell culture system by incubating, soaking, flooding or as a premix during gel preparation.

21. The method according to any of the claims 11 to 20, wherein the step of identifying compounds that cause physiological and / or pathophysiological differences in the brain cells exposed to said compound, identifies compounds that are capable of prophy lactica Uy avoiding and / ortreating neurological disorders dependent on or caused by changes of the extracellular viscoelasticity of the CNS.

22. A method for ex vivo personalized oncology testing, comprising:(a) obtaining a cancer cell biopsy from a patient;(b) dissociating the biopsy into multicellular aggregates or single cells;(c) suspending the dissociated cells in the hydrogel composition;(d) casting the cell-laden hydrogel into a culture well or microfluidic chamber and initiating gelation;(e) exposing the hydrogel-embedded cells to one or more candidate agents; and(f) evaluating cellular responses.

23. The method according to claim 22 wherein evaluating cellular responses comprises one or more of: (i) measuring cell viability via ATP luminescence or live / dead fluorescence assays; (ii) quantifying proliferation via EdU incorporation or Ki-67 immunostaining; (iii) detecting apoptosis via cleaved caspase-3 immunofluorescence; (iv) profiling gene expression by RT- qPCR of sensitivity- or resistance-associated transcripts; and (v) performing time-lapse or high- content imaging to assess morphological changes and invasion.

24. The method according to claim 22 or 23 further comprising analyzing the data obtained in step (f) to determine, for each tested agent, (i) an IC50value or percent inhibition as a measure of therapeutic efficacy, and (ii) off-target cytotoxicity in co-cultured non-tumor cells as a measure of potential adverse effects.

25. The method according to claim 22 to 24, further comprising integrating the efficacy and toxicity data into a computational decision-support framework to generate a personalized treatment recommendation for the patient.

26. The method according to claim 22 to 25, wherein the hydrogel-based assay yields a correlation coefficient (R2) greater than 0.8 between in vitro drug response and clinical outcomes including tumor regression, progression-free survival, or observed toxicities.

Images