Dose regimens for the glutaminyl cyclase inhibitor varoglutamstat

Abstract

The present invention is concerned with dose regimens for administering the glutaminyl cyclase inhibitor varoglutamstat, and pharmaceutical compositions containing the same, to a subject, providing an optimized therapeutic window for therapeutic uses thereof and methods of treatment employing them.

Description

[0001] DOSE REGIMENS FOR THE GLUTAMINYL CYCLASE INHIBITOR

[0002] VAROGLUTAMSTAT

[0003] Field of the Invention

[0004] The present invention is concerned with dose regimens for administering the glutaminyl cyclase inhibitor varoglutamstat, and pharmaceutical compositions containing the same, to a subject, providing an optimized therapeutic window for therapeutic uses thereof and methods of treatment employing them.

[0005] Background of the Invention

[0006] Glutaminyl cyclases (QC and isoQC, EC 2.3.2.5) catalyze the intramolecular cyclization of N- terminal glutamine (Gin, Q), and, at a lower rate, also glutamate (Glu, E) residues into pyroglutamic acid (pGlu; pE), liberating ammonia or water, respectively. A QC was first isolated by Messer from the latex of the tropical plant Carica papaya in 1963 (Messer, M. (1963), Nature 4874: 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. (1987), J. Biol. Chem. 262: 8532-8536; Fischer, W. H. and Spiess, J. (1987), Proc. Natl. Acad. Sci. USA 84: 3628-3632). For mammals, the conversion of Gin into pGlu by QC was shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. (1987), J. Biol. Chem. 262: 8532- 8536; Fischer, W. H. and Spiess, J. (1987), Proc. Natl. Acad. Sci. USA 84: 3628-3632). In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in bovine pituitary, supporting the suggested function in post-translational peptide hormone maturation (Bockers, T. M. et al. (1995), J. Neuroendocrinol. 7: 445-453).

[0007] WO 2008 / 034891 discloses novel glutaminyl-peptide cyclotransferase-like proteins (QPCTLs), which are isoenzymes of glutaminyl cyclase (isoQC), and isolated nucleic acids coding for these isoenzymes. Isoenzymes of glutaminyl cyclase were discovered in several mammalian species including humans. It was confirmed that the two enzymes, namely QC (also known as QPCT) and the isoenzyme isoQC (also known as QPCTL) are different. QC and isoQC are expressed in several tissues and organs of the body, including brain, lung, heart, liver and kidney. Crystal structures of both QC and isoQC for in silico screening are also known (WO 2012 / 022804 and WO 2012 / 059413).

[0008] QC and isoQC are closely related single-zinc metalloenzymes, which exhibit nearly identical substrate specificity in vitro, and the major difference refers to subcellular localization: QC is secreted from expressing cells; isoQC is a resident enzyme of the golgi complex (Cynis et al. (2008), J. Mol. Biol. 379: 966-980). It was shown that recombinant human QC as well as QC activity from brain extracts catalyze both, the N-terminal glutaminyl as well as glutamate cyclization. Most striking is the finding, that cyclase-catalyzed Glui -conversion is favored around pH 6.0 while Gl -conversion to pGlu-derivatives occurs with a pH-optimum of around 8.0.

[0009] Physiological substrates of QC and isoQC in mammals are, e.g. amyloid beta-peptides (3-40), (3- 42), (11-40) and (11-42), ABn, ADan, Gastrin, Neurotensin, FPP, CCL2, CCL7, CCL8, CCL13, CCL16, CCL18, Fractalkine, Orexin A, [Gln3]-glucagon(3-29), [Gln5]-substance P(5-l l) and the peptide QYNAD (WO 2008 / 034891 and W02010 / 026209), and also hormones such as thyreotropin-releasing hormone (TRH) and GnRH (Goren et al. (1977), Mol. Pharmacol. 13: 606- 614; Abraham & Podell (1981), Mol. Cell. Biochem. 38: 181-190; Awade et al. (1994), Proteins 20: 34-51). Furthermore, several proteins participating in the establishment of extracelluar matrix (ECM) are either proven substrates for QC / isoQC-catalyzed post-translational modifications or can be postulated to be likely substrates in analogy due to comprising N-terminal Gin or Glu residues - and this structural feature seems to be quite conserved for the species human, rat and mouse: e.g. collagens (Bornstein et al. (1970), Biochem. 9: 4699-4706; Bornstein (1969), Biochem. 8: 63-71; Kang et al. (1967), Biochem. 6: 788-795; Rauterberg et al. (1972), Eur. J. Biochem. 27: 231-237; Hoerlein et al. (1979), Eur. J. Biochem. 99: 31-38; Weil et al. (1990), J. Biol. Chem. 265: 16007- 16011; Cowan et al. (2022), Drug Test Anal. 14: 808-819), fibronectin (Garcia-Pardo et al. (1983), J. Biol. Chem. 258: 12670-12674) and fibromodulin (Oennerfjord et al. (2004), J. Biol. Chem. 279: 26-33).

[0010] The QC metalloenzyme is upregulated in the brains of Alzheimer’s disease patients. QC generates pyroglutamate A0 (pGlu-A ), a modified, pathogenic form of the peptide, by catalyzing the cyclization of an exposed glutamate at the N-terminus of A0. The enzyme has been reported to be highly expressed in affected cortical regions in AD; pGlu-A0 resulting from this enzymatic conversion has been found to be toxic, highly aggregation-prone, and a major component of amyloid plaques in humans (Morawski, M. et al. (2014), J. Alzheimers Dis. 39: 385-400; Frost, J. L. et al. (2013), Am. J. Pathol. 183: 369-81). In preclinical work, QC-inhibitors have been reported to reduce amyloid pathology and improve performance in learning and memory tests in various mouse models (Schilling, S. et al. (2008), Nat. Med. 14: 1106-11; Hoffmann, T. et al. (2017), J. Pharmacol. Exp. Ther. 362: 119-130.). Thus, varoglutamstat represents a small-molecule approach for reducing pGlu-A0 generation in treatment of Alzheimer’s Disease.

[0011] CCL2 (MCP-1), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL16, CCL18 and fractalkine (CX3CL1) are chemotactic cytokines (chemokines) which attract and activate leukocytes and play a fundamental role in inflammation and in numerous pathophysiological conditions.

[0012] It was found that the isoenzyme of glutaminyl cyclase (isoQC) is an important regulator of monocyte infiltration under inflammatory conditions (Cynis et al. (2011), EMBO Mol. Med. 3: 545-558). Cynis reported that isoQC-inhibitors effectively inhibit CCL2 maturation, and that CCL2 plays a pivotal role in several conditions, including atherosclerosis, pancreatitis, Alzheimer's disease, MS and cancer. It was shown that the chemotactic activity of CCL2 depends on a modified N-terminus of the polypeptide, particularly the formation of a pyroglutamate (pE)-residue protecting against proteolytic degradation in vivo. The N-terminal pE of CCL2 can be post- translationally formed by both, glutaminyl cyclase (QC, QPCT) or its isoenzyme iso-glutaminyl cyclase (isoQC, QPCTL) (Schilling et al. (2003), J. Biol. Chem. 278: 49773-49779; Cynis et al. (2008), J. Mol. Biol. 379: 966-980; Cynis et al. (2011), EMBO Mol. Med. 3: 545-558). The N- terminal pE modification makes the protein resistant against degradation by aminopeptidases, and it is important for the chemotactic potency of CCL2 (Van Damme et al. (1999), Chem. Immunol. 72: 42-56). Artificial elongation or degradation leads to a drastic decrease of function although CCL2 still binds to its receptor (CCR2) (Proost et al. (1998), J. Immunol. 160: 4034-4041; Zhang et al. (1994), J. Biol. Chem. 269: 15918-15924; Masure et al. (1995), J. Interferon Cytokine Res. 15: 955-963; Hemmench et al. (1999), Biochem. 38: 13013-13025).

[0013] Fibrosis is a progressive and potentially fatal process that can occur in numerous organ systems, characterized by the excessive deposition of extracellular matrix (ECM) proteins such as collagens and fibronectin. Fibrosis affects normal tissue architecture and impedes organ function. Defined by the pathological accumulation of ECM proteins, fibrosis results in scarring and thickening of the affected tissue - in essence it represents an exaggerated wound healing response which can interfere with normal organ function (Neary etal. (2015), Fibrogenesis Tissue Repair 8: article 35).

[0014] Collagens are the most abundant protein in the ECM. Fibronectins are glycoproteins that connect cells with collagen fibers in the ECM, allowing cells to move through the ECM. Fibromodulin participates in the assembly of the collagen fibers of the ECM.

[0015] With collagens as potential substrates for QC activity (this term combines both, enzyme activities of QPCT and QPCTL), QC activity can be relevant at two different stages of collagen deposition: either by acting on N-terminally located Glu and Gin on the respective N-propeptides, or, upon extracellular cleavage of the N-propeptide, on an N-terminally located Glu or Gin of the main collagen domain. To date, such a post-translational modification has been described in the literature mainly for collagen I, but for collagen III as well.

[0016] Renal fibrosis is characterized by excessive deposition of ECM, leading to destruction of normal kidney architecture and loss of renal function. The activation of myofibroblasts plays a key role in this process. (Yuan et al. (2019), Adv. Exp. Med. Biol. 1165: 253-283).

[0017] As one mode-of-action, QC-inhibitors (this term combines both, inhibition of QPCT and / or QPCTL) are supposed to suppress the progression of inflammation-induced renal dysfunction by inhibiting especially the CCL2 / CCR2 axis, but also inflammatory signals exerted by other monocyte chemoattractant molecules being substrates for glutaminyl cyclases like e.g. CCL7, CCL8, CCL13 and CX3CL1. Chronic inflammation, characteristic of CKD, is often the trigger for a fibrotic process. The inflammatory process is transmitted through epithelial and endothelial cells, which give rise to inflammatory mediators including cytokines and chemokines among others, which in turn lead to the recruitment of inflammatory cells: lymphocytes, polymorphonuclear leukocytes, eosinophils, basophils, mast cells, and macrophages. These inflammatory cells release, among others, transforming growth factor beta 1 (TGF-01), a potent fibrogenic factor that induces the activation of fibroblasts, increasing the synthesis of extracellular matrix (ECM) proteins (Panizo et al. (2021), Int. J. Mol. Sci. 22: 408-426). Innate immune cells are key contributors to kidney inflammation and fibrosis. Infiltration of the renal parenchyma by innate immune cells is governed by multiple signaling pathways. Since the discovery of the chemokine fractalkine (CX3CL1) and its receptor, CX3CR1 over twenty years ago, a wealth of evidence has emerged linking CX3CL1-CX3CR1 signaling to renal pathologies in both acute and chronic kidney diseases (AKI and CKD, respectively). Although acute autoimmune kidney disease is often successfully treated with immunomodulatory medications, there is a notable lack of treatment options for patients with progressive fibrotic CKD. CX3CL1-CX3CR1 interactions mediate important events in the intra-renal pathophysiology of CKD progression, particularly via recruitment of innate immune cells into the kidney. QC-inhibitors act on the CX3CL1-CX3CR1 system and offer therefore an attractive alternative for the treatment of fibrotic CKD. Chemokine CC motif ligand 7 (CCL7), also known as monocyte chemotactic protein (MCP)-3, is a chemotactic factor for monocytes and neutrophils. Elevated CCL7 expression is observed in cardiovascular disease, diabetes mellitus, and kidney disease. (Chang et al. (2022), Cardiovasc. Diabetol. 21: 185-192) It has been suggested that CCL7 may promote the progression of atherosclerosis and aortic aneurysm and play a significant role in the inflammatory events underlying most vascular diseases, diabetes mellitus, and kidney disease by attracting macrophages and monocytes to amplify inflammatory processes and contribute to the disease progression. However, there are currently no target drugs or small molecule drugs against CCL7 available. As a substrate of QC / isoQC, the post-translational modification and activity profile of CCL7 is vulnerable to enzyme inhibition by QC-inhibitors, which thus offer an attractive alternative for the treatment of forms of CKD.

[0018] Thus, QC-inhibitors act on post-translational modifications on all of e.g. CCL2, CCL7, CX3CL1, which thus offer an attractive alternative for the treatment of forms of CKD. Given a predominance of proven and potential substrates for QC activity as part of the establishment of extracellular matrix (ECM), including several pro-collagens and collagens like collagen I and collagen III among others, fibronectin and fibromodulin, and of the potential targets for QC activity in chemoattractant processes for myofibroblast precursors (CCL2 / CCR2 and CCL21 / CCR7), QC-inhibitors exert anti -fibrotic effects as a second arm of their mode-of-action.

[0019] In animal models, a glomerular filtration rate (GFR) can be measured, e.g. by transcutaneous measurements of FITC-Sinistrin clearance for GFR estimation in mice.

[0020] In clinical practice, however, direct measurement of GFR is rather intricate, therefore creatinine clearance or estimates of creatinine clearance based on the serum creatinine level, or serum Cystatin C levels are used to estimate GFR (eGFR). (cf. e.g. Pei etal. (2013), PLoS ONE 8: e57852, and references cited therein) Creatinine is produced naturally by the body (creatinine is a breakdown product of creatine phosphate, a small molecule, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the peritubular capillaries in small amounts such that creatinine clearance overestimates actual GFR by 10% to 20%. Futhermore, creatinine levels are also influenced by muscle mass, which is quite variable. This margin of error is acceptable, considering the ease with which creatinine concentrations can be measured from serum samples. Unlike precise GFR measurements involving e.g. constant infusions of inulin, creatinine is already at a steady-state concentration in the blood, and so measuring creatinine levels is much less cumbersome. Estimated GFR (eGFR) is now recommended by clinical practice guidelines and regulatory agencies for routine evaluation of GFR whereas measured GFR (mGFR) is recommended as a confirmatory test when more accurate assessment is required.

[0021] There are alternative methods and formulae used in practice to calculate the eGFR, e.g MDRD (Modification of Diet in Renal Disease) and CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration), with methods or variants thereof being dependend on either creatinine or cystatin C measurements alone or of a combination of both. In the example hereinbelow, the so-called "4- variable MDRD" has been used, which estimates GFR using four variables: serum creatinine, age, ethnicity, and gender. The GFR is used to describe the severity of a CKD. For most patients, a GFR over 60 ml / min / 1.73m2is considered adequate. The severity of chronic kidney disease (CKD) is described by six stages; the most severe three stages are defined only by the eGFR value itself (as shown below; stages 3, 4 and 5), whereas definition of the first three, less severe stages also depends on whether there is other evidence of kidney disease (e.g., proteinuria):

[0022] 0) Normal kidney function - GFR above 90 ml / min / 1.73 m2and no proteinuria

[0023] 1) CKD1 - GFR above 90 ml / min / 1.73 m2with evidence of kidney damage

[0024] 2) CKD2 (mild) - GFR of 60 to 89 ml / min / 1.73 m2with evidence of kidney damage

[0025] 3) CKD3 (moderate) - GFR of 30 to 59 ml / min / 1.73 m2

[0026] 4) CKD4 (severe) - GFR of 15 to 29 ml / min / 1.73 m2

[0027] 5) CKD5 (kidney failure) - GFR less than 15 ml / min / 1.73 m2

[0028] Some people add CKD5D for those stage 5 patients requiring dialysis; many patients in CKD5 are not yet on dialysis.

[0029] Given the two postulated arms of QC-inhibitor mode-of-action, modulation of inflammatory signals and of ECM deposition, QC-inhibitors offer a unique option for treatment of this variety of kidney diseases.

[0030] Certain inhibitors of glutaminyl cyclase (QC), also named glutaminyl-peptide cyclotransferase (QPCT), and its iso-form isoQC (QPCTL) are described in WO 2011 / 029920. WO2011029920A1 also discloses a general synthesis description of varoglutamstat as free base, and mentions several pharmaceutically acceptable salts derived from a general addition of respective acids. No specific salt form of varoglutamstat is disclosed in WO2011029920A1 , though. WO 2011 / 029920 describes a broad range of compounds and discloses 235 example compounds, and varoglutamstat ((S)-l- (lH-benzo[d]imidazol-5-yl)-5-(4-propoxyphenyl)imidazolidin-2-one) is disclosed therein as one. WO 2011 / 029920 further describes the use of said compounds of formula I in the treatment of a disease or disorder selected from the group consisting of Kennedy’s disease, duodenal cancer with or without Helicobacter pylori infections, colorectal cancer, Zolliger-Ellison syndrome, gastric cancer with or without Helicobacter pylori infections, pathogenic psychotic conditions, schizophrenia, infertility, neoplasia, inflammatory host responses, cancer, malign metastasis, melanoma, psoriasis, impaired humoral and cell-mediated immune responses, leukocyte adhesion and migration processes in the endothelium, impaired food intake, impaired sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance or impaired regulation of body fluids, multiple sclerosis, the Guillain-Barre syndrome, chronic inflammatory demyelinizing polyradiculoneuropathy, mild cognitive impairment, Alzheimer’s disease, Familial British Dementia, Familial Danish Dementia, neurodegeneration in Down Syndrome and Huntington’s disease, rheumatoid arthritis, atherosclerosis, pancreatitis and restenosis.

[0031] WO 2024 / 256618 Al discloses the hydrochloride salt to be a preferred chemical entity, and a polymorph thereof.

[0032] The glutaminyl cyclase inhibitor varoglutamstat is the compound (S)-l-(lH-benzo[d]imidazol-5- yl)-5-(4-propoxyphenyl)imidazolidin-2-one and can be represented by the following structural formula I:

[0033] The synthesis of varoglutamstat is described in WO 2011 / 029920 Al on pp. 152 to 155, which is incorporated herein in its entirety.

[0034] A first clinical phase II study with varoglutamstat has been conducted in subjects with biomarker- proven Alzheimer’s disease (AD) (SAPHIR; Scheltens et al. (2018), Alzheimer's Res. Ther. 10: 107-120). This was a randomized, double-blind, placebo-controlled trial. The aim was to scrutinize the maximal tolerated dose, and to determine level of target inhibition and treatment-related pharmacodynamic effects (safety, tolerability and efficacy) of varoglutamstat, which was administered at 800 mg twice daily (BID) for 11 weeks in subjects with mild cognitive impairment or mild dementia due to AD. The 120 enrolled subjects were treatment-naive at the start of the study. After 1 week of treatment with 400 mg BID, patients were switched to 800 mg BID for 11 weeks. Patients were randomized 1 : 1 to either varoglutamstat or placebo.

[0035] There were slightly more patients with a serious adverse event (SAE) in the varoglutamstat group compared to placebo, although statistically not significant: In the varoglutamstat group, eight subjects reported 13 SAEs and in the placebo group, five subjects reported five SAEs. The SAEs reported were heterogeneous in nature and showed no pattern pointing to a specific effect of varoglutamstat. Six subjects in the varoglutamstat group discontinued treatment due to the SAEs and none in the placebo group.

[0036] The number of subjects with treatment emergent adverse events (TEAEs) was not significantly different between study groups, although the varoglutamstat-treated subjects reported slightly more TEAEs: In the varoglutamstat group, 45 subjects reported 135 TEAEs (3.0 TEAEs per reporting subject), while 40 subjects in the placebo group reported 103 TEAEs (2.6 TEAEs per reporting subject).

[0037] More subjects treated with varoglutamstat discontinued treatment due to adverse events, mostly related to gastrointestinal and skin / subcutaneous tissue disorders. With regard to discontinuations due to TEAEs, none in placebo group was opposed by 20 discontinuations under varoglutamstat treatment.

[0038] The majority of AEs were classified as mild or moderate in severity, with a shift towards moderate or severe within the varoglutamstat group (combined 46% of reported TEAEs in varoglutamstat group versus 22% in placebo). Subjects in the varoglutamstat group reported TEAEs most frequently in the system organ class (SOC) categories of gastrointestinal disorders (n = 21), infections and infestations (n = 17), and skin and subcutaneous tissue disorders (n = 15). The most frequently reported TEAEs in the placebo group were in the SOC categories of gastrointestinal disorders (n = 12) and infections and infestations (n = 17). Five placebo subjects reported skin and subcutaneous tissue disorders. Thus, the greatest differences between incidences of TEAEs were observed in the SOC categories of gastrointestinal disorders and skin and subcutaneous tissue disorders. Within these two categories, subjects in the varoglutamstat group reported mostly nausea, diarrhea and constipation, and rash and urticaria. Subjects in the placebo group reported mostly nausea, abdominal pain upper and rash in these categories. AEs in the SOC category of infections and infestations showed no difference between treatment arms.

[0039] Varoglutamstat-related TEAEs within the SOC of skin and subcutaneous tissue disorders were heterogeneous (reddening of skin / exanthema, rash, urticaria) and occurred in week 2 to week 8 after the start of treatment, with already around 50% of TEAEs occuring until day 28 of treatment. There is currently no clear explanation for this observation, but it was suggested that these events observed with varoglutamstat may be caused by a hypersensitivity reaction to the drug or one of its metabolites.

[0040] In summary, the results showed an overal good safety and tolerability profile with a comparable number of subjects reporting TEAEs or SAEs under varoglutamstat treatment or placebo, with TEAEs in varoglutamstat group tending to be of higher severity, and more subjects treated with varoglutamstat discontinuing treatment due to AEs especially in the first 8 weeks of the exposure, though.

[0041] In this clinical study, varoglutamstat treatment resulted in a significant reduction in glutaminyl cyclase activity measured in CSF samples collected from all subjects at end of treatment (EOT; Scheltens et al. (2018), Alzheimer's Res. Ther. 10: 107-120): upon 11 weeks of treatment with 800 mg BID, the mean relative QC activity (as determined according to the general assay principle as described in Example 1 of this invention and stated relative to the respective subject’s QC activity from pretreatment measurement) decreased to 39% in CSF at EOT (p < 0.001), whereas the mean relative QC activity within the Placebo group remained constant at 99.9%. It was concluded that the maximal tolerated dose of varoglutamstat had been reached (800 mg BID). However, the increased level of severity observed for TEAEs reported and notably the high number of subjects treated with varoglutamstat discontinuing treatment due to AEs especially in the first 8 weeks of exposure strongly limits the usability of the dose regimen used in this clinical study for future studies.

[0042] Thus, for the benefit of the patient to be treated with QC-inhibitors, a carefully adjusted dose regimen is required, addressing time course and dose levels implemented in an optimized uptitration protocol, final maximum dose, and once daily vs. twice daily dosing options.

[0043] Description of the invention

[0044] The present invention generally solves this problem by providing an extended (slow) uptitration regimen, potentially starting even with sub-pharmacological doses of varoglutamstat together with applying overall lower maximum doses which still reach a certain target occupancy for fostering efficacy, thus improving adherence to treatment.

[0045] In a first aspect, the invention provides solutions for optimized uptitration protocols and maximum dose selection for balancing target occupancy and safety.

[0046] As stated above, even though there is currently no clear explanation for the increased TEAE occurrence early on during treatment, it was suggested that these events observed with varoglutamstat may be caused by a hypersensitivity reaction to the drug or one of its metabolites. If this assumption were true, a slower uptitration schedule at the start of the treatment may allow for an adaptation of the immune system to the drug and reduce the number and severity of adverse events substantially.

[0047] Thus, a multicenter, randomized, double-blind, placebo-controlled, parallel group dose finding study including 259 human subjects selected for Mild Cognitive Impairment and Mild Dementia due to Alzheimer’s Disease was conducted, in which patients were treated with varoglutamstat hydrochloride versus placebo incorporating a significantly slowed down and thus extended uptitration strategy (VIVIAD; Example 1). Furthermore, the question was addressed which maximum dose level might be required to get efficacy whilst maintaining a good therapeutic window. The highest dose was selected as 600 mg BID, and to be already below the 800 mg BID used within the Phase Ila study (SAPHIR) described above. Furthermore, the new study was intended to compare safety aspects up to a DSMB (Data Safety Monitoring Board) decision for 300 vs. 600 mg BID, also allowing for a post-study evaluation of different levels of target occupancy in serum and CSF samples. The uptitration was performed carefully and slowly, starting at a sub-pharmacological dose of only 50 mg once daily, taking already 8 weeks until reaching 300 mg BID, and another 4 weeks (in total 12 weeks) to reach the highest dose envisaged, 600 mg BID. Uptitration steps were selected as follows: 50 mg once daily (2 weeks), 50 mg BID (2 weeks), 150 mg BID (4 weeks), 300 mg BID (4 weeks for uptitration). Thus, varoglutamstat or placebo tablets were administered orally once daily in weeks 1 and 2 and twice daily orally from week 3 onwards. The total treatment duration was between 48 and 96 weeks. Subjects were randomized 1:1 :1 (placebo, 300 mg, 600 mg; all BID) for the first 90 subjects included. Subjects randomized between the 90th randomized subject and the DSMB decision were randomized 1: 1 for 300 mg BID or placebo, and 1 : 1 on placebo and 600 mg BID as decided by the DSMB afterwards. Furthermore, all patients randomized to 300 mg BID at the time of the DSMB decision received the chosen dose (600 mg BID) afterwards up to week 48 to 96.

[0048] In order to allow a comparison of safety aspects between VIVIAD and SAPHIR studies, TEAE reporting was summarized for 11 weeks of treatment on high dose (600 mg BID in VIVIAD vs. 800 mg BID in SAPHIR), thus generally covering 23 weeks from baseline for VIVIAD versus 12 weeks from baseline in SAPHIR (which represented the EOT in SAPHIR), taking into account the respective uptitration phases (12 weeks for VIVIAD, 1 week in SAPHIR) (Table 2).

[0049] Comparing the percentage of subjects reporting any TEAEs, numbers were elevated in SAPHIR for the treatment group (800 mg BID) vs. placebo (75 vs. 67%, respectively), the difference of which was diminished in VIVIAD after extended uptitration and 11 weeks on high dose (600 mg BID in this study) reaching comparable levels (62 vs. 59%). A surprisingly strong improvement for the extended uptitration protocol is visualized by the percentage of discontinuation due to TEAEs reported for verum groups, which dropped from 33% in SAPHIR for verum group to 0% in VIVIAD. This success for the new dosing regimen was further documented by comparing numbers and percentages of the severity grades “moderate” and “severe” for TEAEs: moderate TEAEs occurred more often in verum then in placebo in either study, but the factor over placebo was diminished for VIVIAD to 1.46 (vs. 1.65 in SAPHIR), and severe TEAEs were decreased in VIVIAD to an identically low level as observed in the respective placebo group (both around 2.5% only), whereas severe TEAEs were reported in 2% of placebo subjects in SAPHIR as well, but the percentage within the verum group was clearly higher at 13%.

[0050] Similarly, percentages of subjects reporting serious TEAEs, number of SAEs (serious adverse events), and number of serious TEAEs on verum treatment decreased strongly within VIVIAD compared to SAPHIR, approaching levels observed within the respective placebo group.

[0051] In SAPHIR, most prominent differences for verum treatment vs. placebo in reported TEAEs were observed in Medical Dictionary for Regulatory Activities (MedDRA) system organ classes (SOC) gastrointestinal disorders and skin and subcutaneous tissue disorders. A slower uptitration was envisaged to affect the appearance of such adverse events within the treatment group, and its impact was unexpectedly substantial: for the gastrointestinal disorders SOC, the difference in ratios per subject of treatment vs. placebo were decreased from 0.15 in SAPHIR to 0.05 in VIVIAD, and from 0.17 in SAPHIR to 0.05 in VIVIAD for the skin and subcutaneous tissue disorders SOC.

[0052] In conclusion, the slower and extended uptitration combined with a lower maximum dose of 600 mg vs. 800 mg BID as administered in SAPHIR resulted in a clear diminuation of moderate but especially of severe and serious TEAEs as well as SAEs reported for the period up to 11 weeks on high dose. Furthermore, accumulation of TEAEs on verum within the two SOCs gastrointestinal disorders and skin and subcutaneous tissue disorders as observed in SAPHIR was clearly attenuated by treatment with 600 mg BID following a 12 week uptitration phase.

[0053] Such an outcome is only meaningful, though, if the maximum dose level is still sufficient to provide relevant target inhibition and thus potential for efficacy. QC activity was determined in serum and CSF samples from patients treated with placebo or varoglutamstat 2-6 h post-dose at week 48 of treatment, and is reported relative to the respective pretreatment level of same patient to be set to 100% QC activity. Since a steady state of exposure and QC secretion for a given dose is expected to be established within several days, it is assumed that inhibition of QC activity as evaluated with the assay chosen is comparable after 11 weeks vs. 36 weeks of treatment on a given dose level, which refers to week 23 vs. week 48 of total treatment duration. The assay is set to monitor any impact of blood concentration of varoglutamstat being present within this time period 2-6 h postdose on the activity of QC enzyme being simultaneously present within the serum. Mind that QC- inhibition as determined by this assay is expected to understimate the actual level of enzyme inhibition in vivo as for this assay, a 1:3 dilution is performed on samples prior to measurement, thus the varoglutamstat concentration in such samples is decreased by this factor. The relative QC activity (as referenced to the pretreatment QC activity determined for these subjects) in assay from serum samples collected 2-6 h post-dose revealed a slight increase in relative QC activity (median) at week 48 in placebo group (to 110%) and a dose-dependent decrease at week 48 (down to 66% for 300 mg treatment and to 37% for 600 mg treatment) (Figure 7B). With regard to relative QC activity in assay from CSF samples, its median proved to be stable over time within the Placebo group (95% at week 48), and a dose-dependent decrease was found at week 48 (down to 73% for 300 mg treatment and to 59% for 600 mg treatment) (Figure 7A). Thus, treatment of patients with 600 mg BID instead of 800 mg BID resulted in an increase of remaining enzyme activity in CSF from around 39% for 800 mg to 59% for 600 mg treatment. With a target engagement in circulation resulting in a remaining QC activity of around 37% applying 600 mg BID, it was concluded that the overall target inhibition is still suitable for achieving efficacy for modulating respective target proteins of QC / isoQC enzymes for systemic applications, at least outside the brain.

[0054] This was confirmed by data for estimated glomerular filtration rate (eGFR), which was calculated from serum creatinine concentrations using the MDRD formula. As a result of serum creatinine analysis in human subjects as shown in Example 1 hereinbelow, it has been demonstrated that the treatment with varoglutamstat increases the eGFR significantly when compared to untreated control subjects. The annualized eGFR change from baseline significantly increased within the treatment group, while the annualized eGFR change fell under the baseline for the placebo group. The difference between the treatment group and the placebo group for the annualized eGFR change in the total population tested was highly significant (see Figure 6 A). It has also been found that the treatment effect on the annualized eGFR change from baseline was dose-dependent (see Figure 6B), which reflects the dose-dependency of the inhibition of QC activity within the circulation.

[0055] With this promissing results at hand, it was now the next requirement to evaluate a potentially shortened uptitration period, as any week of treatment on a sub-pharmacological dose is considered a loss for the benefit of the patient. Hence, another clinical study was designed to evaluate the efficacy of 600 mg BID in patients with early Alzheimer’s Disease, but this time implementing a quicker uptitration phase of just 150 mg BID for 4 weeks, followed by 300 mg BID for 4 weeks, so that the final dose level of 600 mg BID was reached after 8 weeks already, by omitting the very low dose starting levels of 50 mg once and twice daily (intermediate uptitration phase; Example 2; VIVA-MIND).

[0056] In this study, relative QC activity was determined from serum collected from subjects 2-6 h postdose in week 24, thus upon 16 weeks of treatment on high dose of 600 mg BID, using the same assay as described in Example 1. From these samples, placebo group maintained its QC activity level as measured pretreatment (median 103%), whereas 600 mg BID treatment resulted in a decrease of QC activity to 12% of the respective pretreatment activity (Figure 8). Mind that mean timepoint of post-dose blood sampling for these subjects can strongly affect the outcome of QC activity measurements as maximum serum concentration Cmaxof varoglutamstat is usually reached at 1-1.5 h pre-dose (Lues et al. (2015), Alzheimers Dementia Transl. Res. Clin. Intervent. 1: 182- 195), thus the sampling window of 2-6 h reflects already the descending arm of exposure past Cmax with an effective half-life ti / 2eff of 2-3 h.

[0057] Analyzing the impact of the implementation of an intermediate uptitration phase, omitting the sub- pharmacological starting doses of 50 mg, first once then twice daily, surprisingly showed no deterioration of safety aspects in VIVA-MIND vs. VIVIAD (Example 1) (Table 2). Again, TEAE reporting was summarized for 11 weeks of treatment on high dose (600 mg BID), thus generally covering 19 weeks from baseline for VIVA-MIND, taking into account the uptitration phase of 8 weeks.

[0058] Comparing percentage of subjects reporting any TEAEs, general numbers were somewhat elevated in VIVA-MIND compared to VIVIAD - but the numbers were comparable for placebo and verum group (73 vs 71%, respectively). Three discontinuations due to TEAEs were observed, which is higher than in VIVIAD (7 vs. ~1%), but still clearly lower as in SAPHIR (33%).

[0059] The competitiveness of the intermediate uptitration protocol was further documented by comparing numbers and percentages of the severity grades “moderate” and “severe” for TEAEs: moderate TEAEs occurred more often in verum then in placebo in either study, but the factor over placebo was diminished for VIVA-MIND even further to 1.26, compared to the already improved factor of 1.46 for VIVIAD, and severe TEAEs were reported for similar percentages for placebo and verum group in VIVA-MIND as well as in VIVIAD.

[0060] Similarly, percentages of subjects reporting serious TEAEs, number of SAEs (serious adverse events), and number of serious TEAEs on verum treatment were very similar to those of VIVIAD and thus decreased strongly compared to SAPHIR.

[0061] With regard to ratios per subject reported within the MedDRA system organ classes (SOCs) gastrointestinal disorders and skin and subcutaneous tissue disorders, which were identified in SAPHIR to produce most prominent differences for verum treatment over placebo in reported TEAEs, such ratios were identically low for VIVIAD and VIVA-MIND within the gastrointestinal disorders SOC, and comparably low in the skin and subcutaneous tissue disorders SOC, and thus clearly diminished for verum treated groups in either setting compared to SAPHIR (Table 2).

[0062] Concluding from the data of this aspect of the invention, implementation of a slow and extended uptitration protocol (12 weeks to reach high dose) combined with a somewhat decreased maximum dose of 600 mg BID resulted in a clear attenuation of treatment emergent adverse events (TEAEs), combined with still excellent levels of QC-inhibition as monitored in serum and CSF samples 2-6 h post-dose, which proved to be efficacious in significantly improving eGFR, a marker for kidney function. Surpisingly, the extended uptitration protocol prevented any patient on verum to discontinue due to a TEAE, which was not expected in this clarity given a discontinuation rate of 33% due to TEAEs in SAPHIR (only 1 week of uptitration). Certain tightening of the uptitration protocol to a duration of 8 weeks unexpectedly resulted in a very similar and bening safety profile as observed with the extended uptitration protocol, only a few discontinuations were now observed again. However, providing a benefit for the patient by avoiding elongated uptitration periods on sub-pharmacological doses, this intermediate uptitration protocol still provides a very suitable alternative for clinical application. Thus, uptitration protocols and maximum dose selection as described in Examples 1 and 2 provide excellent solutions for balancing target occupancy and safety for treatment of human subjects with QC-inhibitors like varoglutamstat.

[0063] For certain indications, depending on the substrates to be modulated by QC-inhibition required to get efficacy, higher exposure levels might still be advantageous, e.g. for addressing brain pathology. In such cases, a high dose of 800 mg BID shall still be envisaged as applied in SAPHIR, but now combined with a slow to intermediate uptitration protocol.

[0064] With regard to uptitration protocols, even the intermediary protocol is still considered to be quite careful with regard to the time allowed for system adjustment of 4 weeks. Depending on the indication at hand and the life-cycle of relevant QC substrates to be addressed within its respective pathology, state steady balances between QC inhibitor dosing and potentially adjusted substrate levels as well as potential systematic responses to the latter can be assumed to be adjusted and established even within one week time. Thus, on a case by case evaluation for different indications and patient populations envisaged, respective uptitration steps of verum doses can be expected to be beneficial for the overall treatment adherence when each performed indepentdently from the other steps at one, two, three or four week intervals.

[0065] Accordingly, in preferred embodiments of the first aspect, the invention provides varoglutamstat ((S)-l-(lH-benzo[d]imidazol-5-yl)-5-(4- propoxyphenyl)imidazolidin-2-one) or a pharmaceutically acceptable salt, solvate or polymorph thereof, including all tautomers thereof, or a pharmaceutical composition comprising said varoglutamstat, for use in the treatment of a disease, wherein said treatment comprises an uptitration phase and a treatment phase of varoglutamstat administration, and wherein said varoglutamstat is administered to a subject in a daily uptake of 10 mg to 1800 mg, preferably 10 mg to 1600 mg, more preferably 10 mg to 1500 mg.

[0066] Preferably, when the treatment phase comprises twice daily administration of at least 300 mg varoglutamstat, said uptitration phase comprises once daily administration of 50 mg varoglutamstat for 1-4 weeks, followed by twice daily administration of 50 mg varoglutamstat for 1-4 weeks, followed by twice daily administration of 150 mg varoglutamstat for 1-4 weeks, and followed by twice daily administration of 300 mg varoglutamstat for 1-4 weeks to a subject.

[0067] In a preferred embodiment of the first aspect, the uptitration phase comprises once daily administration of 50 mg varoglutamstat for 2 weeks, followed by twice daily administration of 50 mg varoglutamstat for 2 weeks, followed by twice daily administration of 150 mg varoglutamstat for 4 weeks, and followed by twice daily administration of 300 mg varoglutamstat for 4 weeks to a subject, and the treatment phase comprises twice daily administration of 300 mg or twice daily administration of 600 mg varoglutamstat to a subject.

[0068] In another preferred embodiment, when the treatment phase comprises twice daily administration of at least 300 mg varoglutamstat, said uptitration phase comprises twice daily administration of 150 mg for 1-4 weeks, followed by twice daily administration of 300 mg varoglutamstat for 1-4 weeks, even more preferably both steps for 4 weeks.

[0069] The treatment phase of the first aspect preferably comprises twice daily administration of 300 mg to 800 mg varoglutamstat, more preferably twice daily administration of 300 mg to 750 mg, most preferably twice daily administration of 600 mg varoglutamstat to a subject.

[0070] In a second aspect, the invention provides support for applying the QC-inhibitor varoglutamstat just once daily in certain indications.

[0071] Given that the glutaminyl cyclases are relevant enzymes for post-translational modification of way more than a 150 potential substrates, an appropriately chosen balance between maximum exposure and exposure time profile of QC-inhibitor concentrations might result in certain selectivity for certain substrate activities given that different substrates are expected to have a different time protocol for their expression, conversion, and / or their cellular homeostasis to exert their intended pharmacological purpose. Accordingly, it was speculated that for modulation of certain substrate activities in pathological conditions even a once daily dosing regimen might suffice, compared to the twice daily dosing regimen realized for the treatment of Alzheimer’s Disease patients (Examples 1 and 2). Based on the unexpected and strong beneficial impact on eGFR data in patients with early Alzheimer’s Disease, it was postulated to evaluate such a potential within the indication area of kidney diseases.

[0072] The adenine-diet induced ADI-CKD model in mice was selected as relevant in vivo animal model. In this model, a metabolite of adenine crystalizes within the kidney and leads to tissue injury resulting in an inflammatory response and tubulointerstitial fibrosis, eventually also affecting the glomerulus and thus the glomerular filtration rate, and having metabolic impact as well. (Yang et al. (2024), Renal Failure 46: article 2336128) QC-inhibitors were thus evaluated in such a model (Example 3), the overall results of which will be described elsewhere - for the purpose of this invention, a potential for achieving efficacy upon once daily dosing combined with a beneficial impact on safety is of relevance. A treatment impact within such mice was compared for varoglutamstat (PQ912) being administered at either 100 or 200 mg / kg BID for three weeks vs. 200 mg / kg QD for the same time.

[0073] The mouse model performed robustly and opened up a good therapeutic window between undiseased and diseased Vehicle-treated groups. A highly significant increase of Collagen IV (Col IV, Col4) deposition in the kidney upon disease induction was observed, which was significantly reduced upon treatment with PQ912 at either 200 mg / kg once daily (QD) or twice daily (BID) (Figure 9). BID dosing at 100 mg / kg was not able to affect Col IV readout significantly.

[0074] Hematologic parameters were evaluated to monitor safety aspects of the different dosing regimens for PQ912 in these mice. Unexpectedly, an impact of PQ912 treatment on lymphocyte count was observed, which did not correlate with total dose per day, but was rather driven by BID dosing compared to QD dosing. The decrease of lymphocytes under PQ912 BID treatment was diminishing lymphocyte numbers even below the levels of healthy control animals (Group 1) (Figure 10). Even though these findings did not reach significance levels (which were assessed in this case against Group 1, non-diseased control animals with vehicle administration), this trend may lead to unwanted side effects.

[0075] Consequently, once daily dosing of PQ912 in ADI-CKD mice at a dose of 200 mg / kg provides evidence of efficacy on disease relevant parameters and simultaneously seems to provide a larger safety window when compared to twice daily dosing with PQ912 (either 100 mg / kg or 200 mg / kg, BID).

[0076] Furthermore, these findings emphasize the different potential for QC-inhibitor application in different indications with regard to applied dosing regimens, as conversion of selected substrates out of the large set of potential QC / isoQC substrates might be best affected either by a rather continuous inhibitor presence throughout the day or rather by a certain time coverage per day followed by a “drug holiday” of rather low to ineffective inhibitor concentrations. With regard to kidney diseases, results from the disease relevant ADI-CKD model indicate that once daily treatment might open up a larger therapeutic window while still being efficacious.

[0077] Allometric scaling from such a treatment dose (200 mg / kg, PO, QD) in mice according to Nair and Jacob (2016), J. Basic Clin. Pharmacy 7: 27-31 results in a human equivalent dose (HED) of 1056 mg per day for a patient of 65 kg. Thus, reasonable treatment of human kidney disease patients with a benign risk-benefit-ratio is postulated for once daily dosing as mono-treatment of 450 to 1200 mg, preferably as 600-1050 mg, most preferably as 1050 mg + / - 150 mg.

[0078] BID dosing is deduced to be adequately effective at 300 to 750 mg / kg, preferably at 600 mg / kg + / - 150 mg BID as HED.

[0079] Accordingly, in preferred embodiments of the second aspect, the invention provides varoglutamstat ((S)-l-(lH-benzo[d]imidazol-5-yl)-5-(4- propoxyphenyl)imidazolidin-2-one) or a pharmaceutically acceptable salt, solvate or polymorph thereof, including all tautomers thereof, or a pharmaceutical composition comprising said varoglutamstat, for use in the treatment of a disease, wherein said treatment comprises an uptitration phase and a treatment phase of varoglutamstat administration, and wherein said varoglutamstat is administered to a subject in a daily uptake of 10 mg to 1800 mg, such as 10 mg to 1600 mg or 10 mg to 1500 mg, preferably of 450 mg to 1200 mg, even more preferably of 600 mg to 1200 mg.

[0080] In some embodiments, the treatment phase of the second aspect comprises once daily administration of 600 mg to 1200 mg, such as once daily administration of 900 mg to 1200 mg varoglutamstat, preferably once daily administration of 600 mg to 1050 mg varoglutamstat, most preferably once daily administration of 1050 mg to a subject.

[0081] Preferably, said uptitration phase comprises once daily administration of 50 mg varoglutamstat for 1-2 weeks, followed by once daily administration of 150 mg varoglutamstat for 1-4 weeks, and followed by once daily administration of 300 mg varoglutamstat for 1-4 weeks to a subject. In case of an envisaged final dose level of 900 mg and above, another uptitration step is incorporated at once daily 600 mg for 1-4 weeks.

[0082] In another preferred embodiment, said uptitration phase comprises once daily administration of 150 mg for 1-4 weeks, followed by once daily administration of 300 mg varoglutamstat for 1-4 weeks. In case of an envisaged final dose level of 900 mg and above, another uptitration step is incorporated at once daily 600 mg for 1-4 weeks.

[0083] Dose levels as provided throughout the entire description of the invention always refer to varoglutamstat free base.

[0084] A third aspect of the invention describes protocols for QC-inhibitor down-titration at an end of treatment. Building upon the notion of systemic adaptation to QC / isoQC-inhibition to be required for dedicated molecular pathways within the body to avoid safety issues like hypersensitivity reactions, it is comprehensible that such adaptations might involve certain feedback loops, resulting in up- or down-regulation of either the QC substrate in question or other molecular entities as compensating actions for e.g. a loss of function, either of QC enzymes or their substrates due to missing post- translational modifications. Such an idea calls for the requirement of a down-titration at any end of treatment with a QC-inhibitor, either at end of treatment (EOT) of a clinical study, or - upon approval for treatment - at discontinuation of treatment. Such down-titration is assumed to be meaningful in 150 or 300 mg steps, preferably in 150 mg steps starting from the general treatment dose and keeping the general dose regimen (BID or QD). Thus, when starting from a 600 mg dose, down-titration by one step every 1-4 weeks seems to be indicated (to 450, then 300, then 150 mg), preferably every 2-4 weeks, most preferably every 4 weeks.

[0085] Thus, in further embodiments of the first and second aspects, the dose regimens provided by the invention further comprise a down-titration phase.

[0086] Preferably, said down-titration phase comprises, when starting from an administered 600 mg dose in the treatment phase, administration of 450 mg varoglutamstat once or twice daily according to the dosing frequency maintained on high dose treatment phase for 1 to 4 weeks, followed by administration of 300 mg varoglutamstat once or twice daily, respectively, for 1 to 4 weeks, followed by administration of 150 mg varoglutamstat once or twice daily, respectively, for 1 to 4 weeks.

[0087] Varoglutamstat as used herein

[0088] Generally, varoglutamstat according to the various aspects and embodiments of the present invention is the compound (S)-l-(lH-benzo[d]imidazol-5-yl)-5-(4- propoxyphenyl)imidazolidin- 2-one, which can be represented by the following structural formula I:

[0089]

[0090] In a preferred embodiment, said varoglutamstat is varoglutamstat hydrochloride.

[0091] In a further preferred embodiment, said varoglutamstat is a polymorph of said varoglutamstat hydrochloride. WO 2024 / 256618 Al discloses the hydrochloride salt of varoglutamstat and its polymorph as well as methods of their production and characterization. The teachings of WO 2024 / 256618 Al are hereby incorporated by reference in their entirety with regard to varoglutamstat, varoglutamstat hydrochloride and the polymorph of varoglutamstat hydrochloride as well as methods of their production and characterization. The following embodiments relate to the characteristics of the polymorph of varoglutamstat hydrochloride. These embodiments are described in detail in WO 2024 / 256618 Al, which is hereby incorporated by reference.

[0092] With regard to the polymorph of varoglutamstat hydrochloride, said polymorph is characterized by X-ray powder diffraction peaks (2[Theta]) selected from one or more of the following: 9.5±0.2°, and 24.8±0.2°.

[0093] Preferably, said polymorph of varoglutamstat hydrochloride is characterized by X-ray powder diffraction peaks (2[Theta]) selected from one or more of the following: 9.5±0.2°, 21.3±0.2°, 22.6±0.2°, and 24.8±0.2°. More preferably, said polymorph of varoglutamstat hydrochloride is characterized by X-ray powder diffraction peaks (2[Theta]) selected from one or more of the following: 5.8±0.2°, 9.5±0.2°, 16.9±0.2°, 17.2±0.2°, 18.9±0.2°, 20.7±0.2°, 21.3±0.2°, 21.7±0.2°, 22.6±0.2°, and 24.8±0.2°

[0094] Even more preferably, said polymorph of varoglutamstat hydrochloride is characterized by X-ray powder diffraction peaks (2[Theta]) selected from one or more of the following: 5.8±0.2°, 9.5±0.2°, 11.3±0.2°, 12.4±0.2°, 15.8±0.2°, 16.9±0.2°, 17.2±0.2°, 18.9±0.2°, 20.2±0.2°, 20.7±0.2°,

[0095] 21.3±0.2°, 21.7±0.2°, 22.6±0.2°, 23.8±0.2°, 24.8±0.2°, 26.3±0.2°, 27.2±0.2°, 28.3±0.2°,

[0096] 28.8±0.2°, 29.4±0.2°, 30.1±0.2°, 31.2±0.2° and 33.8±0.2°

[0097] Most preferably, said polymorph of varoglutamstat hydrochloride is characterized by an X-ray diffraction spectrum as shown in Figure 1.

[0098] In one embodiment of the invention, said polymorph of varoglutamstat hydrochloride is characterized by a differential scanning calorimetry (DSC) thermogram as shown in Figure 2.

[0099] In a preferred embodiment of the invention said polymorph of varoglutamstat hydrochloride is characterized by an DSC endotherm with an onset temperature of 243 °C and with a peak at 251 °C.

[0100] In one embodiment of the invention, said polymorph of varoglutamstat hydrochloride is characterized by a dynamic vapor sorption (DVS) curve as shown in Figure 4.

[0101] In one embodiment of the invention, said polymorph of varoglutamstat hydrochloride characterized by a thermogravimetric analysis (TGA) thermogram as shown in Figure 3.

[0102] In one embodiment of the invention said polymorph of varoglutamstat hydrochloride is characterized by one mass loss of 3.0% with onset / endset temperatures of 190 / 215°C before the main thermal decomposition of said varoglutamstat hydrochloride.

[0103] In one embodiment of the invention said polymorph of varoglutamstat hydrochloride characterized by a1H-NMR spectrum as shown in Figure 5. In a preferred embodiment of the invention, said polymorph of varoglutamstat hydrochloride is characterized by an achiral purity of >95%, preferably >96% or >97%, more preferably >98% or >99%, most preferably >99.5% or >99.8%, wherein achiral purity is determined by HPLC analysis.

[0104] In a further preferred embodiment of the invention, said polymorph of varoglutamstat hydrochloride is substantially free of amorphous material.

[0105] In a further preferred embodiment, the hydrochloride salt of varoglutamstat shows a degree of crystallinity of >50 %, when calculated with formula I:

[0106] % Crystallinity = 100 x A / (A + B - C) (Formula I) wherein

[0107] A is the sum of the net areas of all the peaks arising from the diffraction of the crystalline fraction of the sample;

[0108] B is the area under the diffractogram generated by the sample itself (excluding area A); and

[0109] C is the area of the background noise (due to air scattering, fluorescence, equipment, etc.) which is measured by recording the diffractograms of the (empty) sample holder that was used for recording the diffractograms of the tested samples.

[0110] Assays for determining the achiral purity of the polymorph of the hydrochloride salt of PQ912 and the crystallinity of the hydrochloride salt of PQ912 are described in WO 2024 / 256618 Al, which is hereby incorporated by reference in its entirety.

[0111] Therapeutic uses

[0112] In a fourth aspect, the present invention provides the use of varoglutamstat, as described herein, for use in methods of preventing, alleviating or treating of various diseases. Physiological substrates of QC and / or isoQC in mammals are, among others, e.g. amyloid betapeptides (3-40), (3-42), (11-40) and (11-42), ABri, ADan, Gastrin, Neurotensin, FPP, GnRH, TRH, CCL2, CCL7, CCL8, CCL13, CCL16, CCL18, Fractalkine, Orexin A, [Gln3]-glucagon(3-29), [Gin5] -substance P(5-l l),the peptide QYNAD, and CD47. For further details see Table 1. Table 1: Amino acid sequences of physiological active peptides with an N-terminal glutamine or glutamate residue, which are prone to be cyclized to final pGlu - and precursors thereof, which are cleaved under physiological conditions to liberate a glutamate at the N-terminus

[0113]

[0114] Glutamate is found in positions 3, 11 and 22 of the amyloid beta-peptide. Among them the mutation from glutamic acid (E) to glutamine (Q) in position 22 (corresponding to amyloid precursor protein APP 693, Swissprot P05067) has been described as the so-called Dutch type cerebroarterial amyloidosis mutation.

[0115] The beta-amyloid peptides with a then terminal pyroglutamic acid residue resulting from conversion of residues in position 3, 11 and / or 22 upon N-terminal cleavage up to the respective amino acid have been described to be more cytotoxic and hydrophobic than the amyloid betapeptides 1-40(42 / 43) (Saido T.C. 2000 Medical Hypotheses 54(3): 427-429).

[0116] The multiple N-terminal variations, e.g. Abeta(3-40), Abeta(3-42), Abeta(l l-40) and Abeta (11- 42), can be generated from the full length peptides Abeta(l-40) and Abeta(l-42) by the beta- secretase enzyme beta-site amyloid precursor protein-cleaving enzyme (BACE) by proteolysis at different sites (Huse J.T. et al. 2002 J. Biol. Chem. 277 (18): 16278-16284), and / or by aminopeptidase or dipeptidylaminopeptidase processing. In all cases, cyclization of the then N- terminally occuring glutamic acid residue is catalyzed by QC.

[0117] Transepithelial transducing cells, particularly the gastrin (G) cell, co-ordinate gastric acid secretion with the arrival of food in the stomach. Recent work showed that multiple active products are generated from the gastrin precursor, and that there are multiple control points in gastrin biosynthesis. Biosynthetic precursors and intermediates (progastrin and Gly-gastrins) are putative growth factors; their products, the amidated gastrins, regulate epithelial cell proliferation, the differentiation of acid-producing parietal cells and histamine-secreting enterochromaffin-like (ECL) cells, and the expression of genes associated with histamine synthesis and storage in ECL cells, as well as acutely stimulating acid secretion. Gastrin also stimulates the production of members of the epidermal growth factor (EGF) family, which in turn inhibit parietal cell function but stimulate the growth of surface epithelial cells. Plasma gastrin concentrations are elevated in subjects with Helicobacter pylori, who are known to have increased risk of duodenal ulcer disease and gastric cancer (Dockray, GJ. 1999 J Physiol 15 315-324).

[0118] The peptide hormone gastrin, released from antral G cells, is known to stimulate the synthesis and release of histamine from ECL cells in the oxyntic mucosa via CCK-2 receptors. The mobilized histamine induces acid secretion by binding to the H(2) receptors located on parietal cells. Recent studies suggest that gastrin, in both its fully amidated and less processed forms (progastrin and glycine-extended gastrin), is also a growth factor for the gastrointestinal tract. It has been established that the major trophic effect of amidated gastrin is for the oxyntic mucosa of stomach, where it causes increased proliferation of gastric stem cells and ECL cells, resulting in increased parietal and ECL cell mass. On the other hand, the major trophic target of the less processed gastrin (e.g., glycine-extended gastrin) appears to be the colonic mucosa (Koh, T.J. and Chen, D. 2000 Regul Pept 9337-44).

[0119] Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia that specifically modulates neurotransmitter systems previously demonstrated to be misregulated in this disorder. Clinical studies in which cerebrospinal fluid (CSF) NT concentrations have been measured revealed a subset of schizophrenic patients with decreased CSF NT concentrations that are restored by effective antipsychotic drug treatment. Considerable evidence also exists concordant with the involvement of NT systems in the mechanism of action of antipsychotic drugs. The behavioral and biochemical effects of centrally administered NT remarkably resemble those of systemically administered antipsychotic drugs, and antipsychotic drugs increase NT neurotransmission. This concatenation of findings led to the hypothesis that NT functions as an endogenous antipsychotic. Moreover, typical and atypical antipsychotic drugs differentially alter NT neurotransmission in nigrostriatal and mesolimbic dopamine terminal regions, and these effects are predictive of side effect liability and efficacy, respectively (Binder, E. B. et al. 2001 Biol Psychiatry 50 856-872).

[0120] Fertilization promoting peptide (FPP), a tripeptide related to thyrotrophin releasing hormone (TRH), is found in seminal plasma. Recent evidence obtained in vitro and in vivo showed that FPP plays an important role in regulating sperm fertility. Specifically, FPP initially stimulates nonfertilizing (uncapacitated) spermatozoa to "switch on" and become fertile more quickly, but then arrests capacitation so that spermatozoa do not undergo spontaneous acrosome loss and therefore do not lose fertilizing potential. These responses are mimicked, and indeed augmented, by adenosine, known to regulate the adenylyl cyclase (AC) / cAMP signal transduction pathway. Both FPP and adenosine have been shown to stimulate cAMP production in uncapacitated cells but inhibit it in capacitated cells, with FPP receptors somehow interacting with adenosine receptors and G proteins to achieve regulation of AC. These events affect the tyrosine phosphorylation state of various proteins, some being important in the initial "switching on", others possibly being involved in the acrosome reaction itself. Calcitonin and angiotensin II, also found in seminal plasma, have similar effects in vitro on uncapacitated spermatozoa and can augment responses to FPP. These molecules have similar effects in vivo, affecting fertility by stimulating and then maintaining fertilizing potential. Either reductions in the availability of FPP, adenosine, calcitonin, and angiotensin II or defects in their receptors contribute to male infertility (Fraser, L.R. and Adeoya-Osiguwa, S. A. 2001 Vitam Horm 63, 1-28).

[0121] A number of studies have underlined in particular the crucial role of MCP-1 for the development of atherosclerosis (Gu, L., et al., (1998) Mol.Cell 2, 275-281; Gosling, J., et al., (1999) J Clin.Invest 103, 773-778); rheumatoid arthritis (Gong, J. H., et al., (1997) JExp.Med 186, 131-137; Ogata, H., et al., (1997) J Pathol. 182, 106-114); pancreatitis (Bhatia, M., et al., (2005) Am. J Physiol Gastrointest. Liver Physiol 288, G1259-G1265); Alzheimer’s disease (Yamamoto, M., etal., (2005) Am.J Pathol. 166, 1475-1485); lung fibrosis (Inoshima, I., et al., (2004) Am.J Physiol Lung Cell Mol.Physiol 286, L1038-L1044); and graft rejection (Saiura, A., et al., (2004) Arterioscler. Thromb. Vase. Biol. 24, 1886-1890). Furthermore, MCP-1 might also play a role in gestosis (Katabuchi, H., et al., (2003) Med Electron Microsc. 36, 253-262), as a paracrine factor in tumor development (Ohta, M., et al., (2003) Int.J Oncol. 22, 773-778; Li, S., et al., (2005) J Exp.Med 202, 617-624), neuropathic pain (White, F. A., et al., (2005) Proc. Natl. Acad.Sci.U.S.A) and AIDS (Park, I. W., Wang, J. F., and Groopman, J. E. (2001) Blood 97, 352-358; Coll, B., et al., (2006) Cytokine 34, 51-55).

[0122] MCP-1 levels are increased in CSF of AD patients and patients showing mild cognitive impairment (MCI) (Galimberti, D., et al., (2006) Arch.Neurol. 63, 538-543). Furthermore, MCP-1 shows an increased level in serum of patients with MCI and early AD (Clerici, F., et al., (2006) Neurobiol. Aging 27, 1763-1768). Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A / MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided (Beck A. et al. 2001, J Pept Res 57(6):528-38.).

[0123] Orexin A is a neuropeptide that plays a significant role in the regulation of food intake and sleepwakefulness, possibly by coordinating the complex behavioral and physiologic responses of these complementary homeostatic functions. It plays also a role in the homeostatic regulation of energy metabolism, autonomic function, hormonal balance and the regulation of body fluids.

[0124] Moreover, increased levels of the pentapeptide QYNAD were identified in the cerebrospinal fluid (CSF) of patients suffering from multiple sclerosis or Guillain-Barre syndrome compared to healthy individuals (Brinkmeier H. et al. 2000, Nature Medicine 6, 808-811). There is a big controversy in the literature about the mechanism of action of the pentapeptide Gln-Tyr-Asn-Ala- Asp (QYNAD), especially its efficacy to interact with and block sodium channels resulting in the promotion of axonal dysfunction, which are involved in inflammatory autoimmune diseases of the central nervous system. But recently, it could be demonstrated that not QYNAD, but its cyclized, pyroglutamated form, pEYNAD, is the active form, which blocks sodium channels resulting in the promotion of axonal dysfunction. Sodium channels are expressed at high density in myelinated axons and play an obligatory role in conducting action potentials along axons within the mammalian brain and spinal cord. Therefore, it is speculated that they are involved in several aspects of the pathophysiology of inflammatory autoimmune diseases, especially multiple sclerosis, the Guillain-Barre syndrome and chronic inflammatory demyelinizing polyradiculoneuropathy.

[0125] Furthermore, QYNAD is a substrate of the enzyme glutaminyl cyclase (QC, EC 2.3.2.5), which is also present in the brain of mammals, especially in human brain. Glutaminyl cyclase effectively catalyzes the formation of pEYNAD from its precursor QYNAD.

[0126] Hatherly et al. discovered that CD47 contains a glutamine residue at the N-terminus (“Paired Receptor Specificity Explained by Structures of Signal Regulatory Proteins Alone and Complexed with CD47”. Molecular Cell 31, 266-277, July 25, 2008). CD47 is therefore a potential substrate of QPCT / QPCTL upon cleavage of the CD47 signal sequence, which has been confirmed as such. Furthermore, Murata et al. reported in 2014 on the CD47-SIRPa signaling system and its physiological roles and therapeutic application (The Journal of Biochemistry, Volume 155, Issue 6, June 2014, Pages 335-344). CD47, an immunoglobulin superfamily protein, is a ligand for SIRPa, with the two proteins constituting a cell-cell communication system (the CD47-SIRPa signaling system). SIRPa is particularly abundant in the myeloid lineage of hematopoietic cells such as macrophages or dendritic cells (DCs), whereas CD47 is expressed ubiquitously. For example, interaction of CD47 on red blood cells with SIRPa on macrophages is thought to prevent the phagocytosis of the former cells by the latter cells, determining the lifespan of red blood cells. Recent studies further indicate that this signaling system plays important roles in engraftment of hematopoietic stem cells as well as in tumor immune surveillance through regulation of the phagocytic activity of macrophages. In the immune system, the CD47-SIRPa interaction is also important for the development of a subset of CDl lc+DCs as well as organization of secondary lymphoid organs. Finally, the CD47-SIRPa signaling system likely regulates bone homeostasis by osteoclast development. Such emerged functions of the CD47-SIRPa signaling system thus nourish multiple therapeutic strategies for the treatment of cancer, autoimmune diseases and bone disorders. Inhibitors of QC or isoQC may interfere with the CD47-SIRPa signaling system in decreasing the amount of CD47 binding to SIRPoc, and therefore may be useful in the treatment or prevention of cancer, proliferative disorders, atherosclerosis, fibrotic diseases, and infectious diseases.

[0127] The concept that glutaminyl cyclase (and its isoenzyme) is an enzymatic modifier of the CD47- SIRPa axis and a target for cancer immunotherapy has been described in detail within respective recent literature and is here incorporated by reference (Logtenberg, M.E.W. et al., Nature Med. 2019, 25, 612-619; Wu, Z. et al., Cell Res. 2019, 29, 502-505; Logtenberg, M.E.W. et al., Immunity 2020, 52, 742; Bresser, K. et al., Oncoimmunology 2022, 11, e2049486; Raaben, M. et al., EP 3 747 438 Al). Likewise, combinations of a QPCT / QPCTL inhibitor with antibodies targeting a tumor-associated antigen has been described for treatment of solid as well as hematologic tumors (Raaben, M. et al., EP 3 747437 Al).

[0128] Furthermore, other substrates of QPCT and QPCTL described above, like CCL2 and CCL7, have likewise been described to contribute to the fate of tumors, and inhibition of their N-terminal conversion by glutaminyl cyclases to exhibit anti -tumoral potential (da Silva, R.B. et al., Nature Immunol 2022, 23, 568).

[0129] An indication for the involvement of QC / isoQC in kidney diseases was recently published by Kanemitsu et al. (Kanemitsu etal., Naunyn-Schmiedeberg's Archives of Pharmacology 2020, 394, 751-761), who reported that the chronic treatment with the (iso-)glutaminyl cyclase inhibitor PQ529 is a novel and effective approach for treating glomerulonephritis in chronic kidney disease. Three- week repeated administration of PQ529 (30 and 100 mg / kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. It was suggested that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2 / CCR2 axis.

[0130] Kidney disease, or renal disease, technically referred to as nephropathy, represents a damage to or a disease of a kidney. Nephritis is an inflammatory kidney disease with several subtypes according to the location of the inflammation. Nephrosis is non-inflammatory kidney disease. Nephritis and nephrosis can give rise to nephritic syndrome and nephrotic syndrome, respectively. Kidney disease usually causes a loss of kidney function to some degree and can result in kidney failure, the complete loss of kidney function. Kidney failure is known as the end-stage of kidney disease (end-stage renal disease, ESRD), where dialysis or a kidney transplant is the only treatment option.

[0131] Chronic kidney disease (CKD) is defined as prolonged kidney abnormalities (functional and / or structural in nature) that last for more than three months. Acute kidney disease is now termed acute kidney injury (AKI) and is marked by a sudden reduction in kidney function over seven days.

[0132] Persistent, low-grade inflammation has been recognized as an important component of CKD, playing a unique role in its pathophysiology and being accountable in part for cardiovascular and all-cause mortality, as well as contributing to the development of protein-energy wasting. A variety of factors contribute to chronic inflammatory status in CKD, including increased production and decreased clearance of pro-inflammatory cytokines, oxidative stress and acidosis, chronic and recurrent infections, including those related to dialysis access, altered metabolism of adipose tissue, and intestinal dysbiosis. Inflammation directly impacts the glomerular filtration rate (GFR) in CKD (Akchurin & Kaskel (2015), Blood Purif. 39: 84-92).

[0133] It is known that there are risk factors that may cause and promote the progression of CKD. High blood pressure (hypertension) and diabetes mellitus (type 1 and type 2, T1D and T2D, respectively) are the two most common causes of CKD. Other causes and conditions that affect kidney function and can cause chronic kidney disease include:

[0134] • Glomerulonephritis. This type of kidney disease involves damage to the glomeruli, which are the filtering units inside the kidneys.

[0135] • Polycystic kidney disease. This is a genetic disorder that causes many fluid-filled cysts to grow within the kidneys, reducing the ability of kidneys to function.

[0136] • Membranous nephropathy. This is a disorder where the body’s immune system attacks the waste-filtering membranes in the kidney.

[0137] • Obstructions of the urinary tract from kidney stones, an enlarged prostate or cancer.

[0138] • Vesicoureteral reflux. This is a condition in which pee flows backward back up the ureters to the kidneys. • Nephrotic syndrome. This is a collection of symptoms which indicate kidney damage.

[0139] • Recurrent kidney infection (pyelonephritis).

[0140] • Diabetes-related nephropathy. This is damage or dysfunction of nephrons, caused by diabetes mellitus. Diabetic nephropathy is also called Diabetic kidney disease (DKD) and slowly damages the kidneys' filtering system.

[0141] • Lupus and other immune system diseases which cause kidney problems, including polyarteritis nodosa, sarcoidosis, Goodpasture syndrome and Henoch-Schbnlein purpura.

[0142] • Hereditary diseases caused by mutation of genes, like Fabry disease and Alport syndrome.

[0143] • Connective tissue diseases (CTD), collagenoses, collagenopathies.

[0144] Accordingly, the present invention provides the use of varoglutamstat, as described herein, for the preparation of a medicament for the prevention or alleviation or treatment of a neurodegenerative disease selected from the group consisting of mild cognitive impairment, Alzheimer’s disease, Familial British Dementia, Familial Danish Dementia, neurodegeneration in Down Syndrome, and Huntington’s disease, or a diseases selected from Kennedy’s disease, ulcer disease, duodenal cancer with or w / o Helicobacter pylori infections, colorectal cancer, Zolliger-Ellison syndrome, gastric cancer with or without Helicobacter pylori infections, pathogenic psychotic conditions, schizophrenia, infertility, neoplasia, inflammatory host responses, cancer, malign metastasis, melanoma, psoriasis, rheumatoid arthritis, atherosclerosis, pancreatitis, restenosis, impaired humoral and cell-mediated immune responses, leukocyte adhesion and migration processes in the endothelium, impaired food intake, impaired sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance or impaired regulation of body fluids, multiple sclerosis, the Guillain-Barre syndrome, chronic inflammatory demyelinizing polyradiculoneuropathy and periodontitis. In another embodiment, the present invention provides the use of the varoglutamstat, as described herein, for the preparation of a medicament for the prevention or alleviation or treatment of a proliferative disease selected from the group consisting of leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), lymphoma, B-cell lymphoma, T-cell lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (NHL), hairy cell lymphoma, Burkett’s lymphoma, multiple myeloma (MM), myelodysplastic syndrome, lung cancer, adenocarcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mediastinum cancer, peritoneal cancer, mesothelioma, gastrointestinal cancer, gastric cancer, stomach cancer, bowel cancer, small bowel cancer, large bowel cancer, colon cancer, colon adenocarcinoma, colon adenoma, rectal cancer, colorectal cancer, leiomyosarcoma, breast cancer, gynaecological cancer, genito-urinary cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, testicular cancer, seminoma, teratocarcinoma, liver cancer, kidney cancer, bladder cancer, urothelial cancer, biliary tract cancer, pancreatic cancer, exocrine pancreatic carcinoma, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma (HNSCC), skin cancer, squamous cancer, squamous cell carcinoma, Kaposi's sarcoma, melanoma, malignant melanoma, xeroderma pigmentosum, keratoacanthoma, bone cancer, bone sarcoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, thyroid gland cancer, thyroid follicular cancer, adrenal gland cancer, nervous system cancer, brain cancer, astrocytoma, neuroblastoma, glioma, schwannoma, glioblastoma, or sarcoma; a fibrotic disease: scleroderma, idiopathic pulmonary fibrosis, liver cirrhosis, lung fibrosis, bladder fibrosis, heart fibrosis, pancreas fibrosis, or myelofibrosis; an infectious disease; an infectious disease caused by a virus, bacterium, or protozoan; an infectious disease caused by a pathogen selected from: a lentivirus, human T-lymphotropic virus (HTLV), an hepadna virus, hepatitis B virus, a herpes virus, human papilloma virus, la crosse virus, Yersinia sp., Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Franciscella sp., Helicobacter sp., Helicobacter pylori, Pasturella sp., Vibrio sp., Vibrio cholerae, Vibrio parahemolyticus, Legionella sp., Legionella pneumophila, Listeria sp., Listeria monocytogenes, Mycoplasma sp., Mycoplasma hominis, Mycoplasma pneumoniae, Mycobacterium sp., Mycobacterium tuberculosis, Mycobacterium leprae, Rickettsia sp., Rickettsia rickettsii, Rickettsia typhi, a Plasmodium, a Trypanosoma, a Giardia, a Toxoplasma, and a Leishmania.

[0145] In another embodiment, the present invention provides the use of the varoglutamstat, as described herein, for the preparation of a medicament for the prevention or alleviation or treatment of a kidney disease, wherein said kidney disease is an acute kidney disease (AKD) or a chronic kidney disease (CKD). Such CKD may be accompanied by a persistent inflammation. In a further embodiment, said kidney disease represents a condition selected from the group consisting of diabetic nephropathy (DKD), Focal Segmental Glomerulosclerosis (FSGS), glomerulonephritis, polycystic kidney disease, membranous nephropathy, obstructions of the urinary tract, vesicoureteral reflux, nephrotic syndrome, recurrent kidney infection (pyelonephritis), lupus (Systemic lupus erythematosus; SLE) and other immune system diseases selected from the group consisting of polyarteritis nodosa, sarcoidosis, Good pasture syndrome and Henoch-Schbnlein purpura.

[0146] In even further embodiments, the present invention provides the use of the varoglutamstat, as described herein, for the preparation of a medicament for the prevention or alleviation or treatment of a condition that may cause CKD and is selected from the group consisting of Alport syndrome, Fabry disease, and connective tissue diseases (CTD).

[0147] In some embodiments, said kidney disease is associated with an impaired glomerular filtration rate (GFR).

[0148] In some embodiments, said subject to be treated is at risk for CKD.

[0149] In some embodiments, said subject is suffering from at least one risk factor for CKD, wherein said risk factor is selected from hypertension or diabetes mellitus (type 1 or type 2 diabetes mellitus) or cardiac disease or an eGFR <60 ml / min / 1.73m2.

[0150] In some embodiments, said subject to be treated is not at risk for CKD.

[0151] In a further embodiment, the invention relates to methods of treatment of the aforementioned diseases and conditions comprising the step of administering of a therapeutically effective amount of the varoglutamstat described herein to a subject in need thereof. Said subject is suitably a mammal, preferably a human.

[0152] In a preferred embodiment, the present invention provides the use of the varoglutamstat described herein in combination with other agents, especially for the treatment of neurodegenerative diseases, and inflammatory diseases such as psoriasis, rheumatoid arthritis, atherosclerosis, pancreatitis, restenosis and multiple sclerosis, and fibrotic diseases such as scleroderma, idiopathic pulmonary fibrosis, liver cirrhosis, lung fibrosis, bladder fibrosis, heart fibrosis, pancreas fibrosis, myelofibrosis, or kidney disease.

[0153] Most preferably, said method and corresponding uses are for the treatment of a disease selected from the group consisting of mild cognitive impairment, Alzheimer’s disease, Familial British Dementia, Familial Danish Dementia, neurodegeneration in Down Syndrome, Parkinson’s disease and Chorea Huntington, comprising the administration of a therapeutically active amount of at least one of varoglutamstat of the invention to a mammal, preferably a human.

[0154] Especially preferred according to the invention is a method and corresponding uses for the treatment of a disease selected from the group consisting of mild cognitive impairment and Alzheimer’s disease.

[0155] Even preferably, the present invention provides a method of treatment and corresponding uses for the treatment of rheumatoid arthritis, atherosclerosis, pancreatitis, and restenosis.

[0156] Even preferably, the present invention provides a method of treatment and corresponding uses for the treatment of periodontitis.

[0157] Even preferably, the present invention provides a method of treatment and corresponding uses for the treatment of solid and hematologic tumors.

[0158] Even preferably, the present invention provides a method of treatment and corresponding uses for the treatment of kidney diseases. Pharmaceutical compositions

[0159] The invention further provides a pharmaceutical composition comprising varoglutamstat as described herein.

[0160] To prepare the pharmaceutical compositions of this invention, the varoglutamstat as described herein can be used as the active ingredient. The active ingredient(s) is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, or parenteral such as intramuscular. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations, such as for example, suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations such as, for example, powders, capsules, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, though other ingredients, for example, for purposes such as aiding solubility or for preservation, may be included.

[0161] Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, an amount of the active ingredient(s) necessary to deliver an effective dose as described above. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, suppository, teaspoonful, and the like, from about 0.03 mg to 100 mg / kg (preferred 0.1 - 30 mg / kg) and may be given at a dosage of from about 0.1 - 300 mg / kg per day (preferred 1 - 50 mg / kg per day) of the active ingredient. The dosages, however, may be varied depending upon the requirement of the patients, the severity of the condition being treated, and the compound being employed. The use of either daily administration or post-periodic dosing may be employed.

[0162] Preferably these compositions are in unit dosage forms from such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol, or liquid sprays, drops, ampoules, autoinjector devices or suppositories; for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the composition may be presented in a form suitable for once-weekly or once-monthly administration. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of varoglutamstat of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective dosage forms such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.

[0163] The tablets or pills of the compositions of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of material can be used for such enteric layers or coatings, such materials including a number of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[0164] This liquid forms in which the compositions of the present invention may be incorporated for administration orally or by injection include, aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions, include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.

[0165] The pharmaceutical composition may contain between about 0.01 mg and 1000 mg, preferably about 5 to 800 mg, more preferably 100 to 600 mg of varoglutamstat of the present invention, and may be constituted into any form suitable for the mode of administration selected. Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. Compositions suitable for oral administration include solid forms, such as pills, tablets, caplets, capsules (each including immediate release, timed release, and sustained release formulations), granules, and powders, and liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0166] Advantageously, varoglutamstat of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

[0167] For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders; lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or betalactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

[0168] The liquid forms in suitable flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

[0169] Varoglutamstat according to the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

[0170] Varoglutamstat according to the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled, varoglutamstat of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamid-ephenol, or polyethyl eneoxidepolyllysine substituted with palmitoyl residue. Furthermore, varoglutamstat of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polyactic acid, polyepsilon caprolactone, polyhydroxy butyeric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[0171] Varoglutamstat according to the present invention may be administered in any of the foregoing compositions and according to dosage regimens established in the art whenever treatment of the addressed disorders is required.

[0172] The daily dosage of the products may be varied over a wide range from 10 to 1800 mg per mammal per day. For oral administration, the compositions are preferably provided in the form of tablets containing, 10, 15, 25, 50, 100, 150, 200, 250, 300, 450, 500 and 600 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. Varoglutamstat according to the present invention may be administered on a regimen of 1 to 4 times per day, preferably once daily or twice daily.

[0173] The daily dosage of the varoglutamstat of the invention may be varied over a wide range from 10 mg to 1800 mg per mammal per day, preferably 150 mg to 1800 mg per mammal per day. Such daily dosage levels are referring to the envisaged final dose level and is not referring to up- or down-titration steps. For oral administration, the compositions are preferably provided in the form of tablets containing, 10, 15, 25, 50, 100, 150, 200, 250, 300, 450, 500, 600, 800 or 1.000 mg of varoglutamstat, incorporated as the respective amount of its hydrochloride or the polymorphthereof for the symptomatic adjustment of the dosage to the patient to be treated. More preferably, for oral administration, the compositions are provided in the form of tablets containing 150 mg, 300 mg, 450 mg or 600 mg of a compound of formula I.

[0174] In a most preferred embodiment, the daily dosage of varoglutamstat of the invention is administered by a once daily dosing, in which the content of varoglutamstat may be varied between 600 mg to 1800 mg, most preferably at a dose of 1050 mg + / - 150 mg (that is in a range from 900 mg to 1200 mg). It has been shown in Example 3 that a once daily dosing of the varoglutamstat of the invention within the respective dose ranges within mice provides evidence of efficacy on disease relevant parameters and simultaneously seems to provide a larger safety window when compared to twice daily dosing.

[0175] In a further most preferred aspect, the invention provides a pharmaceutical composition for once daily administration of varoglutamstat, wherein said pharmaceutical composition comprises 150 to 600 mg of a varoglutamstat, incorporated as the respective amount of its hydrochloride or polymorph thereof and one or more pharmaceutically acceptable diluents, carriers or excipients. In some embodiments, the pharmaceutical composition for once daily administration comprises 150 mg of varoglutamstat, incorporated as the respective amount of its hydrochloride or the polymorph thereof. In some embodiments, the pharmaceutical composition for once daily administration comprises 300 mg of varoglutamstat, incorporated as the respective amount of its hydrochloride or the polymorph thereof. In some embodiments, the pharmaceutical composition for once daily administration comprises 450 mg of varoglutamstat, incorporated as the respective amount of its hydrochloride or the polymorph thereof. In some embodiments, the pharmaceutical composition for once daily administration comprises 600 mg of varoglutamstat, incorporated as the respective amount of its hydrochloride or the polymorph thereof. Preferred in regard to the route of administration of the pharmaceutical composition for once daily administration of varoglutamstat is oral administration. The pharmaceutical composition for once daily administration of varoglutamstat is suitably a tablet.

[0176] Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, may result in the need to adjust dosages.

[0177] In a further aspect, the invention also provides a process for preparing a pharmaceutical composition comprising varoglutamstat, optionally in combination with a pharmaceutically acceptable carrier.

[0178] The compositions are preferably in a unit dosage form in an amount appropriate for the relevant daily dosage.

[0179] Suitable dosages, including especially unit dosages, of varoglutamstat of the present invention include the known dosages including unit doses as described or referred to in reference text such as the British and US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack Publishing Co.), Martindale The Extra Pharmacopoeia (London, The Pharmaceutical Press) (for example see the 31st Edition page 341 and pages cited therein) or the above mentioned publications.

[0180] The present invention is further illustrated by the following non-limiting Figures and Examples. The following Figures and Examples are for the purpose of illustration of the invention only and are not intended in any way to limit the scope of the present invention. It will thus be readily apparent to one skilled in the art that e.g., variations in scale of experiments might have an impact on optimized concentrations and / or time scales for certain parts of the processes. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be falling within the scope of the invention.

[0181] Brief description of the drawings

[0182] With reference to the Figures, these are as follows:

[0183] Figure 1 : XRPD of varoglutamstat hydrochloride (corresponds to Figure 19 of WO 2024 / 256618 Al).

[0184] Figure 2: DSC analysis of varoglutamstat hydrochloride (corresponds to Figure 20 of WO 2024 / 256618 Al).

[0185] Figure 3: TGA analysis of varoglutamstat hydrochloride (corresponds to Figure 21 of WO 2024 / 256618 Al).

[0186] Figure 4: DVS analysis of varoglutamstat hydrochloride (corresponds to Figure 22 of WO 2024 / 256618 Al).

[0187] Figure 5: NMR spectrum to determine the assay of varoglutamstat hydrochloride (corresponds to Figure 16 of WO 2024 / 256618 Al, recorded in DMSO-d6).

[0188] Figure 6: Results of the eGFR slope analysis after 96 weeks treatment of human subjects with varoglutamstat vs. placebo (A), and of the different dose groups investigated (Placebo group vs. treatments groups which received 300 mg varoglutamstat twice daily or 600 mg varoglutamstat twice daily; B). eGFR was calculated with the MDRD formula. The results are shown for the total population of the investigated subjects (Placebo groups vs. treatment groups).

[0189] Figure 7: QC activity determined week 48 in VIVI AD, relative to the subjects’ pretreatment QC activity, depicted by treatment groups placebo, 300 mg BID and 600 mg BID; in cerebrospinal fluid (CSF; A) and serum (B)

[0190] Figure 8: QC activity determined week 24 in serum in VIVA-MIND, relative to the subjects’ pretreatment QC activity, depicted by treatment groups placebo and 600 mg BID Figure 9: Results from ADI-CKD model in mice, histomorphometric parameter Collagen IV, as fractional area percentage of the left kidney.

[0191] Figure 10: Results from ADI-CKD model in mice, hematology, lymphocyte count.

[0192] Examples

[0193] Example 1 - Treatment study in human subjects with varoglutamstat hydrochloride: extended uptitration phase (VIVIAD)

[0194] Overall study description

[0195] In a multicenter, randomized, double-blind, placebo-controlled, parallel group dose finding study, 259 human subjects selected for Mild Cognitive Impairment and Mild Dementia due to Alzheimer’s Disease were treated with varoglutamstat hydrochloride versus placebo, varoglutamstat or placebo tablets were administered orally once daily in weeks 1 and 2 and twice daily orally from week 3 onwards. The total treatment duration was between 48 and 96 weeks. Subjects were randomized 1:1 :1 (placebo, 300 mg, 600 mg all BID; indicated dose refers to varuglutamstat free base) for the first 90 subjects included, and 1: 1 (placebo and dose decided by Data Safety Monitoring Board (DSMB)) from patient 91 onwards. All tablets were taken after a meal.

[0196] Dosis regimen

[0197] Dose in weeks 1 and 2: 50 mg once daily (evening) or placebo

[0198] Dose in weeks 3 and 4: 50 mg BID or placebo

[0199] Dose in weeks 5-8: 150 mg BID or placebo

[0200] Dose in weeks 9-12: 300 mg BID or placebo

[0201] Dose weeks 13 onwards (until DSMB dose decision): First 90 subjects: 300 mg BID or 600 mg BID or placebo 1: 1: 1. Subjects randomized between the 90th randomized subject and the DSMB: 300 mg BID or placebo 1: 1.

[0202] After the DSMB decision, all subjects randomized to varoglutamstat and having completed at least 12 weeks uptitration were switched to the chosen dose (600 mg BID) until study completion (week 48 to 96). Subjects randomized to placebo stayed on placebo.

[0203] Randomization after DSMB decision of further subjects occurred 1:1 for 600 mg or placebo.

[0204] Target engagement by varoglutamstat was evaluated at week 48 by assessing remaining QC activity in serum and CSF samples 2-6 h post dosing.

[0205] Serum creatinine monitored during the study:

[0206] The effect of varoglutamstat on serum biomarkers of renal tissue turnover and the change from baseline in serum biomarkers of renal tissue turnover were monitored; creatinine was assessed at visits, typically in 3 months periods.

[0207] Laboratory methods, Creatinine

[0208] Serum creatinine was quantified using a commercial enzymatic kit (ECRE 2, Siemens Medical Solutions, Denmark) on an AD VIA 1800 instrument (Siemens Healthcare Diagnostics, Denmark) according to manufacturer’s instructions.

[0209] Assay Principle:

[0210] Creatinine is converted to creatine by creatininase and further hydrolyzed to sarcosine which in turn is further decomposed by sarcosine oxidase to form glycine, formaldehyde and hydrogen peroxide. In the presence of peroxidase the product of the oxidative condensation with N-(3- sulfopropyl)-3-methoxy-5-methylaniline (HMMPS) and 4-aminoantipyrine can be quantified by photometric readout. Assessment of eGFR

[0211] The eGFR was estimated using the "4-variable MDRD" method, which estimates GFR using four variables: serum creatinine, age, ethnicity, and gender. For measuring creatinine in pmol / 1, this formula reads as follows: eGFR = 32788 x [Serum Creatininei r'1x AgC*1®’ x [1.212 if Blaek] x [0.742 if Female]

[0212] A baseline eGFR was determined at the beginning of the clinical trial representing the mean value of the eGFR of all subjects of the treatment group and the placebo group. The progression of the eGFR was monitored during the trial duration in about 12 week intervals and visualized as annualized change from baseline, expressed as ml / min / 1.73m2 / yr.

[0213] The BSA adjusted eGFR in mL / min / 1.73m2was determined via measured creatinine level using the MDRD formula. Annualized Rate of change of eGFR over time (expressed as ml / min / 1.73m2 / yr) was estimated via random co-efficients analysis. This is a mixed model applied to repeated eGFR measures over time with subject-specific random effects for intercept and slope (DeVries et al. (2024), Pharm. Stat., doi: 10.1002 / pst.2381). The estimated annualized rate of change was extracted from the model by treatment arm, along with the associated standard error; and the difference between treatment arms in the annualized rate of change was also extracted, along with its standard error, 95% confidence interval and 2-sided p-value.

[0214] SAS 9.4 software was used for these analyses.

[0215] Laboratory methods. QC-activity (LCMS):

[0216] The glutaminyl cyclase (QC) assay principle was adapted from Schilling et al. (2002), Anal. Biochem. 303: 49-56 for measurement of serum or CSF QC activity in a minimal dilution (1:3 dilution of serum or CSF). Glutaminyl (Gln)-7-amino-4-methylcoumarin (Gln-AMC) was used as substrate. The formation of pE-AMC was determined directly by LC-MS / MS. In brief, to 1 part of sample (serum or CSF, e.g. 15 pl) 2 parts of assay buffer (150 mM Hepes pH 7.5 containing 0.15 mM Gln-AMC, e.g. 30 pl) was added to start the reaction. The enzymatic reaction was stopped after 0 and 40 minutes of incubation at 30°C with an excess (e.g. 500 pl) of internal standard (Reserpine) solution in organic solvent (acetonitrile). After vortexing and centrifugation, an aliquot of the sample was further diluted with water (e.g. 200 pl + 200 pl). An aliquot of 5 pL was injected onto the HPLC-MS / MS system. The concentration of pE-AMC in this solution was detemined using a pE-AMC calibration curve.

[0217] QC activity was calculated using the following equation; one unit is defined as formation of 1 pmol pE-AMC per minute under the conditions described above:

[0218] A (U / L) = A[pE-AMC] / At * dilution factor with

[0219] A Activity in U / L

[0220] A[pE-AMC] difference of pE-AMC concentration determined at t = 40 min and t = 0 min in [pmol / L]

[0221] At Incubation time from start of enzymatic reaction by addition of substrate (Gln-

[0222] AMC) to stop of reaction by addition of excess of acetonitrile solvent (usually 40 min) dilution factor corresponds to sample dilution during enzyme reaction (= 3)

[0223] The lower and upper limits of quantification were 17.5 and 1750 mU / L, respectively.

[0224] QC activity data was normalized to the individual pretreatment (screening or baseline) values. Results:

[0225] The results for the calculation of the annualized eGFR changes from baseline are shown in Figure 6, and are provided as mL / min / 1.73m2 / yr with 95% confidence intervals indicated.

[0226] “yr” means year.

[0227] *, **, or *** mean different levels of significant difference between the treatment group and placebo, ns means “not significant” difference; *: p < 0.05, **: p < 0.01, ***: P < 0.001 (p-values indicated in the respective Figure).

[0228] Within the overall population, eGFRfor placebo treated subjects showed a slope for the annualized eGFR change over baseline of -1.6 mL / min / 1.73m2 / yr (which reflects a decline of eGFR with time), compared to a positive slope of the annualized eGFR change over baseline for varoglutamstat-treated subjects by +1.9 mL / min / 1.73m2 / yr (which reflects a beneficial increase of eGFR with time), thus a difference between both treatment cohorts of 3.5 mL / min / 1.73m2 / yr was identified in favor of varoglutamstat-treatment (Figure 6A).

[0229] Dissecting the varoglutamstat-treated subject population for their respective dose level, 300 mg treated subjects experienced an increase of eGFR with a slope for the annualized eGFR change over baseline of +1.9 mL / min / 1.73m2 / yr compared to +4.2 mL / min / 1.73m2 / yr for the 600 mg treated sub-cohort, thus revealing dose-dependency of the beneficial effect on eGFR under treatment (Figure 6B).

[0230] The relative QC activity in assay from serum samples collected 2-6 h post-dose revealed a slight increase in rel. QC activity (median) at week 48 in Placebo group (110%) and a dose-dependent decrease at week 48 (down to 66% for 300 mg treatment and to 37% for 600 mg treatment; Figure 7B). 28 subjects with low outlier at pretreatment measurement and missing data were removed for this analysis. Mind: Due to inhibitor dilution in assay, inhibition of QC activity under treatment would be stronger in undiluted samples. A few outliers were observed in either treatment group with increased QC activity. The relative QC activity in assay from CSF samples collected 2-6 h post-dose revealed a stable QC activity (median) over time within the Placebo group (95% at week 48) and a dose-dependent decrease at week 48 (down to 73% for 300 mg treatment and to 59% for 600 mg treatment; Figure 7A). Mind: Due to inhibitor dilution in assay, inhibition of QC activity under treatment would be stronger in undiluted samples. A few outliers were observed in Placebo and 600 mg treatment group with increased QC activity.

[0231] TEAE summary SAPHIR VIVIAD VIVA-MIND

[0232] Subjects with serious TEAEs (%) 3 (5) 8 (13) 2 (1.7) 4 (3.1) 0 1 (2.4)

[0233] Number of SAEs (per Subject) 5 (0.083) 13 (0.22) 3 (0.026) 4 (0.031) 1 (0.023) 1 (0.024)

[0234] Number of serious TEAEs (per Subject) 3 (0.05) 13 (0.22) 3 (0.026) 4 (0.031) 0 1 (0.024)

[0235] Deaths 0 0 0 0 0 0

[0236] MedDRA SOC — preferred term (ratio per

[0237] Subject)

[0238] Gastrointestinal disorders 12 (0.20) 21 (0.35) 20 (0.17) 29 (0.22) 9 (0.20) 10 (0.24)

[0239] Skin and subcutaneous tissue disorders 5 (0.083) 15(0.25) 8 (0.068) 15 (0.12) 3 (0.068) 5 (0.12)

[0240] Table 2 Comparison of treatment emergent adverse events (TEAEs) for the three clinical studies SAPHIR (Scheltens et al. (2018), Alzheimer's Res. Ther. 10: 107-120), VIVI D (Example 1), and VIVA-MIND (Example 2) at a comparable timepoint of

[0241] 11 weeks on final high dose, each following respective uptitration phases. N, number of subjects in population; SAE, serious adverse event; SOC, system organ class; TEAE, treatment emergent adverse event. In summary, the data revealed a pharmacologically relevant drug exposure to be reached upon 600 mg BID treatment which resulted in a decrease of QC activity in serum and CSF of 63% and 41%, respectively, which was meaningful as it affected a significant increase in eGFR on drug treatment in the overall population, compared to a general annual decline of eGFR within the respective population on placebo treatment. Furthermore, the treatment effect exerted by varoglutamstat proved to be dose-dependent, which was likewise mirrored by a reduced QC-inhibition in serum by 300 mg BID treatment compared to 600 mg BID. Combining this effective dose of 600 mg BID with a slow and careful uptitration over 12 weeks and starting from low and sub-pharmacological doses of 50 mg once and twice daily resulted in a clear decrease in discontinuations due to TEAEs, reported moderate and severe TEAEs, serious TEAEs and SAEs, and clearly diminished TEAEs in critical SOCs as identified during SAPHIR, gastrointestinal disorders and skin and subcutaneous tissue disorders, thus resulting in a bening safety profile combined with efficacious exposure levels.

[0242] Example 2 - Treatment study in human subjects with varoglutamstat hydrochloride: intermediate uptitration phase (VIVA-MIND)

[0243] Overall study description

[0244] In a multicenter, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of varoglutamstat in patients with early Alzheimer’s Disease, 109 human subjects were treated with varoglutamstat hydrochloride versus placebo (N = 52 vs. N = 57, respectively), varoglutamstat or placebo tablets were administered orally twice daily. The total treatment duration was between 16 and 72 weeks. Subjects were randomized 1 :1 (placebo, 600 mg all BID; indicated dose refers to Varuglutamstat free base). All tablets were taken after a meal.

[0245] Dosis regimen

[0246] Dose in weeks 1-4: 150 mg BID or placebo

[0247] Dose in weeks 5-8: 300 mg BID or placebo Dose starting from week 9 until EOT : 600 mg BID or placebo

[0248] Target engagement by varoglutamstat was evaluated at week 24 by assessing remaining QC activity in serum samples collected 2-6 h post dosing relative to the subjects’ pretreatment level.

[0249] Laboratory methods, QC activity, as described in Example 1.

[0250] Results:

[0251] The relative QC activity in assay from serum samples collected 2-6 h post-dose revealed a stable QC activity (median) over time at week 24 within the placebo group (103%) and a strong decrease within the treatment group (600 mg BID) at week 24 (down to 12%) (Figure 8). Mind: Due to inhibitor dilution in assay, inhibition of QC activity under treatment would be stronger in undiluted samples.

[0252] For a comparison of treatment emergent adverse events (TEAEs) for the three clinical studies SAPHIR (Scheltens et al. (2018), Alzheimer's Res. Ther. 10: 107-120), VIVIAD (Example 1), and VIVA-MIND (Example 2) at a comparable timepoint of 11 weeks on final high dose, each following respective uptitration phases, cf. Table 2, Example 1.

[0253] In summary, the data revealed a pharmacologically relevant drug exposure to be reached upon 600 mg BID treatment which resulted in a decrease of QC activity in serum of 88%, which was even more pronounced than observed in VIVIAD upon treatment at same dose level. Shortening the slow uptitration protocol of Example 2 by 4 weeks and omitting the sub-pharmacological dosing steps of 50 mg once and twice daily resulted in a similarly bening safety profile with regard to reported moderate and severe TEAEs, serious TEAEs and SAEs, and clearly diminished TEAEs in critical SOCs as identified during SAPHIR, gastrointestinal disorders and skin and subcutaneous tissue disorders. Only a small number of discontinuations due to TEAEs were additionally observed compared to Example 2, resulting numbers (7%), however, were still very clearly below respective numbers observed in SAPHIR (33%), and where thus not considered to be critical but to emphasize the potential for an intermediary uptitration protocol to be realized for the benefit of the patient.

[0254] Example 3 - Comparison of dosing regimens of varoglutamstat (PQ912) in ADI-CKD mouse model (adenine-diet induced chronic kidney disease)

[0255] Male C57BL / 6JRj mice (11 weeks old) were randomized into 5 groups (n=10 per group) based on body weight at day -3: 1) Control - vehicle (PO, BID), 2) CKD - vehicle (PO, BID), 3) CKD - PQ912 (100 mg / kg, PO, BID), 4) CKD - PQ912 (200 mg / kg, PO, QD), 5) CKD - PQ912 (200 mg / kg, PO, BID). Vehicle used comprised 0.8% (w / v) Methylcellulose (400 cP) and 0.25% (v / v) Tween80 in water. PQ912 was used as a hydrochloride salt, the dose level is indicated for the free base, though. On study day -3, all groups were switched to control diet (S9352-E064, Ssniff, Germany), and from study day 1 and throughout the study, groups 2 to 5 were switched to a diet containing 0.2% adenine (S9352-E060, Ssniff, Germany) to induce CKD. At termination, hematology data was generated from terminal blood resulting from cardiac puncture, and the left kidney was further processed for histomorphometric assessment of fibrosis (collagen IV; Col IV; Col4).

[0256] Hematology

[0257] Blood samples were collected in EDTA tubes, and 22 pL of well-mixed blood is loaded into an EDTA microcapillary tube. Red blood cells, mean cell volume for RBC, hematocrit, hemoglobin, mean cell hemoglobin, mean cell hemoglobin concentration, platelets, white blood cells, lymphocytes, and neutrophil granulocytes were measured using the Exigo H400 (Boule) hematology analyzer. Histological staining procedures

[0258] In brief, glass slides with paraffin embedded sections were deparaffinated in xylene and rehydrated in series of graded ethanol.

[0259] Immunohistochemistry using single chromogen:

[0260] Immunohistochemistry (IHC) was performed using standard procedures. Briefly, after antigen retrieval and blocking of endogenous peroxidase activity, slides were incubated with primary antibody. The primary antibody was detected using a polymeric HRP-linker antibody conjugate. Next, the primary antibody was visualized with DAB as chromogen. Finally, sections were counterstained in hematoxylin and cover slipped. Slides were scanned under a 20x objective in a ScanScope AT slide scanner (Aperio).

[0261] Quantitative image analysis of kidney (Quantitative assessment of immunoreactivity)

[0262] IHC-positive staining was quantified by image analysis using the VIS software (Visiopharm, Denmark). VIS protocols were designed to analyze the virtual slides in two steps:

[0263] Statistics

[0264] ANOVA with Dunnett’s test one-factor linear model, each Group compared to Group 2 (CKD - vehicle). *: p < 0.05, **: p < 0.01, ***: P < 0.001, ****: p < 0.0001

[0265] Results:

[0266] The mouse model performed robustly and opened up a good therapeutic window between Group 1 and 2. A highly significant increase of Col IV deposition in the kidney upon disease induction was observed, which was significantly reduced upon treatment with PQ912 either 200 mg / kg once daily (QD) or twice daily (BID) (Figure 9).

[0267] As to hematology: Unexpectedly, an impact of PQ912 treatment on lymphocyte count was observed, which did not correlate with total dose per day, but was rather driven by BID dosing compared to QD dosing. The decrease of lymphocytes under PQ912 BID treatment was even getting lymphocyte numbers below the levels of healthy control animals (Group 1) (Figure 10). Even though these findings did not reach significance levels (which were assessed in this case against Group 1), this trend may lead to unwanted side effects.

[0268] Conclusions: - once daily dosing of PQ912 in ADI-CKD mice at a dose of 200 mg / kg provides evidence of efficacy on disease relevant parameters and simultaneously seems to provide a larger safety window when compared to twice daily dosing with PQ912 (either 100 mg / kg or 200 mg / kg, BID).

[0269] Allometric scaling from such a treatment dose (200 mg / kg, PO, QD) in mice according to Nair and Jacob (2016), J. Basic Clin. Pharmacy 7: 27-31 results in a human equivalent dose

[0270] (HED) of 1056 mg per day for a patient of 65 kg. Thus, reasonable treatment of human kidney disease patients with a benign risk-benefit-ratio is postulated for once daily dosing of 1050 mg + / - 150 mg.

Claims

CLAIMS1. Varoglutamstat ((S)-l-(lH-benzo[d]imidazol-5-yl)-5-(4- propoxyphenyl)imidazolidin-2- one) or a pharmaceutically acceptable salt, solvate or polymorph thereof, including all tautomers thereof, or a pharmaceutical composition comprising said varoglutamstat, for use in the treatment of a disease, wherein said treatment comprises an uptitration phase and a treatment phase of varoglutamstat administration, and wherein said varoglutamstat is administered to a subject in a daily uptake of 10 mg to 1800 mg.

2. Varoglutamstat or pharmaceutical composition for use according to claim 1, wherein said varoglutamstat is administered to a subject in a daily uptake of 10 mg to 1600 mg.

3. Varoglutamstat or pharmaceutical composition for use according to claim 1, wherein said varoglutamstat is administered to a subject in a daily uptake of 10 mg to 1500 mg.

4. Varoglutamstat or pharmaceutical composition for use according to any one of claims 1 to 3, wherein the treatment phase comprises twice daily administration of at least 300 mg varoglutamstat and wherein said uptitration phase comprises once daily administration of 50 mg varoglutamstat for 1 to 4 weeks, followed by twice daily administration of 50 mg varoglutamstat for 1 to 4 weeks, followed by twice daily administration of 150 mg varoglutamstat for 1 to 4 weeks, and followed by twice daily administration of 300 mg varoglutamstat for 1 to 4 weeks to a subject.

5. Varoglutamstat or pharmaceutical composition for use according to any one of claims 1 to 3, wherein the treatment phase comprises twice daily administration of at least 300 mg varoglutamstat and wherein said uptitration phase comprises twice daily administration of 150 mg for 1 to 4 weeks, followed by twice daily administration of 300 mg varoglutamstat for 1 to 4 weeks.

6. Varoglutamstat or pharmaceutical composition for use according to any one of the preceding claims, wherein the treatment phase comprises twice daily administration of 300 mg to 800 mg varoglutamstat.

7. Varoglutamstat or pharmaceutical composition for use according to any one of the preceding claims, wherein the treatment phase comprises twice daily administration of 300 mg or twice daily administration of 600 mg varoglutamstat to a subject.

8. Varoglutamstat or pharmaceutical composition for use according to claim 7, wherein said uptitration phase comprises once daily administration of 50 mg varoglutamstat for 2 weeks, followed by twice daily administration of 50 mg varoglutamstat for 2 weeks, followed by twice daily administration of 150 mg varoglutamstat for 4 weeks, and followed by twice daily administration of 300 mg varoglutamstat for 4 weeks to a subject, and wherein said treatment phase comprises twice daily administration of 300 mg or twice daily administration of 600 mg varoglutamstat to a subject.

9. Varoglutamstat or pharmaceutical composition for use according to any one of claims 1 to 3, wherein the treatment phase comprises once daily administration of 150 mg to 600 mg varoglutamstat, preferably once daily administration of 150 mg or 300 mg or 450 mg or 600 mg varoglutamstat to a subject.

10. Varoglutamstat or pharmaceutical composition for use according to claim 9, wherein the treatment phase comprises once daily administration of at least 300 mg varoglutamstat, and wherein the uptitration phase comprises once daily administration of 50 mg varoglutamstat for 1 to 4 weeks, followed by once daily administration of 150 mg varoglutamstat for 1 to 4 weeks, and followed by once daily administration of 300 mg varoglutamstat for 1 to 4 weeks to a subject.

11. Varoglutamstat or pharmaceutical composition for use according to claim 9, wherein the treatment phase comprises once daily administration of at least 300 mg varoglutamstat, and wherein the uptitration phase comprises once daily administration of 150 mg varoglutamstat for 1 to 4 weeks, and followed by once daily administration of 300 mg varoglutamstat for 1 to 4 weeks to a subject.

12. Varoglutamstat or pharmaceutical composition for use according to any one of claims 1 to 3, wherein said treatment phase comprises once daily administration of 600 mg to 1200 mg varoglutamstat, such as once daily administration of 900 mg to 1200 mg varoglutamstat to a subject.

13. Varoglutamstat or pharmaceutical composition for use according to any one of claims 1 to 3, wherein said treatment phase comprises once daily administration of 600 mg to 1050 mg varoglutamstat, preferably once daily administration of 1050 mg to a subject.

14. Varoglutamstat or pharmaceutical composition for use according to claim 12 or 13, wherein said uptitration phase comprises once daily administration of 50 mg varoglutamstat for 1 to 4weeks, followed by once daily administration of 150 mg varoglutamstat for 1-4 weeks, followed by once daily administration of 300 mg varoglutamstat for 1-4 weeks, and followed by once daily administration of 600 mg varoglutamstat for 1-4 weeks to a subject.

15. Varoglutamstat or pharmaceutical composition for use according to claim 12 or 13, wherein said uptitration phase comprises once daily administration of 150 mg varoglutamstat for 1-4 weeks, followed by once daily administration of 300 mg varoglutamstat for 1-4 weeks, and followed by once daily administration of 600 mg varoglutamstat for 1-4 weeks to a subject.

16. Varoglutamstat or a pharmaceutically acceptable salt, solvate or polymorph thereof, including all tautomers thereof, or a pharmaceutical composition comprising said varoglutamstat, for use in the treatment of a disease, wherein said treatment comprises once daily administration of 600 mg to 1800 mg varoglutamstat to a subject.

17. Varoglutamstat or pharmaceutical composition for use according to any one of the preceding claims, wherein said treatment further comprises a down-titration phase.

18. Varoglutamstat or pharmaceutical composition for use according to claim 17, wherein, when at least 600 mg varoglutamstat is administered daily in the treatment phase to a subject, said down-titration phase comprises administration of 450 mg varoglutamstat for 1 to 4 weeks, followed by administration of 300 mg varoglutamstat for 1 to 4 weeks, followed by administration of 150 mg varoglutamstat for 1 to 4 weeks, and wherein the dosing frequency in the down-titration phase is continued as selected for the treatment phase, once or twice daily.

19. Varoglutamstat or pharmaceutical composition for use according to any one of the preceding claims, wherein said varoglutamstat is varoglutamstat hydrochloride.

20. Varoglutamstat for use according to any one of the preceding claims, wherein said varoglutamstat is a polymorph of varoglutamstat hydrochloride.

21. Pharmaceutical composition for use in the treatment of a disease, said pharmaceutical composition comprising a varoglutamstat according to any one of the preceding claims, wherein said pharmaceutical composition comprises 50 to 600 mg of varoglutamstat and one or more pharmaceutically acceptable diluents, carriers or excipients.

22. The pharmaceutical composition for use according to any one of the preceding claims, said pharmaceutical composition comprising 50 mg, 150 mg, 300 mg, 450 mg or 600 mg of varoglutamstat, preferably 150 mg, 300 mg, 450 mg or 600 mg varoglutamstat.

23. The pharmaceutical composition for use according to any one of the preceding claims, wherein said pharmaceutical composition is administered orally.

24. The pharmaceutical composition for use according to any one of the preceding claims, wherein said pharmaceutical composition is a tablet.

25. Varoglutamstat or pharmaceutical composition for use according to any one of the preceding claims, wherein said disease to be treated is selected from the group consisting of neurodegenerative diseases, inflammatory diseases, infectious diseases, proliferative diseases and tumors, fibrotic diseases, and kidney diseases.

26. Varoglutamstat or pharmaceutical composition according to any one of claims 1 to 24 for use in the treatment of kidney diseases.

27. Varoglutamstat or pharmaceutical composition for use according to claim 25, wherein said neurodegenerative disease is mild cognitive impairment and / or Alzheimer’s disease.

28. Varoglutamstat or pharmaceutical composition for use according to claim 25, wherein said inflammatory disease is selected from the group consisting of psoriasis, rheumatoid arthritis, atherosclerosis, pancreatitis, restenosis, and multiple sclerosis.

29. Varoglutamstat or pharmaceutical composition for use according to claim 25, wherein said infectious disease is periodontitis.

30. Varoglutamstat or pharmaceutical composition for use according to claim 25, wherein said tumors are solid and hematologic tumors.

31. Varoglutamstat or pharmaceutical composition for use according to claim 25, wherein said fibrotic disease is selected from the group consisting of scleroderma, idiopathic pulmonary fibrosis, liver cirrhosis, lung fibrosis, bladder fibrosis, heart fibrosis, pancreas fibrosis, and myelofibrosis.

32. Varoglutamstat or pharmaceutical composition for use according to claim 25, wherein said disease is an acute kidney disease (AKD; acute kidney injury, AKI) or a chronic kidney disease(CKD), such as a condition selected from the group consisting of diabetic nephropathy, Focal Segmental Glomerulosclerosis (FSGS), glomerulonephritis, polycystic kidney disease, membranous nephropathy, obstructions of the urinary tract, vesicoureteral reflux, nephrotic syndrome, recurrent kidney infection (pyelonephritis), lupus and other immune system diseases selected from the group consisting of polyarteritis nodosa, sarcoidosis, Goodpasture syndrome and Henoch-Schbnlein purpura.

33. Varoglutamstat or pharmaceutical composition for use according to claim 25, 26 or 32, wherein said kidney disease is a CKD that is accompanied by a persistent inflammation.

34. Varoglutamstat or pharmaceutical composition for use according to claim 25, 26, 32 or 33, wherein said kidney disease is kidney fibrosis, such as fibrotic CKD.

35. Varoglutamstat or pharmaceutical composition for use according to any one of claims 25, 26, 32 or 33, wherein said kidney disease is diabetic nephropathy.

36. Varoglutamstat or pharmaceutical composition for use according to any one of claims 25, 26, 32 or 33, wherein said kidney disease is Focal Segmental Glomerulosclerosis (FSGS).

37. Varoglutamstat or pharmaceutical composition for use according to any one of claims 25, 26, 32 or 33, wherein said kidney disease is a condition selected from the group consisting of glomerulonephritis, polycystic kidney disease, membranous nephropathy, obstructions of the urinary tract, vesicoureteral reflux, nephrotic syndrome, recurrent kidney infection (pyelonephritis), lupus and other immune system diseases selected from the group consisting of polyarteritis nodosa, sarcoidosis, Goodpasture syndrome and Henoch-Schbnlein purpura.

38. Varoglutamstat or pharmaceutical composition for use according to any one of claims 25, 26 and 33 to 37, wherein said kidney disease is associated with an impaired glomerular filtration rate (GFR).

39. Varoglutamstat or pharmaceutical composition for use according to claim 38, wherein said subject is at risk for CKD.

40. Varoglutamstat or pharmaceutical composition for use according to claim 38 or 39, wherein said subject is suffering from at least one risk factor for CKD, wherein said risk factor is selected from hypertension or diabetes mellitus (type 1 or type 2 diabetes mellitus) or cardiac disease or an eGFR <60 ml / min / 1.73m2.

41. Varoglutamstat or pharmaceutical composition for use according to any one of claims 1 to 38, wherein said subject is not at risk for CKD.

42. Varoglutamstat or pharmaceutical composition for use according to claim 25 or 32, wherein said kidney disease is caused by a condition selected from the group consisting of Alport syndrome, Fabry disease, and connective tissue diseases (CTD).

43. Varoglutamstat or the pharmaceutical composition for use according to any one of the preceding claims, wherein said subject is a mammal, preferably a human.

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