Dose regimen for vicariabine

Abstract

The present invention relates to (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl) methyl] triazolo [4, 5-d] pyrimidin-7-yl] pyrrolidin-3-ol for use in the treatment of diabetic retinopathy, and methods of treatment thereof.

Description

Background Art

[0001] Diabetes affects 463 million people worldwide, and the number of people with diabetes is expected to increase to 700 million by 2045 (IDF 2019; Nair et al., 2016). Diabetic retinopathy (DR) affects approximately one-third of people with type 1 and type 2 diabetes (Yau et al., 2012). Chronic hyperglycemia leads to retinal microvascular changes, inflammation, and neurodegeneration (Fong et al., 2004). At any stage of DR, patients may develop diabetic macular edema (DME), which is the most common cause of central vision loss in patients with diabetes (Resnikoff et al., 2004). DME, along with advanced DR (i.e., proliferative diabetic retinopathy [PDR]), impairs vision and may lead to blindness (Leasher et al., 2016). Overall, DR is estimated to be the most common cause of new cases of blindness among working-age adults worldwide (American Diabetes Association, 2002; Yau et al., 2012).

[0002] Unlike in other parts of the world, patients with DR in the United States can be treated with intravitreal (IVT) injections of anti-vascular endothelial growth factor (VEGF) drugs, regardless of disease severity or the presence of visual impairment, PDR, or DME (Lucentis USPI; Eylea USPI). However, clinical practice trends indicate that a large proportion of patients with nonproliferative diabetic retinopathy (NPDR) and DME that causes mild visual impairment are not easily treated (Cantrell et al., 2020). Instead, treatment initiation is delayed until the disease has progressed, retinal anatomy has been further damaged, and vision has worsened. Although patients with severe NPDR do not yet experience overt visual impairment, their underlying retinopathy is progressive. Moderate to severe NPDR, if left untreated, may progress to vision-threatening disease in approximately 41% and 58% of patients within 1 and 2 years, respectively (Regeneron Pharmaceuticals, Inc. 2020). Therefore, there remains an unmet need for the development of novel therapies that provide non-invasive treatment options for severe NPDR and prevent its progression to visual impairment.

[0003] Chronic hyperglycemia can cause DR or DME through pathological mechanisms that are not fully understood but appear to involve four major pathways: the polyol pathway, the advanced glycation end product pathway, the protein kinase C pathway, and the hexosamine pathway (Brownlee 2005). All of these pathways lead to increased oxidative stress, inflammation, vascular dysfunction, and neurodegeneration (Fong et al. 2004). Müller cells, microglia, and endothelial cells produce chemokines that induce leukostasis, diapedesis, influx of systemic immune cells (such as monocytes) into the retina, and increased production of cytokines including VEGF, tumor necrosis factor-, interleukin (IL)-1, IL-6, metalloproteinases, and angiopoietin-2. These inflammatory mediators then lead to a breakdown of the endothelial cell-cell junctions that form the blood-retinal barrier. Retinal microglia are activated in the early stages of the disease process, and apoptosis of ganglion cells and amacrine cells is increased. In the retinal capillaries, hyperglycemia also causes pericyte dropout and basement membrane thickening, all of which promote increased vascular leakage. These factors lead to vascular lesions and ultimately vision loss (Das et al., 2015).

[0004] Based on the extent of microvascular degeneration and associated ischemic damage, DR can be broadly classified into two stages: (a) NPDR, characterized by microaneurysms and possible intraretinal hemorrhages, hard exudates, and cotton-wool spots; and (b) PDR, whose hallmarks include retinal neovascularization, iris neovascularization (NVI), and vitreous hemorrhage (Wilkinson et al., 2003). Another complication of DR is DME, which may occur in all patients with severe DR and is the most common cause of visual loss in DR patients. DME is caused by diabetes-induced rupture of the blood-retinal barrier, followed by leakage of fluid and circulating proteins into the neural retina through the blood vessels. This leads to abnormal thickening of the retina and often results in cystoid macular edema (Frey and Antonetti 2011; Zhang et al., 2014; Stitt et al., 2016). Hyperglycemic conditions also lead to retinal ischemia, which induces increased production of VEGF, leading to vascular leakage, neovascularization, and PDR.

[0005] Current therapies and unmet medical needs

[0006] Intraocular treatment modalities for diabetic eye disease include laser photocoagulation and IVT of anti-VEGF or steroid agents. Current treatment modalities including laser photocoagulation and / or IVT of anti-VEGF are focused on treating advanced disease after the development of PDR or DME. Anti-VEGF agents are approved in the United States for the treatment of all forms of DR (Lucentis USPI; USPI), is approved elsewhere for the treatment of DME and PDR (Lucentis SmPC), and is first-line therapy for most eyes with centrally involved DME. IVT injections of steroids can also be effective for the treatment of DME (Campochiaro et al., 2012; Boyer et al., 2014); however, the use of IVT steroids is often limited by more frequent ocular side effects such as cataracts and glaucoma. According to the International Council of Ophthalmology's 2017 Guidelines for Diabetic Eye Care, NPDR requires close monitoring and laser photocoagulation may be considered; however, the use of anti-VEGF is not mentioned (Wong et al., 2018). Although anti-VEGF therapies for DR in the United States have broad labeling (Lucentis USPI; Although the USPI is limited, clinical practice trends indicate that a large proportion of patients with NPDR with or without DME causing mild visual impairment are not readily treated (PAT Survey; Cantrell et al., 2020), and instead, treatment initiation is delayed until disease progression, further damage to retinal anatomy, and worsening vision occur. In current practice, several factors may contribute to the delay in treatment for moderate to severe NPDR and could potentially be addressed with oral RO6868847. These factors include:

[0007] Invasive nature of available treatments: All currently available DR / DME ocular treatments (e.g., laser, IVT injections) require invasive procedures and carry uncommon but serious risks.

[0008] Additionally, patients report anxiety about starting the IVT injection procedure (Senra et al., 2017). Especially considering that DM is a systemic condition and DR is often bilateral, the sponsor developed RO6868847 for oral administration to provide a non-invasive alternative to the IVT injection procedure and associated risks to improve the patient experience.

[0009] High frequency of treatment / visits with IVT therapy: Anti-VEGF therapy for DR / DME is initiated at least in the first year, which means that high-frequency treatment and / or follow-up visits are required to confirm and maintain efficacy (Lally et al., 2016). As an oral medication, RO6868847 can be self-administered continuously, and its effect is not affected by the application and elimination of IVT drugs. As such, it is conceivable that the follow-up interval for RO6868847 will be longer than that for IVT anti-VEGF therapy, which will reduce the burden on patients and the healthcare system.

[0010] Low perceived urgency for treatment: DR has a slower disease progression and is considered relatively treatable compared to neovascular age-related macular degeneration (AMD), and retinal damage associated with disease progression is incorrectly assumed to be largely reversible. Extrapolated indications from pivotal trials of anti-VEGF therapy for DME (Brown et al., 2013; Schmidt-Erfurth et al., 2014; Heier et al., 2016) suggest that, although delaying therapy may result in a reduction in retinal thickness (structural benefit) comparable to immediate therapy, delaying anti-VEGF therapy appears to reduce the likelihood of visual recovery (functional benefit). There is consensus that early detection and treatment of DR / DME can minimize or prevent the risk of disease progression and visual loss (Nair et al., 2016).

[0011] Lack of consensus on how to treat: As shown by the 2018 PAT Survey (Singh 2018), there is a lack of consensus on the use of anti-VEGF therapy for patients with good visual acuity (BCVA>

[0012] 73 letters) of DR, there is no clear consensus. New evidence from the PANORAMA trial (Regeneron Pharmaceuticals, Inc. 2020) has recently been released, suggesting that a treatment regimen of aflibercept injected every two months (after 5 months of treatment) can improve DR.

[0013] Additionally, the Diabetic Retinopathy Clinical Research Network's clinical trial Protocol W (Protocol W) demonstrated that anti-VEGF therapy could prevent vision-threatening complications in eyes at high risk for PDR.

[0014] Although patients with moderate-to-severe NPDR do not experience significant visual impairment, the underlying retinal disease is progressive (Morello 2007). If left untreated, moderate-to-severe NPDR may progress to vision-threatening disease, with approximately 41% of patients developing the disease within 1 year and 58% developing the disease within 2 years (Regeneron Pharmaceuticals, Inc. 2020). Therefore, there remains an unmet need for the development of novel therapies that provide non-invasive treatment options for early intervention before irreversible retinal damage occurs.

[0015] In conclusion, patients with NPDR in clinical practice are often not treated immediately with IVT anti-VEGF therapy for a variety of possible reasons. Delaying DR therapy in these patients may be disadvantageous because this is a progressive disease. Many of the given reasons for delaying treatment with IVT anti-VEGF therapy could potentially be addressed by RO6868847, an oral small molecule agonist with high specificity for cannabinoid receptor 2 (CB2). Oral small molecule agonists such as RO6868847 (also known as RG7774 or vicasinabin) may be suitable for treating patients with all stages of NPDR and patients with advanced complications of early stage DR (PDR, DME) for whom IVT anti-VEGF injections are not approved / reimbursed or administered in clinical practice. DR is often bilateral, and based on its intended oral route, RO6868847 may benefit both eyes simultaneously. A number of studies have shown that cannabinoids can inhibit the production of cytokines in both innate and adaptive immune responses in animal models and in human cell culture (Klein 2005). Inhibition of proinflammatory cytokine and chemokine production indicates that synthetic CB2 agonists may have anti-inflammatory effects and therefore be useful in treating chronic inflammatory diseases such as DR.

[0016] RO6868847 is a white to off-white powder with medium to good solubility in organic solvents and is slightly soluble in aqueous solution. RO6868847 is quite stable to temperature stress in solid state and solution and is stable to light. For Phase I, oral film-coated tablets containing 0.05mg, 0.75mg, 10mg or 100mg RO6868847 drug substance for clinical use have been developed. Three placebo film-coated tablets have been prepared to match the active film-coated tablets in size and appearance: one size matches the active 0.05mg and 0.75mg film-coated tablets, and one size matches the active 10mg and 100mg film-coated tablets respectively.

[0017] Rationale for RO6868847 treatment

[0018] RO6868847 is a highly selective CB2 agonist intended for oral treatment of all stages of NPDR and early-stage complications of DR (PDR, DME). CB2 is primarily expressed in immune cells, including microglia in the retina. In the eye, activation of CB2 by RO6868847 has been shown to produce anti-inflammatory effects by inhibiting leukocyte adhesion and microglial activation, reducing vascular permeability and thereby preserving endothelial barrier function. In addition, RO6868847 is highly selective for CB2, thereby excluding the psychotropic effects of central CB1 activation (Topol et al., 2010). Current data suggest that RO6868847 exhibits the highest selectivity compared to well-known CB2 agonists (Soethoudt et al., 2017). Oral administration may benefit DR patients by achieving systemic and bilateral retinal exposure. Based on preclinical evidence, systemic exposure may target leukocytes to prevent attachment to retinal endothelial cells, whereas retinal exposure may activate CB2 receptors in microglia and maintain them in an inactive, ramified state. In addition, preclinical evidence suggests that systemic exposure may have benefits for other diabetic complications such as diabetic nephropathy, which was demonstrated to be improved in a nonclinical model identical to that used for the retinal microglia analysis (Zoja et al., 2016). In summary, with the advantages of this noninvasive oral administration and the expected bilateral benefit, physicians may more easily initiate treatment for all DR patients who, in current practice, may only receive deferred IVT therapy. Additionally, the use of a highly selective CB2 agonist is not expected to result in CB1 modulation and the attendant side effects. The development program for RO6868847 was initiated with the goal of providing a treatment option for patients with moderate to severe NPDR, for whom photocoagulation and intravitreal anti-VEGF therapy are often deferred in current practice.

[0019] The key challenge in selecting the dose range for clinical development of RO6868847 is that the predicted human pharmacologically active dose range is wide, ranging from 0.22 mg to 42 mg per day, based on different nonclinical animal models. In addition to nonclinical in vivo data, CB2 receptor target engagement (TE) information from human whole blood was used to select the clinical dose range. Since CB2R-TE on microglia in the eye cannot be monitored in vivo, the observed human plasma PK is used instead, combined with affinity data derived from peripheral ex vivo CB2R-TE on B cells to predict the CB2R-TE of RO6868847, a type of circulating immune cell that expresses CB2 receptors. CB2R-TE in the retina was estimated, assuming that the plasma concentration of RO6868847 reflects the concentration in the retina, and assuming that the binding affinity of RO6868847 to CB2 receptors on B cells (as assessed in human blood using an ex vivo target engagement assay) represents binding to CB2 receptors in the retina. Surprisingly, in vitro experiments evaluating CB2R-TE in whole blood samples from elderly and age- and demographically-matched diabetic and non-diabetic subjects revealed that approximately 3-fold higher concentrations of RO6868847 were required to achieve similar effects of CB2R-target engagement as observed in the blood of young healthy participants.

[0020] In a Phase I single ascending dose (SAD) and multiple ascending dose (MAD) study (BP40387), CB2R-TE was measured in the blood of healthy participants receiving single and multiple 0.75 mg to 300 mg doses of RO6868847. Based on clinical RO6868847 pharmacokinetic (PK) and pharmacodynamic (PD; measured by CB2R-TE) data and taking into account the differences between healthy and diabetic subjects, the lower end of the 0.75 mg to 300 mg dose range is expected to reach approximately up to half the maximum CB2R-TE, while the high end of the dose range is expected to reach the maximum CB2R-TE. These doses are surprisingly higher than the predictions based on non-clinical models.

[0021] In addition to the CB2R-TE whole blood assay, an in vitro LPS whole blood immunoassay was applied using blood from healthy participants in the MAD study who received multiple 300 mg doses of RO6868847 6. Surprisingly, signals of attenuated LPS-induced cytokine release were only observed at the high end of the dose range, suggesting an additive pharmacological effect supporting the use of doses that provide exposure in the high exposure range.

[0022] In summary, the clinical dose range was established based on a combination of three datasets; nonclinical biomarker readouts of CB2 for target engagement and effects on LPS-induced cytokine production in blood from healthy participants, elderly diabetic patients, and age- and demographically matched controls; and clinical and biomarker data obtained from healthy participants in the Phase I clinical study. Summary of the invention

[0023] A first aspect of the present invention relates to the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use as an agonist of cannabinoid receptor 2 in patients at a dose of between 0.75 mg and 300 mg.

[0024] A second aspect of the present invention relates to a method of use of an agonist of cannabinoid receptor 2 in treatment, the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient, particularly a patient in need thereof, at a dose of between 0.75 mg and 300 mg.

[0025] A third aspect of the present invention relates to the use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol in the manufacture of a medicament as an agonist of cannabinoid receptor 2 at a dose between 0.75 mg and 300 mg.

[0026] Figure 1 Shown is the ratio of CB2 surface expression on B cells to T cells in whole blood from healthy participants following exposure to increasing concentrations of RO6868847.

[0027] Figure 2 IC50 values ​​of RO6868847 in B cells from diabetic patients and age-matched healthy participants are shown.

[0028] Figure 3 IC50 determinations of the effects of RO6868847 on G-CSF, IFNγ, IL1β, and TNFα levels in an in vitro LPS whole blood immunoassay in healthy participants are presented.

[0029] Figure 4 IC50 determinations of the effects of RO6868847 on G-CSF, IFNγ, IL1β, and TNFα levels in LPS whole blood immunoassay in diabetic patients and matched healthy participants in vitro are presented.

[0030] Figure 5 Shown are mean (+SD) CB2 receptor target engagement (% change from baseline) on B cells versus time following 14 days of multiple oral dosing of 20 mg to 300 mg RO6868847 or placebo.

[0031] Figure 6 Baseline normalized TNF-α levels and individual median plasma Cavg,ss at day 14 for RO6868847 are shown.

[0032] Figure 7 Baseline normalized G-CSF levels and individual median plasma Cavg,ss on Day 14 for RO6868847 are shown.

[0033] Figure 8 Baseline normalized IL-1β levels and individual median plasma Cavg,ss on Day 14 for RO6868847 are shown.

[0034] Fig. 9 Baseline normalized IFNγ levels and individual median plasma Cavg,ss on day 14 for RO6868847 are shown.

[0035] Fig.10 Free exposure of disease markers to the active S-dimer RO6868847 in several animal models is shown. AUC 0-24 = area under the concentration-time curve from 0 to 24 h; cyno = cynomolgus monkey; GLP = good laboratory practice; K i = equilibrium dissociation constant; LCNV = laser-induced neovascularization; LPS = lipopolysaccharide; NOAEL = no observed adverse effect level; OD = optical density; RO6868847 = vecanabine; STZ = streptozotocin. Disease markers (intense fluorescence area (mm) in different in vivo PD models 2 ), OD retinal plasma fluorescein ratio, per mm 2 The number of leukocytes, or the total length of processes (in mm)) was expressed as a percentage of the control value (100% represents vehicle-treated diabetic controls and 0% represents vehicle-treated healthy controls), as the free AUC of the active S-epimer RO6868847 0-24 The rat K i (33.3nM = 12ng / mL) is deduced to be the following free AUC 0-24 : Free AUC 0-24 =12′24=286ng·hr / mL. The green area represents the estimated human pharmacological activity exposure range.

[0036] Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:

[0037] The term "active pharmaceutical ingredient" or "API" refers to a compound or molecule in a pharmaceutical composition that possesses a specific biological activity.

[0038] "Chronic degenerative disease" refers to a disease in which the function and / or structure of the affected tissue or organ deteriorates over time. Examples of chronic degenerative diseases are, but are not limited to, neurodegenerative inflammatory disorders, atherosclerosis, liver fibrosis or microvascular diabetic complications.

[0039] "Diabetes mellitus" or "DM" refers to various forms of glucose metabolism disorders with different causes and symptoms. A common feature is a relative or absolute insulin deficiency. The diabetic disease is characterized by a permanent increase in blood glucose levels (hyperglycemia) or an inappropriate utilization of the glucose provided. Diabetes mellitus is subdivided into type I (insulin-dependent; IDDM) and type II (non-insulin-dependent; NIDDM). Chronic complications specific to diabetes and associated with diabetes include microangiopathy, such as retinopathy, nephropathy, and neuropathy; polyneuropathy; diabetic foot; bone, supporting tissue, and connective tissue disorders; and macroangiopathy, especially coronary heart disease, cerebral circulatory disorders, and peripheral arterial occlusive disease.

[0040] "Diabetic retinopathy" or DR refers to fundus microvascular disease that occurs in diabetes. Forms of diabetic retinopathy include non-proliferative retinopathy (NPDR) (background retinopathy), such as retinal hemorrhage, microaneurysms, hard exudates, retinal edema with vision loss, and proliferative retinopathy (PDR), in which cotton wool spots and angiogenesis additionally occur on and in front of the retina due to retinal ischemia caused by vascular occlusion and are accompanied by vitreous hemorrhage. Proliferative retinopathy may lead to tractional retinal detachment, neovascular glaucoma and blindness. The term DR encompasses all stages of PDR and NPDR.

[0041] “Diabetic macular edema,” or DME, refers to swelling of the macula in people with diabetes caused by leaky blood vessels in the retina. DME is the leading cause of vision loss in people with diabetic retinopathy. People with diabetes have a 10% lifetime risk of developing DME. DME affects up to 30% of people who have had diabetes for more than 20 years. If untreated, DME can cause moderate to severe vision loss.

[0042] "Individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0043] “Microvascular diabetic complications” refer to diabetic retinopathy, nephropathy, and neuropathy that can lead to kidney failure, peripheral arterial disease, or limb amputation.

[0044] "Non-proliferative diabetic retinopathy" or "NPDR" refers to the early stage of diabetic retinopathy, which is characterized by edema in the central retina, firm exudates, and lipids leaking from abnormal blood vessels, resulting in blurred central vision. It can also include blood vessel occlusion, restricted retinal blood supply, and increased macular edema.

[0045] "Patient" refers to a human (such as a male or female human) in need of treatment with RO6868847. DETAILED DESCRIPTION

[0046] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0047] Unless otherwise indicated, the nomenclature used in this application is based on the IUPAC systematic nomenclature.

[0048] Various features and embodiments of the present invention are disclosed herein, however, based on the teachings provided, other features, modifications and equivalents of the present invention will be apparent to those skilled in the relevant art. The present invention as described is not limited to the examples and embodiments provided, and those skilled in the art will know various alternative equivalents. As used herein, the singular forms "a", "an" and "the" include the plural, unless the context clearly dictates otherwise. For example, "an" individual will also include "a plurality of individuals".

[0049] According to the present invention, (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol refers to a compound of formula (I)

[0050]

[0051] Also known as RO6868847, Vicanabine, CAS No. 1433361-02-4, methods for preparing and using the compound are described in WO2022 / 106669 and WO2012EP71788.

[0052] In one embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use as an agonist of cannabinoid receptor 2 in a patient at a dose between 0.75 mg and 300 mg.

[0053] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treating or preventing chronic degenerative diseases in patients at a dosage between 0.75 mg and 300 mg.

[0054] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treating or preventing neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications in patients at a dose between 0.75 mg and 300 mg.

[0055] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol, which is used in a patient at a dose between 0.75 mg and 300 mg for treating or preventing microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

[0056] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treating or preventing non-proliferative diabetic retinopathy (NPDR) in a patient at a dose between 0.75 mg and 300 mg.

[0057] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treating or preventing proliferative diabetic retinopathy (PDR) in a patient at a dose between 0.75 mg and 300 mg.

[0058] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treating or preventing diabetic macular edema (DME) in a patient at a dose between 0.75 mg and 300 mg.

[0059] In a specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treating or preventing diabetic nephropathy (DN) in a patient at a dose between 0.75 mg and 300 mg.

[0060] In a more specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treatment or prevention as described herein above, wherein the patient is a human (such as a male or female human).

[0061] In a more specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in the treatment or prevention as described herein above, wherein it is administered orally.

[0062] In a more specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in treatment or prevention as described herein above, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose between 0.75 mg and 300 mg, in particular orally.

[0063] In a more specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in the treatment or prevention as described herein above, wherein it is administered once a day.

[0064] In a more specific embodiment, the present invention provides (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use in the treatment or prevention as described herein above, wherein it is administered in combination with other therapies, in particular in combination with laser, anti-VEGF therapy or other therapies for diabetic retinopathy.

[0065] In one embodiment, the present invention provides a method of treatment using an agonist of cannabinoid receptor 2, the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0066] In a specific embodiment, the present invention provides a method for treating or preventing a chronic degenerative disease, the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0067] In a specific embodiment, the present invention provides a method for treating or preventing neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications, the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0068] In a specific embodiment, the present invention provides a method for treating or preventing microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy, the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0069] In a specific embodiment, the present invention provides a method for treating or preventing non-proliferative diabetic retinopathy (NPDR), the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0070] In a specific embodiment, the present invention provides a method for treating or preventing proliferative diabetic retinopathy (PDR), the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0071] In a specific embodiment, the present invention provides a method for treating or preventing diabetic macular edema (DME), the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0072] In a specific embodiment, the present invention provides a method for treating or preventing diabetic nephropathy (DN), the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient (particularly a patient in need thereof) at a dose between 0.75 mg and 300 mg.

[0073] In a more specific embodiment, the present invention provides a method for treatment or prevention as described herein above, particularly wherein the patient is a human (such as a male or female human).

[0074] In a more specific embodiment, the present invention provides a method for treatment or prevention as described herein above, wherein it is administered orally.

[0075] In a more specific embodiment, the present invention provides a method for treatment or prevention as described herein above, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose of between 0.75 mg and 300 mg, in particular orally.

[0076] In a more specific embodiment, the present invention provides a method for treatment or prevention as described herein above, wherein it is administered once a day.

[0077] In a more specific embodiment, the present invention provides a method for treatment or prevention as described herein above, wherein it is administered in combination with other treatments, in particular in combination with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

[0078] In one embodiment, the present invention provides the use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol in the manufacture of a medicament as an agonist of cannabinoid receptor 2 at a dose between 0.75 mg and 300 mg.

[0079] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament in a dose between 0.75 mg and 300 mg for the treatment or prevention of chronic degenerative diseases.

[0080] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament at a dose between 0.75 mg and 300 mg for the treatment or prevention of neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

[0081] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament at a dose between 0.75 mg and 300 mg for the treatment or prevention of microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

[0082] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament at a dose between 0.75 mg and 300 mg for the treatment or prevention of non-proliferative diabetic retinopathy (NPDR).

[0083] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament at a dose between 0.75 mg and 300 mg for the treatment or prevention of proliferative diabetic retinopathy (PDR).

[0084] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament for treating or preventing diabetic macular edema (DME), wherein the dosage of the compound is between 0.75 mg and 300 mg.

[0085] In a specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for the manufacture of a medicament at a dose between 0.75 mg and 300 mg for the treatment or prevention of diabetic nephropathy (DN).

[0086] In a more specific embodiment, the present invention provides the use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol as described herein above, particularly wherein the patient is a human (such as a male or female human).

[0087] In a more specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol as described herein above, wherein it is administered orally.

[0088] In a more specific embodiment, the present invention provides the use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol as described herein above, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose between 0.75 mg and 300 mg, in particular orally.

[0089] In a more specific embodiment, the present invention provides the use of compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol as described herein above, wherein it is administered once a day.

[0090] In a more specific embodiment, the present invention provides the use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol as described herein above, wherein it is administered in combination with other therapies, in particular in combination with laser, anti-VEGF therapy or other therapies for diabetic retinopathy.

[0091] Examples

[0092] Nonclinical studies

[0093] CB2 in vitro pharmacology

[0094] Pharmacological studies were performed in vitro and in vivo to investigate the potency, selectivity and activity of RO6868847. In vivo pharmacological effects were determined in two different diabetic animal models, including streptozotocin (STZ)-induced rat type 1 diabetes and hereditary type 2 diabetes mouse models, as well as two acute models with acute inflammatory stimulation (LPS injection) or laser injury (CNV). Additional in vitro pharmacological studies were also performed to explore the pharmacological effects of RO6868847 and the suitability of the assay as a biomarker for target engagement and pharmacodynamic effects (PD) in human whole blood obtained from healthy volunteers or diabetic patients.

[0095] RO6868847 binds to human CB2 with a Ki of 51.3 nM and 62.8 nM when measured with Chinese hamster ovary (CHO) cells expressing recombinant CB2 or with U968M cells expressing endogenous CB2. Among the several species tested, RO6868847 showed the highest affinity for human and cynomolgus monkey CB2, with comparable Ki values ​​of 51.3 nM and 66.8 nM, respectively. Table 3 summarizes the affinity of RO6868847 for the CB2 receptor. Based on the physiological expression levels of the endogenous system and the receptor activity state, the human Ki of 62.8 nM in U698M cells is recommended for human dose prediction.

[0096]

[0097] CB2 = cannabinoid receptor 2; CHO = Chinese hamster ovary; K i = equilibrium dissociation constant.

[0098] a IC 50 Refers to the molar concentration of inhibitor that displaces 50% of the radioligand. The reported IC 50 Values ​​are means ± SE of three independent experiments, each performed in duplicate.

[0099] bKi refers to the equilibrium dissociation constant of the unlabeled ligand.

[0100] Table 3: Binding potency of RO6868847 to CB2

[0101] Selectivity of CB2 relative to CB1 and other targets

[0102] The functional potency of RO6868847 against different CB2 species and the selectivity relative to the closely related CB1 were determined by inhibiting cAMP accumulation in Chinese hamster ovary cells stably expressing recombinant CB2. RO6868847 is a potent full agonist against human CB2 and shows similar potency against CB2 in other species including cynomolgus monkeys, rats and mice. Table 4 summarizes the potency against different CB2 species.

[0103]

[0104] cAMP = cyclic adenosine monophosphate; CB2 = cannabinoid receptor 2; CHO = Chinese hamster ovary; E max =Maximum effect.

[0105] a EC 50 It is the molar concentration of agonist that activates 50% of the maximal effect.

[0106] Reported EC 50 Values ​​are means ± SE of four independent experiments, each performed in duplicate.

[0107] b The maximum activity of RO6868847 max Normalized to the effect produced by 10 μM CP55'940.

[0108] Table 4: Agonist potency of RO6868847 against forskolin-stimulated cAMP inhibition in stably transfected CHO cells

[0109] RO6868847 showed no affinity for human CB1 and no receptor activation or inhibition was observed at the highest concentration tested, 10 μM, resulting in a binding selectivity of >195-fold and a functional selectivity of >3600-fold relative to human CB1 (see Table 5).

[0110]

[0111] cAMP = cyclic adenosine monophosphate; CB1 = cannabinoid receptor 1; CB2 = cannabinoid receptor 2; K i = equilibrium dissociation constant.

[0112] a Ki is the equilibrium dissociation constant of the unlabeled ligand.

[0113] b EC 50 It is the molar concentration of agonist that activates 50% of the maximal effect.

[0114] c IC 50 It refers to the molar concentration of agonist that inhibits 50% of the maximal effect induced by 10 μM CP55'940.

[0115] Note: n represents the number of independent experiments, and each experiment was performed in triplicate.

[0116] Table 5: CB2 selectivity of RO6868847 relative to human CB1

[0117] At 30 μM concentration, the in vitro pharmacological properties of RO6868847 were evaluated on a plate including 131 targets and 57 CNS and abuse potential specific targets. RO6868847 showed high selectivity for CB2 in all plates. Further in vitro determinations used radioligand binding assays to evaluate the selectivity of RO6868847 for 78 additional receptors and ion channels, wherein RO6868847 showed excellent selectivity for CB2. In functional cell assays, the activity of RO6868847 was not detected in 15-lipoxygenase, MAGL, diacylglycerol lipase, or FAAH enzymes of the endocannabinoid pathway in both humans and mice (10 μM in mice, up to 500 μM in human targets). These results indicate that the possibility of off-target effects of RO6868847 is low, and therefore it is considered unnecessary to conduct subsequent studies of functional assays.

[0118] Example 1: In vitro target engagement by monitoring CB2 surface expression on peripheral B cells in whole blood from healthy participants and diabetic patients

[0119] To monitor target engagement of RO6868847 in humans, a flow cytometric assay (1078220) was developed using an in-house monoclonal anti-human CB2 antibody in combination with established blood cell markers (CD45, CD14, CD3, and CD19). This assay was used to assess CB2 surface expression on peripheral leukocyte subsets in whole blood from healthy participants, confirming findings published by Castaneda et al. (Castaneda et al., 2013) that B cells showed low but robust CB2 surface expression, while no CB2 surface expression was detected in other peripheral blood cell populations (such as T cells, granulocytes, or monocytes). Therefore, B cells were chosen to monitor CB2 surface expression in order to evaluate any possible drug effects on this measurement in peripheral blood. Like most GPCR agonists, RO6868847 is expected to trigger CB2 receptor internalization upon CB2 activation, resulting in reduced surface detection. To establish this measurement as a surrogate target engagement biomarker, freshly isolated whole blood samples from healthy participants (n=9) were incubated in vitro with eight different increasing concentrations of RO6868847 at 37°C in duplicate for 1 hour and subsequently subjected to flow cytometry (1078220). Data for individual donors (n=9) are expressed as the ratio of CB2 surface expression on B cells to T cells and are normalized to the same ratio in whole blood of corresponding donors without drug incubation (see Figure 1 ), which revealed a concentration-dependent reduction in CB2 surface expression on B cells after RO6868847 treatment.

[0120] Using data from 9 individual donors, the mean IC50 (TE) ± SD = 96.5 ± 27.1 nM and the corresponding mean Emax = 55.0 ± 4.6% (34.6 ng / mL) were estimated. In addition, this in vitro data shows that monitoring target engagement in human plasma using this flow cytometric assay may be feasible in human peripheral blood and was therefore used in a Phase I study of BP40387 in healthy participants.

[0121] The availability of this in vitro target engagement assay also enabled the inventors to explore target engagement-exposure responses in blood samples obtained from diabetic patients (type 1 and type 2) and age, sex and BMI matched healthy controls (Table 6)(1097891). This population was chosen because it is similar to the envisioned target patient population for the proof-of-concept Phase II trial, which encompasses diabetic patients according to the definitions of the World Health Organization and / or the American Diabetes Association.

[0122]

[0123] BMI=body mass index; CRP=C-reactive protein; HbA1c=hemoglobin A1c; SD=standard deviation; nd=not determined.

[0124] If not stated otherwise, medians and interquartile ranges are given (in brackets). Significance levels were calculated using the Mann-Whitney U-test *relative to age, sex and BMI matched healthy controls.

[0125] Table 6: Baseline clinical characteristics of diabetic participants and matched healthy participants

[0126] These experiments showed that RO6868847 also induced a dose-dependent internalization of CB2 receptors on the surface of B cells in whole blood obtained from diabetic patients and matched healthy participants, compared to CB2 receptor levels not exposed to RO6868847 ( Figure 2 ).

[0127] However, by comparing the corresponding mean IC50±SD values ​​of CB2 receptor levels in the three groups of participants (Table 7), the data show that patients with diabetes (DM) and matched healthy participants may require approximately 2-3 times higher exposure to achieve half-maximal internalization of CB2 receptors on B cells compared to previously analyzed healthy participants (one-way ANOVA with Tukeys multiple comparison correction, p=0.0575). This was taken into account during dose selection for the Phase 2 study.

[0128]

[0129] Note: One diabetic patient was excluded from the mean IC 50 and E max In addition to the calculation of individual IC 50 is an outlier (about 10 higher).

[0130] Table 7: Comparison of IC50 (CB2 receptor on B cells) in participants with diabetes (DM) and matched healthy participants with previous data ( Figure 1 )

[0131] Example 2: In vitro pharmacodynamic effects of RO6868847 in LPS immunoassay in whole blood from healthy volunteers and diabetic patients

[0132] In order to explore any putative anti-inflammatory pharmacodynamic effects mediated by the CB2 agonist RO6868847 in potentially clinically relevant human samples, an ex vivo whole blood lipopolysaccharide (LPS) immunoassay was performed. Heparinized whole blood from healthy participants (n = 8) was incubated in vitro for 1 hour at 37°C with eight increasing concentrations of RO6868847 (0 sample - 30 μM) and exposed to LPS (100 ng / ml). After 18 hours at 37°C, multiple ELISAs based on Luminex beads were used to analyze changes in a series of chemokines and cytokines in the supernatant. These experiments revealed dose-dependent changes in specific chemokine and cytokine levels after LPS stimulation in whole blood (IFNγ, IL1β, TNFα decreased and G-CSF increased), and thus indicated the overall anti-inflammatory / immunomodulatory downstream pharmacodynamic effects of CB2 agonists in vitro at high exposure.

[0133] In addition, this in vitro LPS whole blood immunoassay was also used for samples from diabetic (type I and II) and matched healthy participants (Table 6). To explore whether the exposure (0-30 μM)-response relationship of RO6868847 and the corresponding changes in cytokine levels may be altered by any chronic inflammatory conditions in diabetic (DM) and / or elderly healthy participants (HP), who are expected to be the study population for the Phase II proof-of-concept study.

[0134] In diabetic patients and matched healthy participants, a dose-dependent attenuation of IFNγ by RO6868847 was observed, as well as IL1β responses in a few participants at high exposures ( Figure 4 ), which confirms previous in vitro observations in healthy participants ( Figure 3 However, for G-CSF and TNFα levels, the high variability observed and the fact that only EC50 / IC50 values ​​could be calculated for one or two individual samples did not allow for an overall drug response across the explored exposure range ( Figure 4 , Table 8).

[0135]

[0136] The numbers in brackets indicate the EC values ​​that can be fitted. 50 or IC 50 Curves show number of subjects versus total number of subjects per group. n.calc.=not calculated.

[0137] Mean and SD were calculated only when n ≥ 3.

[0138] Table 8

[0139] Overall, the data support that RO6868847 also has general immunomodulatory / anti-inflammatory effects in the elderly and in patients with diabetes. Whether these effects are similar but to different degrees, or whether the differences are more significant, due to less sensitive immune responses, remains to be explored.

[0140] Exposure response analysis based on in vivo pharmacodynamics

[0141] Several animal models have demonstrated effects of vecanabine relevant to DR pathophysiology, including inhibition of ocular leukocytostasis (rodent diabetic and nondiabetic models), reduction of retinal hyperfluorescence (rodent laser injury model), and preservation of retinal permeability, function, and microglial morphology (rodent diabetic model). The diversity of models tested (including acute challenge models versus disease amelioration models) and the lack of recommended nonclinical DME / DR models with validated clinical translatability present challenges in estimating exposure to pharmacological activity in humans.

[0142] Fig.10 Disease markers in different in vivo PD models are shown as percentage of control values, as free AUC of the active S-epimer vecanabine 0-24 Exposures are derived from satellite animals or extrapolated from previous PK or PD studies in the same species.

[0143] Based on this comprehensive assessment, the pharmacologically active exposure range observed in rats spanned 2 logs, with the upper limit (unbound AUC 0-24 [S-dimer] = 546 ng·hr / mL) corresponds to the full efficacy at 10 mg / kg in the rat STZ-induced type 1 diabetes model, and the lower limit (unbound AUC 0-24 [S-dimer] = 2.8 ng·hr / mL) corresponds to a 50% reduction in disease markers (low fluorescence areas) in the laser-induced NV rat model. This results in a plasma concentration close to that of the in vitro rat K i Unbound AUC 0-24 This represents 52% of the upper limit of pharmacologically active exposure observed in rats.

[0144] Impact on humans

[0145] Completed and ongoing Phase I / II clinical studies of RO6868847 are summarized as follows. All studies in the clinical development program are described in detail in Appendix 4 and are conducted in accordance with the principles of Good Clinical Practice (GCP). Study BP40387 is a Phase I study conducted in healthy male and female participants and includes 5 parts. Part 1 (SAD) consists of an adaptive, single ascending dose, investigator / participant unaware, randomized, placebo-controlled, parallel study to investigate the safety, tolerability and pharmacokinetics of RO6868847. Part 2 (FE) is designed as an open-label, randomized, two-stage crossover, single-dose study to investigate the effect of food on the pharmacokinetics of RO6868847. Part 3 (MAD) is an adaptive, multiple ascending dose, investigator / participant unaware, randomized, placebo-controlled, parallel study to investigate the safety, tolerability and pharmacokinetics of RO6868847 administered continuously for 14 days (once daily). Part 4 (DDI-CYP induction) was a non-randomized, open-label, fixed-sequence, two-period study to explore RO6868847-mediated CYP induction, 300 mg once daily for 14 days, using midazolam, repaglinide, and bupropion as probe substrates for CYP3A, CYP2C8, and CYP2B6, respectively. Part 5 (DDI-transporter-mediated inhibition) was a non-randomized, open-label, fixed-sequence, four-period study to investigate the inhibition of drug transporters by RO6868847, using metformin and atorvastatin as probe substrates for OCT2 and MATE2-K, and OATP1B1, respectively. Overall, in Part 1 (SAD), a total of 33 healthy participants received a single oral dose of RO6868847 ranging from 0.75 mg to 300 mg, and 12 participants received placebo under fasting conditions. In Part 2 (FE), 8 participants received a single oral dose of 100 mg RO6868847 once after an overnight fast of at least 10 hours and once within 30 minutes of starting a high-calorie, high-fat breakfast. In Part 3 (MAD), a total of 30 participants received multiple oral doses of 6 mg to 300 mg RO6868847 QD for 14 days, and 10 participants received placebo.

[0146] Clinical pharmacokinetics

[0147] In the study of BP40387, RO6868847 was rapidly absorbed under fasting conditions and reached a median maximum plasma concentration (Tmax) between 1 and 4 hours. For Cmax and AUCInf / tau up to 100 mg, no significant deviation from dose proportionality was observed. However, for doses exceeding 100 mg, Cmax and AUCInf / tau seemed to increase in a manner slightly less than the dose ratio. After a single dose, the AUCtau of the steady-state drug was very consistent with the AUCInf, indicating that there was no relevant PK time dependence. After multiple doses of RO6868847 were administered once a day, steady state was reached on average about the second day after administration, and only slight accumulation occurred within the dose range studied. After single and multiple doses, renal excretion of RO6868847 was very small, accounting for an average of less than 5% of the administered dose. Overall, food had no relevant effect on the pharmacokinetics of RO6868847.

[0148] In part 3 (MAD), co-administration of a single oral dose of 0.100 mg midazolam with multiple oral doses of 6 mg to 300 mg RO6868847 (once daily for 14 days) showed that RO6868847 was a weak inducer of CYP3A at the 300 mg administration dose, but not at the lower doses tested. In study BP40387, single and multiple oral doses of RO6868847 resulted in reversible, dose-dependent, and sustained CB2 receptor target engagement on B cells in the blood.

[0149] Pharmacokinetics after single and multiple oral administration in healthy subjects Single-dose plasma pharmacokinetics

[0150] In part 1 (SAD) of the study BP40387, RO6868847 was generally rapidly absorbed in the fasting state; however, Tmax appeared to change with increasing dose. At 100 mg and 300 mg RO6868847, individual Tmax ranged from 1 hour to 6 hours. The main plasma PK parameters of RO6868847 are summarized in Table 20. AUCinf and Cmax increased approximately in a dose-proportional manner up to 100 mg of RO6868847; however, beyond 100 mg to 300 mg RO6868847, Cmax and AUCinf increased in a slightly less than dose-proportional manner. After reaching peak plasma concentration (Cmax), RO6868847 plasma concentrations decreased in a monoexponential manner, with the apparent terminal elimination half-life of RO6868847 ranging from 6.95 hours to 18.5 hours. In addition, half-life variability increased with increasing doses because individual participants exhibited a biphasic decline after peak plasma concentrations.

[0151]

[0152] T max Median (minimum, maximum) of , otherwise Geo mean (Geo mean CV%)

[0153] Table 20: Summary of plasma pharmacokinetic parameters of RO6868847 after single oral dose administration under fasting conditions

[0154] In Part 2 (FE) of Study BP40387, consistent with delayed gastric emptying due to prolonged meals, time to peak plasma concentration (Tmax) was slightly longer following dosing following a high-fat, high-calorie breakfast. In the fed state, median Tmax was reached at 4 hours post-dose compared to 3 hours post-dose in the fasted state. On average, Cmax in the fed state was not affected by food, and AUCInf was only slightly increased compared to the fasted state. Single-Dose Urine Pharmacokinetics

[0155] Following a single oral dose administration in part 1 (SAD) of study BP40387, RO6868847 could be quantified in the urine; however, the fraction of the administered dose recovered in the urine as RO6868847 within 72 hours post-administration (Fe) was low, ranging in geometric means from 2.16% to 4.27% over the dose range studied.

[0156] Multiple-dose plasma pharmacokinetics

[0157] In part 3 (MAD) of the study BP40387, RO6868847 was rapidly absorbed, with overall individual peak plasma concentration times (Tmax) reaching between 1 and 4 hours after dosing for all dose groups (except the 6 mg dose group on day 1, whose Tmax ranged from 0.5 hours to 3 hours) on days 1, 7, and 14 (Table 21). Table 21 summarizes the plasma PK parameters of RO6868847 at steady state on day 14. At the end of the 14-day treatment period, RO6868847 plasma concentrations decreased monoexponentially for the lowest study dose of 6 mg RO6868847, but for doses exceeding 6 mg, a biexponential decline was observed. The elimination phase was characterized by a geometric mean apparent terminal elimination half-life ranging from 8.27 hours to 39.0 hours within the dose range explored. Statistical analysis indicated that for doses exceeding 100 mg, Cmax and AUCtau at steady state increased in a manner slightly less than dose proportional. However, this effect was more pronounced for Cmax, especially at a dose of 300 mg RO6868847. After multiple doses, steady state was reached approximately after the second day of treatment, with slight accumulation of both Cmax and AUCtau, independent of dose (Table 21). Statistical analysis of logarithmic transformation cumulative ratios indicated no evidence of time-dependent pharmacokinetics.

[0158]

[0159] T max Median (minimum, maximum) of , otherwise Geo mean (Geo mean CV%). ND = Not determined

[0160] Table 21: After once daily oral dose administration of RO6868847 under fasting conditions on Day 14

[0161] Summary of plasma PK parameters of RO6868847

[0162] Multiple-dose urine pharmacokinetics

[0163] At steady state, RO6868847 could be quantified in the urine; however, over the dosing interval (24 hours), the recovery of Fe in the urine as RO6868847 was low, ranging in geometric means from 0.738% to 1.85% over the dose range studied.

[0164] Safety in Phase 1 BP40387

[0165] In the BP40387 study, at least 1 AE was reported by 11 participants (24.4%) in Part 1, 1 participant (12.5%) in Part 2, 16 participants (40%) in Part 3, 12 participants (70.6%) in Part 4, and 2 participants (11.1%) in Part 5 (see Table 22). One participant (5.9%) in Part 4 of the study was withdrawn by the investigator due to an AE. AEs reported by more than 2 participants in Part 1 were medical device site erythema (6 participants in each PT) and vascular procedure complications (2 participants in each PT), AEs reported in Part 3 were medical device site erythema (5 participants in each PT), constipation (3 participants in each PT), medical device site papule, medical device site pruritus, vascular procedure complications, headache (2 participants each), and AEs reported in Part 4 were constipation (2 participants in each PT). In Study BP40387, in Part 4 (DDI-CYP Induction), one SAE of severe intensity (marked change in T-wave morphology compared with baseline) and moderate intensity (complication of tachycardia) was reported in a subject treated with 300 mg vecanabine QD. The event was considered by the investigator to be related to vecanabine and resolved without sequelae within one week after cessation of treatment.

[0166] Example 3

[0167] In Example 2, target engagement of RO6868847 was assessed on circulating B cells from whole blood samples using a newly developed exploratory flow cytometric assay to monitor RO6868847-induced CB2 receptor internalization on B cells. Figure 5The average CB2 receptor target engagement versus time curves after multiple doses of RO6868847 in Example 3 are shown, excluding data for the 6 mg dose as this data is not reportable. Reversible, dose-dependent, and sustained CB2 receptor target engagement was observed after multiple doses of up to 300 mg RO6868847 as described in Example 2. After treatment was stopped, CB2 receptor surface expression returned to baseline in approximately 1 week. At a dose of 100 mg RO6868847, target engagement appeared to reach a plateau, while at higher doses of RO6868847, target engagement increased only slightly. In Example 2, proinflammatory cytokine production was induced by ex vivo stimulation with LPS in freshly obtained whole blood samples to measure downstream markers of RO6868847-mediated CB2 receptor internalization and explore its pharmacological properties. Serum levels of TNF-α, G-CSF, IL-1β, and interferon-γ were assessed in participants in Study BP40387 to investigate changes in these cytokines following repeated dosing of up to 300 mg of RO6868847 once daily for 14 days. Under steady-state conditions, concentration-dependent effects were observed for TNF-α, G-CSF, and IL1-β, respectively, compared to baseline. Figure 6 , Figure 7 and Figure 8 However, IFN-γ did not show any detectable trend within the investigated concentration range of RO6868847 (see Fig. 9 ). It should be noted that the IL1-β analysis was negatively impacted by a large proportion of non-reportable baseline data (see Figure 8 ), especially at the highest tested dose of 300 mg RO6868847. Significant differences were observed for these measures. However, this was expected and no specific predefined statistical analysis was performed due to the small cohort size of this exploratory evaluation.

[0168] in conclusion

[0169] Safety and Tolerability

[0170] RO6868847 was also safe and well tolerated in healthy male and female participants following single oral doses up to 300 mg.

[0171] RO6868847 was generally well tolerated after multiple oral doses up to 300 mg of RO6868847 were administered once daily for 14 days in healthy male and female participants, with the only exception of one participant who experienced a concurrent SAE with an unexplained transient increase in plasma trough concentrations approximately 4- to 5-fold higher than the mean of the remaining participants in the 300 mg RO6868847 dose group.

[0172] There was no dose-related increase in the incidence or severity of reported AEs following single and multiple doses of RO6868847, and no series of adverse events were observed.

[0173] No specific questions were found regarding clinical laboratory parameters, ECG, vital signs, C-SSRS, Bowdle VAS, Bond and Lader VAS, TBNK assay, or neurological examination.

[0174] Pharmacokinetics and pharmacodynamics

[0175] RO6868847 is rapidly absorbed, and no significant deviation from dose proportionality of AUC and Cmax was observed up to 100 mg RO6868847 after single and multiple oral dose administration. However, for doses above 100 mg RO6868847, AUC and Cmax appeared to increase in a slightly less than dose proportional manner.

[0176] After multiple doses of RO6868847 were administered once daily, steady state was reached on average approximately after the second day of dosing, with only slight accumulation occurring.

[0177] Overall, food had no relevant effect on the pharmacokinetics of RO6868847.

[0178] Renal excretion of RO6868847 was minimal following single and multiple dose administration, averaging less than 5% of the administered dose.

[0179] • Single and multiple doses of RO6868847 administration resulted in a dose-dependent, persistent but reversible internalization of CB2 receptors on B cells in the blood.

[0180] • Multiple dose administration of RO6868847 appears to attenuate ex vivo LPS-induced production of proinflammatory cytokines (ie, TNF-α, G-CSF, and IL1-β) in whole blood compared to baseline in an overall dose-dependent manner.

[0181] Aspects of the Invention

[0182] Aspect 1. A compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use as an agonist of cannabinoid receptor 2 in a patient at a dose between 0.75 mg and 300 mg.

[0183] Aspect 2. The compound according to Aspect 1, which is used for treating or preventing chronic degenerative diseases.

[0184] Aspect 3. The compound according to aspect 1 or 2, which is used for treating or preventing neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

[0185] Aspect 4. The compound according to any one of aspects 1 to 3, for use in treating or preventing microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

[0186] Aspect 5. A compound according to any one of aspects 1 to 4, for use in treating or preventing non-proliferative diabetic retinopathy (NPDR).

[0187] Aspect 6. A compound according to any one of aspects 1 to 5, particularly wherein the patient is a human (such as a male or female human).

[0188] Aspect 7. The compound according to any one of aspects 1 to 6, wherein it is administered orally.

[0189] Aspect 8. The compound according to any one of aspects 1 to 7, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose of between 0.75 mg and 300 mg, in particular orally.

[0190] Aspect 9. The compound according to any one of aspects 1 to 8, wherein it is administered once a day.

[0191] Aspect 10. The compound according to any one of aspects 1 to 9, wherein it is administered in combination with other treatments, in particular in combination with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

[0192] Aspect 11. A method of treatment using an agonist of cannabinoid receptor 2, the method comprising administering (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol to a patient, particularly a patient in need thereof, at a dose between 0.75 mg and 300 mg.

[0193] Aspect 12. The method according to aspect 11, which is used to treat or prevent chronic degenerative diseases.

[0194] Aspect 13. The method according to aspect 11 or 12, which is used to treat or prevent neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

[0195] Aspect 14. The method according to any one of aspects 11 to 13, which is used to treat or prevent microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

[0196] Aspect 15. The method according to any one of aspects 11 to 14, for treating or preventing non-proliferative diabetic retinopathy (NPDR).

[0197] Aspect 16. The method according to any one of aspects 11 to 15, in particular wherein the patient is a human (such as a male or female human).

[0198] Aspect 17. The method according to any one of aspects 11 to 16, wherein it is administered orally.

[0199] Aspect 18. The method according to any one of aspects 11 to 17, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose of between 0.75 mg and 300 mg, in particular orally.

[0200] Aspect 19. The method according to any one of aspects 11 to 18, wherein it is administered once a day.

[0201] Aspect 20. The method according to any one of aspects 11 to 19, wherein it is administered in combination with other treatments, in particular in combination with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

[0202] Aspect 21. Use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol in the manufacture of a medicament, wherein the compound is an agonist of cannabinoid receptor 2 in a dose between 0.75 mg and 300 mg.

[0203] Aspect 22. The use according to Aspect 21, for the manufacture of a medicament for treating or preventing a chronic degenerative disease.

[0204] Aspect 23. The use according to aspect 21 or 22, for the manufacture of a medicament for treating or preventing neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

[0205] Aspect 24. The use according to any one of Aspects 21 to 23, for the manufacture of a medicament for treating or preventing microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

[0206] Aspect 25. Use according to any one of aspects 21 to 24, for the manufacture of a medicament for the treatment or prevention of non-proliferative diabetic retinopathy (NPDR).

[0207] Aspect 26. The use according to any one of aspects 21 to 25, in particular wherein the patient is a human (such as a male or female human).

[0208] Aspect 27. The use according to any one of aspects 21 to 26, wherein it is administered orally.

[0209] Aspect 28. Use according to any one of aspects 21 to 27, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose of between 0.75 mg and 300 mg, in particular orally.

[0210] Aspect 29. The use according to any one of aspects 21 to 28, wherein it is administered once a day.

[0211] Aspect 30. Use according to any one of aspects 21 to 28, wherein it is administered in combination with other treatments, in particular in combination with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

Claims

1. The compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol for use as an agonist of cannabinoid receptor 2 in patients at a dose of between 0.75 mg and 300 mg.

2. The compound according to claim 1, for use in the treatment or prevention of chronic degenerative diseases.

3. The compound according to claim 1 or 2, for use in the treatment or prevention of neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

4. A compound according to any one of claims 1 to 3 for use in the treatment or prevention of microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

5. A compound according to any one of claims 1 to 4 for use in the treatment or prevention of non-proliferative diabetic retinopathy (NPDR).

6. The compound according to any one of claims 1 to 5, particularly wherein the patient is a human (such as a male or female human).

7. The compound according to any one of claims 1 to 6, wherein it is administered orally.

8. The compound according to any one of claims 1 to 7, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose between 0.75 mg and 300 mg, in particular orally.

9. The compound according to any one of claims 1 to 8, wherein it is administered once a day.

10. The compound according to any one of claims 1 to 9, wherein it is administered in combination with other treatments, in particular in combination with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

11. A method for treating with an agonist of cannabinoid receptor 2, the method comprising: (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol is administered to patients, particularly those in need thereof, at a dose of between 0.75 mg and 300 mg.

12. The method according to claim 11, for treating or preventing chronic degenerative diseases.

13. The method according to claim 11 or 12, for treating or preventing neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

14. The method according to any one of claims 11 to 13, for treating or preventing microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

15. The method according to any one of claims 11 to 14, for the treatment or prevention of non-proliferative diabetic retinopathy (NPDR).

16. The method according to any one of claims 11 to 15, in particular wherein the patient is a human (such as a male or female human).

17. A method according to any one of claims 11 to 16, wherein it is administered orally.

18. The method according to any one of claims 11 to 17, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose of between 0.75 mg and 300 mg, in particular orally.

19. The method according to any one of claims 11 to 18, wherein it is administered once a day.

20. The method according to any one of claims 11 to 19, wherein it is administered in combination with other treatments, in particular with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

21. Use of the compound (3S)-1-[5-tert-butyl-3-[(1-methyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol in the manufacture of a medicament, wherein the compound is an agonist of cannabinoid receptor 2 at a dose between 0.75 mg and 300 mg.

22. The use according to claim 21, for the manufacture of a medicament for treating or preventing a chronic degenerative disease.

23. The use according to claim 21 or 22, for the manufacture of a medicament for treating or preventing neurodegenerative inflammatory diseases, atherosclerosis, liver fibrosis or microvascular diabetic complications.

24. The use according to any one of claims 21 to 23, for the manufacture of a medicament for the treatment or prevention of microvascular diabetic complications, wherein the microvascular diabetic complications are diabetic retinopathy, diabetic macular edema or non-proliferative diabetic retinopathy.

25. Use according to any one of claims 21 to 24, for the manufacture of a medicament for the treatment or prevention of non-proliferative diabetic retinopathy (NPDR).

26. Use according to any one of claims 21 to 25, in particular wherein the patient is a human (such as a male or female human).

27. The use according to any one of claims 21 to 26, wherein it is administered orally.

28. Use according to any one of claims 21 to 27, wherein the patient is a human (such as a male or female human), wherein the compound is administered in a dose of between 0.75 mg and 300 mg, in particular orally.

29. The use according to any one of claims 21 to 28, wherein it is administered once a day.

30. Use according to any one of claims 21 to 28, wherein it is administered in combination with other treatments, in particular with laser, anti-VEGF therapy or other treatments for diabetic retinopathy.

31. The invention as hereinbefore described.

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