Liposome formulations comprising ati receptor blockers and uses thereof

Abstract

The present disclosure provides a liposome formulation comprising a lipid membrane comprising at least one liposome-generating phospholipid and a sterol; and an in-liposome water compartment encapsulating at least one AT1 receptor blocker (ARB) and a pH-dependent ionizable anion; the liposomes have an effect on administration of a subject in need of an effect that does not result in greater than 50% reduction in the average blood pressure of the subject compared to administration of the same amount of ARB in free form. The liposomes may be used for systemic administration, such as by injection or for pulmonary administration, such as by inhalation.

Description

[0001] This application is a divisional application of the application with application number 202180017153.5 (PCT application number PCT / IL2021 / 050336), application date March 25, 2021, and invention title "Liposomal Formulations Containing ATI Receptor Blockers and Their Uses". Technical Field

[0002] This disclosure relates to drug delivery systems, and in particular, liposomal drug delivery systems. Background Art

[0003] References considered relevant to the background of the presently disclosed subject matter are listed below:

[0004] V.P. Chauhan, I.X. Chen, R. Tong, M.R. Ng, J.D. Martin, K. Naxerova, M.W. Wu, P. Huang, Y. Boucher, D.S. Kohane, R. Langer, R.K. Jain, Reprogramming the microenvironment with tumor - selective angiotensin blockers enhances cancer immunotherapy, Proc. Natl. Acad. Sci. U.S.A. 166 (2019) 10674–10680. doi:10.1073 / pnas.1819889116

[0005] Y. Zhu, L. Wen, S. Shao, Y. Tan, T. Meng, X. Yang, Y. Liu, X. Liu, H. Yuan, F. Hu, Inhibition of tumor - promoting stroma to enforce subsequently targeting AT1R on tumor cells by pathological inspired micelles, Biomaterials. 161 (2018) 33 - 46. doi:10.1016 / j.biomaterials.2018.01.023

[0006] M.R.Golder, J.Liu, J.N.Andersen, M.V.Shipitsin, F.Vohidov, H.V.T.Nguyen, D.C.Ehrlich, S.J.Huh, B.Vangamudi, K.D.Economides, A.M.Neenan, J.C.Ackley, J.Baddour, S.Paramasivan, S.W.Brady, E.J.Held, L.A.Reiter, J.K.Saucier-Sawyer, P.W.Kopesky, D.E.Chickering, P.Blume-Jensen, J.A.Johnson, Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs, Nat.Biomed.Eng.2(2018)822 - 830.doi:10.1038 / s41551-018-0279-x T.Xia, Q.He, K.Shi, Y.Wang, Q.Yu, L.Zhang, Q.Zhang, H.Gao, L.Ma, J.Liu, Losartan loaded liposomes improve the antitumor efficacy of liposomal paclitaxel modified with pHsensitive peptides by inhibition of collagen in breast cancer, Pharm.Dev.Technol.23(2018)13 - 21.doi:10.1080 / 10837450.2016.1265553

[0007] International Patent Application Publication No. WO15155773

[0008] The acknowledgement of the above references herein should not be construed as an admission that these references are in any way relevant to the patentability of the presently disclosed subject matter.

[0009] Background:

[0010] Angiotensin II (Ang II) is the main effector peptide of the renin-angiotensin system (RAS). Ang II binds to two receptor subtypes, the type 1 and type 2 Ang II (AT1 and AT2) receptors, which are members of the G protein-coupled receptor superfamily (GPCRs). AT1 receptor blockers (ARBs) are highly selective for the AT1 receptor and can block the harmful effects of Ang II, such as vasoconstriction, aldosterone release, sodium and water retention, sympathetic activation, and cell proliferation, and are used clinically as antihypertensive drugs. However, ACE and AT1R play important roles in cancer development: (1) cell migration, invasion, and metastasis; (2) TGFβ-mediated induction of extracellular matrix proteins leads to fibroblast differentiation, resulting in increased mechanical stress; (3) effects on tumor vascular endothelial cells lead to tumor hypoxia, accompanied by increased vasoconstriction; and (4) secretion of cytokines, which in turn causes M2-macrophage polarization and inhibits the lytic activity of CD8+ T cells. Therefore, ARBs have the potential to affect these activities.

[0011] ARBs can also enhance the activity of immune checkpoint inhibition (ICI). The local RAS in the cancer microenvironment has been found to have profound effects, inducing immunosuppression by enhancing the immunosuppressive activities of macrophages, myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs). This effect is reversed by treatment with angiotensin receptor blockers (ARBs).

[0012] ARBs can also be used as a possible treatment for coronavirus infection. The coronavirus S (spike) protein uses ACE2 as a receptor for host cell entry. The S protein binds to the catalytic domain of ACE2 with high affinity. This binding triggers a conformational change in the coronavirus S protein, allowing host cell protease (TMPRSS2) to carry out proteolytic digestion [Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181(2):271–80e8. Epub 2020 / 03 / 07. https: / / doi.org / 10.1016 / j.cell.2020.02.052 PMID: 32142651; PubMed Central PMCID: PMC7102627]. It has been demonstrated that the binding of the coronavirus spike protein to its cellular binding site ACE2 leads to downregulation of ACE2, which in turn leads to overproduction of the related enzyme ACE of angiotensin, and less ACE2 is able to convert it into the vasodilator heptapeptide angiotensin (1-7). This in turn causes lung injury because the binding of angiotensin II to the AT receptor leads to increased pulmonary vascular permeability, thereby mediating increased pulmonary pathology.

[0013] Two complementary mechanisms of using ARBs occur: blocking the over-angiotensin-mediated AT receptor activation caused by viral infection, and upregulating ACE2, thereby reducing the angiotensin produced by ACE and increasing the production of the vasodilator angiotensin 1-7 [D. Gurwitz, Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics., Drug Dev.Res. (2020) 2–5. doi:10.1002 / ddr.21656]. Therefore, the administration of ARBs is a method for treating COVID-19 infection.

[0014] In summary, ARBs demonstrate a wide variety of effects, including inhibiting angiogenesis, affecting the TME, and altering the immune environment.

[0015] However, the clinical application of ARBs in cancer therapy is limited by systemic side effects such as hypotension. To avoid or minimize unwanted systemic physiological effects, it is necessary to selectively target ARBs to tumors.

[0016] V.P. Chauhan et al. (2019) described a nano - formulation consisting of valsartan combined with a pH - sensitive polymer in nano - form, resulting in a nano - ARB that eliminates the blood - pressure - lowering effect of valsartan while increasing the degree of TME normalization.

[0017] Y. Zhu et al. (2018) described a nano - formulation of telmisartan (an angiotensin II type 1 (AT1) receptor antagonist) based on chitosan - glycolipid micelles.

[0018] M.R. Golder et al. (2018) described a nano - formulation of telmisartan with brush - arm star polymers.

[0019] T. Xia et al. (2018) described liposomal losartan (a selective angiotensin II type 1 (AT1) receptor antagonist) based on soy - phosphatidylcholine (Soy - PC), but it is very leaky.

[0020] Finally, WO15 / 155773 described liposomal formulations for systemic administration. Summary of the Invention

[0021] The present disclosure is based on the development of nano - formulations to overcome the obstacles associated with the systemic delivery of ARBs. This is achieved through the development of injectable PEGylated nano - liposomal formulations or inhalable nano - liposomal formulations loaded with at least one ARB.

[0022] A unique feature of the nano - liposomal formulations encapsulating ARBs is that they lack the blood - pressure - lowering side effects of ARBs (e.g., when delivered in free form).

[0023] As further discussed hereinafter, the disclosed formulations can effectively treat cancer, diabetic retinopathy (which is the main cause of blindness in working - age elderly people), and other indications that require systemic delivery of ARBs. Such liposomes are preferably suitable for administration by injection.

[0024] In some other aspects, the disclosed formulations can effectively treat viral infections, especially respiratory viral infections. According to this aspect, the liposomes are preferably suitable for administration by inhalation, as further discussed hereinafter.

[0025] Thus, according to a first aspect herein referred to as the "injectable liposomes aspect", there is disclosed a liposome comprising a lipid membrane and an aqueous compartment within the liposome, the lipid membrane comprising at least one liposome-forming phospholipid and a sterol; and the aqueous compartment within the liposome encapsulating at least one angiotensin II type 1 receptor blocker (ARB) and a pH-dependent ionizable anion;

[0026] wherein

[0027] the weight ratio between the at least one liposome-forming phospholipid and the sterol is between 3:1 and 2:1;

[0028] the liposome has an ARB to phospholipid molar ratio in the range of 0.02 to 1.0 (the ratio also taking into account a lipid polymer if the lipid membrane includes a lipid polymer); and

[0029] the liposome has the effect for systemic administration to a subject in need of the effect and does not cause a reduction in the mean blood pressure of the subject greater than 50% compared to systemic administration of the ARB in free form in the same amount.

[0030] The disclosed injectable liposomes have shown to meet several prerequisites for a clinically viable formulation based on liposomes for systemic delivery. One is regarding sufficient drug loading levels; the second is maintaining the ARB within the liposome while circulating in the blood; the third is releasing the drug at a rate and level sufficient to cause a desired therapeutic effect at the target site; and the fourth is achieving a pharmaceutically acceptable product in terms of stability during the shelf life.

[0031] Also disclosed herein, according to a second aspect herein referred to as the "inhalable liposomes aspect", is a liposome comprising a lipid membrane and an aqueous compartment within the liposome, the lipid membrane comprising at least one liposome-forming phospholipid and a sterol; and the aqueous compartment within the liposome encapsulating at least one angiotensin II type 1 receptor blocker (ARB); wherein the liposome has an average size between 50 nanometers and 600 nanometers, and wherein the liposome has a local effect for inhalation administration to the respiratory tract of a subject and does not cause a reduction in the mean blood pressure of the subject greater than 50% compared to inhalation of the ARB in free form in the same amount.

[0032] Also disclosed herein are formulations comprising the liposomes, which are suitable for systemic administration when referred to the injectable liposomes or suitable for administration by inhalation when referred to the inhalable liposomes; and methods of treatment comprising administering to a subject in need of the treatment the liposomes disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To better understand the subject matter disclosed herein and to illustrate how it may be practiced in practice, a number of embodiments will now be described by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0034] Figure 1 is a graph showing the percentage of valsartan incorporated into liposomes after incubation for 10 minutes at different D / L molar ratios.

[0035] Figure 2 is a graph showing the incorporation kinetics of liposomes with 15% HPCD or without HPCD at a D / L of 0.2.

[0036] Figure 3 is a graph showing the loading efficiency of valsartan incorporated into liposomes with or without HPCD (15% and 25%) as a function of the D / L molar ratio; with single addition and batch addition of the drug.

[0037] Figure 4 is a graph showing the concentration of liposomal valsartan after incubation at 37 °C for 48 hours.

[0038] Figure 5 is a graph showing free valsartan applied to a Sepharose column (1 mg / ml and 0.5 mg / ml).

[0039] Figure 6 is a graph showing the elution of free and liposomal valsartan from a Sepharose column.

[0040] Figure 7 is a graph showing the percentage of liposomal valsartan after incubation at 37 °C for 24 hours in the presence of 50% serum.

[0041] Figure 8 is a graph showing the blood pressure of mice determined using a CODA monitoring device after administration of free valsartan or liposomal valsartan (25 mg / kg).

[0042] Figure 9 is a graph showing the concentration of candesartan incorporated over time of incubation.

[0043] Figure 10 is a graph showing the concentration of candesartan incorporated at different D / L ratios after a 15-minute incubation time.

[0044] Figure 11 is a graph showing the percentage of liposomal candesartan after incubation in saline at 37 °C for 24 hours.

[0045] Figure 12 It is a graph showing liposomal candesartan after incubation in 50% serum at 37 °C for 24 hours. Detailed implementation mode

[0046] This disclosure is based on the development of several formulations that include injectable liposomes encapsulating AT1 receptor blockers (ARBs). In some examples, the developed injectable liposomes are pegylated (PEGylated) nanoliposomes containing valsartan or candesartan. These liposomes, particularly the nanoliposomes containing valsartan, have been tested in vivo for their lack of effect on blood pressure to ensure that the formulations have the ability to concentrate in tumors and avoid any effect on systemic blood pressure.

[0047] The non-limiting examples provided herein demonstrate the high loading, long-term stability, and sustained release in serum of valsartan and candesartan.

[0048] When referring to valsartan, it is understood to mean the compound (2S)-3-methyl-2-[pentanoyl-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]amino]butanoic acid, having the structure of Formula I:

[0049]

[0050] When referring to candesartan, it is understood to mean the compound 2-ethoxy-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]benzimidazole-4-carboxylic acid, having the structure of Formula II:

[0051]

[0052] In addition, in the same example, the ARB can be the compound of formula III, 5-(1,1,2,2,2-pentafluoroethyl)-2-propyl-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]imidazole-4-carboxylic acid:

[0053]

[0054] Specifically, but not limited to, valsartan shows high loading efficiency when incorporated into PEGylated nanoliposomes, which present a trans-membrane calcium acetate gradient with and without 15 and 25% HPCD in the aqueous phase of the liposome solution. The valsartan formulation shows stable loading at 4°C for at least 15 months (stability studies are ongoing). When diluted with glucose and incubated at 37°C for 24 hours, the liposomes are also stable. Valsartan formulations containing 15 and 25% HPCD showed that 80 to 82% of the valsartan remained in the liposomes after incubation for 24 hours in the presence of 50% serum compared to the zero-time content (94 to 96%). However, the valsartan liposomes without HPCD retained only 48% of the valsartan as liposomes.

[0055] In addition, but not limited to, candesartan (although insoluble in aqueous solution media) shows high loading when incorporated into liposomes from a dispersion in phosphate buffer, and the liposomes exhibit a trans-membrane calcium acetate gradient with and without 15 and 25% HPCD. The candesartan concentration in the liposome dispersion reaches a maximum of ~3.4 mg / ml. Liposomes with or without HPCD in the aqueous phase of the liposome showed no release in the presence of 50% serum.

[0056] The non-limiting examples provided herein also show the effect of liposomal valsartan (in liposomes containing 15% HPCD) on the mean blood pressure (MBP) of mice compared to free valsartan. Free valsartan caused a decrease in MBP 2 hours after injection, while the valsartan liposomal formulation showed no effect on MBP, demonstrating an unexpected advantage of using liposomal ARBs.

[0057] Based on the present disclosure, and thus according to the broadest scope, there is provided a liposome comprising a lipid membrane comprising at least one liposome-forming phospholipid and a sterol; and an aqueous compartment within the liposome encapsulating at least one angiotensin II type 1 receptor blocker (ARB) and a pH-dependent ionizable anion.

[0058] In terms of the injectable liposome, there is provided a liposome comprising a lipid membrane and an aqueous compartment within the liposome, the lipid membrane comprising at least one liposome-forming phospholipid and a sterol; the aqueous compartment within the liposome encapsulating at least one angiotensin II type 1 receptor blocker (ARB) and a pH-dependent ionizable anion; wherein

[0059] the weight ratio between the liposome-forming lipid and the sterol is between 3:1 and 2:1;

[0060] the liposome has a molar ratio of ARB to phospholipid in the range of 0.02 to 1.0; and

[0061] the liposome has the effect for systemic administration to a subject in need of the effect, and compared with systemic administration of the ARB in free form at the same dose, does not cause the average blood pressure of the subject to decrease by more than 50%.

[0062] In addition, in terms of the inhalable liposome, there is provided a liposome comprising a lipid membrane and an aqueous compartment within the liposome, the lipid membrane comprising at least one liposome-forming phospholipid and a sterol; the aqueous compartment within the liposome encapsulating at least one angiotensin II type 1 receptor blocker (ARB); wherein the liposome has an average size between 50 nanometers and 600 nanometers, and wherein the liposome has the effect for inhalation administration to the respiratory tract of a subject, and compared with inhalation of the ARB in free form in the same amount, does not cause the average blood pressure of the subject to decrease by more than 50%.

[0063] Sometimes and according to some examples, the inhalable liposome has an average size between 100 nanometers and 400 nanometers, sometimes between 50 nanometers and 300 nanometers, sometimes between 50 nanometers and 200 nanometers, sometimes between 100 nanometers and 300 nanometers.

[0064] In some examples, the inhalable liposome has an average size falling within any range between 50 nanometers and 500 nanometers.

[0065] In some examples, the inhalable liposome has an average size of approximately 300 nanometers.

[0066] In the context of the present invention, the term "liposome forming phospholipids" mainly refers to glycerophospholipids or sphingomyelins that form vesicles in water, such as but not limited to liposomes, as further discussed hereinafter.

[0067] When referring to glycerophospholipids, it should be understood as lipids having a glycerol backbone, in which at least one, preferably two, hydroxyl groups of the head group are substituted by one or two acyl, alkyl or alkenyl chains, phosphate groups, or any combination of the above, and / or their derivatives, and may contain a chemically reactive group (such as amine, acid, ester, aldehyde or alcohol) at the head group, thereby providing a polar head group for the lipid. The sphingomyelins consist of a ceramide unit with a phosphocholine moiety attached to position 1, and are thus actually N-acyl sphingosine. The phosphocholine moiety in sphingomyelins contributes to the polar head group of the sphingomyelins.

[0068] In the liposome-forming lipids, the length of the acyl, alkyl or alkenyl chains is generally between 14 and about 24 carbon atoms, and has different degrees of saturation, i.e., fully, partially or non-hydrogenated naturally occurring lipids, semi-synthetic or fully synthetic lipids, and the saturation level may affect the rigidity of the liposomes formed therefrom (usually lipids with saturated chains are more rigid than lipids of the same chain length with unsaturated chains, especially lipids with cis double bonds).

[0069] In some examples, the liposomes contain a single type or a combination of liposome-forming lipids.

[0070] In some preferred examples, the lipid membrane consists of a single liposome-forming lipid.

[0071] In some examples, the liposome-forming lipid is a phospholipid. When the liposome-forming lipid is a phospholipid, its amount in the liposome can be determined as organic phosphorus by the modified Bartlett method [Shmeeda H, Even-Chen S, Honen R, Cohen R, Weintraub C, Barenholz Y. 2003. Enzymatic assays for quality control and pharmacokinetics of liposome formulations: comparison with nonenzymatic conventional methodologies. Methods Enzymol 367:272–92].

[0072] In some examples, the liposome-forming lipid is a choline phospholipid, such as diacylglycerol-phosphocholine (the acyl, alkyl or alkenyl chains are as defined above).

[0073] In some other examples, the liposome-forming lipid is di-lauroyl-sn-glycero-2-phosphocholine (DLPC). In some examples, the liposome-forming lipid is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some examples, the liposome-forming lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some examples, the liposome-forming lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some examples, the liposome-forming lipid is 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some examples, the liposome-forming lipid is 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC). In some examples, the liposome-forming lipid is 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-ditricosanoyl-sn-glycero-3-phosphocholine.In some examples, the liposome-forming lipid is 1,2-di(tetracosanoyl)-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC). In some examples, the liposome-forming lipid is 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). In some examples, the liposome-forming lipid is 1,2-di(oleoyl)-sn-glycero-3-phosphocholine (DOPC) or dilauroyl-sn-glycero-2-phosphocholine (DLPC).

[0074] In some examples, the liposome-forming phospholipid is an ionizable lipid, such as those described by Buschmann, M.D. et al., [Buschmann, M.D. et al. Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines 2021, 9, 65. https: / / doi.org / 10.3390 / vaccines, the content of which is incorporated herein by reference] and has a pKa below pH 7. For example, the ionizable phospholipid can be any one of the following structures:

[0075]

[0076]

[0077] In some examples, the liposome-forming phospholipid comprises at least hydrogenated soy phosphatidylcholine (HSPC).

[0078] In a preferred embodiment, particularly with respect to the injectable liposomes described, the liposome-forming lipid consists of hydrogenated soy phosphatidylcholine (HSPC) and optionally a lipid polymer, as further detailed below.

[0079] In some other preferred embodiments, particularly with respect to the inhalable liposomes, the liposome-forming lipid consists of DPPC.

[0080] In some examples, the liposome comprises a sterol, such as and sometimes preferably cholesterol.

[0081] In some examples, the liposome comprises a lipid polymer. The lipid polymer comprises a lipid modified at its head group with a polymer moiety (PEG) having a molecular weight equal to or higher than 750 Da. The head group can be polar or non-polar, to which a large (>750 Da) flexible hydrophilic polymer is attached. The attachment of the hydrophilic polymer head group to the lipid region can be a covalent or non-covalent attachment, however, preferably through the formation of a covalent bond (optionally through a linker).

[0082] Although the lipid modified to form the lipid polymer can be neutral, negatively charged, and positively charged, i.e., there is no limitation to a specific (or no) charge. For example, neutral distearoyl glycerol and negatively charged distearoyl phosphatidylethanolamine, both covalently attached to methoxypoly(ethylene glycol) (mPEG or PEG) of Mw 750, 2000, 5000, or 12000 [Priev A, et al. Langmuir 18, 612-617 (2002); Garbuzenko O., Chem Phys Lipids 135, 117-129 (2005); M.C. Woodle and D.D. Lasic Biochim.. Biohys. Acta, 113, 171-199. 1992].

[0083] The most commonly used and commercially available lipids for derivatizing lipid polymers are lipids based on phosphatidylethanolamine (PE), typically distearoylphosphatidylethanolamine (DSPE). A specific family of lipid polymers used in the present invention includes methoxy PEG-DSPE (with PEG chains of different lengths), wherein the PEG polymer is linked to the primary amino group of DSPE via a carbamate bond. The molecular weight of the head group of the PEG moiety is preferably from about 750 Da to about 20,000 Da. More preferably, the molecular weight is from about 750 Da to about 12,000 Da, and most preferably from about 1,000 Da to about 5,000 Da. A specific PEG-DSPE used herein is one in which PEG has a molecular weight of 2,000 Da, referred to herein as 2000 PEG-DSPE or 2k PEG-DSPE (M.C. Woodle and D.D. Lasic Biochim.Biohys.Acta, 113, 171-199.1992).

[0084] Regarding the injectable liposomes, in one specific embodiment within the context of the present disclosure, attention is paid to the liposomes comprising at least hydrogenated soy phosphatidylcholine (HSPC), a lipid polymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]( 2k PEG-DSPE) and cholesterol.

[0085] In some embodiments, particularly when referring to the injectable liposomes, the liposome membrane comprises between 0.5 mole% and 10 mole% of the lipid polymer. Sometimes, the liposome membrane comprises at least 0.5 mole% of the lipid polymer; sometimes, at least 1 mole%; sometimes, at least 2 mole%; sometimes, at least 3 mole%; sometimes, at least 4 mole%; sometimes, at least 5 mole%; sometimes, at least 6 mole%; sometimes, at least 7 mole%; sometimes, at least 8 mole%. Sometimes, the liposome membrane comprises at most 8 mole% of the lipid polymer, sometimes, at most 7 mole%; at most 6 mole%; at most 5 mole%; at most 4 mole%; at most 3 mole%; at most 2 mole%.

[0086] In some examples, particularly when referring to the injectable liposome aspect, the lipid membrane comprises hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and mPEG-DSPE. When using this combination of components, a particular molar ratio comprises a molar ratio of the hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and mPEG-DSPE of HSPC:cholesterol:mPEG-DSPE being approximately 55:40:5.

[0087] In the context of the present disclosure, when referring to a "pH-dependent ionizable anion", it is to be understood as any anion derived from a salt that is charged under suitable pH conditions. Thus, it can be understood that the anion may actually be in a non-ionized form within the liposome, and thus when in an ionized form, it is retained within the liposome, while when in a non-ionized form, it will pass through the lipid membrane and leak out from the lipid inner core of the liposome. This will depend on the internal pH, i.e., the pH in the aqueous compartment within the liposome. The salt is a salt having a high solubility (at least 250 mM), carrying an anion having a pKa higher than 3.5 and a logD at pH 7 in the range of about -2.5 and about 1.5, preferably in the range of about -1.5 and about 1.0. In some examples, the pH-dependent ionizable anion is selected from the group consisting of acetate, benzoate, and formate. In some examples, the anion is an organic anion, such as choline. In one example, the anion is acetate.

[0088] The cation in the salt serves as a counterion for the ARB carried in the liposome. As a weak amphoteric acid, suitable counter cations can be organic cations and inorganic cations. In some examples, the counter cation is selected from the group consisting of calcium, magnesium, and sodium. In some examples, the cation counteracts the pH-dependent ionizable anion, preferably having a very low permeability coefficient, preferably <10 -11 for acetate (which is typically the driving force for remote loading of ARB into the liposome).

[0089] In some other examples, the counterion comprises a cationic polymer. Non-limiting examples of cationic polymers include dextran spermine, dextran spermidine, aminoethyl dextran, trimethyl ammonium dextran, diethylaminoethyl dextran, polyethyleneimine dextran, and the like.

[0090] In some specific examples, the counterion is calcium. In some examples, the calcium ion is derived from any one of calcium formate, calcium acetate, and calcium benzoate.

[0091] In some other examples, the counterion is sodium, such as one derived from sodium acetate, sodium formate, and sodium benzoate.

[0092] In some embodiments, the liposome contains calcium acetate or sodium acetate, preferably calcium acetate.

[0093] In some examples, the molar ratio between the ion and the lipid ranges from about 0.1 to about 0.5, sometimes from about 0.2 to 0.4, and further sometimes the molar ratio is about 0.3 ± 0.05.

[0094] For the ARB itself, such as valsartan or candesartan, its entrapment amount in the liposome is particularly important because it is one of the prerequisites for a clinically acceptable liposomal formulation. To evaluate ARB entrapment, the ratio of ARB to lipid is determined and compared with an initial ratio (before encapsulation). For this purpose, the liposomes loaded with ARB are usually purified after loading ARB to remove the unencapsulated ARB. Then, the amount of ARB and the amount of lipid in the liposome are determined by conventional methods. Based on the determined amounts of ARB and lipid, various parameters are determinable and important for characterizing the liposome: "ARB loading", which is the number of grams or moles of ARB per gram or mole of lipid; and "entrapment efficiency", expressed as a percentage of ARB encapsulation, as a function of the initial preloading ratio; and "ARB to lipid molar ratio", which is the number of moles of ARB per mole of lipid after removing the unencapsulated ARB.

[0095] The amount of ARB in the liposomes can be determined using various chromatographic techniques. In some examples, the concentration of the ARB compound is determined using a high-performance liquid chromatography (HPLC) / UV method. To calculate the in-liposome concentration of the ARB, the aqueous in-liposome capture volume (as described above) that can be calculated from the in-liposome calcium concentration is also required. The concentration of ARB-liposomes in the formulation is determined by the HPLC method. Dividing this concentration by the in-liposome capture volume will yield the in-liposome ARB concentration.

[0096] In some examples, the ARB is carried in the range of 2 to 10 milligrams per milliliter (mg / ml) of the liposome dispersion. In some examples, the ARB is carried at least 2 mg / ml; sometimes at least 3 mg / ml, sometimes at least 4 mg / ml, sometimes at least 5 mg / ml, sometimes at least 6 mg / ml, sometimes at least 7 mg / ml, sometimes at least 8 mg / ml. In some examples, the ARB is carried at most 10 mg / ml, sometimes at most 9 mg / ml, sometimes at most 8 mg / ml, sometimes at most 7 mg / ml, sometimes at most 6 mg / ml.

[0097] In some more specific examples, the ARB is carried in the range of 2 to 5 mg / ml of the liposome dispersion.

[0098] In some examples, the ARB-to-phospholipid molar ratio is determined. In this regard, it is noted that when the lipid membrane contains a lipid polymer, the ARB-to-phospholipid ratio also takes into account the lipid polymer and thus the ARB-to-phospholipid ratio includes two lipids, the lipid polymer, and at least one other PC.

[0099] In some examples, the ARB / phospholipid molar ratio ranges from 0.02 to 1.0; sometimes at least 0.03, sometimes at least 0.04, sometimes at least 0.05, at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.15, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, or at least 0.55, or at least 0.6, or at least 0.65, or at least 0.7, or at least 0.75, or at least 0.8, or at least 0.85, or at least 0.9, or at least 0.95, or at least 1.0. In some examples, the molar ratio is at most 1.0, or at most 0.9, or at most 0.8, or at most 0.7 or at most 0.6, or at most 0.5, or at most 0.4, or at most 0.3.

[0100] In some examples, the ARB-to-phospholipid molar ratio ranges from 0.1 to 0.5.

[0101] In some examples, the ARB to phospholipid molar ratio is between 0.2 and 0.4.

[0102] In some examples, the liposomes, particularly those in the context of injectable liposomes, contain at least one cyclodextrin (CD) compound in the aqueous compartment within the liposome.

[0103] CD compounds are considered to be cyclic oligosaccharides composed of (α-1,4)-linked α-D-glucopyranose units, and contain a lipophilic central cavity and a hydrophilic outer surface. In the context of the present disclosure, the CD can be a naturally occurring CD, as well as derivatives of the naturally occurring CD. Naturally occurring CDs include α-, β- or γ-cyclodextrin (αCD, βCD or γCD) composed of six, seven and eight glucopyranose units respectively. When referring to derivatives of the naturally occurring CD, it should be understood to be any cyclic oligosaccharide composed of (α-1,4)-linked α-D-glucopyranose units, which has a lipophilic central cavity and a hydrophilic outer surface.

[0104] In some examples, the CD is 2-hydroxypropyl-β-cyclodextrin (HPβCD).

[0105] In some examples, the CD is 2-hydroxypropyl-γ-cyclodextrin (HPγCD).

[0106] In some examples, the CD is sulfobutyl ether (SBE) cyclodextrin.

[0107] In a preferred example, the CD is HPβCD.

[0108] The liposomes disclosed herein contain an amount of CD sufficient to stabilize the ARB within the liposome, even in the presence of serum. Without being bound by theory, it is believed that HPCD interacts with the ARB compound in a manner that affects the leakage of ARB from the liposome, possibly through complexation.

[0109] In some examples, the molar ratio of the CD (preferably HPCD) to phospholipid is between 0.05 and 0.5. In some examples, the molar ratio of the CD to phospholipid is between 0.075 and 0.4, or between 0.1 and 0.3. The molar ratio of the CD to phospholipid can be derived from the following assumptions: in 5% liposome volume, the HPCD concentrations in the formulations are 7.5 mg / ml and 12.5 mg / ml respectively, for formulations containing 15% and 25% HPCD (when only liposomal HPCD remains after dialysis).

[0110] In some examples, the molar ratio of ARB to CD is determined and the liposomal formulation is defined. In some examples, the molar ratio of ARB to CD is between 0.5 and 2.0, sometimes between 0.6 and 1.9, and sometimes between 0.7 and 1.5. Similar to the above, the molar ratio of ARB to CD can be derived from the following assumptions: in 5% liposome volume, the 15% and 25% formulations contain HPCD concentrations of 7.5 mg / ml and 12.5 mg / ml respectively.

[0111] When referring to the inhalable liposomes, and according to some examples, the lipid membrane comprises or consists of: dipalmitoylphosphatidylcholine (DPPC) and cholesterol with a DPPC:cholesterol molar ratio ranging from 100 / 0 to 55 / 45.

[0112] A preferred example relates to liposomes as described in ALIS (Arikayc), which consists of dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a weight ratio of 2:1 and a molar ratio of 1:1.

[0113] The liposomes can be of any form or size.

[0114] In some examples, the liposomes are multilamellar or oligolamellar vesicles.

[0115] In some examples, the liposomes are multivesicular vesicles.

[0116] In some other examples, the liposomes are unilamellar vesicles.

[0117] The liposomes can be small, medium, large, or even giant. When referring to small liposomes, it is understood to have an average size in the range of about 20 nanometers to 100 nanometers; when referring to medium-sized liposomes, it is understood to have an average size in the range between about 100 nanometers and 200 nanometers; when referring to large liposomes, it is understood to have an average size of more than about 200 nanometers; when referring to giant liposomes (usually large unilamellar or multilamellar vesicles), it is understood to refer to those liposomes larger than 1 micrometer (μm).

[0118] In some examples, especially when referring to the injectable liposomes, the liposomes are small unilamellar vesicles (SUVs). In some examples, the injectable SUVs have a size distribution between 20 nanometers and 100 nanometers; sometimes between 20 nanometers and 100 nanometers, sometimes between 40 nanometers and 100 nanometers or 50 to 100 nanometers.

[0119] In some examples, the injectable liposomes have an average size between 60 and 90 nanometers; sometimes between 70 nanometers and 80 nanometers; sometimes about 77 ± 5.0 nanometers.

[0120] In some other examples, especially when referring to the inhalable liposomes, the liposomes can have an average size below 600 nm. In some examples, the inhalable liposomes are unilamellar. Thus, the inhalable liposomes can have a size below 100 nm and thus be SUVs; or can have a size above 100 nm and thus be LUVs. In some examples, the inhalable liposomes have an average size between about 50 nm and 600 nm, sometimes about 300 ± 20 nm.

[0121] The liposomes are stable. In fact, it has been found that when in a physiologically acceptable medium, under storage conditions at 4°C and in serum, the liposomes encapsulating ARB are significantly stable.

[0122] When referring to stability in the context disclosed herein, it is understood that after storage (at 4°C) for at least one month, compared to the initially loaded ARB, no more than 20%, sometimes no more than 10% of the ARB compound is released into the storage medium. In some examples, the stability of the liposomes is characterized in that after storage at 4°C for at least 3 months, no more than 10% of the ARB is released into the surrounding medium during storage. In some examples, the stability of the liposomes is characterized in that after storage at 4°C for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 12 months, no more than 10% of the ARB is released into the surrounding medium.

[0123] The stability is determined by one or both of the chemical and physical stabilities under storage conditions (4 °C, in buffer).

[0124] In this case, among other things, the chemical stability can be examined by one or more of the following parameters:

[0125] a) Measure the pH value of the dispersion (pH meter);

[0126] b) Phospholipid (PL) acyl ester hydrolysis by measuring the change in non-esterified (free) fatty acids (NEFA) released during PL hydrolysis [Barenholz et.al. From Liposomes: a practical approach, 2 nd Edn., RRC New ed, IRL Press Oxford, 1997], or by thin layer chromatography (TLC) [Barenholz, Y. and Amsalem, S. In: Liposome Technology 2 nd Edn., G. Gregoriadis (Ed.) CRC Press, Boca Raton, 1993, vol.1, pp:527-616], or by HPLC method.

[0127] Among other things, the physical stability of the liposomes can be examined by one or more of the following parameters:

[0128] a) Determine the liposome size distribution by dynamic light scattering (DLS).

[0129] b) The level of free (unassociated / aggregated) components.

[0130] c) Zeta potential.

[0131] d) % Loading of the drug. Drug loading %.

[0132] The liposomes disclosed herein are stable by at least one or more stability parameters.

[0133] The liposomes can be prepared according to the remote loading technique. The preparation of the injectable liposomes can use the calcium acetate (CA) gradient method [Clerc S, Barenholz Y. 1995. Loading of amphipathic weak acids into liposomes in response to transmembrane calcium acetate gradients. Biochim Biophys Acta 1240:257–265].

[0134] For example, the required molar ratio of lipids, such as 55:40:5 of HSPC:cholesterol:mPEG DSPE, is mechanically hydrated by stirring at 65 °C in a 1:9 weight ratio with 200 mM calcium acetate pH 5.5. The size of the liposome dispersion is reduced by stepwise extrusion. Then, dialysis is performed against a 10% sucrose solution using a regenerated cellulose membrane. In the case of liposomes containing HPCD, the lipids are hydrated with 200 mM calcium acetate pH 5.5 containing the required % (w / w) of HPCD.

[0135] Then, remote loading is carried out by incubating the solution or dispersion of ARB at 65 °C for 3 minutes to 30 minutes, where the volume ratio of the liposome dispersion will yield the required ARB / phospholipid molar ratio, preferably the ARB / phospholipid molar ratio is 0.02 to 1.0 as described above.

[0136] In some examples, the ARB / phospholipid molar ratio is at most 1.0; sometimes, the ARB / phospholipid molar ratio is at most 0.09; sometimes, the ARB / phospholipid molar ratio is at most 0.08; sometimes, the ARB / phospholipid molar ratio is at most 0.07; sometimes, the ARB / phospholipid molar ratio is at least 0.06.

[0137] The ARB loading solution or dispersion is prepared in 200 mM phosphate buffer pH 6.3.

[0138] When referring to the aspect of the inhalable liposomes, it is understood to cover liposomes that are particularly used for local delivery of the ARB to the respiratory tract. In other words, the inhalable liposomes are suitable for local delivery. It has been envisaged that, compared to the inhalation of the same amount of free-form ARB, the inhalable liposomes suitable for local delivery provide their effect without causing the mean blood pressure of the subject to decrease by more than 50%, which is similar to the low or no effect of the injectable liposomes on MBP.

[0139] Therefore, the inhalable liposomes disclosed herein are particularly suitable for treating conditions along the respiratory tract, such as infections.

[0140] In some examples, the inhalable liposomes disclosed herein are suitable for treating viral infections, such as infections caused by coronaviruses. One interesting condition is acute respiratory distress syndrome (ARDS).

[0141] In the case of ARDS, a major inflammation occurs, leading to a process called extravasation through leaky vasculature in the lung (ELVIS), and thus the infected lung should receive high doses of liposomes. The IC50 values of valsartan and candesartan for the AT1 receptor are 60 and 3 nM, corresponding to 30 and 1.3 ng / ml, respectively. Assuming a tidal volume (the volume that enters and leaves the lungs with each breath, from a normal quiet inhalation to a normal quiet exhalation) of 0.5 L, 15 micrograms (μg) and 0.65 micrograms should be administered. These amounts can be achieved through the multiple liposome concentrations obtained based on the inhalable liposomes.

[0142] In the case of coronary treatment, two liposome formulations (injectable liposomes and inhalable liposomes) allow access to the lungs from the inside (blood) and the outside (inhalation). Antiviral treatment using the inhalable liposomes will be further discussed below.

[0143] The preparation of these liposomes for inhalation can be found in Shirley, M., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs, 2019. 79(5): p. 555 - 562, which is incorporated herein by reference.

[0144] The present invention also provides a method of using a formulation for a treatment, the formulation comprising the liposomes encapsulating at least one ARB compound as described herein and a physiologically acceptable carrier.

[0145] In the context of the present invention, a physiologically acceptable carrier refers to any carrier that can be used to prepare a pharmaceutical formulation, which is generally safe, non - toxic and not otherwise undesirable biologically.

[0146] In some examples, the formulation comprises a physiologically acceptable carrier suitable for administration by injection or infusion. This is particularly relevant in relation to the injectable liposomes disclosed herein. In some examples, the administration is by any of intravenous (i.v.), intramuscular (i.m.), intraperitoneal (i.p.) and subcutaneous (s.c.) injection.

[0147] In some other examples, the formulation comprises a physiologically acceptable carrier suitable for administration by inhalation. To this end, the liposomes may be in suspension or may be pre-lyophilized into a dry powder.

[0148] The formulation can be used to treat any condition in which delivery of at least one ARB compound provides a therapeutic benefit.

[0149] As appreciated, ARBs are highly selective for the AT1 receptor and can block the deleterious effects of Ang II, such as vasoconstriction, aldosterone release, sodium and water retention, sympathetic activation, and cell proliferation.

[0150] In addition to their known and clinically used antihypertensive drug activity, ARBs have also been shown to improve cancer treatment in several prospective and retrospective studies. At the level of the tumor microenvironment, ARBs were found to affect cancer-associated fibroblasts (CAFs). CAFs can either inhibit or enable anti-tumor immunity, suggesting that they can be reprogrammed between these states. ARBs can reprogram CAFs into a quiescent state. Additionally, ARBs may reduce immunosuppression and enhance the efficacy of cancer immunotherapy.

[0151] Furthermore, ARBs may have an effect on the angiotensin-converting enzyme 2 (ACE2) receptor. ACE2 has recently received extensive attention because it is the binding site for SARS-CoV-2, the strain associated with the current COVID-19 pandemic and its activity. Specifically, it has been demonstrated that the binding of the coronavirus spike protein to its cellular binding site ACE2 results in the downregulation of ACE2, which in turn leads to the overproduction of the related enzyme ACE of angiotensin, while less ACE2 is able to convert it into the vasodilator heptapeptide angiotensin. This, in turn, can cause lung injury because the binding of angiotensin II to the AT receptor leads to an increase in pulmonary vascular permeability, thereby mediating an increase in lung pathology.

[0152] Therefore, when liposomes encapsulating ARBs are used to combat viral infections, two complementary mechanisms occur: blocking the over-angiotensin-mediated activation of AT receptors caused by viral infection, and upregulating ACE2, thereby reducing the angiotensin produced by ACE and increasing the production of the vasodilator angiotensin. Thus, the administration of ARBs is a method for treating COVID-19 infections.

[0153] In view of the above, and according to some examples, the liposomes of the formulation disclosed herein comprise their use in treating cancer, i.e., as an anti-cancer treatment. The anti-cancer treatment is particularly relevant to the injectable liposome aspect of the present disclosure.

[0154] According to some other examples, the liposomes of the formulations disclosed herein include their use for treating viral infections, i.e., as an antiviral treatment. The antiviral treatment is particularly relevant in the context of the inhalable liposomes disclosed herein.

[0155] The present disclosure also provides a method of treatment, the method comprising administering to a subject in need of an ARB liposomes encapsulating at least one ARB, the liposomes as defined herein, and the amount of the at least one ARB being effective to achieve the desired treatment.

[0156] The amount of the at least one ARB is designed to be sufficient to provide a therapeutic effect when administering (systemically or locally) the at least one ARB to a subject, but does not exhibit a significant effect on the average blood pressure of the treated subject.

[0157] An amount sufficient or effective to achieve a desired therapeutic effect upon administration should be understood to include at least one therapeutic effect known to be achieved by or associated with an ARB, rather than a potential effect on blood pressure.

[0158] In the context of the present disclosure, when referring to effects other than the effect on blood pressure, it should be understood that the liposomes disclosed herein are either those that can be administered by injection or those that can be administered by inhalation; exhibiting their primary effect on a medical condition rather than an effect related to reducing blood pressure.

[0159] In some examples, compared to the effect of the same dose of the free form of the ARB in the same mode of administration (e.g., injection, inhalation), if any, the effect on blood pressure is less than 50%. Sometimes, compared to the effect of the same amount of the free form of the ARB in the same mode of administration (e.g., injection, inhalation), the effect is less than 40%. In addition, sometimes, compared to the effect of the same amount of the free form of the ARB in the same mode of administration (e.g., injection, inhalation), the effect is less than 30%. However, sometimes, compared to the effect of the same amount of the free form of the ARB in the same mode of administration (e.g., injection, inhalation), the effect is less than 20%. In addition, sometimes, compared to the effect of the same amount of the free form of the ARB in the same mode of administration (e.g., injection, inhalation), the effect is less than 10%.

[0160] In other words, the effect of the liposomes when administered to a subject in need of the effect of an ARB to treat a condition, whether by injection or by inhalation, compared to the same amount of the free form of the ARB, provided to the subject by the same mode of administration, does not cause the average blood pressure of the subject to decrease by more than 50%; compared to the same amount of the free form of the ARB delivered by the same mode of administration, sometimes not more than 40%, sometimes not more than 30%, and even not more than 20%.

[0161] Thus, for example, when referring to injecting the liposomes, for example for treating cancer, the effect of the liposomes on cancer cells is demonstrated, while the effect on the blood pressure of the subject is less than 50%, sometimes less than 40%, less than 30%, less than 20% or even less than 10%, compared to the effect of treating with the same drug without the liposomes. The non-limiting examples presented below support the above view, as they show that the liposomal ARB has no effect on MBP compared to the free drug which decreased from 105 to 0 mmHg.

[0162] In some examples, the effect on blood pressure, if any, is considered to be statistically non-significant (medically non-significant).

[0163] The amount of ARB delivered by the pharmaceutical formulation depends on various parameters known to those skilled in the art and can be determined based on appropriately designed clinical trials (dose range studies), and those skilled in the art will know how to appropriately conduct such trials to determine the effective amount. The amount depends in particular on the type and severity of the disease to be treated and the treatment regimen (mode of systemic administration), the sex and / or age and / or weight of the subject to be treated, etc.

[0164] In view of the above, in the context of the present disclosure, when referring to treatment with the liposomes disclosed herein, it should be understood to cover improving the unwanted symptoms associated with a disease, preventing their manifestation before they occur, slowing the progression of the disease, slowing the worsening of the symptoms, enhancing the onset of a remission of a disease, slowing the irreversible damage caused in the progressive chronic phase of a disease, delaying the onset of the progressive phase, reducing the severity of a disease or curing the disease, increasing the survival rate or recovering more rapidly from the disease, preventing the occurrence of the disease, or a combination of two or more of the above.

[0165] The present invention will now be described by way of non-limiting examples.

[0166] Description of non-limiting examples

[0167] Example 1 - Preparation of Liposomal Formulation

[0168] Materials and methods

[0169] Materials:

[0170] The materials used to prepare the formulation are shown in Table 1.

[0171] Table 1: Materials used to prepare the formulation

[0172]

[0173]

[0174] Method:

[0175] Preparation of calcium acetate liposomes:

[0176] Prepare nanoliposomes (hereinafter referred to as "calcium acetate liposomes") by mechanically hydrating LipidMix with a weight ratio of HSPC: cholesterol: mPEG DSPE of 3:1:1 and 200 mM calcium acetate at pH 5.5 at 65 °C. When the liposomes contain HPCD, the hydration solution additionally contains 15% (w / w) or 25% (w / w) HPCD. Gradually extrude through a polycarbonate membrane using a Northern Lipids extruder (Burnaby) to reduce the size of the liposome dispersion, and dialyze with 10% sucrose solution.

[0177] Liposome size:

[0178] Determine the particle size using dynamic light scattering method, performed using a Zetasizer Nano Series ZEN3600F (Malvern Instruments, Malvern, UK). The size of the nanoliposomes is in the range of 73 to 83 nm, and PDI < 0.05.

[0179] Valsartan analysis method:

[0180] The valsartan analysis method (HPLC) is implemented based on the USP method.

[0181] The chromatographic conditions are described as follows:

[0182] Mobile phase - Acetonitrile: DDW: Glacial acetic acid volume ratio is 50:50:0.1

[0183] Column - Phenomemex C18, 150 x 4.6 mm

[0184] Detector - UV 230 nm, 25 nm

[0185] Flow rate - 1 ml / min

[0186] Injection volume - 20 μl

[0187] Column temperature - 30 °C

[0188] Candesartan analytical method:

[0189] The candesartan analysis method (HPLC) is implemented based on the USP method.

[0190] The chromatographic conditions are described as follows:

[0191] Mobile phase - Acetonitrile:DDW:Trifluoroacetic acid volume ratio is 550:450:1

[0192] Column - Phenomemex C8, 150x 4.6mm

[0193] Detector - UV 254nm, 282nm

[0194] Flow rate - 1ml / min

[0195] Injection volume - 20μl

[0196] Column temperature - 30℃

[0197] Results:

[0198] Valsartan:

[0199] Valsartan loading:

[0200] The chemical structure of valsartan is shown in Formula I below.

[0201]

[0202] Valsartan has a carboxyl group that ionizes at a relevant pH value (3.2 - 8.8), and within this pH range, it is in equilibrium with the non-ionized species. Therefore, valsartan is loaded into calcium acetate liposomes (HSPC: Cholesterol: 2000 MPEG-DSPE, 3:1:1). Since these liposomes need to be highly stable in circulation, liposomes containing 15% HPCD or 25% HPCD in their aqueous phase within the liposome are also used to perform loading tests on liposomes that exhibit a transmembrane gradient of calcium acetate. HPCD can prevent the rapid release of the drug in serum and allow for slow and controlled drug release [J.D. Martin, H. Cabral, T. Stylianopoulos, R.K. Jain, Improving cancer immunotherapy using nanomedicines: progress, opportunities and challenges, Nat. Rev. Clin. Oncol. 17 (2020) 251–266].

[0203] The loading of valsartan is carried out by dissolving the drug in 200 mM phosphate buffer at pH 6.3 and adding it to the liposome dispersion at 65℃. The loading efficiency is tested using a Dowex anion exchanger, which has previously been shown to effectively absorb free valsartan but not liposome-bound drug.

[0204] Table 2A provides the concentration of the liposomal valsartan (milligrams per milliliter) (mg / ml), and Table 2B provides the D / L molar ratio of the liposomal valsartan obtained under three different loading conditions:

[0205] Condition A - Add all the drugs at once and then incubate for 10 minutes;

[0206] Condition B - Add all the drugs at once and then incubate for 3 minutes;

[0207] Condition C - Add the drugs in batches.

[0208] All liposomes are calcium acetate liposomes, with or without (w / o) HPCD.

[0209] Table 2A: Concentration of liposomal valsartan (mg / ml) obtained under different conditions

[0210]

[0211] Table 2B: D / L molar ratio of liposomal valsartan with or without (w / o) HPCD

[0212]

[0213] Figure 1 Shows the percentage of loading of valsartan into these liposomes after incubation for 10 minutes at different D / L molar ratios.

[0214] For a D / L of 0.1, a high loading of ~100% was obtained. The loading efficiency decreased with increasing D / L ratio, and was more pronounced for liposomes showing a transmembrane calcium acetate gradient and without HPCD inside the liposomes.

[0215] Figure 2 Represents the kinetics of loading into liposomes with 15% HPCD or without HPCD at a D / L of 0.2. For calcium acetate liposomes, the loading was highest (81%) when the incubation was terminated after 2 minutes and decreased to 8% over a 30-minute incubation period. For liposomes containing 15% HPCD, the loading was found to be stable within the first 20 minutes, ranging from 80 to 89%. A decrease was observed after 30 minutes of incubation, resulting in a loading of 74%.

[0216] To strive to improve the loading efficiency, the loading into the liposomes while adding the drug solution in batches was tested. In this experiment, incubation was carried out with valsartan at a D / L ratio of 0.1 for 3 minutes. Incubation with a D / L of 0.2 was carried out by adding an additional drug solution after 3 minutes, followed by incubation for another 2 minutes. For D / L of 0.3 and 0.4, as described for D / L of 0.2, more drug solution was added to newly add the drug in the added part and incubated for 2 minutes (the total incubation time for D / L of 0.4 was 9 minutes). As Figure 3 shown, the loading was substantially increased by adding the drug in batches compared to the loading obtained after incubating with all the drug at once for 10 minutes.

[0217] Liposomes containing 25% HPCD in the aqueous phase within the liposomes also loaded valsartan. Figure 3 The double black lines in

[0218] show these results. Loading was carried out after incubating for 3 minutes in a part. The highest loading efficiency was obtained compared to all other test conditions.

[0219] Valsartan release:

[0220] When incubating at 37 °C, the release of valsartan liposomes was tested after first diluting in glucose. The test was carried out using valsartan-loaded liposomes with and without HPCD. The liposomes used were those that loaded valsartan at a D / L molar ratio of 0.1.

[0221] The liposomes were diluted 10-fold in glucose and placed in an incubator at 37 °C. After 1, 4, 24, and 48 hours, samples were taken from the incubation and the liposome fraction was separated using a Dowex ion exchanger. As Figure 4 described, no release from the liposomes was obtained during the 48-hour incubation.

[0222] Release in 50% serum:

[0223] Separation of the free and liposome fractions of the drug in the presence of serum requires separation by size exclusion chromatography (SEC). For this, Sepharose CL4B was used. The separation method needs to be applicable to each of the mixtures of free drug and liposome drug. The elution curve of free valsartan at a concentration of 0.5 mg / ml was tested through the column and it was eluted only in the late part, thus allowing the separation of free valsartan from liposome valsartan eluted in the early part.

[0224] The elution curves of free valsartan described in Figure 6 were also examined. Having a method applicable to separating liposomes and free valsartan allows for the release test of valsartan from liposomes in the presence of serum.Figure 7 These results are shown.

[0225] It was found that valsartan was slowly released from calcium acetate liposomes, and after 24 hours, only 45% of the drug remained in the liposomes. The release of liposomes exhibiting a transmembrane calcium acetate gradient and HPCD was much slower, with 87% of the drug remaining in the liposomes after 24 hours. This value was similar to the % liposomes found at t = 0 (84%) and lower than the % liposomes found after 4 hours (96%). Repeating this assay and incubating for 24 hours showed similar results for 80 to 86% of liposomal valsartan. The release of valsartan in liposomes containing 25% HPCD (D / L 0.2) was similar to that of 15% HPCD, showing 82% liposomal valsartan after 24 hours of incubation.

[0226] Loading stability during storage:

[0227] The loaded valsartan content of valsartan liposomes with and without HPCD at different D / L molar ratios stored at 4°C for 5 months was detected and summarized in Table 3. In Table 3, these formulations were based on either calcium acetate alone within the liposomes or formulations containing 15% HPCD with an increased ARB / phospholipid (D / L) molar ratio in the initial incubation.

[0228] It was found that the loading was stable over time and even increased during storage, as expected for remotely loaded liposomes.

[0229] Table 3: Loading stability of valsartan liposome formulations stored at 4°C

[0230]

[0231]

[0232] *Internal reference

[0233] **During initial incubation

[0234] ND = Not determined

[0235] Activity of liposomal valsartan on mouse blood pressure

[0236] The development of liposomal ARBs was aimed at delivering the drug to tumors and exerting its activity there while avoiding the systemic effects of the drug on blood pressure. Therefore, in vivo studies tested the effect of free valsartan (25 mg / kg dose) on the mean blood pressure (MBP) of mice. MBP was measured using a CODA monitoring device that allowed blood pressure measurement on the mouse tail.

[0237] Four mice were tested before dosing and at 2, 24, and 48 hours after dosing. For each mouse, at least 3 measurements (up to 10) were recorded at each time point.

[0238] Figure 8 The results obtained are shown. At 2 hours after dosing, free valsartan caused a decrease in MBP of ~35 (approximately 35) units. MBP returned to baseline at t = 24 hours. During the time points tested, the same dose of liposomal valsartan (15% HPCD formulation) had no effect on MBP.

[0239] Candesartan:

[0240] Candesartan loading:

[0241] The chemical structure of candesartan is shown in formula II below.

[0242]

[0243] Candesartan has a carboxyl group that ionizes at relevant pH values (1.6 - 8.8), and within this pH range it is in equilibrium with the unionized species (similar to valsartan). Thus, candesartan was loaded into liposomes presenting a transmembrane calcium acetate gradient, which liposomes have or lack HPCD in their aqueous phase within the liposome, as previously described for valsartan.

[0244] As previously mentioned, the solubility of candesartan is very limited (much lower than that of valsartan), and it has the highest affinity for the AT1 receptor [Bhuiyan, M.A.; Shahriar, M.; Nagatomo, T. Binding Affinity of Candesartan, Losartan, Telmisartan and Valsartan with Angiotensin II Receptor 1 Subtype. Bangladesh Pharm J. 2013, 16, 10–14, doi:10.3329 / bpj.v16i1.14484].

[0245] Therefore, candesartan was dispersed in phosphate buffer at pH 6.3 at a concentration of 10 mg / ml, and this dispersion was used for loading. Since these liposomes need to be highly stable in circulation, liposomes presenting a transmembrane calcium acetate with 15% and 25% HPCD also in their aqueous phase within the liposome were also tested for loading, as it was previously found that this increases the stability of nanoliposomes in serum.

[0246] Candesartan was incorporated into liposomes at 65 °C and tested over an incubation time of 5 to 60 minutes. Loading was carried out from a dispersion at a 1:1 D / L molar ratio of 0.4. After loading, the obtained liposomes were centrifuged and the total drug concentration after centrifugation was measured in the supernatant phase. For liposomes lacking HPCD and liposomes including HPCD in the aqueous phase within their lipid bodies, the D / L molar ratios after centrifugation (excluding the precipitate) were 0.24 and 0.28, respectively.

[0247] Figure 9 The loading concentration during the incubation time is shown

[0248] Figure 10 The loading concentration within the initial D / L molar ratio (0.2 - 0.4) tested is presented. The loaded candesartan concentration was in the range of 2.6 to 3.7 mg / ml.

[0249] Candesartan release:

[0250] Release in saline:

[0251] The release of liposomes containing only calcium acetate, or 15% or 25% HPCD loaded with candesartan, was tested after dilution 20-fold with saline at 37 °C. The results obtained are described in Figure 11 It. The surprising result is that candesartan is released from the liposomes over time and the release increases with the increase in the HPCD content within the liposomes.

[0252] Release in 50% serum:

[0253] Candesartan formulations of liposomes showing transmembrane calcium acetate without HPCD within the liposomes and with 25% HPCD within the liposomes were diluted 15-fold with 50% serum. At t = 0 and t = 24 hours, the samples were loaded onto a Sepharose column to separate free and liposomal candesartan. The results are as Figure 12 shown.

[0254] Specifically, Figure 12 it is shown that in the presence of serum, after 24 hours of incubation, the content of liposomal candesartan did not decrease for both formulations.

[0255] These results are surprising because a significant release of candesartan was obtained in saline and was higher with the increase in the HPCD concentration within the liposomes. In most cases, in serum, compared to saline, faster drug release is induced, and the fact that no candesartan release was shown in serum is unexpected.

[0256] Example 2: In vivo studies - Valsartan and Candesartan

[0257] The efficacy of the disclosed formulation was tested in a 4T1 breast cancer model and compared with Doxil according to the following steps:

[0258] - The therapeutic efficacy of Doxil alone or in combination with the lead liposomal-ARB formulation

[0259] - Efficacy in the 4T1 breast cancer model compared to immune checkpoint inhibition (ICI) therapy: Determine the efficacy of ICI as such and its combination with the liposomal ARB formulation. The immune checkpoint cocktail therapy used was anti-PD-1 (BioXcell) and anti-CTLA-4 (BioXcell).

[0260] - Efficacy in the human adenocarcinoma (HT29) model compared to ICI therapy: Determine the efficacy of ICI as such and its combination with the liposomal ARB formulation. The immune checkpoint cocktail therapy used was anti-PD-1 (BioXcell) and anti-CTLA-4 (BioXcell).

[0261] - Pharmacokinetic studies, including biodistribution of the lead formulation in tumors and diseased mice compared to free drug. Determine drug concentrations in plasma and tumors.

[0262] Example 3: Inhalable liposomal valsartan and candesartan for the treatment of coronavirus

[0263] The inhaled liposomal formulation for the treatment of acute respiratory distress syndrome (ARDS), including coronavirus complications, is based on DPPC and cholesterol.

[0264] In the case of ARDS, a major inflammation occurs, leading to a process called extravasation via leaky vasculature (ELVIS), so the infected lungs should receive a high dose of liposomes. The IC50 of valsartan and candesartan for the AT1 receptor are 60 and 3 nM, corresponding to 30 and 1.3 ng / ml respectively. Assuming a tidal volume (the volume that enters and leaves with each breath, from a normal quiet inhalation to a normal quiet exhalation) of 0.5 L, 15 micrograms (μg) and 0.65 micrograms should be administered. These amounts can be achieved by depending on multiple liposome concentrations.

[0265] The nano-liposomes for inhalation are prepared by the same remote loading method as above, using a transmembrane calcium acetate gradient.

[0266] The lipid composition for the inhalation preparation comprises dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a weight ratio of 2:1 and a molar ratio of 1:1. Intraliposomal HPCD in the concentration range of 0 to 30 is used to achieve the desired control of the rate of ARB release from the liposomes. The size of the inhaled liposomes is about 300 nanometers.

Claims

1. Liposome, characterized in that: The liposome comprises a lipid membrane, the lipid membrane comprising at least one liposome-forming phospholipid and a sterol; and an aqueous compartment within the liposome encapsulating at least one AT1 receptor blocker, i.e., ARB, and a pH-dependent ionizable anion; wherein the ARB is selected from a group consisting of Formula I and Formula II, and Formula I is (2S)-3-methyl-2-[pentanoyl-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]amino]butyric acid, i.e., valsartan: and Formula II is 2-ethoxy-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]benzimidazole-4-carboxylic acid, i.e., candesartan; the weight ratio between the at least one liposome-forming phospholipid and the sterol is between 3:1 and 2:1; the liposome has a molar ratio of ARB to phospholipid in the range of 0.02 to 1.0; and the ARB is selected to provide an effect by systemic administration of the liposome to a subject in need of the effect, without causing the mean blood pressure of the subject to decrease by more than 50% compared to systemic administration of the same amount of ARB in free form.

2. The liposome according to claim 1, characterized in that: The lipid membrane comprises a lipid polymer.

3. The liposome according to claim 1 or 2, characterized in that: The at least one liposome-forming lipid comprises hydrogenated soy-phosphatidylcholine, i.e., HSPC, in addition to a lipid polymer or consists of hydrogenated soy-phosphatidylcholine if the lipid polymer is present in the lipid membrane.

4. The liposome according to any one of claims 1 to 3, characterized in that: The sterol is cholesterol.

5. The liposome according to any one of claims 1 to 4, characterized in that: The aqueous compartment within the liposome encapsulates at least one cyclodextrin compound, i.e., CD compound.

6. The liposome according to claim 5, wherein: The at least one CD is 2-hydroxypropyl-β-cyclodextrin, i.e., HPβCD.

7. The liposome according to any one of claims 1 to 6, characterized in that: The pH-dependent ionizable anion is acetate.

8. The liposome according to claim 1, characterized in that: The aqueous compartment within the liposome of the liposome comprises the valsartan; acetate, as the pH-dependent ionizable anion; and HPCD; the molar ratio of the valsartan to the liposome-forming phospholipid and the lipid polymer, if present, is between 0.02 and 1.

0.

9. The liposome according to claim 1, wherein: The aqueous compartment within the liposome of the liposome comprises the valsartan; acetate, as the pH-dependent ionizable anion; and HPCD; the molar ratio of the valsartan to the HPCD is between 0.5 and 2.

0.

10. The liposome according to claim 1, characterized in that: The aqueous compartment within the liposome of the liposome comprises the candesartan and acetate, as the pH-dependent ionizable anion; the molar ratio of the candesartan to at least one liposome-forming phospholipid and the lipid polymer, if present, is between 0.02 and 1.

0.

11. The liposome according to claim 1, wherein: The aqueous compartment within the liposome of the liposome comprises the candesartan and acetate, as the pH-dependent ionizable anion; and HPCD; the molar ratio of the candesartan to the HPCD is between 0.5 and 2.

0.

12. The liposome according to any one of claims 1 to 11, characterized in that: The lipid membrane comprises a combination of the following: HSPC, cholesterol, and N-(carbonyloxy-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, namely 2000 PEG-DSPE.

13. The liposome according to claim 12, wherein: The lipid membrane contains HSPC: cholesterol: 2000 The molar ratio of PEG-DSPE is 55:40:

4.

14. The liposome according to any one of claims 1 to 13, characterized in that: The liposome is a small unilamellar vesicle.

15. A preparation used in a treatment method, characterized in that: The formulation comprises a liposome according to any one of claims 1 to 14 and a physiologically acceptable carrier.

16. The preparation used according to claim 15, characterized in that: The treatment is an anti-cancer treatment.

17. The preparation used according to claim 16, characterized in that: The method is for treating a disease in need of at least one ARB for treatment.

18. The preparation used according to claim 15 or 16, characterized in that: The preparation is a dosage form suitable for injection.

19. Liposome, characterized in that: The liposome comprises a lipid membrane, the lipid membrane comprising at least one liposome-forming phospholipid and a sterol; and an aqueous compartment within the liposome encapsulating at least one AT1 receptor blocker, namely ARB; the liposome having an average size between 50 nanometers and 600 nanometers, The ARB is selected from a group consisting of Formula I and Formula II, wherein Formula I is (2S)-3-methyl-2-[pentanoyl-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]amino]butyric acid, namely valsartan: and Formula II is 2-ethoxy-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]benzimidazole-4-carboxylic acid, namely candesartan; wherein the ARB is selected to provide an effect by inhalation administration of the liposome to a subject's respiratory tract, without causing the average blood pressure of the subject to decrease by more than 50% compared to inhalation of the same amount of the ARB in free form.

20. The liposome according to claim 19, wherein: The liposome is used in a method for treating a disorder along a subject's respiratory tract.

21. The liposome according to claim 19 or 20, characterized in that: The at least one liposome-forming lipid comprises dipalmitoylphosphatidylcholine, namely DPPC, or consists of dipalmitoylphosphatidylcholine.

22. The liposome according to any one of claims 19 to 21, characterized in that: The sterol is cholesterol.

23. The liposome according to any one of claims 19 to 22, characterized in that: The lipid membrane comprises or consists of DPPC and cholesterol in a molar ratio of 1:

1.

24. The liposome according to any one of claims 19 to 23, characterized in that: The liposome is in the form of a suspension.

25. The liposome according to any one of claims 19 to 24, characterized in that: The liposome is in dry form.

26. The liposome according to claim 25, wherein: The liposome is in the form of a lyophilized powder.

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