EP4199906A1

EP4199906A1 - Lipid delivery systems for delivery of oxaliplatin palmitate acetate

Info

Publication number: EP4199906A1
Application number: EP21762817.1A
Authority: EP
Inventors: Simon Benita; Taher Nassar; Amit BADIHI; Meital NAIM
Original assignee: Bionanosim BNS Ltd
Current assignee: Bionanosim BNS Ltd
Priority date: 2020-08-20
Filing date: 2021-08-18
Publication date: 2023-06-28
Also published as: WO2022038605A1; IL300676A

Abstract

The technology concerns lipid-based delivery systems comprising OPA and a lipid- based material.

Description

LIPID DELIVERY SYSTEMS FOR DELIVERY OF OXALIPLATIN PALMITATE ACETATE

TECHNOLOGICAL FIELD

The present disclosure concerns lipid delivery systems for delivery of oxaliplatin palmitate acetate.

BACKGROUND

Cancers become more widespread as world population is aging [1]. However, marked progress in cancer therapies has been achieved in the last decades. This is mostly due to the development of immunotherapies combined with chemotherapy, and more particularly with platinum (Pt)-based drugs [2,3], despite significant side effects due to nonspecific damage to normal cells [4]. In order to overcome at least one of the drawbacks associated with Pt-based therapeutics, new Pt-based drugs are explored. Following the clinical success of cisplatin, a Pt(II) drug approved by the FDA in 1979, development of Pt anticancer agents has attracted the attention of researchers. Cleare and Hoeschele [5] have shown that in order for Pt complexes to play an active role in cancer therapy, causing structural distortion of the double stranded DNA, thereby leading to apoptosis, they must be neutral square-planar Pt(II) complexes with two cis-oriented inert amine or chelating diamine ligands and two semi-labile cis-oriented ligands bound to the Pt oxygen donors. As reported by Gibson [6], despite many years of intensive research, all approved Pt drugs conform to such structure-activity relationship. Although very few Pt agents have been used successfully as drugs, the clinical significance of Pt compounds in cancer therapy is well recognized.

Currently, three Pt(II) anticancer drugs are clinically used worldwide in 50- 70% of cancer patients [7-9]; these are cisplatin, carboplatin (approved in 1989), and oxaliplatin (approved in 2002). Unfortunately, despite the resurgence in their use for cancer therapy since 2007 [10], therapeutic outcomes of Pt(II) drugs are seriously affected owing to severe side effects attributed to the reactivity of the Pt(II) compounds with biological nucleophiles prior to reaching the cancerous tissues, as well as inherent or acquired resistance [11]. Pt(IV) complexes with two additional axial groups may have advantages over the reactive Pt(II) species. They are inert in plasma, may reach cancerous lesions in their Pt(IV) form and be activated into their Pt(II) analogs only inside the cancerous cells. Unfortunately, clinical evaluation of several Platinum(IV) complexes showed rapid elimination, less or equal efficacy than Pt (II) drugs and wide inter- and intra-variable oral bioavailability. Therefore, no Pt(IV) drug has yet reached the market. [12-15].

Oxaliplatin (OXA), a 1,2-diaminocyclohexane (DACH) derivative of cisplatin, is a third-generation Pt(II) drug, active against several lines of colon, ovarian and lung cancer cells. However, the use of Oxaliplatin is limited due to severe side effects such as neurotoxicity, hematologic and gastrointestinal toxicities, neutropenia and intrinsic or acquired resistance [10,16]. OPA (Oxaliplatin palmitate acetate), a Pt(IV) chemical entity derived from OXA and containing both lipophilic and hydrophilic axial ligands, demonstrated at least a 20-time better efficiency in killing cancer cells [17]. OPA showed significantly higher tumor growth inhibition compared to OXA in both orthotopic and xenograft mice tumor models. A detailed description of OPA synthesis has been previously reported [22].

PUBLICATIONS

[1] E. Biskup et al., Ann. Palliat. Med. 2019, 9(3), 3.8.2019

[2] D. Shaloam et al., Eur. J. Pharmacol. 2014, 740, 364-378

[3] S. Brown et al., Br. J. Cancer 2018, 118, 312-324

[4] C. Mohanty et al., Curr. Drug Deliv. 2011, 8(1), 45-58

[5] M. J. Cleare et al., Bioinorg. Chem. 1973, 2, 187-210

[6] D. Gibson, J. Inorg. Biochem. 2019, 191, 77-84

[7] E. Cvitkovic, Semin. Oncol. 1999, 26(6), 647-662

[8] P. J. O'Dwyer et al., Drugs 2000, 59 Suppl 4, 19-27

[9] V. Dieras et al., Ann. Oncol. 2002, 13(2), 258-266

[10] L. Kelland, Nat. Rev. Cancer 2007, 7(8), 573-584

[11] M. Galanski et al., Curr. Med. Chem. 2005, 12(18), 2075-2094

[12] S. Theiner et al., Dalton Trans. 2018, 47(15), 5252-5258

[13] A. Najjar et al., Curr. Pharm. Des. 2017, 23(16), 2366-2376

[14] D. Gibson, Dalton Trans. 2016, 45(33), 12983-12991

[15] T. C. Johnstone et al., Chem. Rev. 2016, 116(5), 3436-3486

[16] I. Kostova, Anticancer Drug Discov. 2006, 1(1), 1-22 [17] A. Abu Ammar et al., J. Med. Chem. 2016, 59(19), 9035-9046

[18] E. Jerremalm et al., J. Pharm. Sci. 2009, 98(11), 3879-3885

[19] P. Allain, Drug Metab Dispos. 2000, 28(11), 1379-1384

[20] S. S. Shord et al., Anticancer Res. 2002, 22(4), 2301-2309

[21] T. Alcindor et al., Curr Oncol. 2011, 18(1), 18-25

[22] PCT patent publication WO2015/166498

GENERAL DESCRIPTION

Oxaliplatin palmitate acetate (OPA) has demonstrated significantly higher tumor growth inhibition compared to OXA in both orthotopic and xenograft mice tumor models of ovarian, pancreatic, lung and liver. However despite its demonstrated capabilities, OPA was prematurely eliminated before cellular uptake. Even when incorporated in a variety of acceptable nanoparticles, proper retention of OPA in the oil core was not observed. Thus, the inventors of the invention disclosed herein have embarked on the development of a suitable delivery system that would hold or contain OPA over long periods of time and efficiently deliver the drug to a patient. Unlike the nanoparticles proposed in the past, it was surprisingly found that only lipid-based nanocarriers could be loaded with significant amounts of OPA while maintaining their stability over time.

OPA (Oxaliplatin palmitate acetate) is a Pt(IV) organic complex having the following structural formula:

The delivery systems of the present disclosure are based on nanocarriers that comprise at least one lipid material. In the context of the present disclosure, lipids are organic molecules typically comprising a polar “head” and one or more nonpolar “tails”, such that they can be arranged spontaneously into organized structures, typically with the polar heads (that are hydrophilic) oriented toward a water-based medium and their nonpolar tails (that are hydrophobic) shielded from the water. Such structures may be micelles, bilayers or liposomes.

Thus, in one of its aspects, the present disclosure provides a lipid-based delivery system comprising OPA and a lipid-based material, wherein the delivery system is in a form of a nanocarrier. A depiction of the delivery system is provided in

Scheme 1 below:

Scheme 1

Without wishing to be bound by theory, as shown in Scheme 1, the arrangement of OPA in the lipid-based structure is highly dependent on its nature. As an amphiphilic molecule, its hydrophilic moieties are located closer to the interface of the liposomes, and its lipophilic palmitic moieties strongly anchored in the bilayers by inter- and intra-molecular attraction forces. This interaction holds OPA in place, preventing its rapid unwanted release. This interaction is referred to herein as “intercalation” or “embedment” or “incorporation”.

Thus in another aspect, there is provided a lipid-based molecular assembly (herein assembly or nanocarrier) intercalating or incorporating a plurality of OPA molecules. The OPA molecules are intercalated between neighboring lipid molecules as depicted in Scheme 1.

The lipid-based assembly is a molecular assembly of lipid molecules (at least one lipid) selected from phospholipids, glycerolipids, glycerophospholipids, sphingolipids, and mixtures thereof. In some embodiments, the at least one lipid is a phospholipid, which may be fully saturated, unsaturated or partially hydrogenated. The phospholipid may additionally or alternatively be derived from a natural source or may be partially or fully synthetic. Non-limiting examples of phospholipids include phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), as well as lipid derivatives thereof, such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylglycerol (DPPG), and others.

In dialiphatic phospholipids, the aliphatic chains can be of various chain lengths, comprising a number of carbon atoms ranging between 12 and 22 carbon atoms, e.g., having a C 12 to C22 aliphatic chain(s).

In some embodiments, the aliphatic chain has at least 18 carbon atoms. Thus, the at least one phospholipid, being fully saturated, unsaturated or partially hydrogenated may be distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

In some embodiments, the at least one phospholipid is not dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC).

Sphingolipids, by some embodiments, can include lipids having two fatty acid chains, one of which is the hydrocarbon chain of sphingosine. Such also include, for example, glycosphingolipids, which are sphingolipids with one or more sugar residues.

Assemblies of the invention are nanocarriers, namely a particulate material that is biocompatible and sufficiently resistant to chemical and/or physical destruction, such that a sufficient amount of the nanocarriers remains substantially intact after administration to a human or an animal and for a time period sufficient to reach the desired target tissue (organ). Generally, the nanocarriers are spherical in shape, having an average diameter of up to 500 nm (nanometers). Where the shape of the nanocarrier is not spherical, the diameter refers to the longest dimension of the nanocarrier.

In some embodiments, the nanocarriers have an average diameter of between about 20 nm and about 500 nm. In some embodiments, the average diameter of the nanocarrier is between about 100 and 200 nm. In other embodiments, the average diameter is between about 200 and 300 nm. In further embodiments, the average diameter is between about 300 and 400 nm, the average diameters between 400 and 500 nm. In other embodiments, the average diameter is between about 50 and 400 nm. In further embodiments, the average diameter is between about 50 and 300 nm. In further embodiments, the average diameter is between about 50 and 200 nm. In further embodiments, the average diameter is between about 50 and 100 nm.

The nanocarriers may each be substantially of the same shape and/or size. In some embodiments, the nanocarriers have a narrow diameter distribution. In other words, no more than 0.01% to 10% of the particles have a diameter greater than 10% above or below the average diameter noted above, and in some embodiments, such that no more than 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, or 9% of the nanocarriers have a diameter greater than 10% above or below the average diameters noted above.

OPA may be intercalated or incorporated in the lipid shell of lipid assembly, as depicted in Scheme 1 and as will be further detailed below. Hence, by some embodiments, the assembly or nanocarrier may be in a form of a lipid bilayer assembly (e.g. a liposome), a lipid nanocapsule or a lipid nanosphere.

According to some embodiments, the lipids are selected to form a nanocarrier having a lipid bilayer structure. The bilayer structure comprises two layers of lipids, typically arranged such that their hydrophilic heads are appositively directed (directed away from each other) to form external sheets of hydrophilic surfaces, while the hydrophobic tails of the lipids are sandwiched between the two surfaces of the bilayer. The bilayer may be formed or may be provided as a closed spherical bilayer assembly, i.e., as a liposome. Thus, in some embodiments, the molecular assembly or nanocarrier is in the form of a liposome.

The liposome is a closed bilayer structure made of the at least one lipid, and OPA intercalated or incorporated between the lipid molecules in the assembly, as exemplified by the structure of Scheme 1. Without wishing to be bound by theory, OPA is lipophilic in nature, and hence may be associated with the lipid bilayer, e.g. incorporated, intercalated or embedded within the lipid bilayer or partially dissolved therein (dispersed at the molecular level and/or partly dispersed as small molecule aggregates within the bilayer). Typically, the liposome is a unilamellar liposome, namely structured out of a single lipid bilayer. However, multilamellar liposomes, being liposomes constructed out of two or more concentric lipid bilayers, can also be used in the context of the present disclosure. According to some embodiments, the bilayer structure (e.g. the liposome) comprises at least one phospholipid. According to other embodiments, the bilayer structure comprises at least one phospholipid and at least one sterol.

Sterols are steroid alcohols and are typically considered a type of lipid. Sterols are derived from steroids, and have a fused rings core structure in which one of the hydrogen atoms is substituted with a hydroxyl group at the 3-position of the A-ring. Sterols are added to the lipids forming the lipid bilayer typically to decrease the bilayer permeability and hence increase its stability. The sterols may be selected from cholesterol, cholesteryl, cholesteryl hemisuccinate, cholesteryl sulfate and other derivatives of cholesterol and combinations thereof.

According to some embodiments, the liposome comprises at least one lipid and at least one sterol, wherein the weight ratio between the lipids and the sterols in a nanocarrier is in the range of between about 1:0.05 and about 1:5.

In other embodiments, the weight ratio between the lipids and the sterols in nanocarrier may be in the range of between about 1:0.1 and about 1:5, between about 1:0.2 and about 1:5, between about 1:0.3 and about 1:5, between about 1:0.4 and about 1:5, between about 1:0.5 and about 1:5, between about 1:0.6 and about 1:5, between about 1:0.7 and about 1:5, between about 1:0.8 and about 1:5, between about 1:0.9 and about 1:5, or even between about 1:1 and about 1:5.

In some other embodiments, the weight ratio between the lipids and the sterols in nanocarrier may be in the range of between about 1:0.05 and about 1:4.5, between about 1:0.05 and about 1:4, between about 1:0.05 and about 1:3.5, between about 1:0.05 and about 1:3, between about 1:0.05 and about 1:2.5, between about 1:0.05 and about 1:2, between about 1:0.05 and about 1:1.5, or even between about 1:0.05 and about 1:1.

In further embodiments, the weight ratio between the lipids and the sterols in nanocarrier may be in the range of between about 1:0.1 and about 1:4.5, between about 1:0.3 and about 1:4, between about 1:0.5 and about 1:3, or even between about 1:0.7 and about 1:2.5.

The lipid composition of the bilayer may further comprise one or more surfactants. The surfactant(s) can be hydrophilic, hydrophobic, amphiphilic, cationic, anionic, or non-ionic, depending on the lipids used. According to some embodiments, the lipid composition comprises at least one non-ionic surfactant. According to some embodiments, the surfactant(s) may be selected from polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, and polyoxyeyhylene esters of saturated and unsaturated castor oil, ethoxylated monglycerol esters, ethoxylated fatty acids and ethoxylated fatty acids of short and medium and long chain fatty acids and others.

The surfactant(s) may be at least one of the polyoxyethylenes, ethoxylated (20EO) sorbitan mono laurate (T20), ethoxylated (20EO) sorbitan monostearate/palmitate (T60), ethoxylated (20EO) sorbitan mono oleate/linoleate (T80), ethoxylated (20EO) sorbitan trioleate (T85), castor oil ethoxylated (20EO to 40EO); hydrogenated castor oil ethoxylated (20 to 40EO), ethoxylated (5-40 EO) monoglyceride stearate/plamitate, polyoxyl 35 and 40 EOs castor oil. According to other embodiments, the hydrophilic surfactant may be selected from polyoxyl 35 castor oil, polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), Mirj S40, oleoyl macrogolglycerides, poly glyceryl- 3 dioleate, ethoxylated hydroxyl stearic acid (Solutol HS15), sugar esters such as sucrose monooleate, sucrose monolaurate, sucrose mono stearate, polyglycerol esters such as decaglycerol monooleate or monolaurate, hexaglycerol monolaurate or mono oleate, etc.

In some embodiments, the surfactant may be at least one of polyethylene glycol 15-hydroxystearate (Solutol HS 15), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80).

The lipid composition may further comprise, by some embodiments, at least one oil at a concentration which does not affect the bilayer structure of the nanocarrier. The at least one oil may be selected from mineral oil, paraffinic oils, vegetable oils, glycerides, fatty acids, esters of fatty acids, liquid hydrocarbons and alcohols thereof, and others.

According to some embodiments, the oil may be selected from medium-chain triglycerides (MCT), long chain triglycerides such as fish oil, safflower oil, soybean oil, cottonseed oil, sesame oil, castor oil, olive oil, and others.

Alternatively, the bilayered nanoparticles may be of a substantially uniform composition not featuring a distinct core/shell structure. These nanocarriers are herein referred to as lipid nanospheres, and comprise a lipid matrix into which OPA is embedded. The lipid matrix of such nanospheres can comprise one or more lipids as disclosed herein. The lipid matrix may also comprise small quantities of injectable oils, e.g. at a quantity between about 0.1 wt% and about 10 wt% of the lipid matrix total weight. Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, com oil, olive oil, fish oil, safflower oil, castor oil. Suitable fatty acids for use in parenteral formulations in small quantities are unsaturated fatty, oleic acid (18: 1), linoleic (18: 2) and linolenic acid (18:3), long- chain omega-3 fatty acids (e.g. docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA)) and others, as well as medium chain fatty acid from C8 to C12, octanoic acid, caprylic acid, etc.

In some embodiments, the nanocarriers may be surface-associated with at least one non-active agent. The term surface-associated means a chemical or a physical association of a nanocarrier component(s) to a non-active agent(s) that extends outwards from the surface of the nanocarrier. The term refers to any association between the surface of the nanocarrier and the non-active agent, e.g. ionic bonding, electrostatic bonding, covalent bonding, dipole-dipole interaction, hydrophilic interaction, van der Waal's interaction, hydrogen bonding, physical anchoring, adsorption, or any other suitable attachment mechanism of the non-active agent to the surface of the nanocarrier.

The non-active agent (non-therapeutic agent) may be selected to modulate at least one characteristic of the nanocarrier, such characteristic may for example be one or more of size, polarity, hydrophobicity/hydrophilicity, electrical charge, reactivity, chemical stability, clearance rate, distribution, targeting and others.

In some embodiments, the non-active agent is a substantially linear carbon chain having at least 5 carbon atoms, and may or may not have one or more heteroatoms in the linear carbon chain. In other embodiments, the non-active agent is selected from polyethylene glycols (PEG) of varying chain lengths, fatty acids, amino acids, aliphatic or non-aliphatic molecules, aliphatic thiols, aliphatic amines, and others. The non-active agent may or may not be charged.

According to some embodiments, the non-active agent is polyethylene glycol (PEG). In such embodiments, the PEG may have an average molecular weight in the range of between about 2,000 and 5,000 Da (Daltons).

In other embodiments, the nanocarrier may be non-PEGylated, i.e. the non- active agent is different from PEG.

By some embodiments, the nanocarrier is lyophilized. At times, especially when lyophilization of the nanocarriers is desired, a cryoprotectant may be added to protect and improve the stability of the nanocarriers during the lyophilization process. According to some embodiments, the cryoprotectant may be selected from lactose, maltose, trehalose, sorbitol, mannitol, sulfobutyl-ether-P-cyclodextrin, polyvinyl alcohols, high molecular weight poloxamers, high molecular weight hyaluronic acid, etc.

In some embodiments, in a system of the invention, the assembly constructed of lipid molecules comprises a phospholipid that is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof. This system may be lyophilized.

Thus, a liposome is provided that comprises at least one phospholipid and OPA, wherein said at least one phospholipid is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

In some embodiments, the liposome is surface decorated with a plurality of non-active materials, as defined, e.g., polyethylene glycol (PEG).

In some embodiments, the liposome comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, OPA and N-(Carbonyl-methoxypolyethyl- eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-

PEG2000), wherein optionally the molar ratio of DSPC:cholesterol:OPA:DSPE- PEG2000 is 5:3:2:0.5, or 5:3:l:0.5, 5:3:0.75:0.5, or 5:3:0.5:0.5 respectively.

In some embodiments, the liposome comprises hydrogenated soy phosphatidylcholine (HSPC), Cholesterol, OPA and (N-(Carbonyl-methoxypolyethyl- eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE-

PEG2000), wherein optionally the molar ratio of HSPC:Cholesterol:OPA:DSPE- PEG2000 is 5:3:2:0.5, or 5:3:l:0.5, 5:3:0.75:0.5, or 5:3:0.5:0.5 respectively.

The liposome may comprise hydrogenated soy phosphatidylcholine (HSPC), Cholesterol, OPA and dipalmitoylphosphatidylglycerol sodium salt (DPPG-Na), optionally at a molar ratio of 3 :2: 1 : 1.

As exemplified herein the liposome may be prepared by thin-film hydration or by ethanol injection, as exemplified herein.

By another one of its aspects, this disclosure provides oxaliplatin palmitate acetate (OPA) loaded lipid-based nanocarrier.

According to another aspect, there is provided a lipid-based nanocarrier consisting of a lipid material and oxaliplatin palmitate acetate (OPA). In some embodiments, the nanocarrier is in the form of a lipid bilayer or a liposome. In other embodiments, the nanocarrier is in the form of a uni-lamellar liposome.

By some embodiments, the lipid is selected from at least one phospholipid, at least one sterol, and combinations thereof.

According to some embodiments, the lipid formulation comprises at least one phospholipid and at least one sterol. By an embodiment, the weight ratio between the lipids and the sterol is in the range of between about 1:0.05 and about 1:5.

According to other embodiments, the nanocarrier is surface-associated with at least one non-active agent, e.g. polyethylene glycols (PEG).

In another one of its aspects, this disclosure provides a composition comprising a lipid-based delivery system or a lipid-based nanocarrier as described herein. Typically, the composition is a pharmaceutical composition.

As used herein, pharmaceutical composition comprises a therapeutically effective amount of OPA, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g. tris- HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, surfactants (e.g. Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g. glycerol, polyethylene glycerol), anti-oxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g. thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g. lactose, mannitol), etc. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils).

Formulations suitable for parenteral administration include aqueous and nonaqueous formulations, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly (ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid.

The lipid-based delivery systems of the present disclosure may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4^th ed., pages 622-630 (1986).

In some embodiments, the composition is suitable for administration by injection.

In some embodiments, the composition is suitable for intravenous administration.

In other embodiments, the composition is suitable for topical administration, i.e. directly onto at least a portion of a subject's skin (human's or non-human's skin) so as to achieve a desired systemic or local effect. A topical composition comprising the delivery system or nanocarrier of this disclosure may be in any suitable form, e.g. a cream, a lotion, an ointment, an emulsion, a gel, a suspension, a solution, a liquid, an aerosol, a foam, etc.

In further embodiments, the composition is suitable for ocular administration, e.g. administrated topically to the conjunctiva or the eyelid or administrated parenterally, e.g. intraocular injection to the anterior, posterior and vitreous chambers. The composition may be of any suitable topical delivery form, such as a solution, a suspension, a paste, a cream, a foam, a gel, an ointment, a spray, drops, etc. By another aspect, there is provided a lipid-based delivery system, a lipid- based nanocarrier, or a composition as described herein, for use in delivery of OPA to a patient in need thereof.

By a further aspect, there is provided a lipid-based delivery system, a nanocarrier or composition as described herein, for use in treating or delaying progression of a proliferative disorder.

In yet another aspect, there is provided use of a lipid-based delivery system, a nanocarrier, or a composition as described herein, for the preparation of a medicament for treating or delaying progression of a proliferative disorder.

By yet a further aspect of the disclosure provides a method for delivering OPA to a subject in need thereof, the method comprising administering an effective amount of a lipid-based delivery system, a nanocarrier, or a composition as described herein.

A further aspect of the disclosure provides a method for treating or delaying or preventing the progression of a proliferative disorder, the method comprising administering an effective amount of a lipid-based delivery system, a nanocarrier, or a composition as described herein.

The term proliferative disorders encompass diseases or disorders that effect a cellular growth, differentiation or proliferation processes. In some embodiments, the proliferation disorder is cancer. The term cancer as used herein encompasses any neoplastic disease which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. Cancer may refer to either a solid tumor or tumor metastasis.

Non-limiting examples of cancer are ovary cancer, and pancreatic cancer, squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Solid cancers appear in many forms, for example, breast cancer, prostate cancer, sarcomas, and skin cancer. One form of skin cancer is melanoma. In some embodiments, the cancer is selected from lung cancer, colon cancer, pancreatic cancer and ovarian cancer.

The term treatment as used herein refers to the administering of a therapeutic amount of the composition of the present disclosure which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease (also referred to herein as “delaying the progression”), slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease from occurring or a combination of two or more of the above.

The term effective amount as used herein is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender, etc.

In some embodiments, the effective amount of the OPA is provided in the form of a lipid-based delivery system, a nanocarrier, or composition as disclosed herein, and administrated by one or more of the following routes: dermal, ocular, rectal, transmucosal, transnasal, intestinal, parenteral, intramuscular, subcutaneous, intramedullary injections, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

The term subject refers to a mammal, human or non-human.

The phrases “ranging/ranges between ” a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. It should be noted that where various embodiments are described by using a given range, the range is given as such merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.

As used herein, the term about is meant to encompass deviation of ±10% from the specifically mentioned value of a parameter, such as temperature, pressure, concentration, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figs. 1A-1G are cryo-TEM images of Blank Lip.l (Figs. 1A-1C) and OPA Lip.l. (Figs. 1D-1G). Lipid composition of DSPC:cholesterol:OPA:DSPE-PEG2000 at ratios 5:3:l:0.5.

Figs. 2A-2D are TEM micrographs of uranyl acetate negatively stained non- PEGylated NCs at different areas of the grid: Freshly prepared samples NC7 (Figs. 2A-2B), NC7 nanocapsules (Fig. 2C), NE3 nanoemulsion (Fig. 2D) - after 3 months of lyophilization and reconstitution of the aqueous dispersion.

Figs. 3A-3B are cryo-TEM micrographs of non- PEGylated liposomes containing OPA before (Fig. 3A) and after (Fig. 3B) lyophilization. Lipids composition was DSPC:Chol at ratio 2:1.

Figs. 4A-4B are cryo-TEM micrographs of PEGylated liposomes containing OPA, before (Fig. 4A) and after (Fig. 4B) lyophilization. Lipids composition was HSPC:Chol:DSPE-PEG2000 at ratio 12:4:3. (scale bar = 100 nm).

Figs. 5A-5B are cryo-TEM micrographs of non-

PEGylated liposomes containing OPA before (Fig. 5A) and after (Fig. 5B) lyophilization. Lipids composition was DSPC:Chol at ratio 2:1.

Figs. 6A-6B show mean plasma concentration of OPA (Fig. 6A) and OXA (Fig. 6B) following 1.25, 5 and 20 mg/kg IV administrations. Values are mean ± SD. N=3.

Fig. 7A-7B show Pt distribution in the rat (N=4) in the rat (n=4) whole blood, plasma and hematocrit after applying OPA Solution IV dosage equivalent to 5 mg/kg OPA (Fig.7A) and OPA Liposomes PEGylated (OPA-LIP5 LYO) IV dosage equivalent to 5 mg/kg OPA (Fig.7B).

Figs. 8A-8B show mean plasma concentration of OPA (Fig. 8A) and oxaliplatin as metabolite (Fig. 8B), following 7.5 mg/kg IV administrations of OPA- containing liposomes. Values are mean ± SD. N=3.

Fig. 9 shows Pt (pg/ml) in rat (n=3) plasma after applying various formulations of OPA, IV dosage equivalent to 5 mg/kg OPA.

Fig. 10 shows the mean whole blood and plasma Pt Concentration (pg/mL) following dual IV administrations of OPA-containing mPEG-liposomes (of OPA-LIP- 5 (LYO), 5 mg/kg at t=0 and 72 hours). Dashed lines are estimated Pt levels base on previous data. Values are mean ± SD. N=3

Figs. 11A-11D show plasma concentrations (ng/mL) and organ biodistribution (ng/g) of OPA (Figs. 11A-11B) and one of its metabolites, oxaliplatin (Figs. 11C-11D), 1 and 4 hours after a single IV administration of mPEG-liposomal formulation (60 mg/kg), Bare-liposomal formulation (60 mg/kg) and OPA solution (15 mg/kg) to BALB/c female mice. N=3, Values are mean ± SE.

Fig. 12 shows mean body weights of mice in different groups during treatment.

Fig. 13 shows mean body weight changes of mice in different groups during treatment.

Fig. 14 shows mean body weights of mice in different groups during treatment.

Fig. 15 shows mean body weight changes of mice in different groups during treatment.

Fig. 16 shows tumor volumes of mice in different groups during treatment of Hep3B model in balb/c nude mice.

Fig. 17 shows mean body weights of mice in different groups during treatment of Hep3B model in balb/c nude mice.

Fig. 18 shows mean body weight changes of mice in different groups during treatment of Hep3B model in balb/c nude mice.

Fig. 19 shows survival curves of mice in different groups during treatment in mouse liver cancer model Hep3B.

Fig. 20 shows tumor volumes of mice in different groups during treatment in mouse liver cancer model Hep3B. Fig. 21 shows mean body weights of mice in different groups during treatment in mouse liver cancer model Hep3B.

Fig. 22 shows mean body weight changes of mice in different groups during treatment in mouse liver cancer model Hep3B.

Fig. 23 shows survival curves of mice in different groups during treatment in mouse liver cancer model Hep3B.

Figs. 24A-D show how OPA liposomes and Avastin combination arrest tumor growth and extends survival in ovarian cancer xenograft orthotopic mouse model. For tumor development, luciferase transfected SKOV3-luc cells (2 * 106 cells in 100 pL of PBS) were injected into intraperitoneal cavity of mice. The Tumor growth was measured and quantified by IVIS every week. (Fig. 24A) Longitudinal detection and quantification of tumor growth. Tumor size is expressed as luminescence intensity of the dorsal images, expressed in radiance units (photons/s/cm2/sr). Results are presented as mean ± S.E.M. (Fig. 24B) Body weight follow-up beginning from tumor inoculation (day 0) through the study period. Changes were recorded as a percentage of the initial body weight observed on the day of tumor cells injection (100% at day 0). (Fig. 24C) Kaplan-Meier survival curve from tumor cells injection day until death. (Fig. 24D) Bioluminescent monitoring of orthotopic ovarian SKOV3-luc cancer cells expressing the luciferase gene. Bioluminescent images were acquired 10 min after intraperitoneal injection with luciferin.

Fig. 25 shows an illustration of the thin-film hydration method for the preparation of OPA Liposomes.

Fig. 26 is an illustration of the ethanol injection method for the preparation of OPA Liposomes.

Figs. 27A-B provide Cryo-TEM images of Blank Lip.l. Fig. 27A) image at 1 pm scale and Fig. 27B) image at 100 nm scale.

Figs. 28A-B provide Cryo-TEM images of OPA Lip.l. Fig. 28A) image at 1 pm scale and Fig. 28B) image at 100 nm scale.

DETAILED DESCRIPTION OF EMBODIMENTS

MATERIALS AND METHODS

In the following experimental sections, various lipid components were utilized: Lipoid PC 14:0/14:0 (DMPC), Lipoid PC 16:0/16:0 (DPPC), Lipoid PC 18:0/18:0 (DSPC), Lipoid PE 18:O/18:O-PEG 2000 (DSPE-mPEG2000, sodium salt), Lipoid PG 16:0/16:0 (DPPG, sodium salt), Lipoid S PC-3 (HSPC) (all manufactured by Lipoid GmbH).

Preparation of OPA liposomes using this-film hydration method

Lipid film preparation using tert-butanol

All the lipids of the OPA liposome preparation and OPA were weighed (see Results section below) and transferred to a round-bottom flask. Tert-butanol was added to the lipids' mixture and heated for few minutes at 50°C until all ingredients were completely dissolved. Afterwards, the round-bottom flask was frozen under rotation in ice cold ethanol bath, followed by an overnight lyophilization. Consequently, a thin film of lipid cake was obtained and further hydrated, with an appropriate amount of 5% dextrose solution or water pre-heated to 60°C, under rotation for 1 hour at 60°C. After the rotation, large multilamellar vesicles (MLV) were obtained and further down-sized to small unilamellar vesicles (SUV) using tipsonication homogenization (Ultrasonic processor, VCX 750, Sonics & Materials, Inc.) for 6 min, at 40% amplitude.

Lipid film preparation using chloroform

All the lipids of the OPA liposome preparation and OPA were weighed (see Results section below) and transferred to a round-bottom flask. Chloroform was added to the lipids' mixture. After a complete dissolution of all ingredients, chloroform was evaporated using a Rotary-evaporator instrument at 100 rpm without heating. Consequently, a thin lipid film was formed on the round-bottom flask and was further hydrated, with an appropriate amount of 5% dextrose solution or water pre-heated to 60°C, under rotation for 1 hour at 60°C. After the rotation, large multilamellar vesicles (MLV) were obtained and were further down-sized to small unilamellar vesicles (SUV) using tip-sonication homogenization (Ultrasonic processor, VCX 750, Sonics & Materials, Inc.) for 6 min, at 40% amplitude.

Preparation of OPA liposomes by ethanolic injection

PEGylated and Non-PEGylated liposomes were prepared according to the well-established ethanolic injection method (Table 1). The organic phase was 10 ml ethanol and the aqueous phase was 20 ml water. OPA, hydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-methoxypoly ethylene glycol 2000)- 1,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG 2000-DSPE), and cholesterol were dissolved in 10 ml ethanol. The aqueous phase and organic phase were heated to 60°C. The organic phase was added dropwise to the aqueous phase while stirring at 800 rpm on a magnetic stirrer. Immediately after addition the mixture was moved to a stirrer at room temperature and continuously stirred at 800 rpm for 30 minutes. Ethanol was evaporated using a rotary evaporator. Liposomes were stored at 4°C.

Immediately after addition, the mixture was moved to a stirrer at room temperature and continuously stirred at 800 rpm for 30 minutes. Ethanol was evaporated using a rotary evaporator. Liposomes were stored at 4°C.

Table 1: OPA liposome formulations (PEGylated and non-PEGylated)

Preparation of OPA nanocapsules

PEGylated and Non-PEGylated OPA nanocapsules were prepared according to the well-established solvent interfacial deposition method (Table 2). OPA or [Pt(DACH)(OAc)(OPal) (ox)], PLGA/PEG-PLA, Tween 80 and MCT were dissolved in 9 ml acetone. 50 mg/ml Lipoid E80 solution was prepared in methanol. 1 ml of this solution was mixed with 9 ml acetone with the dissolved ingredients and was stirred at 1000 rpm in a magnetic stirrer for 15 min. The organic phase was added dropwise into 20 ml of aqueous phase of Solutol®HS 15 by a needle with an inner diameter of 0.3 mm and was stirred at 1500 rpm for 30 minutes, followed by the evaporation of acetone using a Rota evaporator (Rotavapor, R300, BUCHI, Switzerland).

Large aggregates in the suspension were precipitated by centrifugation at 4 °C, 4000 rpm for 10 min. the supernatant was collected and the pH of the formulation was adjusted to 7.4. Table 2: OPA nanocapsule formulations (PEGylated and non-PEGylated)

Lyophilization of formulations

Lyophilization was carried out in Epsilon 2-6d Martin Christ lyophilizer (Gef., Germany) to obtain dry powder. Various sugars were investigated as possible cryoprotectant for the freeze-drying process of OPA NCs and liposomes e.g. Mannitol, sucrose, trehalose, dextrose, lactose, captisol and hydroxypropyl-P-cyclodextrin (HPβCD) at various concentrations of 2, 3, 4, 5, 6% w/w or at different weight ratios of 1:0.25, 1:0.5, 1:1, 1:2, 1:4 (liposome ingredients: cryoprotectant). The sugars were weighed and directly added to the formulation batches and stirred on magnetic stirrer for 10 min to mix or were added as a solution to the liposome formulation. At the end of the drying process, the vials were rapidly stoppered under vacuum and stored at room temperature.

Determination of drug content

To determine the OPA content in NCs and liposomes prepared by the ethanolic injection method, it was dissolved in ethanol and diluted with acetonitrile, and the OPA concentration was determined using analytical Dionex HPLC consisting of Dionex 3000 Ultimate auto sampler. Separation was performed on a reverse phase Cis column (5pm, 4.6x250 mm) from Agela Technologies, USA. The mobile phase consisted of water: acetonitrile (10:90 v/v), eluted at a flow rate of 1.0 ml/min. The effluent was monitored using a UV detector at 220 nm.

To determine the OPA content in the liposomes prepared by the thin-film hydration method, liposome sample was diluted x20 with methanol, and the OPA concentration was determined using analytical Thermo HPLC consisting of Thermo Scientific Dionex UltiMate 3000 Autosampler. Separation was performed on a reverse phase C18 column (5pm, 250x4.6mm, Xselect CSH, Waters, USA). The mobile phase consisted of acetonitrile (Eluent A) and water (Eluent B), at a gradient elution (from 60/40 A/B to 80/20 A/B), at a flow rate of ImL/min. The effluent was monitored using a UV detector at 220 nm.

Transmission electron microscopy (TEM) of non-PEGylated NCs

Morphological images were recorded on a TEM system (CM12 TEM, Philips) with an acceleration voltage 100 kV after negative staining using 2% uranyl acetate. A diluted suspension of the formulation (1:10) in water was dropped on carbon-coated copper grids (300-mesh), dried and analyzed.

Cryogenic TEM (cryo-TEM)

Cryo-TEM enables direct imaging of nanostructures in their native, aqueous, environment. The samples were prepared by applying a 3 pL drop onto a glowdischarge TEM grid (300 mesh Cu Lacey substrate, Ted Pella, Ltd.). The excess liquid was blotted, and the specimens were vitrified by a rapid plunging into liquid ethane precooled with liquid nitrogen using Vitrobot Mark IV (FEI). The vitrified samples were examined at -177 °C using FEI Tecnai 12 G2 TWIN TEM operated at 120 kV and equipped with a Gatan model 626 cold stage. The images were recorded by a 4K x 4K FEI Eagle CCD camera in low-dose mode (to reduce radiation damage). TIA (Tecnai Imaging & Analysis) software was used to record the images.

In vitro cytotoxicity

The in vitro cytotoxic effect of OPA, free and in liposomes formulation in comparison to the parent molecule OXA, was evaluated in four cancerous cell lines: BxPC-3 luc (human pancreatic cancer), Scl-1 (Human skin squamous cell cancer), SKOV-3 luc (Human ovarian cancer) and CNS-1 (Rat CNS glioblastoma). Experimental design

Cytotoxicity was determined in the various cancer cell lines using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay. Briefly, cells of the different cell lines were seeded in a sterile 96-well plate (3000 cells/well) in the appropriate growth medium and allowed to attach overnight. Then, the cells were treated with the test drugs (OXA, OPA and OPA liposomes) at increasing drug concentrations (0-7 pM for OPA and OPA liposomes, and 0-25 pM for OXA) at 37°C under 5% CO₂ for 72 hr.

MTT assay

Cytotoxicity was determined by the colorimetric MTT (3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay. At the end of the 72 hr-long incubation, cells were incubated in MTT solution (0.5 mg/mL) in PBS for 1 hr at 37°C. The resulting precipitated formazan was extracted in dimethylsulfoxide (DMSO) and the absorbance was measured at 570 nm and at 690 nm. The difference (OD 570nm - OD 690nm) reflects cell viability. Results were normalized to the untreated samples defined as 100% and shown as mean ± S.D.

In vitro determination of blood to plasma ratio of OPA test in human

Experimental design

Fresh human blood was collected from at least two donors, and Spiked with test compound at 1 pM in triplicates. The blood-compound mixtures were incubated with gentle shaking for 60 minutes at 37°C, and after incubation, aliquots of blood and plasma were removed for determination of analytes. Relative concentrations of OPA and OXA in samples were assessed based on peak area ratio versus internal standard, and the ratio of compound concentrations in whole blood over plasma (KB/P), the respective drug concentrations in the erythrocytes to plasma (KE/P), and %Recovery in blood were calculated.

Data analysis

The ratio of compound concentrations in whole blood over plasma (KB/P), the respective drug concentrations in the erythrocytes to plasma (KE/P), and %Recovery in blood are calculated by the following equations:

Whereas, T60-blood is compound peak area ratios of analyte/intemal standard in whole blood sample at 60 mi, T60-plasma is compound peak area ratios of analyte/intemal standard in plasma sample at 60 min, TO is compound peak area ratios of analyte/internal standard in whole blood sample at time zero, and He is the hematocrit of the whole blood used in the determination. Pharmacokinetics and biodistribution

The pharmacokinetics of intact OPA in male rats

1.1 Animal protocol

9 male SD rats (6-9 weeks, 180-300 gm, 3 groups: 1.25/5/20 mg/kg, N=3) were used in this study (The IACUC number: PK02-001-2017vl.0). Animals were group-housed during acclimation and individually housed during the study. The animal room environment was controlled (target conditions: temperature 18 to 26°C, relative humidity 30 to 70%, 12 h artificial light and 12 h dark). Temperature and relative humidity were monitored daily. Animals were fasted at least 12 h prior to the administration. All animals had access to Certified Rodent Diet ad libitum 4 h post dosing.

1.2 Experimental design

An appropriate amount of OPA was accurately weighed and mixed with the appropriate volume of vehicle to get a clear intravenous (IV) solution as detailed in Table 3. The formulations were prepared on the day of dosing, and rats were dosed (via tail vein) up to 4 h after formulations were prepared. About 200 pl blood per time point (0, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h) was collected from the jugular vein followed by plasma preparation. Blood samples were processed for plasma by centrifugation at approximately 4°C, 3000 g within half an hour of collection. Plasma samples were stored in polypropylene tubes, quick-frozen over dry ice and kept at -70 ± 10°C until LC-MS/MS analysis. Plasma concentration versus time data was analyzed by non-compartmental approach.

Table 3: In vivo Study design in SD rats (Route of administration was IV bolus)

1.3 Instrumental condition

The mass spectrometric analysis was performed with a LC-MS/MS-AR (Sciex

Triple Quad 6500+), and chromatographic separations were carried out on an ACQUITY UPLC HSS T3 1.8 m 2.1 x 50 mm column thermostated at 60°C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), at the flow rate of 0.6 ml/min. The gradient elution program was performed as follows: 0-0.3 min, 0% B; 0.3-0.8 min, 36% B; 0.8-l.lmin 85% B; 1.1-2.3 min; 100% B; 2.3-2.6 min 100% B; 2.6-2.61 min; 0% B; 2.61-2.8 min 0% B. Various internal standards (IS) were used in the study like 100 ng/ml labetalol & 100 ng/ml dexamethasone & 100 ng/ml tolbutamide & 100 ng/ml verapamil & 100 ng/ml glyburide & 100 ng/ml celecoxib in methanol. An aliquot of 40 pl sample was protein-precipitated with 200 pl IS, the mixture was vortex-mixed well and centrifuged at 12 000 rpm for 15 min, 4°C. 5 pl supernatant was injected for LC- MS/MS analysis.

1.4 Data analysis

Plasma concentration versus time data was analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. Co, Cl, Vdss, Cmax, Tmax, TI/₂, AUC(o-t), AUC(o-inf), MRT (o-t), MRT(o-inf) and graphs of plasma concentration versus time profile were reported.

In vivo pharmacokinetics of platinum from OPA solution in SD rats

Animal protocol

Eight male SD rats (6-9 weeks, 274-280 gm, dose: 5 mg/kg, N=4) were used in this study. Animals were group-housed during acclimation and individually housed during the study. The animal room environment was controlled (target conditions: temperature 18 to 26°C, relative humidity 30 to 70%, 12 h artificial light and 12 h dark). Temperature and relative humidity were monitored daily. Animals were fasted at least 12 h prior to the administration. All animals had access to Certified Rodent Diet ad libitum 4 h post-dosing.

Experimental design

Animals were divided into two groups and each half received OPA solution and lyophilized liposomes (OPA-LIP-5 LYO) as intravenous bolus dose equivalent to 5 mg/Kg of OPA.

OPA Solution preparation

25 mg OPA was dissolved in 1 ml of ethanol and Tween 80 mixture (1:1) and diluted with saline to get a clear intravenous (IV) solution of 2.5 mg/ml. The formulations were prepared on the day of dosing. Rats were dosed via a jugular vein (5 mg/kg or 1.3 mg/Rat). Approximately 200 pl blood per time point (0.16, 0.5, 1, 2, 4, 6, 10, and 24 h) was collected from the tail vein followed by whole blood separation and plasma preparation. Blood samples were processed for plasma by centrifugation at approximately 4°C, 8,000 rpm within half an hour of collection. Whole blood (100 pl), Plasma and hematocrit each 50 pl samples were stored in glass scintillation vials and kept at -70 ± 10°C until ICP-MS analysis.

Determination of platinum levels

Pharmacokinetic analysis was performed on blood, plasma and hematocrit. Platinum content of biological samples was analyzed using an inductively coupled plasma mass spectrometer (ICP-MS; Agilent 7500cx, USA, coupled with an I-AS auto sampler). For the digestion, 2 mL of concentrated nitric acid (70%) were added to each sample, the cap was tightened, and the vial was placed over a heating block at 80°C for 4 h. After obtaining a transparent solution, the screwcaps were removed and the vials were continuously heated till complete drying. The samples were reconstituted in 1% HNO₃ aqueous medium, filtered and analyzed for Pt content by ICP-MS.

Pharmacokinetic data analysis

Pharmacokinetic parameters like AUC, Cmax, Tmax, clearance, MRT, ti/2were calculated using PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.

In vivo pharmacokinetics of OPA and oxaliplatin following a single intravenous dose of OPA-containing liposomes in SD rats

Experimental design

3 male SD rats (6-9 weeks, single group: 7.5 mg/kg, N=3) were used in this study. Rats were fasted at least 12 hours prior to administration of OPA and have access to food ad libitum 4 hours post dosing.

A ready-to-use OPA-containing liposomes solution was prepared and characterized at BNS, and shipped under controlled conditions to Wuxi facility (shanghai, china), weighted and mixed with the appropriate volume of vehicle to get a clear IV solution as detailed below. The formulation was re-evaluated on the day of dosing, up to 4 hours before rats were dosed (via tail vein). About 200 pL blood per time point was collected from the jugular vein followed by plasma preparation. Dose formulation and sample analysis was performed by LC-MS/MS method. Plasma concentration versus time data was analyzed by non-compartmental approach. Instrument conditions and data analysis were identical to the detailed above.

Table 4: In vivo Study design in SD rats (Route of administration was IV bolus)

Pharmacokinetics of platinum in male SD rats following IV administration of various OPA formulation solutions, PEGylated liposomes, non-PEGylated liposomes, PEGylated NPs and non-PEGylated NPs as single dose

For in vivo experiments, male rats (Harlan, Jerusalem, Israel) were used and maintained under strict animal care procedures set forth by Authority for Biological and Biomedical Models of the Hebrew University of Jerusalem based on guidelines from the NIH guide for the Care and Use of Laboratory Animals.

1. Animal protocol

15 male SD rats (6-9 weeks, 300-324 gm, dose: 5 mg/kg, N=3) were used in this study. Animals were group-housed during acclimation and individually housed during the study. The animal room environment was controlled (target conditions: temperature 18 to 26°C, relative humidity 30 to 70%, 12 h artificial light and 12 h dark). Temperature and relative humidity were monitored daily. All animals had access to water and Certified Rodent during the experiment.

2. Experimental design

Animals were divided in 5 groups each of 3 and given various formulations as single dose, as intravenous injection equivalent to 5 mg/kg of OPA.

Rats were dosed via jugular vein (5 mg/kg). Approximately 200 pl blood per time point (0.16, 0.5, 1, 2, 4, 6, 10, and 24 h) was collected from the tail vein followed by plasma preparation. Blood samples were processed for plasma by centrifugation at approximately 4°C, 8,000 rpm within half an hour of collection.

Whole blood (100 pl), Plasma and hematocrit each 50 pl samples were stored in glass scintillation vials, and kept at -70 ± 10°C until ICP-MS analysis. IV administration of OPA PEGylated liposomes to healthy male SD rats as double dose

1. Animal protocol

Three male SD rats (6-9 weeks, 300-324 gm, dose: 5 mg/kg, N=3) were used in this study. Animals were group-housed during acclimation and individually housed during the study. The animal room environment was controlled (target conditions: temperature 18 to 26°C, relative humidity 30 to 70%, 12 h artificial light and 12 h dark). Temperature and relative humidity were monitored daily. All animals had access to water and Certified Rodent during the experiment.

2. Experimental design

Animals were given OPA Liposomes (OPA-LIP-5 LYO) double dose, as intravenous injection equivalent to 5 mg/kg of OPA. The second dose was injected after 3 days of the first dose.

Rats were dosed via jugular vein (5mg/kg) focula. Approximately 200 pl blood per time point (24, 48, 72, 96, 120, 144, 168 h) were collected from the tail vein followed by plasma preparation. Blood samples were processed for plasma by centrifugation at approximately 4°C, 8,000 rpm within half an hour of collection. Whole blood (100 pl), Plasma and hematocrit each 50 pl samples were stored in glass scintillation vials, and kept at -70 ± 10°C until ICP-MS analysis.

Plasma pharmacokinetics and bio-distribution of OPA in female BALB/c mice following a single intravenous dose of OPA in various formulations

Experimental design

18 female BALB/c mice (7-9 weeks, six group, N=3) were used in this study. Mice were not fasted prior to administration of OPA and have access to food ad libitum post dosing.

A ready-to-use OPA-containing liposomes solutions and OPA-solution were prepared and characterized at BNS, and shipped to Wuxi facility (shanghai, china), weighted and mixed with the appropriate volume of vehicle to get either a milky or clear IV solution as detailed below. The formulations were re-evaluated on the day of dosing, up to 4hr before mice were dosed (via tail vein). Animals were sacrificed per time point (1 and 4 hours) and 100 μL blood was collected via cardiac puncture followed by plasma preparation. Brain, liver, lung, pancreas and ovaries were harvested and further processed. Dose formulation and sample analysis was performed by LC-MS/MS method. Plasma concentration versus time data was analyzed by non-compartmental approach.

Table 5: Study design

In vivo maximal tolerated dose studies

The maximum tolerated dose (MTD) of different dosing frequency of test article OPA in non-tumor-bearing female BALB/c mice was evaluated.

Table 6.1: Experimental design

Table 6.2: Experimental design During routine monitoring, the animals were checked for any effects of treatments on behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. Body weight were measured once a day after randomization. The last measurement of body weights was taken on the day when the study meets the termination criteria or if a mouse is found moribund.

In vivo antitumor activities

In vivo efficacy study of OPA solution in the treatment of Hep3B human liver cancer xenograft model in female Balb/c nude mice

Table 7: Experimental design for efficacy study (I)

Note: a. The interval between BID dosing was 6 hours. b. The first dose was given on the next day of randomization. In vivo efficacy study of OPA-loaded liposomes in the treatment of SubQ Hep3B human liver cancer xenograft model in female Balb/c nude mice

Table 8: Experimental design for efficacy study (II)

Cell culture

The Hep3B tumor cells were maintained in vitro in MEM medium containing O.OlmM NEAA supplemented with 10% heat inactivated fetal bovine serum, at 37°C in an atmosphere of 5% CO₂ in air. The tumor cells were routinely sub-cultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

Tumor inoculation

Each mouse was inoculated subcutaneously at the right lower region with Hep3B tumor cells (5xl0⁶) in 0.1 ml of Matrigel (1:1) for tumor development.

Randomization

50 mice were enrolled in the efficacy study. All animals were randomly allocated to the 5 different study groups. Randomization was performed based on “Matched distribution” randomization method using multi-task method (StudyDirector™ software, version 3.1.399.19) on Day 1 and treatment was initiated on the same day.

Observation and data collection

After tumor tissue inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (body weights will be measured twice per week), and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals.

Tumor volumes were measured twice per week in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V = (L x W x W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurement were conducted in a Laminar Flow Cabinet.

Preparation of OPA liposomes for the SKOV-3 luc

OPA liposomes were prepared using a modified thin film hydration technique and were composed of DSPC (l,2-Distearoyl-sn-glycero-3-phosphocholine), cholesterol, OPA and DSPE-PEG2000 (N-(Carbonyl-methoxypolyethyl-eneglycol- 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine) at a molar ratio of 5:3:2:0.5, respectively. The lipids and OPA were weighed and transferred to a round -bottom flask. Chloroform was added to the lipid mixture at a total concentration of 12.6 mg/mL lipids and 2.75 mg/mL OPA and heated at 50°C until all the ingredients were completely dissolved. Afterwards, chloroform was evaporated using a rotary evaporator (Rotavapor, R300, BUCHI, Switzerland) at 100 rpm without heating. Consequently, a thin film of lipid cake was obtained and further hydrated, with an appropriate amount of 5% dextrose solution or water pre-heated to 60°C, under rotation for 1 h at 60°C. After the rotation, large multilamellar vesicles (MLV) were obtained and further down-sized to small unilamellar vesicles (SUV) using tipsonication homogenization (Ultrasonic processor, VCX 750, Sonics & Materials, Inc., USA) for 6 min, at 40% amplitude.

Cytotoxicity (MTT & CTG assay)

Cell culture

Cells of different human cell lines (ovarian adenocarcinoma SKOV-3-luc) were cultured in the appropriate cell culture medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin and 100 pg/mL streptomycin (SKOV-3-luc in McCoy's). Cells were kept at 37°C in 5% CO₂ and 95% humidity. Luciferase-transfected cell line SKOV3-luc was purchased from Caliper Life Sciences (Hopkinton, MA, USA). Cytotoxicity

Approximately 6,000 cells/well were seeded in a 96-well plate. The cells were allowed to attach overnight, and fresh medium containing the appropriate dilutions of test compounds was added for a 72 h-long incubation. SKOV-31uc cells were further treated for 1 h with 0.5 mg/mL MTT in phosphate buffer solution (PBS). The developed dye was dissolved in DMSO and absorbance measured at 570 nm by a microplate reader (Cytation 3, BioTek, USA).

Orthotopic mouse model of human ovarian cancer (SKOV3-luc)

Female mice with severe combined immunodeficiency (SCID)-bg, 8 weeks old (18.5-21.5 gm) were used in accordance with NIH regulations. Luciferase transfected SKOV3-luc cells (2 x 10⁶ cells in 100 pL of PBS) were injected directly into intraperitoneal cavity of mice. For tumor validation and tumor growth follow up, bioluminescent imaging was performed every 7 days using a CCCD camera (IVIS, Caliper Life Sciences, Xenogen Corporation, USA). 200 pL of 15 mg/mL firefly D- luciferin was injected intraperitoneally and mice were anesthetized by 3% isoflurane. Animals were placed onto black paper in the IVIS imaging box and imaged dorsally. Luminescence was recorded in radiance units (photons/s/cm²/sr). The mice were randomly allocated to the 7 study groups according to the tumor size, while verifying that all 7 groups have similar initial Mean Luminescence Intensity (MLI). Randomization was performed 4 days prior to treatment initiation (n = 6). The treatment groups were (group 1) vehicle liposomes (dispersed in dextrose); (group 2) free OXA (5mg/kg in dextrose); (group 3) OPA solution (15 mg/kg dissolved in Cremophor EL : ethanol at 1:1 ratio and diluted by 10 in dextrose); (group 4) OPA liposomes (15 mg/kg in dextrose), (group 5) OPA liposomes (30 mg/kg in dextrose), (group 6) OPA liposomes (15 mg/kg in dextrose) and Avastin (10 mg/kg in saline) combination and (group 7) Avastin (10 mg/kg in saline). All the animals were given i.v. bolus doses of the specific formulation to the tail vein, twice a week for four weeks.

Statistical analysis

Data and bars represent the mean ± standard deviation (SD)/standard error mean (S.E.M) of three or more independent experiments. Differences between two groups were analyzed using unpaired Student’s t test. Differences between three or more groups were analyzed using one-way ANOVA. To compare tumor volumes of different groups at a pre-specified day, we first used Bartlett's test to check the assumption of homogeneity of variance across all groups. When the p-value of Bartlett's test was >0.05, we ran one-way ANOVA to test overall equality of means across all groups. If the p-value of the one-way ANOVA was <0.05, we further performed post hoc testing by running Tukey's HSD (honest significant difference) tests for all pairwise comparisons and Dunnett’s tests for comparing each treatment group with the vehicle group. Survival curves were constructed for each group using the Kaplan-Meier method and compared statistically by the log rank (Mantel-Cox) test. All the statistical calculations were performed using GraphPad Prism Statistics Software, version 9. All tests were two-sided unless otherwise specified, and p-values of <0.05 were regarded as statistically significant.

RESULTS

Liposomes prepared by the thin-film hydration method

Initially, the ability to formulate OPA containing liposomes in DSPC -lipoid based formulation was evaluated as described in Table 9.

Table 9: DSPC-lipoid based OPA liposomes

According to Tables 10-11, it can be noted that, regarding appearance, physicochemical properties and pH value, that Blank and OPA Lip. based on DSPC- lipoid formulations were successfully prepared. Moreover, for the evaluation of OPA assay %, methanol was chosen as the suitable diluent for sample preparation due to higher assay % values and peak symmetry.

It can be noted from Fig. 1 that the liposomes exhibit bilayers of phospholipids of small unilamellar vesicles (SUV). Table 10: Characterization of DSPC-lipoid based Blank lip, (without OPA)

Table 11: Characterization of DSPC-lipoid based OPA Lip

According to Table 12, it can be noted that a decrease of up to 15-20% in OPA assay % after 1 week of storage was observed at 25 and 40°C. A decrease of up to 50% in OPA assay % was observed after 2 months of storage at 25°C. Therefore, 4°C was selected as the appropriate storage temperature for extending the stability of OPA Lip. as aqueous suspension.

Table 12: Preliminary stability test of OPA Lip.l

Next, the difference in QPA liposomes formulations prepared by thin-film hydration method using tert-butanol or chloroform for thin-film preparation was examined. 2 additional OPA concentrations were also tested, as described in Table

13.

Table 13: Tert-butanol vs. chloroform in thin-film preparation

As shown in Table 13, no significant effect of tert-butanol or chloroform, used for thin-film preparation, on OPA Lip. formulations was observed.

In order to increase the shelf-life stability of OPA Lip.l (less than 3 weeks at 4 °C), the feasibility of lyophilizing the liposomes was evaluated. In this part, another composition of OPA Lip was tested based on HSPC-lipoid (Table 14). The effect of different cryoprotectants in various weight ratios on the lyophilization process was investigated (Table 15).

Table 14: HSPC-lipoid based OPA liposomes prepared for lyophilization optimization; (OPA Lip .8) Table 15: Lyophilization results of OPA Lip.8 According to Table 15, it can be noted that among many cryoprotectants that were used, HPβCD at a ratio of 1 :2 (added as solution) and 1:4 (added as powder) presented preserved values of mean diameter and PDI before and after lyophilization.

Since the appearance of the liposomes dried powder (cake) was not optimal, additional attempts were made using HPβCD as the cryoprotectant, evaluating also the effect of the liposomes hydration medium. Two additional formulations were prepared using water/phosphate buffer (PBS) as the hydration mediums (Table 16).

Table 16: HSPC-lipoid based OPA Lip.13 and 14

Table 17: Lyophilization results of OPA Lip, 13 and 14 using HPPCD

*relative to TO (prior to Lyo.)

According to Table 17, it could be noted that PBS as the hydration medium was inadequate and resulted in 62.4% assay of OPA in comparison to 105.6% obtained with water as the hydration medium. Moreover, the appearance of the cake after lyophilization with water used as the hydration medium was good and was better than the cake obtained after using 5% dextrose as the hydration medium. Furthermore, 1:2 ratio of Lip:HPβCD (w/w) was sufficient to obtain a good cake. It should be noted that the relatively low assay % observed after lyophilization is due to the dilution of the sample due to contribution of the dried powder volume to the final sample volume after reconstitution (in future studies this was taken into account).

Overall, best results were obtained with water as the hydration medium therefore it was chosen for further development using 1 :2 ratio of HPβCD

Following lyophilization optimization, different formulations of PEGylated liposomes with various lipid compositions and different OPA concentrations were prepared (Table 18). The stability of the different formulations as aqueous suspension (at 4°C) was evaluated up to 3 months and after lyophilization (TO lyo.).

Table 18: OPA Lip. (15- 26) formulations

All formulations were prepared by thin-film hydration (tert-butanol). Downsizing was performed using tip sonication. Hydration medium was water. All formulations were lyophilized using 1:2 ratio of Lip. Ingredients :HPβCD.

As shown in Tables 19-1 to 19-4, OPA liposomal formulations based on various lipid compositions and different OPA concentrations were successfully prepared, all demonstrating good appearance, size and assay% (except for OPA Lip.24 which looked cloudy after preparation). Based on the stability results it could be noted that liposomes based on the shortest lipidic chain DMPC demonstrated the highest instability in aqueous suspension. In addition, although almost all formulations exhibited >80% assay after 3 months (samples were mixed prior to the assay measurement), precipitates were observed indicating physical instability after < 1 month (summarized in Table 20). It should be noted that all formulations were found stable after lyophilization in terms of appearance of the lyophilized cake and after reconstitution, assay % and physicochemical characterization.

Table 19-1: Stability results of DSPC-lipoid based OPA Lip.15-17 as aqueous suspension (aq.) and after lyophilization [T0 Lyo.) _

Table 19-1 (cont.): Stability results of DSPC-lipoid based OPA Lip.15- 17 as aqueous suspension (aq.) and after lyophilization (T0 Lvo.)_ Table 19-2: Stability results of HSPC-lipoid based OPA Lip.18-20 as aqueous suspension tag.) and after lyophilization (T0 Lyo.) _

Table 19-2 (cont.): Stability results of HSPC-lipoid based OPA Lip.18-20 as aqueous suspension (aq.) and after lyophilization (T0 Lyo.)

Table 19-3: Stability results of DPPC-lipoid based OPA Lip.21-23 as aqueous suspension (aq.) and after lyophilization (T0 Lyo.)

Table 19-3 (cont.): Stability results of DPPC -lipoid based OPA Lip.21-23 as aqueous suspension (aq.) and after lyophilization (T0 Lyo.)

Table 19-4: Stability results of DMPC -lipoid based OPA Lip.24-26 as aqueous suspension (aq.) and after lyophilization (T0 Lvo.)

Table 19-4 (cont.): Stability results of DMPC-lipoid based OPA Lip.24-26 as

Table 20: Summary of stability results of PEGylated OPA liposomes as aqueous suspension

In order to increase the shelf-life stability of OPA liposomes, additional formulations were prepared using the same recipe as OPA Lip.15 with two OPA concentrations of 2.5 and 5 mg/ml (OPA Lip.37 and 38, Table 21). For comparison, non-PEGylated liposomes were prepared (OPA Lip. 42a, Table 22). The liposomes were prepared and was then lyophilized. The stability of the liposomes dried powder was evaluated for up to 1 month at 4°C, 25°C and 40°C (accelerated conditions).

Table 21: OPA Lip. 37 and OPA Lip.38 formulations Table 22: non-PEGylated OPA Lip. 42a formulation

As shown in Tables 23 and 24, lyophilization of both PEGylated and non- PEGylated OPA liposomes improved their physicochemical stability compared to aqueous suspension. Liposomes samples of all time points and in all temperatures were easily reconstituted obtaining good appearance and size. The liposomes dried powder was found stable after 1 month of storage at 4°C, 25 °C and even at 40°C (accelerated conditions). OPA assay % was above 90% in all lyophilized formulations after 1 month at 40°C (accelerated conditions), indicating the vital effect of lyophilization on enhancing OPA Lip. stability and extending its shelf-life. The various property magnitudes are listed in Table 25. As can be noted in Figs. 2A-5B, the liposomes exhibit bi-layers of phospholipids.

Table 23: Stability results of OPA PEGylated Lip.37, 38 as dried powder Table 23 (cont.): Stability results of OPA PEGylated Lip.37, 38 as dried powder

Table 24: Stability results of non-PEGylated OPA Lip 42a as dried powder

Table 25: Properties of OPA liposomes and nanocapsules before and after lyophilization Preliminary stability results of OPA Lip. prepared by the ethanol injection method

According to Table 26, it is noted that OPA Lip.EtOH showed promising results regarding appearance, mean diameter and PDI before and after lyophilization and after a storage period of 1 month at room temperature (RT). A decrease in OPA assay % and pH was observed after 1 month of storage at RT (Table 27).

Table 26: Description of HSPC-lipoid based OPA liposomes prepared by ethanol injection

Table 27: Characterization and preliminary stability of lyophilized OPA Lip, prepared by ethanol injection (OPA Lip. EtOH)

(*)-high value of osmolality could be due to ethanol residues after evaporation.

In vitro cytotoxicity

The anti-proliferative activities of OXA, OPA and OPA liposomes were tested in human skin squamous cell carcinoma Scl-1, pancreas adenocarcinoma BxPC-3 luc, ovarian cancer cells SKOV-3 luc and rat glioblastoma CNS-1 cell lines by the MTT assay. The 50% growth inhibitory concentration (IC50) values were calculated and summarized in Table 28. OPA showed a unique potency against different cancer cell lines, with higher cytotoxicity than OXA. Table 28: IC₅₀ (μM) values of OPA, OXA and OPA liposomes in various cancer cell lines following 72 h-long treatment

IC50 values are drug concentrations required to induce 50% cell death and are the means ± SD of two- four independent experiments with quadruplicates in each.

*single experiment

In vitro determination of blood to plasma ratio of OPA test in human

Table 29: OPA summary

Diclofenac was used as a negative control for low affinity to blood cells in this study. Chloroquine was used as a positive control for high affinity to blood cells for Human.

Table 30: Oxaliplatin summary

Table 31: Raw data of hematocrit in whole blood

Pharmacokinetics and bio-distribution

The Pharmacokinetics of intact OPA in male rats

Three PK experiments were performed following administration of OPA at 1.25, 5 and 20 mg/kg in rats. Major PK parameters of intact OPA and oxaliplatin (active metabolite originated from OPA biodegradation) are presented in Table 32 and Figs. 6A-6B. Table 32: Pharmacokinetic parameters of OPA and oxaliplatin following single dose of increasing doses of OPA to male rats (OXA was formed by the biodegradation of

OPA)

The most important finding from the pharmacokinetic study in rats was that OPA is not a prodrug of oxaliplatin in vivo, irrespective of the injected dose. The formation of OXA from the biodegradation of OPA ranges from 5 to 10% (Table 32), based on the calculated ratio of the respective AUC values regenerated from the pharmacokinetic profiles of both drugs presented in Figs. 6A-6B. Furthermore, the terminal elimination of the intact OPA molecule is rapid, in less than an hour for the β ti/2 values of OPA and OXA confirming the behavior of most of the platinum drugs and especially oxaliplatin [18]. Dose proportionality, evaluated from the calculated parameters, was demonstrated between 1.25 and 5 mg/kg but not for the 20 mg/kg. Supra-proportional behavior was recorded between 5 and 20 mg/kg, where, particularly for AUC and ti/2 values of OPA, the data was much more elevated than the proportional values achieved between 1.25 and 5 mg/kg. In addition, it can be noted that there is a marked difference in the behavior of the intact OPA molecule PK profile as compared to the platinum compound of the molecule as shown below.

In vivo pharmacokinetics of platinum from OPA solution in SD rats

Oxaliplatin (OXA) exhibits a complicated PK profile and has several mechanisms of action but cancer cells can develop resistance. OXA exerts its cytotoxic effect mostly through DNA damage. Most papers do not address the PK of OXA per se, but of the Pt content. Shortly after infusion, OXA forms many Pt compounds which bind to blood or cell proteins. Total ultra-filterable plasma Pt is measured by atomic absorption or inductively coupled plasma mass spectrometry (ICP-MS). These techniques result in a codetermination of OXA and other Pt containing complexes due to high propensity of OXA to react with endogenous sulfur-containing compounds. OXA is often administered concomitantly with 5-FU. Its PK is not altered in presence of 5-FUTri-exponential pattern elimination: The ti/2 values for Pt compounds were 0.28,16.3 and 273h whereas the reported elimination ti/2 of intact OXA was 14.1 min and another ti/2 value reported was 45min related to in vivo degradation in blood rather than to elimination. OXA and other Pt compounds are present in plasma and urine. Pt is bound to albumin, y-globulin and hemoglobin. Ih post- infusion, OXA in PUF & urine was 12 and 50% of total Pt respectively. 3 h post-infusion, OXA undetectable, represented 10% of total Pt in urine. Inside red blood cells, 2 Pt compounds were found; Pt peak at Pt-hemoglobin and low molecular species (60 and 40% of total Pt respectively) [18-21].

Platinum (IV) complexes such as OPA exhibit an advantage over Platinum (Il)-based drugs thanks to their kinetic stability in the body. They remain inert in the blood and only once reaching the tumor sites, they are activated in the cancer cells by a reduction process. The ligand coordination spheres affect the lipophilicity and redox behavior in blood and has a significant impact on their accumulation in red blood cells and their degree of kinetic inertness in blood. The most lipophilic platinum (IV) compounds featuring equatorial Chloro ligands showed a pronounced penetration into blood cells and a rapid reductive biotransformation. In contrast, the more hydrophilic platinum (IV) compounds with a carboplatin or oxaliplatin-core exerted kinetics inertness on a pharmacologically relevant time scale (up to 24 h) with notable amounts of the compound accumulated in the plasma fraction [12].

From the results in Fig. 7 that the residence time of Pt compounds regenerated by the metabolism of OPA in plasma is much more prolonged than that of the intact OPA. More importantly OPA penetration in the hematocrit (blood cells) is less pronounced than in plasma and has surprisingly a less rapid reductive biotransformation than OXA despite its higher lipophilicity (Fig.7A). It should be emphasized that as long as OPA remain intact or is transformed into Pt (IV) metabolites, it does not elicit any side-effect or toxicity on healthy blood cells. Furthermore, in both OPA Solution (Fig. 7A) and OPA Liposomes (Fig. 7B) treated groups with the same dose of 5mg/kg, the plasma Pt. level immediately elevated and reached to the peak point (Cmax) at time zero, then reduced sharply; however, significant differences were found in these changes between the solution and liposome treated groups. The area under the curve (AUC) and maximum concentration (Cmax) for Pt in plasma after OPA liposomes were administered, approximately 96.38 pg/mlxh and 7.23 pg/ml compared with 9.98 pg/mlxh and 1.67 pg/ml, for OPA solution (Table 33).

Table 33: PK parameters of Pt. following IV administration OPA Solution and lyophilized Liposomes (OPA-LIP-5) at a dose equivalent to 5 mg/kg OPA to rats

* Extrapolate from Fig.2 & 3 at 10 min

Moreover, in previous published work [17], it was observed that both OPA in solution and in nanocapsules (NCs) were almost equally effective with little superiority to the NCs. The hypothesis was that apparently in the plasma, the NCs are unable to retain efficiently the OPA within the oil core of the NCs owing to the infinite dilution of the NCs in the plasma (sink conditions). Thus, additional formulative approaches were needed to be investigated to retain within the nanocarrier the OPA in the plasma and prevent its release over time in view of its instability in the plasma over very short period of times of less than 60min. Logically, it was attempted to encapsulate OPA within PLGA nanospheres (small nanomatrices). Unfortunately, OPA could not be incorporated within such matrices at significant drug content levels and even low contents of 1 or 2% were incompatible since precipitation and aggregation were observed in the dispersed formulations. Furthermore, it was not possible to prepare nanoemulsions since OPA was not enough soluble within injectable approved oils and precipitated rapidly in the presence of the water continuous phase of the nanoemulsions. Surprisingly, it was possible to prepare appropriate liposomal formulations of OPA at a normal level contents within the bilayers of the phospholipids. Different methods of Liposome manufacturing methods were used, and different formulations were prepared of OPA liposome formulation (PEGylated and non-PEGylated). In addition, OPA nanocapsule formulations (PEGylated and non-PEGylated) were prepared followed by lyophilization of all formulations and characterization of the following properties: particle size, PDI and zeta potential analysis, determination of drug content (HPLC), morphology by Cryo TEM and stability at room temperature.

PK experiment was performed following administration of OPA-containing liposomes at 7.5 mg/kg in rats. Major PK parameters and another active metabolite, oxaliplatin, are presented in Table 34 and Figs. 8A-8B. AUC and tl/2 levels of OPA were markedly elevated, and clearance value was significantly decreased, compared to IV injection of OPA solution (RND-RPT-007). These finding suggest that the PK parameters of OPA, when administered intravenously in liposomal formulation, can be significantly altered, resulting in an extended exposure profile. Table 34: Pharmacokinetic parameters of OPA and oxaliplatin following single dose of OPA-containing liposomes in SD rats

Pharmacokinetics of platinum in male SD rats following IV administration of various OPA formulation solution, PEGylated liposomes, non-pegylated liposomes, PEGylated NPs and non-PEGylated NPs as single dose

The pharmacokinetics of platinum compounds in healthy male SD rats following IV administration of OPA Solution, PEGylated and Non-PEGylated OPA nanocapsules and liposomes were carried out and their respective profiles compared.

It can be noted from the results presented in Fig. 9 that the more prolonged release of Pt compounds in plasma were achieved only by both liposomal formulations, PEGylated and Non-PEGylated. These findings are very encouraging and validate the results achieved with intact OPA liposomes PK studies (Fig. 8). Table 35 exhibit the PK parameters showing Cmax values for the PEGylated Liposomes of 5fold higher than with the NPs and a half life time value of 21.6 hours and a respective AUC value (92.76 pg/mlxh) of more than lOfold the AUC value (8.43 pg/mlxh) of the Pegylated NPS. Liposomes appear to be superior to the NCs in terms of extending the residence time of OPA in the plasma.

Table 35: PK parameters of Pt following IV administration OPA Solution and various other formulations at dose equivalent to 5 mg/kg OPA to rats

IV administration of OPA pegylated liposomes to healthy male SD rats as double dose

It can be noted from Fig. 10 that the OPA molecule apparently remains in the liposomes in the plasma over more than 72 hours and even a second injection after 3 days from the first does enhance the levels of Pt the plasma and whole blood but much less in the hematocrit showing minimal release of OPA from the liposomes.

Table 36: PK parameters of Pt. following IV administration OPA-LIP-5 (LYQ) as a single dose each equivalent to 5 mg/kg OPA to rats

Table 36 shows the various PK parameter values for the first dose only in plasma, whole blood and hematocrit

The summary of mice PK and organ bio-distribution of OPA and Oxaliplatin after a single intravenous dose of OPA-containing liposomes (60 mg/kg) and OPA solution (15 mg/kg) to female mice is described in Table 37 and Figs. 11A-11D. In plasma, OPA-containing mPEG-liposomes have the longest circulation residence time, up to 4-fold higher and 4000-fold higher OPA and oxaliplatin normalized concentrations compared to non-PEG liposomes and IV solution, respectively (mPEG-liposomes> Non PEG-liposomes »> OPA solution).

The pharmacokinetics of OPA and oxaliplatin in rats following IV solution administration was previously tested (Fig. 6), and comparison with the recent data show that, even taking into account lower, 15 mg/kg dose applied in the current experiment and allometric differences between mice and rat species, both OPA and OXA plasma 1- and 4-hour post-dose levels are not too different from those obtained in the earlier study.

Analysis of OPA and oxaliplatin disposition in organs following the administration of the three formulations was performed using organ-to-plasma ratios, which demonstrate significant differences of tested formulations, presenting dose- normalized organs’ exposures of both compounds. It can be noted that organ-to- plasma ratios of oxaliplatin are much higher than those of OPA for both liposomal preparations. For the IV solution, organ-to-plasma ratios of oxaliplatin are higher than that of OPA in the liver and pancreas, but not in the lungs and ovaries.

Overall, it can be clearly noted that higher OPA levels were reported in most organs, but not in the liver, following the PEG-liposomal preparation administration, compared to the non-PEG liposomes and the IV solution. OPA levels in the liver were relatively similar 1 and 4 hours post administration of PEG-liposomes and non-PEG liposomes.

OPA in non-PEGylated liposomes provides highest organ-to-plasma ratios, which may indicate ability of this formulation to provide maximal efficacy of the drug with minimal systemic exposure and, therefore, toxicity, e.g. nephrotoxicity, neurotoxicity, etc. The latter may be predicted as result of lower brain permeability of OPA and OXA following administration of both liposomal formulations, compared to that after OPA solution injection. On the other hand, both liposomal formulations provided high liver exposures. Table 37: Mice PK and organ bio-distribution of OPA and oxaliplatin as metabolite of OPA

*organ/plasma ratio in brackets

PK experiment, including potential tumor-forming organs bio-distribution, was performed following administration of OPA-containing PEG liposomes, non- PEG liposomes and IV solution in mice. Major PK parameters and another active metabolite, oxaliplatin, are reported. Following administration of liposome -based formulations concentrations of OPA in organs were in decreasing order: liver>lung>ovary>pancreas, while lung>liver>pancreas>ovary for oxaliplatin.

Liposomes-based forms of the OPA DP showed distribution patterns different from those of OPA in IV solution and had impact on overall disposition of the OPA as well as derived OXA molecules.

The graphs in Figs.llA-llD demonstrate the advantage of Liposome PEG- formulation in Pt delivery to relevant organs compared to OPA or oxaliplatin solutions. OPA Liposomes can definitely be targeted to treat severe cancer such as ovarian, liver, pancreatic and lung cancers in addition to Glioblastoma multiforme.

In vivo maximal tolerated dose studies

MTD PEG-liposomes

The results of mean body weight and mean body weight changes in the nontumor bearing mice are shown in Figs. 12 and 13, respectively.

OPA Liposomes administered at 15 mg/kg, i.v., BIW for 2 weeks, body weight loss was observed with -2.95% mean BWL% nadir on Day 4. OPA Liposomes administered at 30 mg/kg, i.v., BIW for 2 weeks, body weight loss was observed with -2.00% mean BWL% nadir on Day 10. OPA Liposomes administered at 60 mg/kg, i.v., BIW for 2 weeks, body weight loss was observed with -4.17% mean BWL% nadir on Day 4.

MTD non-PEG-liposomes

OPA Liposomes (Lip-42C) administered at 60 mg/kg, i.v., on Day 1, 4, 8 and 11. Body weight loss was observed with -3.69% mean BWL% nadir on Day 2. In summary, the test article OPA Liposomes (Lip-42C) administered at 60 mg/kg, i.v., was well tolerated by non-tumor-bearing female BALB/c nude mice in this MTD study (Figs. 14-15).

The findings clearly show that liposomes release gradually the active ingredient in a way which does not elicit severe side-effects while maintaining high drug levels over time.

In vivo antitumor activities

Efficacy study (I)

1. Tumor volume The tumor growth curves (mean tumor volume over time) of different groups are shown in Fig. 16.

2. Tumor growth inhibition

The tumor growth inhibition is summarized in Table 38 below.

Table 38: Antitumor activity of test articles in the treatment of Hep3B model in

Balb/c nude mice

Note: a. Mean ± SEM (mice number); b. TGI%= [l-(Ti-TO) / (Ci-C0)] x 100%; c. The p value of Bartlett’s test is 0.544, treatment groups vs. vehicle control was ran by parametric test.

3. Body weight

The results of mean body weight changes in the tumor bearing mice are shown in Fig. 17. In this study, the therapeutic efficacy of different concentrations of test article OPA and Oxaliplatin and Cisplatin were evaluated in the Hep3B model of liver cancer in BALB/c nude mice (Figs. 17-19). Group 2 (OPA, 12mg/kg) showed moderate antitumor efficacy (TGI = 28.79% on day 18) but was not statistically different from the vehicle group (p = 0.078 vs vehicle). Group 3 (OPA, 8mg/kg) demonstrated good antitumor efficacy (TGI = 41.40% on day 18) and was statistically different vs vehicle group (p = 0.00503 vs vehicle). Group 4 (OPA, 60mg/kg) demonstrated moderate antitumor efficacy (TGI = 27.10% on day 18) but was not statistically different vs vehicle group (p = 0.107 vs vehicle). Group 5 (Oxaliplatin, 5mg/kg) demonstrated moderate antitumor efficacy (TGI = 14.42% on day 18) but was not statistically different from the vehicle group (p = 0.634 vs vehicle). Group 6 (Cisplatin, 4mg/kg) demonstrated good antitumor efficacy (TGI =45.61% on day 18) and was statistically different vs vehicle group (p = 0.00175 vs vehicle). In summary, moderate to good anti-tumor efficacy was observed in all treatment groups. Furthermore, the most significant anti-tumor efficacy was observed in OPA, 8mg/kg treated group and Cisplatin, 4mg/kg treated group.

Efficacy study (II)

1. Tumor volume

The tumor growth curves (mean tumor volume over time) of different groups are shown in Fig. 20.

2. Tumor growth inhibition

The tumor growth inhibition is summarized in Tables 39 and 40 below.

Table 39: Antitumor activity of test agents in the treatment of mouse liver cancer model Hep3B on day 18

Note: a. Mean ± SEM (mice number); b. TGI%= [l-(Ti-Tl) / (Ci-Cl)] x 100%; c. The p value of Bartlett’s test is 0.0167, treatment groups vs. vehicle control was ran by Conover’s non-parametric many-to-one comparison test.

Table 40: Antitumor activity of test agents in the treatment of mouse liver cancer model Hep3B on day 22

Note: a. Mean ± SEM (mice number); b. TGI%= [l-(Ti-Tl) / (Ci-Cl)] x 100%; c. The p value of Bartlett’s test is 0.0831, treatment groups vs. vehicle control was ran by Dunnett’s t test

3. Body weight

The results of mean body weight changes in the tumor bearing mice are shown in Figs. 21-23.

In this study, the therapeutic efficacy of test agents were evaluated in the SubQ Hep3B syngeneic human liver cancer model in female BALB/c mice. In summary, good anti-tumor effect was observed in all treatment groups. Furthermore, the best therapeutic responses were observed in group 5 and group 2 treated with Cisplatin at 4mg/k and OPA liposomes 60mg/kg twice a week. Overall the nanoformulation of OPA in liposomes appear to be a very attractive innovative delivery system with a high potential of therapeutic activity in severe life-threatening cancers.

Efficacy of OPA liposomes in SKOV3-luc human ovarian cancer orthotopic mouse model

In this study, the antitumor potential of OPA liposomes at 2 different dosages of OPA (15 and 30 mg/kg) as well as the potential synergistic effect of OPA and Avastin (15 and 10 mg/kg, respectively) combination was evaluated in an orthotopic intraperitoneal model of metastatic ovarian cancer in female SCID-bg mice. Four days following the randomization, animals were treated with the various formulations twice a week up to 25 days by i.v. injection (Fig. 24 and Table 41). The tumor growth in the treatment groups were not significantly arrested compared to vehicle control group, measured on day 29. The observed differences in radiance values were insignificant (p<0.05, one-way ANOVA).

Significant antitumor activity was measured in mice treated with OPA liposomes (15 mg/kg and 30 mg/kg) and the combination of OPA liposome and Avastin compared to the vehicle controls at day 43 (oxaliplatin treated mice did not survive 43 days) (Table 41). The combination treatment was found most effective in delaying tumor growth and resulted in more tumor regression than OPA solution. The observed differences in radiance values between OPA solution and the combination treatment at the 43^th and 50^th days were significant (p=0.045 and 0.008, respectively). The groups treated with OPA liposomes alone demonstrated slightly better antitumor activity to that of the OPA solution treated one but the differences were insignificant

(P>0.05).

Table 41: Antitumor activity of various formulations in SKQV3-luc human ovarian cancer orthotopic model in female SCID-bg Mice on Day 43 [Mean ± SEM (mice number)] .

Note: a. TGI%= [l-(Ti-Tl) / (Ci-Cl)] x 100% (Ti and Ci as the mean tumor volumes of the treatment and vehicle groups on the measurement day; Tl and Cl as the mean tumor volumes of the treatment and vehicle groups on Randomization); b. T/C% =

(Ti) / (Ci) x 100% Kaplan-Meier survival analysis (Fig. 24C) showed that all the groups other than oxaliplatin treated one, significantly prolong the median survival time compared to vehicle liposome group (log-rank test, p < 0.05). The combination of OPA liposome and Avastin improved the survival significantly as indicated by the increased median survival time of 60.5 days compared to 50 days in case of individual treatments of OPA liposome and Avastin (p=0.0174). No significant difference in survival was noted between OPA solution and OPA liposome treated groups. However, we observed significant difference in the median survival between the combination and OPA solution treated group (p=0.0178). Finally, there was an initial decline in body weight following the injections of OPA, 15 mg/kg and the combination-treated groups, but the animals recovered with the progression of the experiment.

OPA Liposome preparation using the Thin-film hydration method

Lipid film preparation using tert-butanol

Lipids and other ingredients of the lipsomal preparation, were weighed and transferred to a round-bottom flask. Tert-butanol was added to the lipids' mixture and heated for few minutes at 50^sC until all ingredients are completely dissolved. Afterwards, the round-bottom flask was frozen under rotation in an ethanol bath for few minutes (Tzabam, medicine school of Hadassah Ein-kerem) followed by an overnight lyophilization (lyophilizer #3, Tzabam, medicine school of Hadassah Ein- kerem). Consequently, a thin-film of lipid cake is obtained and is further hydrated, with an appropriate amount of pre-heated water to 60^sC, under rotation for 1 hour at 60°C. After the rotation, large multilamellar vesicles are obtained and further downsized to small unilamellar vesicles (SUV) using tip-sonication extrusion homogenization method; by inserting a 50 mL tube into an ice bath for 6 min, 40% amplitude.

Lipid film preparation using chloroform

Lipids of the OPA liposomal preparation were weighed and transferred to a round-bottom flask. Chloroform was added to the lipids' mixture. After a complete dissolution of all lipids chloroform is evaporated using a Rota-evaporator instrument at 100 rpm without heating. Consequently, a thin lipid film is formed on the roundbottom flask and is further hydrated, with an appropriate amount of pre-heated water to 60°C, under rotation for 1 hour at 60°C. After the rotation, large multilamellar vesicles are obtained and further down-sized to small unilamellar vesicles (SUV) using tip- sonication extrusion homogenization method; by inserting a 50 mL tube into an ice bath for 6 min, 40% amplitude. An illustration of the process is provided in Fig. 25.

OPA liposome preparation using the Ethanol injection method

The preparation of OPA liposomes using ethanol injection was performed by weighing the ingredients of the liposomal formulation into a 20 mL scintillation vial and dissolving them in 10 mL of ethanol. The aqueous phase was prepared by heating 20 mL of water in a beaker to 60°C. When the temperature of water reaches 55°C; the ethanol solution is heated to 60°C. When the ethanol solution and the aqueous phase reach 60°C, they were removed from heat and mounted on a head-stirrer. The ethanol solution was rapidly injected into the aqueous phase used a needle- syringe 21G and stirred for a 15 min mix at 900 rpm. Afterwards, ethanol was evaporated using a Rota- evaporator instrument and followed by filtering the final preparation using NY 0.1 pm filter, diameter of 30 mm and a glass prefilter. An illustration of the porcess is depicted in Fig. 26.

Physicochemical characterization and morphology

Mean diameter and PDI:

Mean diameter (nm) and polydispersity index (PDI) of the various Liposomal suspensions were characterized using Malvern’s Zeta-sizer (Nano series, Nanos-ZS) at 25°C. Samples were prepared by diluting the liposomal suspension in water by 1:100 in 1 mL of water. Results were the average of 3 measurements per sample (n=3 ± SD) and 14 runs per measure. The instrument measured the mean diameter and PDI, representative of the width of the size distribution.

Cryo-TEM imaging:

Morphological evaluation of aqueous suspensions of Blank/ OPA Lip. was performed using Cryogenic Electron Microscopy (Cryo-TEM). Cryo-TEM observations were assessed using Philips Technai F20 100 KV. Liposomes specimens were prepared by mixing the samples with uranyl acetate for negative staining. pH measurement:

The pH-value of the prepared Lip. was evaluated using a Mettler-Toledo pH- meter. Osmolality:

Osmolality (mOsm/Kg) of the prepared liposomes was evaluated using osmometer 3320 purchased from Advanced Instruments, Inc.

Analytical method development

Description of the HPLC method for OPA assay:

Column: Select®CSH™ C18 5pm, 250x4.6mm

Detector: UV/ PDA

Wavelength: 220nm

Flow rate: ImL/min

Injection volume: 40pL

Column Temperature: 50°C

Mobile Phase: Gradient elution

Eluent A: Acetonitrile

Eluent B: water

Table 42: Gradient mode

OPA liposomes sample preparation

The sample was prepared by diluting the OPA liposomes sample by 20 (50 pl sample + 950 pl of methanol) containing overall 5% of water. Moreover, working stocks were prepared for OPA quantification at three different concentrations as follows:

-125 pg/mL:12.5 mg OPA in 100 mL volumetric flask (5% water in methanol).

-93.5 pg/mL: 18.75 mg OPA in 200 mL volumetric flask (5% water in methanol).

-62.5 pg/mL: 12.5 mg OPA in 200 mL volumetric flask (5% water in methanol). Calculation:

The Assay percentage is calculated by the following equation:

Where:

Asam - Area of peak of due to injected sample solution

Astd - Average Peak Area due to working standard solution

Csam - Concentration of the injected sample solution, pg/mL

Cstd - Concentration of the working standard solution, pg/mL

Lyophilization protocol

OPA liposomes were lyophilized in order to improve their physicochemical stability over storage period. After lyophilization (Lyo.), a dry powder of OPA liposomes is obtained and is further reconstituted in order to conduct all the required measurements including; mean diameter (nm), PDI, OPA assay%, pH, water content% and osmolality (mOsm/Kg).

The lyophilization process was carried out using Christ-lyophilizer, according to the following protocol:

Table 43: Lyophilization protocol for OPA lip. - Program no .9

Water content %:

After the lyophilization protocol, water content of dried-powders was evaluated using Mettler-Toledo Karl-Fischer instrument.

Stability study

Stability study of aqueous OP A liposomal suspensions

The purpose of this stability study was to examine the stability of various OPA liposomal formulations at 4°C over three months in order to identify a stable prototype formulation suitable for efficacy testing and stable over shelf life. OPA liposomes samples were stored in 5 mL clear crimper vials and stored at 4°C.

Table 44: Time intervals for stability study of aqueous (Aq.) OPA Lip, suspensions

Table 45: Stability tests and specifications - Aq. OPA Liposomes

Stability study for lyophilized OPA Liposomes

The purpose of this stability study was to examine the stability of various OPA liposomal formulations after being lyophilized for extending their stability over shelf life compared to aqueous suspensions. OPA liposome samples were stored in 5 mL clear crimper vials and stored at three different temperatures:

1. 4°C ± 2°C (long-term conditions)

2. 25°C ± 2°C / 60% RH ± 5% RH (long-term conditions)

3. 40°C ± 2°C / 75% RH ± 5% RH (accelerated conditions)

Table 46: Time intervals for stability study of lyophilized (Lyo.) OPA liposomes

Table 47: Stability tests and specifications - Lyo. OPA Liposomes

Results

PEGylated OPA Liposomes using Thin-film hydration method

DSPC-lipoid based OPA Liposomes preparation In this part, we prepared two Blank Liposomal preparations and three OPA liposomal preparations of the recipe mentioned below (see Table 48), in an effort to learn about OPA Lip. preparation, to determine the appropriate diluent for analytical analysis of OPA and to determine the optimal storage temperature of OPA Liposomes The concentration of OPA in the recipe below is 2.5 mg/mL.

Table 48: Description of DSPC-lipoid based OPA liposomes recipe 1

Table 49: Characterization of DSPC-lipoid based Blank lip, following recipe 1

*- The osmolality of dextrose solution is 289 mOsm/Kg

According to Table 49, it can be noted that we successfully prepared a Blank Liposomes containing no aggregates, with a mean diameter of ~ 160 nm, PDI< 0.4 and pH ranging between 6.0- 8.0. Therefore, three repetitions of OPA Liposomes were prepared afterwards, following the same recipe. Table 50: Characterization of DSPC-lipoid based OPA Lip, following recipe 1

According to Table 50, it can be noted that we successfully prepared OPA Liposomes, following recipe 1, regarding appearance, physicochemical properties and pH value. Moreover, for the evaluation of OPA assay %, we used three different diluents for the sample preparation; methanol, ethanol and acetonitrile. According to Table 50, we selected methanol to be the optimal diluent for OPA sample preparation due to higher assay % values compared to acetonitrile and higher solubility of OPA compared to ethanol.

Following the preparation of OPA lip. 1 (see Table 50), this preparation was stored at three different temperatures over 2 months for preliminary stability test, and the results were as follows:

Table 51: Preliminary stability test of OPA Lip.1 According to Table 51, it can be noted that a decrease up to 15-20 % in OPA assay % after 1 week of storage was observed at 25 and 40 °C. A decrease up to 50 % in OPA assay % was observed after 2 months of storage at 25 °C. Therefore, 4 °C was selected as the appropriate storage temperature for extending the stability of OPA Liposomes as aqueous suspension.

Tert-butanol vs. chloroform in thin-film preparation of DSPC-lipoid based OPA Liposomes

Two different OPA Liposomal preparations, with two different concentrations of OPA, were prepared using thin-film hydration method using tert-butanol or chloroform for thin-film preparation in an effort to understand the effect of the solvent on thin-film and on final OPA Liposomal preparation.

Table 52: Tert-butanol vs. chloroform in thin-film preparation According to Table 52, it can be noted that no significant effect of tert-butanol or chloroform on OPA lip. preparation was observed.

HSPC-lipoid based OPA Liposomes preparation and lyophilization

OPA Lip.8, HSPC-lipoid based OPA liposomes (see Table 53), was prepared to optimize the lyophilization protocol of OPA Liposomes after the preparation of OPA Lip.8, different cryoprotectants at different ratios relative to Lipids amounts were used. OPA Lip.8 cakes that were obtained after lyophilization were reconstituted and characterized to determine the appropriate type and ratio of cryoprotectant (see Table 54).

Table 53: Description of HSPC-lipoid based OPA liposomes following recipe 2

(OPA Lip.8)

Table 54: Lyophilization results of OPA Lip.8

According to Table 54, it can be noted that among many cryoprotectants that were used, HPβCD at a ratio of 1:2 (in solution) and 1:4 (as a powder) presents preserved values of mean diameter and PDI before and after lyophilization. Therefore, it was selected for further investigation (see Table 55).

Table 55: Lyophilization results of OPA Lip.8 using HPPCD

According to Table 55, it could be noted that we succeeded at lyophilizing OPA Lip. using 1:2 HPβCD; along with preserved properties of appearance, mean diameter, PDI and assay%. Therefore, 1:2 HPβCD will be used as a cryoprotectant for the lyophilization of the coming OPA Lip. preparations.5.2. PEGylated OPA Liposomes using Ethanol injection method

Table 56: Description of HSPC-lipoid based OPA liposomes using ethanol injectionrecipe 3

Table 57: Characterization and preliminary stability of lyophilized OPA Lip.EtOH

(*)- high value of osmolality could be due to ethanol residues after evaporation. According to Table 57, it could be noted that OPA Lip.EtOH showed promising results regarding appearance, mean diameter and PDI before and after lyophilization and after a storage period of 1 month at room temperature (RT). A decrease in OPA assay% and pH was observed after 1 month of storage at RT.

Preliminary Stability of aqueous PEGylated OPA Lip. using Thin-film Hydration method

In this part of the project, we prepared OPA Lip. using Thin-film Hydration method using different types of Lipoids; including DSPC, HSPC, DPPC and DMPC. Three descending concentrations of OPA were used for each lipoid type. Following preparation, we conducted a preliminary stability study for the aqueous suspensions over storage period of 3 months at 4°C.

Preliminary Stability of aqueous PEGylated DSPC-lipoid based OPA Lip. (15- 17) using Thin-film Hydration method

Table 58: Description of OPA Lip. 15- 17

Table 59: Preliminary stability of aqueous OPA Lip.15- 17

According to Table 59, it can be noted that OPA Lip.15- 17 show physical instability as aqueous suspensions, over time, in accordance to OPA concentration. Regarding, physico-chemical characterization and OPA assay %; the obtained values are promising and apply to specifications of Table 55. However, high assay % values could be a consequence of vortexing the sample prior to characterization at the different points of storage period.

Preliminary Stability of aqueous PEGylated HSPC-lipoid based OPA Lip. (18- 20) using Thin-film Hydration method

These OPA Liposomal preparations were prepared using the same recipe mentioned on Table 58; except for replacing DSPC-lipoid with HSPC-lipoid.

Table 60: Preliminary stability of aqueous QPA Lip.18- 20

According to Table 60, it can be noted that OPA Lip.18- 20 show physical instability, after < 1 month, as aqueous suspensions in accordance to OPA concentration. Regarding, physico-chemical characterization and OPA assay %; the obtained values are promising and apply to specifications on Table 45. However, this could be a consequence of vortexing the sample prior to characterization at the different points of storage period.

Preliminary Stability of aqueous PEGylated DPPC-lipoid based OPA Lip. (21- 23) using Thin-film Hydration method

These OPA Lip. preparations were prepared using the same recipe mentioned on Table 58; except for replacing DSPC-lipoid with DPPC-lipoid.

Table 61: Preliminary stability of aqueous OPA Lip.21- 23

According to Table 61, it could be noted that OPA Lip. prepared using DPPC- lipoid show increased physical instability, after 1 week of storage at 4 °C, as aqueous suspensions. Regarding physico-chemical characterization and OPA assay %; the obtained values are promising and apply to specifications on Table 45. However, this could be a consequence of vortexing the sample prior to characterization at the different points of storage period.

Preliminary Stability of aqueous PEGylated DMPC-lipoid based OPA Lip. (24- 26) using Thin-film Hydration method

These OPA Lipsomal preparations were prepared using the same recipe mentioned on Table 58; except for replacing DSPC-lipoid with DMPC-lipoid.

Table 62: Preliminary stability of aqueous OPA Lip.24- 26

According to Table 62, it could be noted that OPA Lip. prepared with DMPC present the highest physical instability as aqueous suspensions compared to OPA Lip. prepared with other lipoids. OPA Lip.24 (2.5 mg/mL) was instable after overnight while OPA Lip.25 and 26 (1.87 and 1.25 mg/mL respectively) were instable after 1 month regarding physical appearance and OPA assay %.

Preliminary Stability of lyophilized PEGylated DSPC-based OPA Lip. using Thin-film Hydration method

Following the same recipe and protocol of OPA Lip.15; we prepared OPA Lip.37 (repetition of OPA Lip.15) with OPA concentration of 2.5 mg/mL and OPA Lip.38 (same recipe of OPA Lip.15) with OPA concentration of 5 mg/mL. Afterwards, these preparations were characterized and lyophilized. Furthermore, a preliminary stability for the lyophilized preparations was carried out (see Table 46 and 47).

Table 63: Preliminary stability of lyophilized OPA Lip.37 and 38

According to Table 63, it could be noted that the physical stability of lyophilized OPA Lip. was significantly improved due to lyophilization even after storage period of 1 month at 40°C (accelerated conditions). Moreover, physico-chemical characterization and OPA assay % were well-preserved after lyophilization over the mentioned storage period.

Preliminary Stability of lyophilized Non-PEGylated OPA Lip. using Thin-film Hydration method

In this part of the project, we prepared a non-PEGylated OPA Liposomal formulation using HSPC and DPPG-NA lipoids. OPA concentration in this preparation is 5 mg/mL. Furthermore, OPA Lip.42a was characterized and lyophilized for further preliminary stability study of the lyophilized preparation (see Table 46 and 47).

Table 64: Description of OPA Lip. 42a

It should be noted, that this formulation was physically instable (precipitated) as an aqueous suspension after an overnight- stand at RT. Therefore, it was crucial to lyophilize it and further evaluate its' stability as a dried-powder.

Table 65: Preliminary stability of lyophilized OPA Lip.42a

According to Table 65, it could be noted that that the physical stability of lyophilized non-PEGylated OPA Lip. was significantly improved due to lyophilization even after storage period of 1 month at 40 °C (accelerated conditions). Moreover, physico-chemical characterization and OPA assay % were well-preserved after lyophilization over the mentioned storage period. Furthermore, regarding stability of the lyophilized preparation over the stability period, no significant difference was observed in non-PEGylated OPA Lip. (see Table 65) compared to PEGylated OPA Lip. 37 and 38 (see Table 63).

Summary

PEGylated OPA Lip. using Thin-film Hydration method

Most of the PEGylated OPA Lip. (15-26) that were prepared and stored as aqueous suspensions at 4 °C were found physically instable after < 1 month. However, the most significant physical instability was observed when using DMPC-lipoid for OPA Lip. preparation compared to the other lipoids (DSPC, HSPC and DPPC). The highest OPA concentration in these studied preparations was 2.5 mg/mL.

Lyophilization of these preparations was proposed in an effort to extend physico-chemical stability of OPA Lip. Different cryoprotectants were used for optimization of the lyophilization process; HPβCD at a ratio of 1:2 (Lipoids amounts in OPA Lip. preparation: HPβCD) was found adequate regarding preservation of physicochemical characteristics and OPA assay % after lyophilization compared to before lyophilization.

Lyophilization of the PEGylated OPA Lip. based on DSPC -lipoid (OPA Lip.37 and 38) successfully improved their physical stability over a storage period of 1 month at 40°C. Moreover, physico-chemical characterization and OPA assay % were well- preserved and apply to specifications during the stability-test period. The highest OPA concentration in these studied preparations was 5 mg/mL.

PEGylated OPA Lip. using Ethanol-injection method

Lyophilized OPA Lip.EtOH showed promising results regarding physicochemical and OPA assay % stability over a storage period of 1 month at RT.

Non-PEGylated OPA Lip. using Thin-film Hydration method

The non-PEGylated OPA Lip.42a preparation with OPA concentration of 5 mg/mL was found physically instable after an overnight- stand at RT. Lyophilization of OPA Lip.42a showed an enhanced physical stability over a storage period of 1 month at 40°C. Moreover, physico-chemical characterization and OPA assay % were well-preserved and apply to specifications during the stability-test period.

Claims

89 CLAIMS:

1. A lipid-based delivery system of oxaliplatin palmitate acetate (OP A), the delivery system comprising a lipid-based assembly and OPA.

2. The delivery system of claim 1, wherein said lipid -based assembly comprises at least one lipid selected from phospholipids, glycerolipids, glycerophospholipids, sphingolipids, and mixtures thereof.

3. The delivery system of claim 1 or 2, wherein the assembly is in the form of a lipid bilayer.

4. The delivery system of claim 3, wherein the assembly is a liposome.

5. The delivery system of claim 4, wherein the liposome is a unilamellar liposome.

6. The delivery system of any one of the preceding claims, wherein said OPA is intercalated in the assembly.

7. The delivery system of claim 3, wherein said OPA is intercalated in said lipid bilayer.

8. The delivery system of any one of claims 1 to 7, wherein said at least one lipid is at least one phospholipid.

9. The delivery system of any one of claims 1 to 8, wherein the assembly comprises at least one phospholipid and at least one sterol.

10. The delivery system of claim 9, wherein the weight ratio between the phospholipids and the sterols in assembly is in the range of between about 1:0.05 and about 1:5.

11. The delivery system of claim 1, wherein the assembly is a lipid nanosphere, said nanosphere comprising a lipid matrix and said OPA is embedded within said matrix.

12. The delivery system of claim 1, wherein the assembly is a nanocapsule having a shell composition comprising said at least one lipid, the shell incorporating said OPA, wherein said nanocapsule is free of a polymeric material.

13. The delivery system of claim 12, wherein said at least one polymeric material is selected from polylactic acid (PLA), polyglycolic acid (PGA), polyhydroxybutyrate, polycaprolactone, poly (orthoesters), poly anhydrides, poly amino acid, poly (alkyl cyanoacrylates), polyphophazenes, copolymers of (PLA/PGA) and asparate or polyethylene-oxide (PEO), and/or copolymers or mixtures thereof. 90

14. The delivery system of any one of claims 1 to 13, wherein said assembly being surface-associated with at least one non-active agent.

15. The delivery system of claim 14, wherein said at least one non-active agent is selected to modulate at least one characteristic of the nanocarrier, said characteristic being selected from size, polarity, hydrophobicity/hydrophilicity, electrical charge, reactivity, chemical stability, clearance rate, distribution and targeting.

16. The delivery system of claim 14 or 15, wherein the non-active agent is polyethylene glycols (PEG).

17. The delivery system of any one of claims 1 to 14, wherein said assembly is non- PEGylated.

18. The delivery system of any one of claims 1 to 17, wherein said assembly having a mean diameter of at most 500 nm.

19. The delivery system of claim 18, wherein said assembly having a mean diameter of between about 20 nm and about 500 nm.

20. The delivery system of any one of claims 1 to 19, further comprising at least one cryoprotectant.

21. The delivery system of any one of claims 1 to 20, wherein the assembly is lyophilized.

22. An oxaliplatin palmitate acetate (OPA) loaded lipid-based nanocarrier.

23. The nanocarrier of claim 22, wherein said lipid-based nanocarrier comprising at least one lipid selected from at least one phospholipid, at least one sterol, and combinations thereof.

24. The nanocarrier of claim 22 or 23, being in the form of a lipid bilayer or a liposome.

25. The nanocarrier of claim 24, wherein the nanocarrier is in the form of a unilamellar liposome.

26. The nanocarrier of any one of claims 22 to 25, wherein the nanocarrier is surface-associated with at least one non-active agent.

27. The nanocarrier of claim 26, wherein said non-active agent is polyethylene glycols (PEG).

28. A composition comprising a lipid-based delivery system according to any one of claims 1 to 21. 91

29. The composition of claim 28, being a pharmaceutical composition.

30. The composition of claim 28 or 29, configured for intravenous administration.

31. A lipid-based delivery system of any one of claims 1 to 21, for use in delivery of OPA to a patient in need thereof.

32. A lipid-based delivery system of any one of claims 1 to 21, for use in treating or delaying progression of a proliferative disorder.

33. A method of treating a proliferative disorder, the method comprising administering an effective amount of lipid-based delivery system of any one of claims 1 to 21, to a subject in need thereof.

34. The method of claim 33, wherein the proliferative disorder is cancer.

35. The method of claim 34, wherein said cancer is selected from ovary cancer, and pancreatic cancer, squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

36. The delivery system of claim 2, wherein the phospholipid is selected from phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), as well as lipid derivatives thereof, such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC) and dipalmitoylphosphatidylglycerol (DPPG).

37. The delivery system of claim 2, wherein the phospholipid comprising an aliphatic chain having at least 18 carbon atoms.

38. The delivery system of claim 37, wherein the phospholipid is fully saturated, unsaturated or partially hydrogenated. 92

39. The delivery system according to claim 2 or 38, wherein the phospholipid is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

40. The delivery system according to claim 1, in a lyophilized form.

41. A liposome comprising at least one phospholipid and OPA, wherein said at least one phospholipid is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

42. The liposome according to claim 41, being surface decorated with a plurality of non-active materials.

43. The liposome according to claim 42, being surface decorated with polyethylene glycol (PEG).

44. The liposome according to any one of claims 41 to 43, comprising 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, OPA and N-(Carbonyl- methoxypolyethyl-eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000).

45. The liposome according to claim 44, wherein the molar ratio of is 5:3:2:0.5, or 5:3:l:0.5, 5:3:0.75:0.5, or 5:3:0.5:0.5 respectively.

46. The liposome according to any one of claims 41 to 43, comprising hydrogenated soy phosphatidylcholine (HSPC), Cholesterol, OPA and (N-(Carbonyl- methoxypolyethyl-eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE-PEG2000).

47. The liposome according to claim 46, wherein the molar ratio is 5:3:2:0.5, or 5:3:l:0.5, 5:3:0.75:0.5, or 5:3:0.5:0.5 respectively.

48. The liposome according to claim 46 or 47, comprising hydrogenated soy phosphatidylcholine (HSPC), Cholesterol, OPA and dipalmitoylphosphatidylglycerol sodium salt (DPPG-Na).

49. The liposome according to claim 48, wherein the molar ratio is 3 :2: 1 : 1.

50. The liposome according to any one of claims 41 to 49, prepared by thin-film hydration or by ethanol injection.