REFERENCE TO RELATED APPLICATION

[0001] The present application claims the priority of U.S. provisional application No.

62/987366, filed on March 10, 2020, the entirety of which is incorporated herein by

5 reference.

BACKGROUND

Technical Field

[0002] The present disclosure relates to a liposome composition, and more particularly 10 to a liposome composition encapsulating a platinum-based precursor and a method for preparing the same.

Related Art

[0003] -di aminedichloropl atinum (11) (CDDP, also known as cisplatin) has been commonly used as a chemotherapeutic agent. However, it is poorly water soluble, and its

15 high toxicity is known to cause various undesirable side effects.

SUMMARY

|0004] To improve solubility and reduce toxicity of CDDP, an embodiment of the present disclosure provides a method for preparing a liposome composition. The method comprises 20 the steps of: providing precursor liposomes encapsulating platinum-based precursors; and incubating the precursor liposomes with a salt solution to convert the platinum-based precursors to platinum-based drugs to form the liposome composition.

[0005] Another embodiment of the present disclosure provides a method for preparing the liposome composition. The method comprises the steps of: providing salt liposomes l encapsulating salts; and mixing the salt liposomes with platinum -based precursors to allow the platinum-based precursors to enter the salt liposomes and interact with the salts so as to convert the platinum-based precursors to platinum -based drugs to form the liposome composition. 10006] Yet another embodiment of the present disclosure provides a method for preparing the liposome composition. The method comprises the steps of: providing precursor liposomes encapsulating platinum-based precursors., and providing salt liposomes encapsulating salts, and mixing the precursor liposomes and the salt liposomes to convert the platinum-based precursors to platinum-based drugs to form the liposome composition. [0007] Still another embodiment of the present disclosure provides a method for preparing the liposome composition. The method comprises the steps of: providing precursor cores encapsulating platinum -based precursors, and providing salt cores encapsulating salts; mixing the precursor cores and the salt cores to convert the platinum- based precursors to platinum-based drugs to form a liposome core; and mixing the liposome core with a first lipid-based formulation to form the liposome composition.

[0008] Yet still another embodiment of the present disclosure provides a liposome composition. The liposome composition is prepared by any of the methods mentioned above. Drug loading of the liposome composition is at least 10%.

[0009] According to the embodiments of the present disclosure, the liposome composition provides an effective solution to improving platinum-based drug solubility and encapsulation efficiency by liposomal particles. By using the methods of the embodiments of the present disclosure, the precursor liposomes can be converted to platinum-based drug encapsulating liposomes with ease and at low cost. The methods also provide an effective tool for enhancing drug loading of liposome compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The disclosure will become more folly understood from the detailed description given herein below for illustration only, and thus not limitative of the disclosure.

[0011] FIG. 1 illustrates a schematic diagram of a preparation of a liposome composition

5 according to an exemplary embodiment of the present disclosure;

[0012} FIG. 2 illustrates a preparation of a CDDP precursor of the liposome composition according to an exemplary embodiment of the present disclosure;

[0013] FIG. 3 illustrates a schematic diagram of a preparation of a liposome composition according to an exemplary embodiment of the present disclosure;

10 [0014| FIG. 4 illustrates a schematic diagram of a preparation of the liposome composition according to an exemplary embodiment of the present disclosure;

[0015] FIG. 5 illustrates a schematic diagram of a preparation of the liposome composition according to an exemplary embodiment of the present disclosure;

[0016] FIG. 6 shows a Cryo-EM image of the liposome composition according to an

15 exem plary embodiment of the present disclosure;

[0017] FIG. 7 shows a size distribution of the liposome composition according to an exemplary embodiment of the present disclosure;

[0018] FIG. 8 shows a pharmacokinetic analysis of the liposome composition in animal models according to an exemplary embodiment of the present disclosure;

20 [0019] FIG. 9 shows changes in tumor growth and tumor volume of human non-small- cell-lung-cancer (NSCLC) adenocarcinoma HI 975 cells xenograft mouse models treated with PBS, CDDP, and LipoCis;

[0020] FIG. 10 shows changes in tumor growth, tumor volume and body weight of A549 xenograft mouse models treated with PBS and LipoCis;

3 [0021] FIG. 11 shows dose-dependent changes In tumor volume, body weight, tumor growth and weight change of lung large cell adenocarcinoma H460 cells xenograft mouse models treated with PBS and LipoCis;

[0022] FIG. 12 shows changes i n tumor volume of human oral squamous cell carcinoma (HOSCC) SAS cells xenograft mouse models treated with PBS, CDDP, and LipoCis; and [0023] FIG 13 shows the tumor reduction effect of the liposome composition according to an exemplary embodiment of the present disclosure in the metastatic SAS cells of

HOSCC mouse models. DETAILED DESCRIPTION

[0024] Referri ng to FIG. 1. In a first embodiment of the present disclosure, a method for preparing a liposome composition is provided. ^' The method may include the steps of: providing precursor liposomes encapsulating platinum -based precursors; and incubating the precursor liposomes in a salt solution to convert the platinum- based precursors to platinum- based drugs to form the liposome composition. Specifically, the precursor liposomes may be prepared by the steps of: hydrating the platinum-based drugs to form the platinum-based precursors, and adding the platinum -based precursors to a lipid bilayer vehicle to form the precursor liposomes ,

[0025] The platinum-based drugs may be hydrated by incubating the platinum -based drugs with silver nitrate ( ), silver sulfate ( ), silver phosphate ( , calcium nitrate ( ), calcium sulfate ( ), calcium phosphate ( ), magnesium ni irate ( ), magnesium sul fate ( ), and/or magnesium phosphate

( .

[0026] In an embodiment., the platinum-based drugs may include at least one platinum- halides bond (e g,, Pt-F. Pt-Cl, Pt-Br, or Pi-I bonds). Some examples of the platinum-based drugs may include cisplatin, triplatin, phenanthriplatin, picoplatin, satraplatin, -diamine diiodo platinum (It), -diamine difluoro platinum (11), and -diamine dibromo platinum

II).

[0027] Referring to FIG. 2. The platinum-based precursors may be monoaqua and/or

5 diaqua forms of the platinum-based drugs; for example, or . The platinum-based precursors may be encapsulated into the lipid bilayer vehicle (e.g., liposomal nanoparticles) due to their water-soluble nature.

[00281 The lipid bilayer vehicle may be prepared by mixing a lipid-based formulation in an organic solvent, such as chloroform, cyclohexane, methanol, ethanol, ethyl acetate, or

10 any combination thereof. The lipid-based formulation may include a combination of choline phospholipids, cholesterols, and polyethylene glycol (PEG)-based compounds. Preferably, the choline phospholipids may include neutral lipids, such as l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dilauroy 1- sn-gly cero-3 -

15 phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-3-phosphocholi ne (DMPC), hexadecyl phosphorylcholine (HePC), 1 -stearoyl -2-oleoyl -sn-gly cero-3 -phosphocholi ne (SOPC), 1 ,2-di phy tanoy I -sn -gly cero-3 -phosphocholine (diPhyPC), or any combination thereof. The PEG-based compounds may be distearoy I phosphatidyl ethanolamine (DSPE> PEG compounds, such as

20 sn-glycero-3-phosphoethanoIamine (DSPE-PEG-200 or DSPE-mPEG-200), DSPE-PEG-

400 (or DSPE-mPEG-400), DSPE-PEG-800 (or DSPE-mPEG-800), DSPE-PEG-1000 (or

DSPE-mPEG- 1000), DSPE-PEG-2000 (or DSPE-mPEG-2000), DSPE-PEG-2500 (or

DSPE-mPEG-2500), DSPE-PEG-3000 (or DSPE-mPEG-3000), D _x SPE-PEG-4000 (or

DSPE-mPEG-4000), DSPE-PEG-5000 (or DSPE-mPEG-5000), DSPE-PEG-6000 (or

25 DSPE-mPEG-6000), DSPE-PEG-10000 (or DSPE-mPE-G- 10000), or any combination

5 thereof. In some embodiments, the PEG-based compounds may be selected from DSPE- PEG-aminoethyl anisamide (DSPE-PEG-AEAA), DSPE-PEG-monocIonal antibodies (DSPE-PEG-mAb), and DSPE-PEG with other ligand moieties.

[0029] The lipid-based formulation may be self-assembled in an aqueous environment via hydrophobic interaction and/or van der Waals interaction to form the lipid bi layer vehicle. In one or more preferred embodiments, neutrality of the lipid-based formulation provides minimum energy bonding to the encapsulated active pharmaceutical ingredients (API) or precursor ^’ s thereof therefore facilitating drug release in vivo. Furthermore, as the neutral lipid bilayer vehicle does not interact with the charged precursors, drug conversion occurred therein would not be affected or hindered.

[0030] In the embodiment, the volume ratio of the lipid bilayer vehicle to the CDDP precursors in the liposome composition may fall within 1 : 1 to 20: 1. The molar ratio of the CDDP precursors to the lipid bi layer vehicle may fail within 0.1:1 to 1:1. The CDDP precursors may be added to the lipid bilayer vehicle at an oil to water ratio of 1 :0.01 to 1 :0.8. The concentration of the platinum -based precursors added to the lipid bilayer vehicle or in the resulting precursor liposomes may fall within a range of 25 laM to 600 roM, preferably 1 .5 mM to 5 niM.

[0031] To convert the platinum-based drugs from the platinum-based precursors, the precursor liposomes may be incubated in the salt solution to allow the salts to enter the precursor liposomes. In the embodiment, the salt solution may include fluoride, chloride, bromide, iodide, or other salts of the halogen group. A concentration of the salt solution may fall within a range of 0.2 M to 4 M; more specifically, when NaCl is used for the conversion, the concentration ofNaC! may fall within a range of 0.4 M to 3,9 M; when KC1 is used, the concentration of KC1 may fall within a range of 0.4 M to 3.0 M In an embodiment, the incubation may be earned out at 4-65 T for 1 -24 h to allow the salts to enter the precursor liposomes and convert CDDPs from diaqua CDDP precursors. For example, the precursor liposomes may be incubated with the salt solution at 4-TC overnight, or at 10-50 ^º C for 2-

15 h followed by cooling to stabilize the structure of the liposome composition.

[0032] In one or more embodiments, the high concentration of the salt solution generates

5 an osmotic pressure that pushes the halogen ions through the lipid bilayer vehicle irreversibly and allows the halogen ions to stay inside of the precursor liposomes, without affecting the stability of the liposome structure. As the halogen ions are being consumed inside the precursor liposomes for API conversion, more halogen ions would continue to diffuse into the precursor liposomes. Such osmosis-based approach presents a cost-effective

10 and time-efficient route for driving drug conversion inside the precursor liposomes.

[0033] In an example, to prepare a liposome composition (abbreviated hereunder as

LipoCis) in which CDDP is the platinum-based drug encapsulated in a lipid bilayer vehicle made of DSPC, cholesterol, and DSPE-PEG-2000, CDDP precursors may be obtained by incubating 0.2-0.4 mmol of CDDP with 0.3-0.4 mmol of silver nitrate ( ) at 25 ^º C 15 for 16-18 h or at 60C for 3-4 h. Thereafter, the LipoCis may be formed firstly by mixing

DSPC, cholesterol, and 40-50:25-50:10-30 w/w% of DSPE-PEG-2000 at 30-60°C under

100-400 rpm for 10-60 min to form the lipid bilayer vehicle. The CDDP precursors may then be added into the lipid bilayer vehicle at the v/v ratio of 1 :0,01 to 1 :0.8 oil-to-water by using a micro-volume dropper at 1 mL/min or by bulk mixing followed by either

20 handshaking or stirring for 15-30 min to form the precursor liposomes. The liposome encapsulating the CDDP precursors was then homogenized for 1-10 passes to reach a liposome size of 60-250 nm. Finally, the CDDP precursors in the liposomes were converted to CDDPs by incubating the precursor liposomes in 0.2-3.9M of potassium chloride (KC1) or sodium chloride (NaCl) at 25-50°C and stirring for about 2-24 b. The resulting LipoCis 25 may be purified by using a tangential flow filtration (TFT) system to remove excess salts

7 and be exchanged into a 10 mM HEPES, 5% glucose buffer (pH 6.5-7.6), a 10 mM HEPES,

0.9% saline buffer (pH 6.5-7.6), a 0.9% saline solution, 5% glucose solution, or ddHaO for storage. The dmg-to-lipid (D/L) ratio of the resulting LipoCis may range up to 0.05-0.8 per mole.

5 10034] Referring to FIG. 3. In a second embodiment, another method for preparing a liposome composition is provided. The method may include the steps of: providing salt liposomes encapsulating salts; and incubating the salt liposomes with platinum-based precursors to allow the platinum-based precursors to enter the salt liposomes and interact with the salts so as to convert the platinum-based precursors to platinum-based drags to

10 form the liposome composition. Specifically, the salt liposomes may be prepared by adding the salts to a lipid bilayer vehicle to form the salt liposomes. Ingredients and preparation details of the second embodiment are similar to those of the first embodiment.

[0035] Referring to FIG. 4. In a third embodiment, yet another method for preparing the liposome composition is provided. The method may include the steps of: providing

15 precursor liposomes encapsulating platinum-based precursors and providing salt liposomes encapsulating salts; and mixing the precursor liposomes and the salt liposomes to convert the platinum-based precursors to platinum-based drugs to form the liposome composition.

Specifically, the precursor liposomes may be prepared by the steps of: hydrating the platinum-based drugs to form the platinum-based precursors; and adding the platinum-based

20 precursors to a lipid bilayer vehicle to form the precursor liposomes. Similarly, the salt liposomes may be prepared by step of: adding the salts to a lipid bilayer vehicle to form the salt liposomes. Ingredients and preparation details of the third embodiment are similar to those of the first embodiment.

[0036) Referring to F.1G. 5. In a fourth embodiment, still another method for preparing

25 the liposome composition is provided. The method may include the steps of: providing

8 precursor cores encapsulating platinum-based precursors and providing salt cores encapsulating salts; mixing the precursor ewes and the salt cores to convert the platinum- based precursors to platinum-based drugs to form a liposome core; and mixing the liposome core with a first lipid-based formulation to form the liposome composition. Specifically, the

5 precursor cores may be prepared by step of: hydrating the platinum-based drugs to form the platinum-based precursors; and adding the platinum-based precursors to a lipid monolayer vehicle to form the precursor cores. Similarly, the salt cores may be prepared by step of: adding the salts to a lipid monolayer vehicle to form the salt cores. Ingredients and preparation details of the fourth embodiment are similar to those of the first embodiment.

10 [0037) In the embodiment, the first lipid-based formulation may include cholesterol and

PEG-based compounds. The first lipid-based formulation may be dissolved in an oiganic solvent (e.g., chloroform, ethanol, ethyl acetate), and then mixed with the liposome cores at

25-45º C for 25-60 min. For example, as illustrated in FIG. 5, after the lipid-based formulation is added, the cholesterol would stabilize the monolayered liposome core and

15 the DSPE-PEG-2000 would coat the liposome core to result in the bilayered liposome composition.

[0038J The lipid monolayer vehicle may be prepared by mixing a second lipid-based formulation in an organic solvent (e.g., chloroform, ethanol, ethyl acetate). The second lipid- based formulation may include DSPC and/or other choline phospholipids. When chloroform

20 or other oil-based solvent is used, the lipid monolayer vehicle could form immediately therein. In a water-miscible system (e.g., EtOH), heating at 45-60 ^º C for about 15-30 min may be required to form the lipid monolayered vehicle.

[0039) As evidenced by the high conversion rate and drug loading shown in Table 1, the methods of the embodiments of the present disclosure as mentioned above can effectively 25 encapsulate and convert the platinum-based precursors to the platinum-based drugs.

9 Accordingly, the calculated drug loading of the liposome composition prepared by the embodiments of the present disclosure could be as high as 62%.

[0040] Table 1. Drug loading (DL) of the LipoCis of the embodiments

(00411 Referring to FIGS. 6 and 7. in an example, CDDP Is encapsulated and precipitated into a lipid bilayer made of DSPC, cholesterol and DSPC-PEG-2000 to form LipoCis nanoparticles (NPs). The LipoCis NPs may be characterized by various approaches. In the example shown in FIGS. 6 and 7, particle size and zeta potential were measured using the Malvern Zetasizer Nano series (Westborough, MA, US). Morphology of the LipoCis NPs was captured using cryogenic electron microscopy (Cry o- EM). The amount of CDDP was measured using high performance liquid chromatography (HPLC) and the Ft content in the LipoCis NPs was validated using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) or inductively coupled plasma-optical emission spectrometry (ICP-QES), The excipient concentration was determined using HPLC evaporative light scattering detectors (ELSD). As shown in FIG. 6, the morphology of the LipoCis NPs as captured by Cryo-EM revealed a fine and homogeneous monodispersity, with an approximate particle size in the range 80-150 nm, which is equivalent to that of dynamic light scattering (DLS) measurement. As shown in FIG. 7, all interaction polymer chromatography (IPC) points of the LipoCis NPs were measured.

(0042] Referring to FIG. 8. A pharmacokinetic analysis of the LipoCis prepared according to the first embodiment of the present disclosure was performed in a rat model. As shown in Table 2, the LipoCis resulted in low in vivo clearance (CL), high area under the curve (AUC), and long circulatoiy time (i.e., Vz and Vss values were lower than those of API). No statistically significant difference was observed between LipoCis and API in their half-life and mean residence time (MRT). The pharmacokinetic results suggested a sustained release of the LipoCis in vivo . |0043] Table 2

(0044] To assess the inhibitory potential of LipoCis on tumor growth, xenograft experiments were conducted for 21 days and the xenograft animal models were monitored daily. In the experiments, human cancer cell lines (lx!O* cells/in 200 uL PRS-Martigel 1 :1 solution) were subcutaneously injected in the right hind legs of Balb/c nude mice. After a considerably sized tumor had appeared, the tumor size is measured daily or every other day and calculated by the formula (lengthxwidthxheight)/2. When the tumor size reached to the desired size (e.g., 100-210 mm ³ , LipoCis samples were intravenously injected into the tumor-bearing mice once per week for 3 weeks,

[0045] Referring to FIGS. 9 to 1 1. Three lung cancer cell lines, including human non- small -cell -lung-cancer adenocarcinoma (NSCLC) H 1975 cells and A549 cells, and human large ceil carcinoma H460 cells, were tested to validate the in vivo efficacy of the LipoCis NPs As shown in FIG. 9, in the H1975 lung adenocarcinoma cell xenograft mice models, LipoCis prepared according to the embodiments of the present disclosure was shown to induce a more significant apoptotic response (by immunostaining studies) as well as stronger tumor inhibitory effects than CDDP As shown in FIGS. 10 to 11, similar results were also observed in the A549 lung adenocarcinoma cells and H460 lung large cell carcinoma cells xenograft mice models.

(0046] Referring to FIGS, 12 and 13, A human oral squamous cell carcinoma (HOSCC) xenograft animal model was also established. 160 uL of 5 x lO ⁶ SAS human oral cancer cells in the presence of 200 p.L matrigel (Corning, Bedford, MA) were subcutaneously injected using a 28-gauge needle at the lower right dorsal flank of 7 to 9- week-old male B ALB/cAnN.Cg-.Foxn l nu nude mouse (from National Laboratory Animal Center, Taipei, Taiwan) The SAS xenograft mice were randomly separated into three groups and treated with (i) phosphate-buffered saline (PBS); (ii) CDDP; and (iii) LipoCis NPs. All of the treatments were administered intravenously. The CDDP or LipoCis NPs were given at a similar dose of 3.0 mg/kg. The treatment procedure was carried out when tumors reached 200.1 mnr ± 3.5 (or 195-210 mm ³ ) Tumor volume was determined as length x width x high x 0.5. The mice were sacrificed on the 12th day. Excised tumors and organs were dissected and fixed in 10% formalin for further experiments. These studies were approved and carried out in strict accordance with the recommendations in the Guide for the Care and Use of the Institutional Animal Care and Use Committee of Chung Yuan Christian University, Chungli, Taoyuan, Taiwan, ROC.

[0047| To examine the efficacy of the LipoCis NPs in vivo , the S AS human oral tumorbearing xenograft models with 200.1*3.5mm ³ tumor volume were randomly clustered into three different treatment groups, including (i) PBS, (ii) CDDP; and (iii) LipoCis. Each group received two cycles of treatment with a 6-day interval between each cycle. As demonstrated by the results shown in FIGS. 9 and 10, LipoCis served a potential implication in growth inhibition of SAS tumors,

10048] According to the embodiments of the present disclosure as mentioned above, the LipoCis provided an effective solution to improving platinum-based drag solubility and encapsulation efficiency by liposomal particles. By using the methods of the embodiments of the present disclosure, the precursor liposomes can be converted to platinum-based drug encapsulating liposomes with ease and at tow cost The methods also provide an effective tool for enhancing drug loading of liposome compositions.

[0049] While the instant disclosure has been described by the way of example and in

5 terms of the preferred embodiments, it is to be understood that the invention need not be limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures.

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