Patent Description:
cis-diaminedichloroplatinum (II) (CDDP, also known as cisplatin) has been commonly used as a chemotherapeutic agent. However, it is poorly water soluble, and its high toxicity is known to cause various undesirable side effects.

To improve solubility and reduce toxicity of CDDP, present disclosure provides a method for preparing a liposome composition. The method comprises 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.

Drug loading of the liposome composition is at least <NUM>%.

According to 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.

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

Referring to <FIG>. A method for preparing a liposome composition is provided. The method comprises 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 are 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.

The platinum-based drugs are hydrated by incubating the platinum-based drugs with silver nitrate (AgNO<NUM>), silver sulfate (Ag<NUM>SO<NUM>), silver phosphate (Ag<NUM>PO<NUM>), calcium nitrate (Ca(NO<NUM>)<NUM>), calcium sulfate (CaSO<NUM>), calcium phosphate (Ca<NUM>(PO<NUM>)<NUM>), magnesium nitrate (Mg(NO<NUM>)<NUM>), magnesium sulfate (MgSO<NUM>), and/or magnesium phosphate (Mg(H<NUM>PO<NUM>)<NUM>).

The platinum-based drugs comprise at least one platinum-halides bond (e.g., Pt-F, Pt-Cl, Pt-Br, or Pt-I bonds). Some examples of the platinum-based drugs may include cisplatin, triplatin, phenanthriplatin, picoplatin, satraplatin, cis-diamine diiodo platinum (II), cis-diamine difluoro platinum (II), and cis-diamine dibromo platinum (II).

Referring to <FIG>. The platinum-based precursors may be monoaqua and/or diaqua forms of the platinum-based drugs; for example, cis-[Pt(NH<NUM>)<NUM>(H<NUM>O)<NUM>](NO<NUM>)<NUM> or cis-[Pt(NH<NUM>)<NUM>(H<NUM>O)<NUM>]<NUM>+. The platinum-based precursors may be encapsulated into the lipid bilayer vehicle (e.g., liposomal nanoparticles) due to their water-soluble nature.

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 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 <NUM>,<NUM>-distearoyl-sn-glycero-<NUM>-phosphocholine (DSPC), <NUM>,<NUM>-dioleoyl-sn-glycero-<NUM>-phosphocholine (DOPC), <NUM>,<NUM>-dipalmitoyl-sn-glycero-<NUM>-phosphocholine (DPPC), <NUM>,<NUM>-dilauroyl-sn-glycero-<NUM>-phosphocholine (DLPC), <NUM>,<NUM>-dimyristoyl-sn-glycero-<NUM>-phosphocholine (DMPC), hexadecyl phosphorylcholine (HePC), <NUM>-stearoyl-<NUM>-oleoyl-sn-glycero-<NUM>-phosphocholine (SOPC), <NUM>,<NUM>-diphytanoyl-sn-glycero-<NUM>-phosphocholine (diPhyPC), or any combination thereof. The PEG-based compounds may be distearoylphosphatidyl ethanolamine (DSPE)-PEG compounds, such as [N-(carbonyl-methoxypolyethylene glycol-<NUM>)-<NUM>,<NUM>-distearoyl-sn-glycero-<NUM>-phosphoethanolamine (DSPE-PEG-<NUM> or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), DSPE-PEG-<NUM> (or DSPE-mPEG-<NUM>), or any combination thereof. In some embodiments, the PEG-based compounds may be selected from DSPE-PEG-aminoethyl anisamide (DSPE-PEG-AEAA), DSPE-PEG-monoclonal antibodies (DSPE-PEG-mAb), and DSPE-PEG with other ligand moieties.

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 bilayer 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 precursors 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.

In the embodiment, the volume ratio of the lipid bilayer vehicle to the CDDP precursors in the liposome composition may fall within <NUM>:<NUM> to <NUM>:<NUM>. The molar ratio of the CDDP precursors to the lipid bilayer vehicle may fall within <NUM>:<NUM> to <NUM>:<NUM>. The CDDP precursors may be added to the lipid bilayer vehicle at an oil to water ratio of <NUM>:<NUM> to <NUM>:<NUM>. 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 <NUM> to <NUM>, preferably <NUM> to <NUM>.

To convert the platinum-based drugs from the platinum-based precursors, the precursor liposomes are 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 <NUM> to <NUM>; more specifically, when NaCl is used for the conversion, the concentration of NaCl may fall within a range of <NUM> to <NUM>; when KCl is used, the concentration of KCl may fall within a range of <NUM> to <NUM>. In an embodiment, the incubation may be carried out at <NUM>-<NUM> for <NUM>-<NUM> 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 <NUM>-<NUM> overnight, or at <NUM>-<NUM> for <NUM>-<NUM> followed by cooling to stabilize the structure of the liposome composition.

In one or more embodiments, the high concentration of the salt solution generates 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 and time-efficient route for driving drug conversion inside the precursor liposomes.

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-<NUM>, CDDP precursors may be obtained by incubating <NUM>-<NUM> mmol of CDDP with <NUM>-<NUM> mmol of silver nitrate (AgNO<NUM>(aq)) at <NUM> for <NUM>-<NUM> or at <NUM> for <NUM>-<NUM>. Thereafter, the LipoCis may be formed firstly by mixing DSPC, cholesterol, and <NUM>-<NUM>:<NUM>-<NUM>:<NUM>-<NUM> w/w% of DSPE-PEG-<NUM> at <NUM>-<NUM> under <NUM>-<NUM> rpm for <NUM>-<NUM> to form the lipid bilayer vehicle. The CDDP precursors may then be added into the lipid bilayer vehicle at the v/v ratio of <NUM>:<NUM> to <NUM>:<NUM> oil-to-water by using a micro-volume dropper at <NUM>/min or by bulk mixing followed by either handshaking or stirring for <NUM>-<NUM> to form the precursor liposomes. The liposome encapsulating the CDDP precursors was then homogenized for <NUM>-<NUM> passes to reach a liposome size of <NUM>-<NUM>. Finally, the CDDP precursors in the liposomes were converted to CDDPs by incubating the precursor liposomes in <NUM>-<NUM> of potassium chloride (KCl) or sodium chloride (NaCl) at <NUM>-<NUM> and stirring for about <NUM>-<NUM>. The resulting LipoCis may be purified by using a tangential flow filtration (TFF) system to remove excess salts and be exchanged into a <NUM> HEPES, <NUM>% glucose buffer (pH <NUM>-<NUM>), a <NUM> HEPES, <NUM>% saline buffer (pH <NUM>-<NUM>), a <NUM>% saline solution, <NUM>% glucose solution, or ddH<NUM>O for storage. The drug-to-lipid (D/L) ratio of the resulting LipoCis may range up to <NUM>-<NUM> per mole.

As evidenced by the high conversion rate and drug loading shown in Table <NUM>, the method of the present disclosure as mentioned above can effectively encapsulate and convert the platinum-based precursors to the platinum-based drugs. Accordingly, the calculated drug loading of the liposome composition prepared by the embodiments of the present disclosure could be as high as <NUM>%.

Referring to <FIG>. In an example, CDDP is encapsulated and precipitated into a lipid bilayer made of DSPC, cholesterol and DSPC-PEG-<NUM> to form LipoCis nanoparticles (NPs). The LipoCis NPs may be characterized by various approaches. In the example shown in <FIG>, 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 (Cryo-EM). The amount of CDDP was measured using high performance liquid chromatography (HPLC) and the Pt content in the LipoCis NPs was validated using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) or inductively coupled plasma-optical emission spectrometry (ICP-OES). The excipient concentration was determined using HPLC evaporative light scattering detectors (ELSD). As shown in <FIG>, 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 <NUM>-<NUM>, which is equivalent to that of dynamic light scattering (DLS) measurement. As shown in <FIG>, all interaction polymer chromatography (IPC) points of the LipoCis NPs were measured.

Referring to <FIG>. 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 <NUM>, the LipoCis resulted in low in vivo clearance (CL), high area under the curve (AUC), and long circulatory 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.

To assess the inhibitory potential of LipoCis on tumor growth, xenograft experiments were conducted for <NUM> days and the xenograft animal models were monitored daily. In the experiments, human cancer cell lines (<NUM>×<NUM><NUM> cells/in <NUM>µL PBS-Martigel <NUM>:<NUM> 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 (length×width×height)/<NUM>. When the tumor size reached to the desired size (e.g., <NUM>-<NUM><NUM>), LipoCis samples were intravenously injected into the tumor-bearing mice once per week for <NUM> weeks.

Referring to <FIG>. Three lung cancer cell lines, including human non-small-cell-lung-cancer adenocarcinoma (NSCLC) H1975 cells and A549 cells, and human large cell carcinoma H460 cells, were tested to validate the in vivo efficacy of the LipoCis NPs. As shown in <FIG>, 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 <FIG>, similar results were also observed in the A549 lung adenocarcinoma cells and H460 lung large cell carcinoma cells xenograft mice models.

Referring to <FIG>. A human oral squamous cell carcinoma (HOSCC) xenograft animal model was also established. <NUM>µL of <NUM> × <NUM><NUM> SAS human oral cancer cells in the presence of <NUM>µL matrigel (Corning, Bedford, MA) were subcutaneously injected using a <NUM>-gauge needle at the lower right dorsal flank of <NUM> to <NUM>-week-old male BALB/cAnN. Cg-Foxn1nu 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 <NUM>/kg. The treatment procedure was carried out when tumors reached <NUM><NUM> ± <NUM> (or <NUM>-<NUM><NUM>). Tumor volume was determined as length × width × high × <NUM>. The mice were sacrificed on the 12th day. Excised tumors and organs were dissected and fixed in <NUM>% 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.

To examine the efficacy of the LipoCis NPs in vivo, the SAS human oral tumor-bearing xenograft models with <NUM>±<NUM><NUM> 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 <NUM>-day interval between each cycle. As demonstrated by the results shown in <FIG> and <FIG>, LipoCis served a potential implication in growth inhibition of SAS tumors.

Claim 1:
A method for preparing a liposome composition, comprising 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,
wherein the precursor liposomes are prepared by hydrating the platinum-based drugs to form the platinum-based precursors; mixing a lipid-based formulation to form a lipid bilayer vehicle; and adding the platinum-based precursors to the lipid bilayer vehicle to form the precursor liposomes,
wherein the platinum-based drugs comprise at least one platinum-halide bond; and
the lipid-based formulation comprises DSPC, cholesterol, and a member selected from a group consisting of DSPE-PEG-<NUM>, DSPE-PEG-<NUM>, DSPE-PEG-<NUM>, DSPE-PEG-<NUM>, and DSPE-PEG-<NUM>.