KR20230152921A - Liposome comprising rapamycin or a derivative thereof and use thereof in therapy - Google Patents

Abstract

The present disclosure relates to lipid-based formulations comprising rapamycin and derivatives thereof, and also to the use of the formulations for the treatment of diseases or conditions, such as cancer, immune-related diseases, etc.

Description

Liposomes containing rapamycin or derivatives thereof and their use in therapy {LIPOSOME COMPRISING RAPAMYCIN OR A DERIVATIVE THEREOF AND USE THEREOF IN THERAPY}

The present disclosure relates to lipid-based formulations comprising rapamycin and its derivatives and their applications.

The mammalian target of rapamycin (mTOR) is a kinase encoded in humans by the MTOR gene. mTOR integrates inputs from upstream pathways, and the mTOR pathway is a central regulator of mammalian metabolism and physiology, with an important role in tissue function. For example, mTOR signaling can lead to diseases related to metabolism, immunity, brain function, aging, and even cancer. Modulation, usually inhibition, of mTOR can improve or treat diseases and conditions. The first known inhibitor of mTOR is rapamycin, from which the name mTOR is derived:

Rapamycin, M.W.: 914.17 g/mol.

Clinical applications of rapamycin include use as an immunosuppressant, treatment of tuberous sclerosis complex, drug-eluting coronary stents, etc. Rapamycin as a BCS Class II drug, i.e., a BCS Class II drug with low water solubility (2.6 μg/mL) and high permeability, exhibits a bioavailability of only 14% to 18%, and thus, this makes it a promising candidate for clinical development as an anticancer agent. interfere with A solution to overcome the low solubility of rapamycin is to develop its analogs or derivatives, i.e. “rapalogs”. Examples of rapallogs include everolimus, temsirolimus, etc. Although everolimus exhibits ~20% bioavailability in tablet form, and temsirolimus can even exhibit up to 100% bioavailability via intravenous infusion, oral Rapalog has low bioavailability, and injectable Rapalog Rogue is highly allergic due to its excipients. Therefore, there is still no clinically available formulation of rapamycin or Rapalog with high bioavailability, high efficacy, and low toxicity.

The present invention relates, in part, to rapamycin or an analogue thereof, or a prodrug or salt of rapamycin, or an analogue thereof, which has excellent stability, excellent tumor inhibition ability and reduced toxicity before and after lyophilization and hydration of rapamycin or an analogue thereof. It is based on the development of specific liposomes.

The present disclosure provides a liposome comprising a lipid component encapsulating rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof, comprising:

Lipid components include cholesterol, phosphatidylcholine (PC), L-α-phosphatidylcholine (EggPC), 1,2-didecanoyl-sn-glycerol-3-phosphocholine (DDPC), 1,2-distearoyl-sn -Glycerol-3-phosphorylethanolamine (DSPE), distearoyl phosphatidylcholine (DSPC), dioleyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2 -Palmitoyl-phosphatidyl acid (DPPA), 1,2-dimyristoyl phosphatidylcholine (DMPC), dioleyl phosphatidylcholine (DOPC), palmitoyl-myristoyl phosphatidylcholine (PMPC), palmitoyl-oleoyl phosphatidylcholine (POPC) ), dioleyl phosphatidylglycerol (DOPG), distearoyl phosphatidyl glycerol (DSPG), dipalmitoyl phosphatidylglycerol (DPPG), dipalmitoyl phosphatidylethanolamine (DPPE), or PEG and combinations thereof. selected,

Rapamycin analogs can inhibit the mammalian target of rapamycin (mTOR);

The amount of lipid component, based on the total dry weight of the liposome, ranges from about 30% (w/w) to 95% (w/w); The amount of rapamycin or an analog thereof, or a prodrug or salt of rapamycin or an analog thereof, based on the total dry weight of the liposome, ranges from about 5% (w/w) to 30% (w/w),

Liposomes are provided.

In one embodiment, the liposome is a poly(ethylene glycol) (PEG)-modified liposome.

In some embodiments, the analog of rapamycin is everolimus, temsirolimus, tacrolimus, prerapamycin, zotarolimus, ridaforolimus, 7-epi-rapamycin, 7-thiomethyl- Rapamycin, 7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin, 7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-desmethyl-rapamycin, and 42-O-(2-hydroxy)ethyl rapamycin, rapamycin oxime, rapamycin amino ester, rapamycin dialdehyde, rapamycin 29-enol, O-alkylated rapamycin derivatives, water-soluble rapamycin ester, alkylated rapamycin Mycin derivatives, rapamycin amidino carbamate, biotin ester of rapamycin, carbamate of rapamycin, rapamycin hydroxy ester, rapamycin 42-sulfonate, 42-(N-carboalkoxy) sulfamate, rapamycin Mycin oxepane isomers, imidazolidinyl rapamycin derivatives, rapamycin alkoxyesters, rapamycin pyrazole, acyl derivatives of rapamycin, rapamycin amide ester, rapamycin fluorinated ester, rapamycin acetal, oxoraphamycin, and rapamycin. is selected from the group consisting of silyl ethers.

In some embodiments, rapamycin or an analog thereof, or a prodrug or salt of rapamycin or an analog thereof, is present in an amount of about 5% (w/w) to 25% (w/w) based on the total dry weight of the liposome. , about 5% (w/w) to 20% (w/w), about 10% (w/w) to 30% (w/w), about 10% (w/w) to 25% (w/w) ), about 10% (w/w) to 20% (w/w), about 15% (w/w) to 30% (w/w), about 15% (w/w) to 25% (w/w) w) or in the range of about 15% (w/w) to 20% (w/w). In some further embodiments, rapamycin or an analog thereof, or a prodrug or salt of rapamycin or an analog thereof, is present in an amount of about 10% (w/w), about 12% (w), based on the total dry weight of the liposome. /w), about 14% (w/w), about 15% (w/w), about 16% (w/w), about 17% (w/w), about 18% (w/w), about 5% (w/w) to 20% (w/w), about 10% (w/w) to 20% (w/w), about 15% (w/w) to 25% (w/w), or It is about 15% (w/w) to 20% (w/w).

In some embodiments, the lipid component is DOPE, DDPC, cholesterol, DSPE, EggPC, HSPC, DPPC, DMPC, DSPC, PC; A combination of DPPC, DDPC, cholesterol and DSPE (or DSPE-PEG); A combination of DOPE, DDPC, cholesterol and DSPE (or DSPE-PEG); A combination of HSPC and DDPC; A combination of DSPC and DDPC; A combination of DOPE and HSPC; or a combination of DOPE and DSPC. In some further embodiments, the lipid component is from about 65% (w/w) to about 95% (w/w) or from 70% (w/w) to about 90%, based on the total dry weight of the liposome. (w/w) DOPE, DDPC, cholesterol, DSPE, EggPC, HSPC, DMPC, DPPC, DSPC, or PC, or about 15% (w/w) to about 65% (w/w) DPPC, about 20 % (w/w) to about 65% (w/w) DDPC, from about 0% (w/w) to about 30% (w/w) cholesterol and from about 15% (w/w) to about 65% (w/w) of a combination of DSPE (or DSPE-PEG), from about 25% (w/w) to about 60% (w/w) DOPE, from about 25% (w/w) to about 70% ( w/w) of DDPC, from about 0% (w/w) to about 20% (w/w) cholesterol and from about 0% (w/w) to about 25% (w/w) of DSPE (or DSPE- PEG), a combination of about 40% (w/w) to about 55% (w/w) HSPC and about 30% (w/w) to about 40% (w/w) DDPC, about A combination of 40% (w/w) to about 55% (w/w) DSPC and about 30% (w/w) to about 40% (w/w) DDPC, about 35% (w/w) A combination of from about 45% (w/w) DOPE and from about 35% (w/w) to about 50% (w/w) HSPC, or from about 35% (w/w) to about 45% (w) /w) of DOPE and about 35% (w/w) to about 50% (w/w) of DSPC. The amount of rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof in the above embodiments is as previously described.

In some further embodiments, the amounts of the lipid component and rapamycin or an analog thereof, or a prodrug or salt of rapamycin or an analog thereof, based on the total dry weight of the liposome, are as described below.

In one embodiment, the average particle size of liposomes ranges from about 100 nm to about 500 nm. In some embodiments, the average particle size of liposomes is about 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm. , 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, or within a range consisting of any two values noted above, e.g. For example, it may be 100 nm to 500 nm, 150 nm to 450 nm, 100 nm to 250 nm, 180 nm to 220 nm, etc.

The present disclosure also provides liposomal formulations comprising the liposomes of the present disclosure and a cryoprotectant.

In one embodiment, the cryoprotectant is a disaccharide. In some embodiments, the cryoprotectant is sucrose or trehalose. In one embodiment, the cryoprotectant is present in an amount of about 80% (w/w), about 84% (w/w), about 87% (w/w), about 90% (w/w) based on the total dry weight of the liposome. w), about 94% (w/w), about 95% (w/w), about 97% (w/w), or within a range consisting of any two values noted above, for example, from about 80% to About 97%, about 84% to about 98%, etc.

The present disclosure also provides treatment of cancer, diabetes, obesity, neurological disease, and genetic disorder in a subject, including administering to the subject a therapeutically effective amount of a liposome of the present disclosure. and/or provide a method of preventing organ transplant rejection.

Figures 1A and 1B show different ratios of particle sizes of liposomal rapamycin formulations.
Figures 2A, 2B, 3A, and 3B show the properties of lyophilized and rehydrated lyophilized products.
Figures 4A and 4B show in vivo toxicity results for Balb/c mice.
Figures 5a and 5b show the results of in vivo experiments on SKOV3 xenograft NOD/SCID mice.
Figures 6a and 6b show the results of in vivo experiments on MDA-MB-231 xenograft NOD/SCID mice.
Figures 7a and 7b show the results of in vivo experiments on CT26 Balb/c mice.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. All patents, applications, published applications and other publications are hereby incorporated by reference in their entirety. In the event that there are multiple definitions for a term, the definition in this paragraph shall prevail, unless otherwise stated.

As used herein, the term “rapalog” refers to a derivative of rapamycin that has inhibitory activity against mTOR.

As used herein, the term “liposome” refers to a microscopic closed vesicle with an internal phase surrounded by a lipid bilayer. Liposomes include small single-membrane liposomes, such as small unilamellar vesicles (SUV), large single-membrane liposomes, such as large unilamellar vesicles (LUV), and larger single-membrane liposomes, such as, giant unilamellar vesicles (GUVs), multilamellar liposomes with multiple concentric membranes, such as multi-lamellar vesicles (MLVs), or liposomes with irregular, non-concentric multiple membranes, such as multivesicular ones. It may be a multivesicular vesicle (MVV). According to the present disclosure, liposome is a general term encompassing a variety of single- and multi-lamellar lipid vehicles formed by the creation of enclosed lipid bilayers or aggregates. Liposomes can generally be characterized as having a vesicular structure with a bilayer membrane containing phospholipids and an internal medium. Liposomes can range in size from a few nanometers to a few micrometers in diameter. Liposomes used according to the present disclosure can be produced by various methods known to those skilled in the art. Additional details regarding the preparation of liposomes are described in U.S. Patent No. 4,342,826 and PCT International Publication No. WO 80/01515, both of which are incorporated by reference.

As used herein, the term “tumor” refers to a neoplasm and includes both benign and malignant tumors. This term particularly includes malignant tumors, which may be solid or non-solid. Tumors may also be further divided into subtypes, such as adenocarcinoma.

As used herein, the term “(therapeutically) effective dose/amount” is a dose/amount sufficient to inhibit the progression of a disease or cause regression, or to alleviate symptoms caused by a disease.

As used herein, the term “mammal” or “mammalian subject” includes farm animals such as cattle, pigs and sheep, as well as pets or animals used in sports such as horses, dogs, and cats.

Rapamycin (C ₅₁ H ₇₉ NO ₁₃ ) is a macrolide compound isolated in 1975 from hygroscopic Streptomyces (Streptomyces hygroscopicus). Rapamycin (otherwise known as sirolimus) is an inhibitor of mTOR that inhibits the activation of T and B cells by inhibiting their response to interleukin-2 (IL-2). . It is an FDA-approved drug for immunosuppression that has both antifungal and antitumor properties.

However, rapamycin has poor solubility and pharmacokinetics, and therefore its therapeutic effect is limited. For BCS class II or IV drugs (i.e., with low solubility), nanocarriers or conjugates may be a promising method to improve their bioavailability. Examples include lipid-based nanocarriers such as liposomes; Polymer-based nanocarriers, such as polymeric micelles; inorganic nanoparticles such as silica nanoparticles; viral nanoparticles; Includes drug conjugates, such as antibody-drug conjugates, etc. Compared to conventional stabilizer or polymer formulations, lipids as vehicles are highly biocompatible and biodegradable. In particular, liposomes can be prepared from amphipathic phospholipids, which can form a barrier to protect hydrophobic drugs from exposure to aqueous or biological environments. They may also affect pharmacokinetics and distribution. However, the bioavailability of liposome-based oral Rapalog is not significantly improved.

Accordingly, the present disclosure provides liposomes specifically for encapsulating rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof for cancer treatment, in order to improve the low bioavailability and allergy problems of stabilizers. Liposomes of the present disclosure provide high bioavailability, high efficacy, and low toxicity.

Liposomes are artificial vesicles composed of concentric lipid bilayers separated by water-compartments and have been extensively investigated as drug delivery vehicles. Because of their structure, chemical composition and colloidal size, which can be well controlled by manufacturing methods, liposomes exhibit several properties that may be useful for a variety of applications. Liposomes are used as carriers for drugs and antigens because they can serve several different purposes. Liposome-encapsulated drugs are not accessible to metabolic enzymes. Conversely, body components (eg, red blood cells or tissue at the injection site) are not directly exposed to the entire dose of drug. The duration of drug action can be extended by liposomes due to the slower release of the drug in the body. Liposomes have direct efficacy, meaning that their targeting option changes the distribution of the drug in the body. Cells absorb liposomes into the cytosol using endocytosis or phagocytosis mechanisms. Moreover, liposomes can protect drugs against degradation (e.g., metabolic degradation). However, liposomes have a potential drawback in their relatively limited ability to sufficiently release certain encapsulated drugs (e.g., anti-cancer drugs).

To improve the quality of liposomal particles, several means can be adopted, such as submicron filtration, lyophilization, etc. In one embodiment, liposomal particles encapsulating rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof, are subjected to submicron filtration to remove precipitated drugs or particles of larger size.

In another embodiment, liposomes encapsulating rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof, are processed by lyophilization. Liposome lyophilization involves (1) freezing the liquid containing liposome particles to form a solid/ice form by lowering the temperature, (2) freeze-concentrating the solid/ice form by reducing the pressure, and (3) increasing the temperature. (4) sublimating the solid/ice form to obtain the crude product, and (4) performing final drying to obtain the final liposomal freeze-dried stable product (e.g., lyo-cake). . Prior to the lyophilization process, a cryoprotectant can be introduced into the liquid containing the liposomal particles to protect the active ingredient (i.e. rapamycin, rapalog). The lyophilized liposomes of the present disclosure are stable for long periods of time (at least 20 weeks) and are suitable for clinical applications. After hydration of lyophilized liposomes, the liposomes can maintain the particle size and stability before lyophilization.

The liposomes of the present disclosure demonstrate excellent stability, excellent tumor inhibition ability, and reduced toxicity before or after lyophilization and hydration of rapamycin or its analogue.

Liposomes of the present disclosure can be administered by any route that effectively transports the liposomes to the appropriate or desired site of action. Preferred modes of administration include intravenous (IV) and intraarterial (IA). Other suitable modes of administration include intramuscular (IM), subcutaneous (SC), and intraperitoneal (IP). Such administration may be by intravenous injection or infusion. Another mode of administration may be perivascular delivery. The formulation may be administered directly or after dilution. Pharmaceutical compositions comprising liposomes of the present disclosure include one or more physiologically acceptable carriers containing excipients and auxiliaries known in the art that facilitate processing of the active ingredients into preparations that can be used pharmaceutically. It can be formulated using .

Examples are provided below to more clearly illustrate the concepts of the present disclosure.

Example

Example 1: Preparation of liposomes of the present disclosure

Preparation of liposomes containing rapamycin

Rapamycin, 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- Didecanoyl-sn-glycero-3-phosphocholine (DDPC), cholesterol and N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phospho Ethanolamine (DSPE-PEG2000) was provided at different dry weight ratios as shown in the table in the specification for the preparation of liposomes containing rapamycin. The raw materials were dissolved in a mixture of chloroform and methanol. The solution was heated to 40°C and the pressure was reduced to remove the organic solvent and form a thin film. After the organic solvent was completely removed, the solution containing the cryoprotectant was added to hydrate the thin film for 1 hour. The particle size of the liposome formulation is shown in Figures 1A and 1B. These results revealed that liposomes encapsulating rapamycin with an appropriate amount of DSPE-PEG2000 exhibited smaller particle sizes.

Preparation of liposomes containing rapamycin (RAP-P)

Rapamycin, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and N-( Methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000) was used at 1:2.4:1.9 for the preparation of liposomes encapsulating rapamycin. It was provided at a dry weight ratio of :1.6. The raw materials were dissolved in a mixture of chloroform and methanol. The solution was heated to 40°C and the pressure was reduced to remove the organic solvent and form a thin film. After complete removal of the organic solvent, a solution containing cryoprotectant was added to hydrate the thin film for 1 hour. The resulting product was lyophilized, and the amounts of rapamycin, DPPC, DDPC, and DSPE-PEG2000 based on the total dry weight of the liposome were 15%, 35%, 27%, and 23%, respectively. The final product is named RAP-P formulation.

Preparation of liposomes containing rapamycin (RAP-E)

Rapamycin, 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and N-(methyl Polyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine at a dry weight ratio of 1:2.4:1.8:1.6 for the preparation of liposomes encapsulating rapamycin. provided. The raw materials were dissolved in a mixture of chloroform and methanol. The solution was heated to 40°C and the pressure was reduced to remove the organic solvent and form a thin film. After complete removal of the organic solvent, a solution containing cryoprotectant was added to hydrate the thin film for 1-2 hours. The resulting product was lyophilized, and the amounts of rapamycin, DPPC, DDPC, and DSPE-PEG2000 based on the total dry weight of the liposome were 15%, 35%, 27%, and 23%, respectively. The final product is named RAP-E formulation.

Specialization

The average particle size and particle size polydispersity index (PI) of the RAP-P formulation were 127.4 +/- 10.8 nm and 1.29 +/- 0.41, respectively. The average particle size and particle size polydispersity index (PI) of the RAP-E formulation were 348.6 +/- 30.9 nm and 0.47 +/- 0.13, respectively. The results show that both RAP-P and RAP-E can be used as suitable formulations for use in administering rapamycin.

Example 2: Different types of liposomal formulations

To investigate the application of liposomal formulations, the RAP-P and RAP-E formulations shown in Example 1 were further processed.

Submicron filtration

RAP-P and RAP-E formulations were filtered through a 0.45 μm membrane to remove large-sized free or precipitated drugs, particles, and impurities, thereby increasing product stability and safety. To validate the data, samples containing different concentrations (0.05 to 5 μg/mL) of rapamycin standard were used in UPLC-MS/MS tests to establish a calibration curve of signal (area) versus rapamycin concentration. I ordered it. The results show that the coefficient of determination (r2) of the linear calibration curve was 0.9999.

The average particle size and PI of the particle size of the RAP-P formulation after filtration were 182.4 +/- 4.3 nm and 1.10 +/- 0.07, respectively. The average particle size and PI of the particle size of the RAP-E formulation after filtration were 219.0 +/- 5.1 nm and 0.58 +/- 0.09, respectively. The results showed that after filtration, both RAP-P and RAP-E formulations exhibited narrower particle sizes and the average particle size of the RAP-E formulation was reduced to about a 37% reduction.

Freeze drying

The RAP-P and RAP-E formulations were lyophilized to obtain lyophilized products. First, the samples were frozen at -45°C. Next, most of the moisture was removed through primary drying by creating a vacuum pressure of 200 mTorr. Then, the temperature was raised to -20°C for secondary drying to remove residual moisture. To evaluate the effectiveness of cryoprotectants, sucrose and trehalose at a final concentration (w/w) of 87% or 94% were introduced into the formulation prior to lyophilization. The average particle size is shown in Figures 2A and 2B, and the results show that both sucrose and trehalose at final concentrations (w/w) of 87% or 94% did not alter or influence the properties of the liposomal particles after lyophilization. does not give

The long-term storage stability of the lyophilized product was also demonstrated. Freeze-dried products of RAP-P and RAP-E formulations containing 87% (w/w) trehalose as cryoprotectant were stored at -20°C (minus 20°C) for 20 weeks, and a portion of the products was stored at 4, After storage for 16, and 20 weeks, they were recovered and rehydrated to 2 mg/mL for characterization. The average particle size and PI of the particle size of the rehydrated product are shown in Figures 3A and 3B. Results showed that the lyophilized product can be stable for at least 20 weeks when stored at -20°C (minus 20°C).

Example 3: In vivo studies

In order to study the applicability of the claimed formulation, its toxicity studies, pharmacokinetics and efficacy were investigated in vivo in mice.

Toxicity Study

Male BALB/c mice, aged 6–8 weeks, were obtained from BioLasco (Ilan, Taiwan). Preliminary toxicity studies were performed with mice administered a single dose of RAP-P and RAP-E formulations, and body weight change was selected as an indicator. Figures 4A and 4B show the results of a study showing that the maximum tolerated dose (MTD) of the RAP-P formulation is 120 mg/kg in mice and the MTD of RAP-E is 220 mg/kg in mice. I'm doing it. For comparison, the 50% lethal dose (LD ₅₀ ) of rapamycin in a previous intravenous formulation was 40 mg/kg in rats, equivalent to 80 mg/kg in mice (Baker, H. ; Sidorowicz, A.; Sehgal, SN; Vezina, C. Rapamycin (ay-22,989), a new antifungal antibiotic. Iii. In vitro and in vivo evaluation. The Journal of antibiotics 1978, 31, 539-545). Rapamycin The liposomal formulation of the invention provides a significantly higher MTD dosage than the LD ₅₀ of previous intravenous injection formulations, making rapamycin beneficial in clinical applications.

Pharmacokinetic studies

Male BALB/c mice, aged 6–8 weeks, were obtained from BioLasco (Ilan, Taiwan). Animals were randomly divided into 2 groups (n=6). Blood was collected from the cheek before injection. After drug administration via tail vein, blood was drawn at 0.083, 0.5, 1, 2, 4, 8, 10, and 24 hours. Blood was extracted with methanol and 0.1 M zinc sulfate. Drug concentration in blood was analyzed by UPLC-MS/MS. Details of the pharmacokinetic data of RAP-P and RAP-E formulations are listed in Table 1 below:

Table 1:

Effect on the treatment of xenograft ovarian tumors in mice

Akt and mTOR phosphorylation is frequently detected in ovarian cancer, and the ovarian cancer cell line SKOV3 is resistant to cis-platinum and doxorubicin. Therefore, SKOV3 was selected as a model tumor to evaluate the therapeutic efficacy of the liposomes of the invention containing rapamycin. SKOV3 xenografts on NOD/SCID mice were used as an animal model and divided into three groups: control group (no drug), comparison group (administration of docetaxel, dose: 4 mg/kg, Q3d*4 ) and the inventive group (administration of RAP-E formulation, dose: 50 mg/kg, Q3d*4). 6-week-old NOD SCID immunodeficient mice (BioLasco, Ilan, Taiwan) were inoculated subcutaneously with 8*10 ⁵ SKOV3 cells (human ovarian cancer cells). After tumor inoculation, tumor size was measured twice a week, and treatment was started when the tumor grew to 80-100 mm ³ . Figure 5A shows tumor volumes on days 0 and 39 for each group. The average inhibition rates for docetaxel and RAP-E formulations were 53% and 62%, respectively; The 39-day tumor inhibition rates of the docetaxel and RAP-E formulations were 57% and 61%; Both groups showed significant tumor volume suppression compared to the control group. Figure 5b shows the change in body weight of mice in each group. The results show that the formulation of the present invention (RAP-E) can achieve comparable efficacy (inhibition of tumor size) to docetaxel, but with lower toxicity.

Effect on the treatment of xenograft mammary tumors in mice

In addition to ovarian cancer, studies have shown that PI3K/AKT/mTOR and the pathway are frequently dysregulated in breast cancer. Upregulated mTORC1 contributes to cell growth, i.e. proliferation, and promotes tumorigenesis. Rapamycin, an inhibitor of mTOR, may also hold promise in the treatment of breast cancer. MDA-MB-231 cells, a triple-negative breast cancer cell line for which no targeted therapy is available, are the only chemotherapy option. However, serious side effects may occur. Therefore, MDA-MB-231 was selected as a model to evaluate the therapeutic efficacy of the liposomes of the present invention containing rapamycin. MDA-MB-231 xenograft on NOD/SCID mice was used as an animal model and divided into three groups: control group (no drug), comparison group (administration of Lipo-Dox, dose: 2 mg/kg, Q3d*4) and the inventive group (administration of RAP-E formulation, dose: 25 mg/kg, Q3d*4). 6-week-old NOD SCID immunodeficient mice (BioLasco, Ilan, Taiwan) were inoculated subcutaneously with 1*10 ⁶ MDA-MB-231 cells (human breast cancer cells). After tumor inoculation, tumor size was measured twice a week, and treatment was started when the tumor grew to 80-100 mm ³ . Figure 6A shows tumor volume on days 0 and 16 for each group. The average inhibition rates of Lipo-Dox and RAP-E formulations were 27% and 34%, respectively; The 16-day tumor inhibition rates of Lipo-Dox and RAP-E formulations were 55% and 52%; Both groups showed significant tumor volume suppression compared to the control group. Figure 6b shows the change in body weight of mice in each group, especially significant weight loss in the group administered Lipo-Dox. The results show that the formulation of the present invention (RAP-E) can achieve comparable efficacy (inhibition of tumor size) to Lipo-Dox, but with lower toxicity.

Comparison of efficacy between 5-FU and formulations of the present invention against mouse colon cancer

The CT26 model on Balb/c mice was used as an animal model and divided into three groups: control group (no drug), comparative group (administration of 5-FU, dose: 25 mg/kg, Q3d*4) and invention. Group (administration of RAP-E formulation, dose: 25 mg/kg, Q3d*4). Six-week-old Balb/c mice (BioLasco, Ilan, Taiwan) were inoculated subcutaneously with 1*10 ⁵ CT26 cells (murine colorectal carcinoma cells). After tumor inoculation, tumor size was measured twice a week, and treatment was started when the tumor grew to 80-100 mm ³ . Figure 7A shows tumor volume on days 0 and 16 for each group. The average inhibition rates of 5-FU and RAP-E formulations were 11% and 37%, respectively; The 16-day tumor inhibition rates of the 5-FU and RAP-E formulations were 26% and 53%, respectively; Both groups showed significant tumor volume suppression compared to the control group. Figure 7b shows the change in body weight of mice in each group, especially significant weight loss in the group administered 5-FU. The results show that the formulation of the present invention (RAP-E) can achieve comparable efficacy (inhibition of tumor size) to 5-FU, but with lower toxicity.

Claims (13)

A liposome containing a lipid component encapsulating rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof,
The lipid components are cholesterol, phosphatidylcholine (PC), L-α-phosphatidylcholine (EggPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-distearoyl- sn-glycero-3-phosphorylethanolamine (DSPE), distearoylphosphatidyl choline (DSPC), dioleyl phosphatidyl ethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), hydrogenated soy phosphatidylcholine : HSPC), 1,2-palmitoyl-phosphatidic acid (DPPA), 1,2-dimyristoyl phosphatidylcholine (DMPC), dioleyl phosphatidylcholine (DOPC), palmitoyl-myristoyl phosphatidylcholine (PMPC), palmitoyl Toyl-oleoyl phosphatidylcholine (POPC), dioleyl phosphatidyl glycerol (DOPG), distearoyl phosphatidyl glycerol (DSPG), dipalmitoyl phosphatidyl glycerol (DPPG), dipalmitoyl phosphatidyl ethanolamine (DPPE), or PEG and these. is selected from the group consisting of combinations of;
Rapamycin analogs can inhibit the mammalian target of rapamycin (mTOR);
The amount of lipid component, based on the total dry weight of the liposome, ranges from about 30% (w/w) to 95% (w/w);
Liposomes, wherein the amount of rapamycin or an analog thereof, or a prodrug or salt of rapamycin or an analog thereof, based on the total dry weight of the liposome, ranges from about 5% (w/w) to 30% (w/w). . In claim 1,
A liposome, wherein the liposome is a poly(ekylene glycol) (PEG)-modified liposome. In claim 1,
Analogues of rapamycin include everolimus, temserolimus, tacrolimus, prerapamycin, zotarolimus, ridaforolimus, and 7-epi-rapa. 7-epi-rapamycin, 7-thiomethyl-rapamycin, 7-epi-trimethoxyphenyl-rapamycin, 7-epi-thio Methyl-rapamycin (7-epi-thiomethyl-rapamycin), 7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-desmethyl-rapamycin Mycin (2-desmethyl-rapamycin), and 42-O-(2-hydroxy)ethyl rapamycin, rapamycin oxime, rapamycin amino ester, rapamycin dialdehyde , rapamycin 29-enols, O-alkylated rapamycin derivatives, water-soluble rapamycin esters, alkylated rapamycin derivatives, rapamycin amidino carbamate, rapamycin Biotin ester, carbamate of rapamycin, rapamycin hydroxyester, rapamycin 42-sulfonates, 42-(N-carbalkoxy)sulfamates , rapamycin oxepane isomer, imidazolidinyl rapamycin derivative, rapamycin alkoxyester, rapamycin pyrazole, acyl derivative of rapamycin, rapamycin amide ester, rapamycin fluorinated ester, rapamycin acetal, oxoraphamycin, and A liposome selected from the group consisting of rapamycin silyl ether. In claim 1,
A liposome, wherein the amount of rapamycin or an analog thereof, or a prodrug or salt of rapamycin or an analog thereof, based on the total dry weight of the liposome, ranges from 10% (w/w) to 25% (w/w). The method according to any one of claims 1 to 4,
Lipid components include DOPE, DDPC, cholesterol, DSPE, EggPC, HSPC, DPPC, DMPC, DSPC, and PC; A combination of DPPC, DDPC, cholesterol and DSPE (or DSPE-PEG); A combination of DOPE, DDPC, cholesterol and DSPE (or DSPE-PEG); A combination of HSPC and DDPC; A combination of DSPC and DDPC; A combination of DOPE and HSPC; or liposomes, which are a combination of DOPE and DSPC. The method according to any one of claims 1 to 4,
The amount of lipid component is from about 65% (w/w) to about 95% (w/w) or from 70% (w/w) to about 90% (w/w), based on the total dry weight of the liposome. DOPE, DDPC, cholesterol, DSPE, EggPC, HSPC, DPPC, DMPC, DSPC, or PC; or from about 15% (w/w) to about 65% (w/w) DPPC, from about 20% (w/w) to about 65% (w/w) DDPC, from about 0% (w/w) to A combination of about 30% (w/w) cholesterol and about 15% (w/w) to about 65% (w/w) DSPE (or DSPE-PEG); About 25% (w/w) to about 60% (w/w) DOPE, about 25% (w/w) to about 70% (w/w) DDPC, about 0% (w/w) to about A combination of 20% (w/w) cholesterol and about 0% (w/w) to about 25% (w/w) DSPE (or DSPE-PEG); a combination of about 40% (w/w) to about 55% (w/w) HSPC and about 30% (w/w) to about 40% (w/w) DDPC; a combination of about 40% (w/w) to about 55% (w/w) DSPC and about 30% (w/w) to about 40% (w/w) DDPC; A combination of about 35% (w/w) to about 45% (w/w) DOPE and about 35% (w/w) to about 50% (w/w) HSPC; A liposome that is a combination of about 35% (w/w) to about 45% (w/w) DOPE and about 35% (w/w) to about 50% (w/w) DSPC. In claim 6,
Liposomes, wherein the amounts of the lipid component and rapamycin or an analogue thereof, or a prodrug or salt of rapamycin or an analogue thereof, based on the total dry weight of the liposome, are as listed below:
The method according to any one of claims 1 to 4,
Liposomes, wherein the average particle size of the liposomes ranges from about 100 nm to about 500 nm. A liposome formulation comprising the liposome of any one of claims 1 to 4 and a cryoprotectant. In claim 9,
A liposomal formulation wherein the cryoprotectant is disaccharide. In claim 9,
Liposomal formulations, wherein the cryoprotectant is sucrose or trehalose. In claim 9,
A liposomal formulation, wherein the amount of cryoprotectant ranges from about 80% to about 97% (w/w) based on the total dry weight of the liposome. A pharmaceutical composition for treating cancer, diabetes, obesity, neurological disease or genetic disorder and/or preventing organ transplant rejection in a subject, comprising a therapeutically effective amount of the liposome of any one of claims 1 to 4.