CN1795867A - Doxorubicin liposome and its preparation method and use - Google Patents

Abstract

The present invention provides a liposomal pharmaceutical composition of doxorubicin for treating a mammal having pancreatic cancer, and a method of making such a composition.

Description

Doxorubicin liposome and its preparation method and use

technical field

The present invention relates to a doxorubicin liposome pharmaceutical composition, a method for manufacturing the doxorubicin liposome pharmaceutical composition, and an application thereof for treating pancreatic cancer.

Background technique

In 2003, approximately 30,000 patients died from pancreatic cancer and approximately 30,700 new cases were diagnosed in the United States, making pancreatic cancer one of the ten most common causes of cancer-related mortality. Surgery, radiation therapy, and chemotherapy are life-prolonging and/or symptom-relieving treatment options for many patients, but are rarely curative (Cancer Facts and Figures, American Cancer Society 2003).

Compared with non-liposomal drugs, liposomal drugs can quite successfully reach the primary tumor and inhibit its metastasis after intravenous injection into animals and humans, and show stronger therapeutic effects and milder side effects . One of the most widely developed liposomal drugs is doxorubicin liposome. In Taiwan, the drug is most commonly known under the brand name Lipo-Dox® (manufactured and sold exclusively by Taiwan Dongyang Pharmaceutical Industry Co., Ltd.), and a similar product known in the United States as Doxil®. . Liposome doxorubicin is often used to treat certain cancers, such as breast cancer, ovarian cancer, and a type of malignancy known as Kaposi's malignancy associated with AIDS.

Preparation of liposomal drugs is difficult for the pharmaceutical industry. Many methods for the preparation of liposomes and/or liposome drugs have been disclosed, such as the invention of Prof. Szoka.

U.S. Patent Nos. 5,077,057, 5,277,914 and 5,549,910 to Francis C. Szoka, Jr. disclose a method for preparing a suspension of liposomes having a defined particle size and encapsulating a useful compound, This compound has poor solubility in water, alcohols or halogenated hydrocarbons. The poorly soluble compound is dissolved in an aprotic solvent such as DMSO (dimethyl sulfoxide) together with a sufficient amount of the appropriate lipid, if desired, may contain a dissolved amount of a lower alcohol (e.g. ethanol) , and then the mixture is extruded or injected into a stirred aqueous solution. The resulting liposome suspension can be dialyzed or concentrated, if desired. Extrusion can be performed using a syringe, porous disk, or any other suitable device having hole sizes from about 0.05 mm to about 5 mm. In Example 2 of these three U.S. patents, Doxorubic was dissolved in DMSO, and added containing egg phosphatidylglycerol (EPG): egg phosphatidylcholine (EPC): cholesterol (Chol) (7: 3: 6) in ethanol solution. The lipid system was prepared by injecting the lipid-doxorubicin mixture into an aqueous phase consisting of 140mM NaCl-10mM Tris-HCl (pH 4.0) at 30°C. The liposome suspension was dialyzed, and the liposome-encapsulated doxorubicin was separated from unencapsulated substances by column chromatography. The particle diameter of the obtained liposome was 277nm, and 41.2% of doxorubicin was encapsulated in the liposome particle.

However, the aforementioned methods cannot eliminate certain problems, such as toxic organic solvents, maintenance of sterile conditions, and liposome uniformity and quality, so the aforementioned methods are not suitable for large-scale production.

The prior art has disclosed a method of using doxorubicin liposomes to treat pancreatic cancer, such as "evaluating the efficacy of Kanglisi (CAELYX, doxorubicin liposomes, Doxil) in the treatment of unresectable pancreatic cancer." Tolerance and Efficacy Phase II Study in Visceral Cancer" (Ann Oncol. 2001 Oct; 12(10): 1399-402). However, this study concluded that CAELYX(R) (liposomal doxorubicin, Doxil(R)) did not see an objective response. However, the regimens used are not effective in treating the disease.

In addition to providing a method for preparing doxorubicin liposome suitable for industrial production, the present invention also provides a method for treating pancreatic cancer with doxorubicin liposome. To evaluate efficacy, tumor size and survival time of pancreatic tumor-bearing rats with and without doxorubicin liposome administration were studied.

All of the above documents and patents, as well as the documents mentioned below, are incorporated in this specification in their entirety by reference.

Contents of the invention

Therefore, an object of the present invention is to provide a doxorubicin liposome pharmaceutical composition and to effectively use the composition for the treatment of pancreatic cancer.

In one aspect, the present invention provides a method of treating a mammal suffering from pancreatic cancer, comprising the step of administering a therapeutically effective amount of a liposomal doxorubicin pharmaceutical composition.

In another aspect, the present invention provides a method for manufacturing a doxorubicin liposomal pharmaceutical composition.

Description of drawings

Figure 1 shows the effect of Lipo-Dox® on tumor volume in rats bearing AR42J pancreatic tumors; and

Figure 2 shows the effect of Lipo-Dox(R) on the survival time of rats bearing AR42J pancreatic tumors.

Detailed ways

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a method for manufacturing doxorubicin liposomes, comprising the steps of: (a) at about 55-65°C, providing a premix comprising: 40-70% distearoyl phospholipid Acylcholine (DSPC), 10-35% cholesterol, and 15-30% methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE) and a premix/alcohol solution, wherein the ratio of the weight of the premixture to the volume of the alcohol solvent is about 1:5, and the alcohols are preferably lower alcohols, such as methanol, ethanol and propanol; (b) the premixture/alcohol solution and 0.2-0.8N ammonium sulfate solution (preferably 0.2N ammonium sulfate solution), mixed in a volume ratio of about 1:2-10 to form a mixture; (c) at 50-70°C, preferably at 60°C , extruding the mixture obtained in step (b) through holes with a pore size of 0.05-0.45 μm to form a pre-liposome suspension; (d) using 5% by weight to 15% by weight of sucrose aqueous solution at room temperature, dialyzing the pre-liposome suspension so as to obtain a liposome suspension; and (e) at 55-65°C, preferably at 60°C, doxorubicin and step (d) from aqueous sucrose The liposome suspension obtained is mixed.

The present invention can be performed at lower pressures (about 40 to 140 psi) but yields higher yields (about 2 to 10 L/min) when compared to conventional processes compared to operating at about 100 to 200 psi and producing A known method with a rate of 1 to 5 L/min is good.

The applicant has filed an invention patent application in mainland China on July 7, 2003 with the title of "liposome suspension manufacturing method and its product", and the application number is 03178458.5, which describes in detail Lipo- Dox) preparation method and composition, the content of this application also serves as a part of the present invention.

Rats with pancreatic cancer were treated intravenously (I.V.) with Lipo-Dox® at a dose of 10 mg/kg. The results are shown in the description below.

Tables 1 and 2 show the changes in body weight, tumor size and survival time after administration of saline or Lipo-Dox(R).

Figure 1 shows the relative tumor volume changes over time after administration of 10 mg/kg normal saline (control group) or Lipo-Dox(R). Treatment was initiated on day 0 and day 13 after tumor implantation in rats. Rats were treated with saline or Lipo-Dox(R) (10 mg/kg) on days 0, 3, 7 and 10. Tumors were measured on the indicated days. Relative tumor volumes are expressed as the _Vt / _V0 index, where _Vt is the tumor volume at the indicated day and _V0 is the volume of the same tumor at the start of treatment.

Fig. 2 shows the survival time of rats after administration of 10 mg/kg normal saline (control group) or Lipo-Dox(R). Treatment was initiated on day 0 and day 13 after tumor implantation in rats. Rats were treated with saline or Lipo-Dox(R) (10 mg/kg) on days 0, 3, 7 and 10.

Table 1 Changes in body weight with or without Lipo-Dox® treatment serial number weight(g) before injection after injection 3 days 7 days 10 days 14 days 17 days 21 days 24 days Normal saline group 303308 180180 180180 220240 230225 death 240 death death death death death death Lide (LIPODOX) group 304305311 170180160 180190170 170180170 120120120 110110110 110110110 110110110 110110110

Table 2 Changes in tumor volume and survival time after or without Lipo-Dox® treatment serial number Tumor volume (mm ³ ) Decrease or increase% reaction Survival time (d) before injection after injection 3 days 7 days 10 days Normal saline group 303308 397.55595.73 2771.723934.75 6885.527601.64 11258.2612050.18 Increased by 391.92% Increased by 291.11% bad bad 1315 Reed (Lipo-Dox®) set 304305311 774.04785.7727.59 1195.91671.621139.37 318.21338.2397.89 000 100% less 100% less 100% less good good good 262727

Note: In Table 1 and Table 2, the numbers "303" and "308" in the normal saline group represent two repeated control groups with the same concentration, and the numbers "304", "305" and " 311" represents three repeated experimental groups with the same composition and content,

Lipo-Dox(R) was highly effective against subcutaneously (s.c.) implanted AR43J pancreatic tumors when administered intravenously on days 0, 3, 7 and 10. At a dose of 10 mg/kg, all animals were cured, and the survival rate of the treated rats showed an increase (see Figures 1 and 2). After administering normal saline intravenously for four times, the body weight of the rats increased steadily, however, the body weight of the rats treated with Lipo-Dox(R) decreased as high as 33.33%. From this result, it can be concluded that the administration of Lipo-Dox(R) is an effective treatment for pancreatic cancer.

Example

The following examples illustrate methods of making, characterizing and using the compositions of the invention. These examples are not intended to limit the scope of the invention.

The preparation of embodiment 1 doxorubicin liposome (doxorubicin (doxorubicin) pharmaceutical composition, force (Lipo-Dox ®)

16.8 grams of PEG-2000-DSPE (purchased from Genzyine Co., the U.S.), 27.4 grams of cholesterol (purchased from NOF Co., Japan) and 38.2 grams of DSPC (purchased from NOF Co., Japan) were added to the 600 ml of ethanol. The mixture was mixed well at 60°C. While stirring the mixture continuously and maintaining the mixture at 60°C, 4 liters of 0.2N aqueous ammonium sulfate solution was added to the mixture. At this temperature, ethanol evaporates almost completely. The mixture was subjected to hole extrusion using a 1.5 liter membrane filter (purchased from Toyo Kaisha, Ltd., Japan). The hole extrusion process consists of:

(1) Using the first filter membrane (142 mm, 0.1 micron), the mixture was filtered 10 times; and

(2) Using a second filter membrane (142 mm, 0.05 micron), the mixture was filtered another 10 times.

The extrusion pressure is maintained at 3 to 10 kg/cm 2 and the flow rate is about 2 to 10 liters/minute. 4500 ml of filtrate was collected and then dialyzed against 30 KD hollow fiber (A/G Technology, UFP-30-C-6A, 30,000 NM, 4800 cm2) against 30 liters of 9 wt % sucrose solution. Residual ethanol was removed by dialysis. The volume of the collected solution was about 3000 ml, and the collected solution was a suspension of liposomes without ethanol.

3000 ml of liposome suspension produced was added to a glass container containing 8000 mg of doxorubicin hydrochloride (red powder), followed by continuous addition of 200 ml of pre-prepared 0.8-1.2N (preferably 0.9 N) the sugarcane powder solution of histidine (histidine-sugar powder solution), the concentration of the sugarcane powder solution is 0.9% by weight. The mixture was placed in a water bath at 60°C and stirred for 30 minutes, then cooled to about 35°C, diluted to 4 liters with 9% sucrose solution, and mixed well.

The product was packaged in sterile glass vials for injectable formulations containing 2.0 mg of doxorubicin hydrochloride/ml.

Example 2 Preparation of AR42J cell suspension

AR42J cell line (ATCC, CRL 1492) derived from diazoserine-induced pancreatic tumor in rats was purchased from ATCC. AR42J cells were cultured in Ham's F-12K (Gibco BRL) medium containing 20% fetal bovine albumin, 2mM L-glucose at 37°C and a humidity environment of 95% oxygen and 5% carbon dioxide. Aminoamide, and 1.5 g/L sodium bicarbonate. Cells were routinely cultured in T-flasks at a concentration of 1×10 ⁶ . By main culture, cells were harvested using trypsin-EDTA (Gibco BRL), and the cell suspension was centrifuged at 1000 rpm for 10 minutes and adjusted to 1×10 ⁷ cells/ml. The viability of AR42J pancreatic tumor cells was assessed using Trypan blue.

Example 3 Implantation of AR42J pancreatic cancer cells

Five Lewis rats (Lewis rats, provided by Taiwan National Science Association) were used to carry out the experiment, each weighing about 160-180 grams, and all the rats were about 3 weeks old. Food and water are provided indefinitely. Animal care and use procedures were in accordance with the Atomic Energy Research Council's Guide for the Care and Use of Laboratory Animals from the Institute of Nuclear Energy Research. One million AR42J cells were suspended in 0.1 ml of culture medium and injected subcutaneously into the right lower limb of rats. Two weeks after implantation, the tumor volume was estimated by the formula: length (mm)×width ² (mm ² )/2. The V _t /V ₀ index represents the relative tumor volume, where V _t is the tumor volume measured on the indicated days and V ₀ is the volume of the same tumor at the beginning of the measurement.

Example 4 Therapeutic effect of drug administration (Lipo-Dox®)

Rats bearing AR42J pancreatic tumors were divided into two groups, two of which were the control group, and the other three were the Lipo-Dox(R) group. Feed normal saline or Lipo-Dox (Lipo-Dox®) to the rats of these two groups respectively, the dosage is 5 to 15 mg/kg, preferably 10 mg/kg), twice a week (on Wednesday and Saturday) , a total of four intravenous injections. Immediately after administration, careful observation was required, such as tumor volume, body weight, activity level, and hair loss, and monitored until no rats survived.

Although the present invention has been described in connection with so-called preferred embodiments, it should be understood that the present invention is not limited to the above-described embodiments and is intended to cover any different embodiments included in the spirit and scope of the invention in its broadest interpretation. improved.

Claims (3)

1. one kind is used for the treatment of the mammiferous Mycocet pharmaceutical composition of suffering from pancreatic cancer, and said composition comprises the Mycocet that effective dose is gone up in a treatment, the dosage scope of said composition be 5 to 15 milligrams/kg body weight, secondary weekly.

2. Mycocet pharmaceutical composition as claimed in claim 1, wherein the dosage of said composition is 10 a milligrams/kg body weight.

3. Mycocet pharmaceutical composition as claimed in claim 1, wherein this Mycocet utilizes following method preparation:

(a) with an alcohols solvent with comprise following chemical compound: 40-70% DSPC, 10-35% cholesterol, and the premix of 15-30% methoxyl group-Polyethylene Glycol-distearyl acyl group PHOSPHATIDYL ETHANOLAMINE combination, to form one premix/alcohol solution, wherein the weight of premix is about 1: 5 with the ratio of the volume of alcohols solvent;

(b) with premix/alcohol solution and 0.2-0.8N ammonium sulfate, with about 1: the 2-10 volume ratio is mixed, to form a mixture;

(c) making the mixture of step (b) gained is the hole extrusion process of 0.05-0.45 μ m through the aperture, to form a preformed liposome liquid suspension;

(d) at room temperature, utilize 5 weight % to 15 weight % aqueous sucrose solutions, this preformed liposome liquid suspension of dialysing obtains to contain the liposome suspension of the liposome particles of suspension with activation; And

(e) doxorubicin is mixed with this liposome suspension that step (d) is obtained.

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