KR20010030599A - Liposomal camptothecin formulations - Google Patents

Abstract

Liposomal encapsulated camptothecin formulations are provided. Liposomal formulations are anti-tumor agents, have improved pharmacokinetics, enhanced efficacy, and provide increased therapeutic indices compared to free drugs and topotecan. The formulations contain liposomes comprising one or more phospholipids and camptothecins or analogs thereof.

Description

Liposomal Camptothecin Formulations {LIPOSOMAL CAMPTOTHECIN FORMULATIONS}

Camptothecin is a five-membered cyclic plant alkaloid originally released from the bark of the Camptotheca accuminata tree native to China (Wall et al., J. Am. Chem. Soc. 94: 388 (1966). The drug contains one conjugated ring that combines quinoline, pyrrolidine and alpha-pyridone and a lactone ring consisting of six members. The naturally occurring camptothecins are optically active, having an asymmetric carbon atom at position 20 of the lactone ring in the "S" configuration. Camptothecin and many of its analogs (hereinafter referred to as camptothecin) are currently the focus of intensive study because of the potent anti-tumor activity exhibited by these compounds both in vitro and in vivo. (E.g., Giovanella et al., Science 246: 1046-1048 (1989)). The cytotoxic effects of camptothecins have also been developed for use as antiviral, antiplasmodium and anti-hemoflagellate agents (Priel et al., US Pat. No. 5,622,959; Friel et al., US Pat. 5,422,344; Atlas, WO 9611005; Wall et al., US Pat. No. 5,614,529; Shapiro et al., US Pat. No. 5,496,830; Pardee, WO 9404160).

Camptothecin drugs are believed to have an anti-tumor effect by binding to Phase Isterase I and reversibly inhibiting its action. This enzyme is required for the synthesis of DNA and RNA in proliferating cells, where it promotes the relaxation of the choco DNA structure that forms during these processes. As a result of the inhibition of the phase isomerase I, the biosynthesis of nucleic acids is strongly inhibited, resulting in DNA rupture and cell death.

Despite the impressive antitumor properties of camptothecins, the use of camptothecins presents many problems. First, some camptothecins are extremely insoluble in aqueous solutions, making the parenteral administration of the drug uncertain. Second, the lactone ring is prone to hydrolysis at the pH of the plasma, resulting in a carboxylate-type drug that significantly lowers the phase satelliteization activity (Fassberg and Stella (1992) J. Pharm.Sci, 81 (7): 676-689; Mi et al., (1995) Biochemistry 34 (42): 13722-13728; Photomesil (1994) Cancer Res. 54: 1431-1439. Page: Slichenmyer et al., (1993) J. Nate. Cancer Inst. 85: 271-291; Saxel et al. (1989) Cancer Res. 49: 5077-5082). In addition, the carboxylate forms are highly toxic, sometimes leading to gastrointestinal toxicity, myelosuppression and bleeding cystitis. Biologically active lactone forms are also toxic.

Because of these application limitations, much effort has been made in the synthesis of semisynthetic camptothecin derivatives with increased water solubility, reduced toxicity and increased resistance to hydrolysis. Two examples include topotecan (Hycamtin ^™ ) (Kingsbury et al., J. Med. Chem. 34: 98 (1991); Boehm et al., European patents approved for the treatment of metastatic ovarian carcinoma) Application No. 321,122) and irinotecan (Miyasaka et al., US Pat. No. 4,604,463) approved for the treatment of colon cancer. Other derivatives of camptothecin and anti-night treatment methods using these derivatives are described by Wall et al. (US Pat. No. 5,340,817); Moonlight (US Pat. No. 5,364,858); Moonlight (US Pat. No. 5,244,903); Monthly (US Pat. No. 5,180,722); Moonlight (US Pat. No. 5,227,380); Moonlight (US Pat. No. 5,049,668); Wani et al. (US Pat. No. 5,122,606); Giovella et al. (US Pat. No. 5,552,154); Moonlight (US Pat. No. 5,401,707); Moonlight (US Pat. No. 5,122,526); Johnson et al. (WO 9311770); Johnson et al. (WO 9214471).

Recently, Luzio et al. (US Pat. No. 5,559,235) disclosed camptothecin derivatives that are moderately soluble in aqueous media and retain in vitro in vitro isomerase I activity and antitumor activity in vivo. This compound, 7- (4-methylpiperazinomethylene) -10, 11-ethylenedioxy-20 (S) -camptothecin dihydrochloride (also known as GI147211C or lurtotecan), is camptothecin More generally less toxic and 5-10 times more potent than topotecan in tumor cell cytotoxicity testing (Emerson et al., (1995) Cancer Research 55: pages 603-609). Several stability samples from GI14721C clinical trial formulations have been reported to contain sulfate-GI147211C precipitates due to residual amounts of sulfate in the potion bottle (Tong et al., (1996) PDA Journal of Pharmacetical Science and Technology 50 ( 50): pages 326-329). As a practical and efficient method of treatment, it is desirable to administer this compound in a manner that provides an improved therapeutic index.

By incorporating the compound into lipid aggregates, ie constructs, such as liposomes or micelles, insoluble, hydrolysable compounds may also be administered in clinical conditions. Liposomes are known to be physiologically compatible and biodegradable delivery systems for a wide range of drugs. In addition, since solvation in an aqueous medium is not required, insoluble compounds may be more concentrated and more readily administered at the site of action than the free drug alone.

Burke (US Pat. No. 5,552,156) discloses liposome-associated camptothecins, wherein the lactone ring of camptothecins is determined to be inserted into the acyl chain of a lipid bilayer. The lactone ring is effectively removed from the internal aqueous environment of the liposomes and protected from hydrolysis. Burke also describes whether the pH of the inner liposome zone can be lowered so that camptothecin derivatives with low affinity for the liposome membrane can remain inside the aqueous phase without undergoing hydrolysis. In vivo tests indicating stability of the formulation were not performed.

Constantinides et al. (WO 95/08986) disclose the preparation of camptothecins and their structurally related analogs in liposomes with at least 80% drug incorporated into lipid bilayers. Pharmacokinetic investigations were performed on the branched phosphatidylcholine, branpy phosphatidylglycerol, and camptothecin formulations. Liposomes are multilayer plates and camptothecins are passively captured. In comparison to the free drug, an increased plasma amount of camptothecin was observed over 4 hours after administration of the liposome preparation. Although the increase in AUC was only about four times higher, a wider plasma concentration-time curve area (AUC) was obtained from liposomes versus free camptothecins.

Camptothecins incorporated into vesicles and liposomes are also described in Slater et al. (WO 9426253) and Castor et al. (WO 9615774).

Summary of the Invention

The present invention provides liposome preparations of camptothecin and structurally related analogs thereof and methods for their preparation. Liposomal formulations have improved pharmacokinetics, enhanced efficacy as antitumor agents and provide improved therapeutic index when compared to free drugs. The formulations contain liposomes comprising one or more phospholipids and camptothecins or analogs thereof (collectively referred to herein as "camptothecins"). In one embodiment, the formulation comprises cholesterol, phosphatidylcholine, excipients (where the excipients are sulfates or citrates) and camptothecins, where some of the camptothecins may precipitate in the aqueous interior of the liposomes in the presence of excipients. Contains liposomes containing. Preferred camptothecins for use in the present invention are GI147211. In addition, the formulations described herein are stable upon storage.

Brief description of the drawings

Figure 1 shows precipitation of GI147211 with various counterion excipients.

2 shows stability in two representative liposome GI147211 lots prepared for comparative efficacy.

Figures 3A-3D show liposome GI147211 cytotoxicity versus GI147211 and topotecan (TP). A) ES-2 (ovarian carcinoma), B) KB (oropharyngeal carcinoma), C) SKON- by GI147211 C, liposomes GI147211 and TP described in ³ H-thymidine addition (CPM) versus concentration [ng / mL] values 3 (ovarian carcinoma), and D) KBV (oropharyngeal carcinoma, vincristine resistance phenotype) tumor cell proliferation. The calculated IC ⁵⁰ values (ng / ml) for each data set are shown at the bottom of each panel graph. ■ = GI147211C, ◆ = liposomal GI147211, and ▼ = TP.

4A-4D diagrams show liposome GI147211 cytotoxicity versus GI147211 and co-liposomes. A) A549 (lung carcinoma), B) Du-145 (prostate carcinoma), C) KB by GI147211 C, liposome GI147211 and co-liposomes described as ³ H-thymidine conjugated (CPM) versus concentration [ng / ml] value (Oropharyngeal carcinoma), and D) LOX (melanoma) tumor cell proliferation. The calculated IC ⁵⁰ values (ng / ml) for each data set are shown at the bottom of each panel graph. All tumor cell types are of human origin. == GI147211C, ▲ = liposome GI147211 (lot #ALM 993-028), and ▼ = coliposomes.

5 shows the pharmacokinetic comparison of HSPC and DSPC containing liposome GI147211 samples, performed in Spraque-Dawley rats. See Table 7 for the characteristics of the formulations.

FIG. 6 is a pharmacokinetic comparison of free drug and liposome GI147211 (ALM 993-030), performed in Sprague-Dawley rats. See Table 7 for the characteristics of the formulations.

FIG. 7 shows the pharmacokinetic comparison of free drug and liposome GI147211 (1 mg / kg) presented by administration to mice via iv, ip or piharut (n = 3, joint data).

8 shows the rate of lipid hydrolysis for liposome GI147211 (citric acid) with or without the addition of ammonium chloride to the final buffer.

FIELD OF THE INVENTION The present invention relates to the field of biochemistry and medicine, and in particular, to novel liposome preparations and methods for preparing the preparations. More specifically, the present invention relates to liposome preparations containing camptothecin and analogs thereof. The present invention also relates to methods of making and using such formulations.

Provided is a preparation comprising camptothecin encapsulated in liposomes and a method of making the same. This formulation has a medicinal purpose including an antitumor or antiviral agent. In addition, liposome preparations have improved efficacy as improved pharmacokinetic, anti-tumor agents and provide improved therapeutic index compared to free drug. The formulation contains liposomes comprising one or more phospholipids and camptothecins. The present invention is also configured to optionally include sterols such as cholesterol and / or cholesterol analogs in liposome preparations. In one embodiment, the formulation contains liposomes including cholesterol, phosphatidylcholine, excipients (where the excipient is sulfate or citrate) and camptothecin. In one embodiment, some of the camptothecins may be precipitated into the aqueous interior of the liposomes by excipients. In addition, the formulations described herein are stable upon storage. In one embodiment, the liposomes are monolayer vesicles of size less than 200 nm, most preferably less than 100 nm, wherein the phospholipid is distearoylphosphatidylcholine (DSPC), containing cholesterol in a 2: 1 molar ratio, and camptote God is GI147211. In a preferred embodiment, the liposomes are 200 nm or less in size, most preferably 100 nm or less, wherein the phospholipid is hydrogenated soy phosphatidylcholine (HSPC), containing cholesterol in a 2: 1 molar ratio, and camptothecin is GI147211. In a preferred embodiment, the molar ratio of lipid: camptothecin is 5: 1 to 100: 1, more preferably 10: 1 to 40: 1 and most preferably 15: 1 to 25: 1 (lipids are phospholipids and Contains both cholesterol). In addition, the liposome formulations of the present invention have improved stability compared to the free drug and other liposome formulations of camptothecin.

As used herein, the term "liposome" refers to a monolayer membrane or a multilayer membrane vesicle as described in US Pat. No. 4,753,788, the contents of which are incorporated herein by reference.

A "monolayer liposome", also called a "monolayer vesicle", is a spherical vesicle comprising one lipid bilayer membrane that defines a single closed aqueous compartment. The bilayer membrane is composed of two layers of lipids, an inner layer and an outer layer (lobe). The outer layer of lipid molecules is arranged with a hydrophilic head facing the outer aqueous system and a hydrophobic tail pointing downwards inside the liposome. The inner layer of lipid lies just below the outer layer, and the lipid is arranged with a head facing the aqueous interior of the liposome and a tail facing the tail of the outer layer of lipid.

"Multilayer liposomes," also called "multilayer vesicles," consist of one or more lipid bilayer membranes, which define one or more closed aqueous compartments. The membranes are arranged concentrically so that the different membranes are separated into aqueous compartments, like onions.

As used herein, the terms "encapsulation" and "entrapment" mean that camptothecin is attached to or associated with liposomes. Camptothecins may be associated with lipid bilayers, present in the aqueous interior of liposomes, or both. In one embodiment, some of the encapsulated camptothecins take the form of salts precipitated inside the liposomes. The drug may precipitate itself inside the liposomes.

As used herein, “excipients”, “counterions” and “counterionic excipients” may initiate or facilitate drug loading, and may also initiate or promote precipitation of camptothecin in the aqueous interior of liposomes. Point. Examples of excipients include, but are not limited to, monovalent anions such as chlorides, acetates, lactobionic acid salts, formates; Divalent anions such as aspartate, succinate and sulfate; And acid, sodium or ammonium forms of trivalent anions such as citrate and phosphate. Preferred excipients are citrate and sulfate.

The term "camptothecin" refers to camptothecin and any some related analog or derivative thereof that exhibits antitumor activity. Camptothecin drugs generally have the same core ring system. Many variations or substituents are found in many camptothecins, preferably such variants or substituents are found in rings A and B. Camptothecin drugs generally have similar structures that may exist in lactone and carboxylate forms as indicated below. As used herein, camptothecin refers to both lactone and carboxylate forms.

Some examples of camptothecin drugs are shown in Table 1.

Other camptothecin drugs have the following structure:

n may be 1 to 4, wherein if R ₁ is C ₁ , the drug is 9-chloro-10,11-methylenedioxycamptothecin; If R ₁ is NH ₂ , the drug is 9-amino-10,11-methylenedioxycamptothecin; And if R ₁ is H, the drug is 10,11-methylenedioxycamptothecin.

Camptothecin GI147211C, (7- (4-methylpiperazinomethylene) -10,11 ethylenedioxy-20 (S) -camptothecin dihydrochloride), has the following structure:

"C" in GI147211C refers to the dihydrochloride salt and "X" in GI147211X refers to the free base.

Camptothecins may have "A" and / or "B" ring substituents. Preferred camptothecins include 7- (4-methylpiperazinomethylene) -10,11-ethylenedioxy-20 (S) -camptothecin (GI1 47211), topotecan and irinotecan (see Table 1). The most preferred camptothecin drug is GI147211. Other camptothecins include, but are not limited to, 9-hydroxy camptothecin, 10-amino camptothecin, 9-hydroxy-10-dimethylaminomethyl camptothecin, 20- (RS) -10, 11-methylenedioxy camptothecin, 9-chloro-10,11-methylenedioxy- (20S) -camptothecin, 7-ethyl-10-hydroxy camptothecin, and 7-ethyl-10-[[ [4- (1-piperidino) -1-piperidino] carbonyl] -oxy] camptothecin.

"Phospholipid" refers to a combination of phospholipids capable of forming any phospholipid or liposome. Phosphatidylcholine (PC), including those obtained from bran, soybean, or other botanical sources or partially or wholly synthesized, or those with varying lengths of lipid chains and unsaturateds are suitable for use in the present invention. This includes, but is not limited to, distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), soybean phosphatidylcholine (soybean PC), hydrogenated phosphatidylcholine (granulated PC), hydrogenated chalcedon phosphatidylcholine (HEPC), dipalmitoyl phosphatidylcholine ( Synthetic, semisynthetic and natural product phosphatidylcholine, including DPPC) and dimyristoyl phosphatidylcholine (DMPC) are phosphatidylcholines suitable for use in the present invention. All these phospholipids are commercially available. Preferred PCs are HSPC and DSPC and most preferred is HSPC.

In addition, phosphatidylglycerol (PG) and phosphate acid (PA) are also suitable phospholipids for use in the present invention, including, but not limited to, dimyristoyl phosphatidylglycerol (DMPG), dilauryl phosphatidylglycerol (DLPG) Dispalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dilauryl phosphatidic acid (DLPA), And dipalmitoyl phosphatidic acid (DPPA). When used in formulations, distearoyl phosphatidylglycerol (DSPG) is the preferred negatively charged lipid. Other suitable phospholipids include phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid containing lauric, myriste stearoyl and palmitic acid chains. Addition of polyethylene glycol (PEG) containing phospholipids may also be present in the present invention.

As used herein, the term “parenteral” refers to intravenous (IV), intramuscular (IM), subcutaneous (SubQ) or intraperitoneal (IP) administration.

The term "improved therapeutic index" means a relatively higher therapeutic index for the drug being released. Treatment index is expressed as the ratio of 50% animal lethal dose to effective dose.

According to the present invention, a method of optionally embedding cholesterol in a liposome preparation is also proposed. Cholesterol is known to improve the stability of liposomes and prevent the loss of phospholipids into lipid proteins in vivo.

Any ratio of lipid to camptothecin is considered by the present invention. Preferred lipids: camptothecins have a molar ratio of 5: 1 to 100: 1, more preferably 10: 1 to 40: 1. Most preferred lipid: camptothecin molar ratio is from 15: 1 to 25: 1. Preferred liposome preparations have a molar ratio of phospholipids to cholesterol in the range of 1.5: 0.5 to 2: 1.5. Most preferred liposome formulations are 2: 1 PC: cholesterol, with or without 1-4 mol% PG. Most preferred liposomes are less than 100 nm. Preferred drug loading efficiency is at least about 70% encapsulated camptothecin percentage. Encapsulation is driven by forces such as molecules in the inner aqueous space of liposomes, molecules in the inner and outer lobules of the membrane bilayer, partially buried in the outer lobules of the bilayer, and molecules outside the liposomes and partially outside the liposomes. Molecules associated with the surface of liposomes.

In general, the preparation of the formulations embodied in the present invention begins with the preparation of the solution that liposomes form. This operation, for example, measures the weight of phosphatidylcholine, optionally cholesterol and optionally phosphatidylglycerol, and places them in a 1: 1 mixture (v / v) or optionally pure chloroform of an organic solvent, preferably chloroform and methanol. This is accomplished by dissolving. This solution is evaporated by, for example, a rotary evaporator, spray dryer or other means to form a solid lipid phase such as a film or powder. The film or powder is then hydrated with an aqueous solution containing an excipient having a pH in the range of 2.0-7.4 to form a liposome dispersion. Preferred aqueous solutions for the purpose of hydration are buffers in the acid, sodium or ammonium form of citrate or sulfate. Preferred buffers are 75 mM, more preferably 50 mM citric acid (pH 2.0 to 5.0), ammonium citrate (pH 2.0 to 5.5), or ammonium sulfate (pH 2.0 to 5.5). One skilled in the art knows that other anionic acid buffers such as phosphoric acid may be used. The lipid film or powder dispersed in the buffer is heated to about 25 ° C. to 70 ° C., depending on the phospholipid used.

Preferably, the multi-membrane liposomes, for example by sonication or homogenizer or by using a thin film evaporator as described in US Pat. No. 4.935.171 or by stirring the dispersion via vibration or vortex mixing, By the use of microfluidization devices such as French presses, monolayer vesicles are formed by applying shear force to an aqueous dispersion of lipid solids. Shear forces can also be applied by injection, cooling and thawing, dialysis removal of detergent solution from lipids, or other known methods used to prepare liposomes. The size of liposomes can be adjusted using various known techniques, including the duration of shear force. Preferably, a homogenizer is used to form a monolayer membrane vesicle having a diameter of 200 nanometers or less at a pressure of 3,000 to 14,000 psi, preferably 10,000 to 14,000 psi and an approximate aggregate transition temperature of the lipid.

Unencapsulated excipients are removed from liposome dispersions by dialysis, complete solution exchange with 9% sucrose, using dialysis, size exclusion column chromatography (Sephadex G-50 resin) or ultrafiltration (100,000-300,000 molecular weight cutting). do. Each preparation of small monolayer liposomes is then actively activated with GI147211 or other camptothecins for about 10-30 minutes against a gradient, such as membrane potential, produced when sodium hydroxide is titrated to an external pH in the range of 5.0 to 6.5. Is loaded. The temperature range during the drug loading period is generally 50 to 70 ° C., and the lipid to drug ratio is 5: 1 to 100: 1. Unencapsulated excipients are removed from liposome dispersions by dialysis, complete solution exchange with 9% sucrose, using dialysis, size exclusion column chromatography (Sephadex G-50 resin) or ultrafiltration (100,000-300,000 molecular weight cutting). do. Samples are generally filtered at 55-65 ° C. through a 0.22 micron filter consisting of cellulose acetate or polyether sulfone.

As mentioned above, camptothecins are generally loaded into liposomes preformed using known loading processes (e.g., BBA 274: 323-335 (1972), Forssen et al., Deamer et al. US Pat. No. 4,946,683; Cramer et al., BBRC 75: 295-301 (1977); see Bally US Pat. No. 5,077,056). Loading can be done by gradient or concentration loading such as pH gradient or ammonium gradient. If a pH gradient is used, it is preferred to start with an internal pH of about pH 2-3. The excipient is the counterion in the loading process, and when contacted with camptothecin inside the liposome, the excipient can cause precipitation of the substantial portion of camptothecin. The drug can also precipitate itself inside the liposomes. Such precipitation may protect the lactone ring of camptothecins from hydrolysis. Excipients such as citrate or sulfate can precipitate camptothecin and can be used inside the liposome with a gradient (pH or ammonia) to facilitate the loading of camptothecin.

Drug loading methods by pH gradients typically involve a low pH of the inner aqueous space of the liposome, which is incompletely neutralized during the drug loading process. This residual internal acid can cause chemical instability in liposome formulations (eg lipid hydrolysis), which limits the shelf life. To remove this residual internal acid, a membrane permeable amine, such as an ammonium salt or alkylamine, may be added after loading of camptothecin in an amount sufficient to reduce the residual internal acid to a minimum. Ammonium salts that can be used include, but are not limited to, ammonium sulfate, ammonium hydroxide, ammonium acetate, ammonium chloride, ammonium phosphate, ammonium citrate, ammonium succinate, ammonium lactobate, ammonium carbonate, ammonium tartrate and ammonium oxalate These include those with monovalent or polyvalent counterions. Analog salts of any alkylamine compounds that are transmembrane may also be used, including, but not limited to, methylamine, ethylamine, diethylamine, ethylenediamine, and propylamine.

Therapeutic use of liposome preparations involves the transport of drugs which are usually toxic in the free form. In the form of liposomes, toxic drugs depart from sensitive tissues that can cause toxicity, targeting the targeted areas where the drug can exhibit therapeutic efficacy. Liposomal preparations can also be used for therapeutic applications that allow the drug to be released slowly over long periods of time, thereby reducing the frequency of drug administration in terms of enhanced pharmacokinetics. Liposomal formulations may also provide a method of forming an aqueous dispersion of hydrophobic drug for intravenous administration.

The delivery route of liposome preparations can also affect their distribution in the body. Other effective dosage forms such as intraarterial injection, inhalant nebulization, oral active preparations, transdermal iontophoresis or suppositories are also conceivable, but passive delivery of liposome preparations involves various routes of administration, such as parenteral methods. Each route produces a difference in the localization of the liposome preparation.

The invention also provides a method of inhibiting the growth of both drug resistant and drug sensitive tumors by transporting a therapeutically effective amount of liposome camptothecin, preferably to a mammalian tumor. Since dosage adjustment methods for camptothecin are well known to medical practitioners, the amount of liposome camptothecin formulations effective for the treatment of the above-mentioned diseases or conditions in mammals and specifically in the human body is obvious to those skilled in the art. do. The optimal amount of formulation and the interval of individual administration are naturally determined by the disease state, form of administration, route and site, and in particular the condition of the patient, which optimal amount is determined by conventional conventional techniques. It will be appreciated by those skilled in the art that the optimal procedure of treatment, ie the number of daily doses for a limited date, can be ascertained by one skilled in the art using conventional procedures of the method of treatment determination.

Methods of inhibiting the growth of tumors associated with all cancers, including multidrug resistant cancers, are also contemplated by the present invention. Cancers for which the aforementioned liposome preparations are particularly effective are ovarian cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer and laryngeal cancer. In addition, the formulations described herein and within the scope of the claims are intended to be used with existing anticancer therapies. For example, the formulations of the present invention may comprise: 1) Taxol (paclitaxel) and platinum complexes for the treatment of ovarian cancer; 2) 5FU and leucovorin or levamisol for treating colorectal cancer; And 3) cisplatin and etoposide for SCLC treatment.

The invention may be more fully understood by reference to the following examples, which are intended to illustrate the invention, but not to limit the invention. Example 1 describes the pharmacokinetics of liposome formulations of GI147211 prepared by three different liposome loading techniques. Example 2 describes a liposome preparation of GI147211 prepared using ammonia to remove gradient loading and liposome internal acidity. Example 3 describes the precipitation of GI147211 salts. Example 4 describes the concentration dependence of precipitation of GI147211 salt using selected excipients. Example 5 describes in vitro efficacy screening and in vivo pharmacokinetics of liposome formulations of GI147211. Example 6 describes the in vivo antitumor efficacy of liposome encapsulation GI147211 as compared to the free drug. Example 7 describes treatment index measurements of liposome formulations of GI147211, Topotecan and GI147211 in two separate xenograft models. Example 8 describes the results of repeated dose efficiency studies of liposome formulations of GI147211 compared to GI147211 released at the same toxic dose. Example 9 compares the free GI147211 with two different liposome formulations of GI147211 in a repeated dose study in the KB tumor xenograft model.

Example 1

Pharmacokinetics of Membrane, Passive and Active Loading Liposome Formulations of GI147211.

Liposomal GI147211 samples were prepared by three different loading techniques: active loading methods for membrane potential generated by drug capture, passive capture and pH gradient in liposome bilayers. Next, the pharmacokinetics of membrane trapped GI147211 liposomes administered at 5 mg / kg in Sprague dauri rats and the released drug were compared. Free GI147211 was administered to mice at 1 mg / kg to compare with passively and actively loaded liposomes.

First, membrane loaded GI147211 liposomes were prepared by covalently phospholipid (DSPC), cholesterol and GI147211 in an organic solvent at a lipid: drug ratio of about 10: 1 (w / w). The solution was dried using nitrogen gas to form a thin film, raised in temperature, and stored in a vacuum desiccator under reduced pressure until use. The lipid film was rehydrated with an aqueous solution, typically 9% sucrose and 10 mM sodium succinate at pH 5.4, sufficient volume to have a lipid concentration of about 50 mg / ml and a GI147211 concentration of about 5 mg / ml. The sample was then sonicated for 10-15 minutes above the aggregate lipid phase transition temperature until the solution was apparently translucent and then filtered through a 0.22 micron filter.

Passively loaded GI147211 liposomes, including negatively charged lipids (DSPG) and / or neutrally charged lipids (DSPC) and cholesterol, were prepared as follows: The drug was dissolved in a 50 mM citric acid solution at 9% sucrose and pH 2.2 at 65 ° C. The aqueous solution of GI147211 was prepared at a drug concentration of about 90 mg / ml. The lipid component was co-dissolved in an organic solvent system to prepare a lipid film or spray dried powder, and then the solution was dried with nitrogen gas and temperature rise to form a film or powder. The drug solution described above was then added, mixed and heated at about 65 ° C., so that the lipid film or powder was hydrolyzed with a 5: 1 ratio of lipid to drug at 150 mg / ml lipid. The sample was then sonicated for about 10-15 minutes above the aggregate lipid phase transition temperature until the solution was apparently translucent. Uncaptured GI147211 was isolated from the liposome encapsulated drug using a Sephadex G-50 column eluted with 9% sucrose. The resulting liposome GI147211 formulation was concentrated to the desired final drug concentration and the pH adjusted to about 5.6-5.7.

Active loading and active loading / samples removed with ammonia were prepared as in Example 2.

The particle size diameter is microtrac ultrafine particle analyzer (Micro Trac Ultrafine) for all of the monolayer membrane vesicles described above, except for a sample of bilayer size distribution having a median diameter of 100 nm or less, ie having a distribution of 7.7% at the peak. Particle Analyzer) was measured to be less than 100nm.

For liposome samples as described in Table 2A, the pharmacokinetics of the released drug was compared to the minimum (three-fold) observed in the area under the curve (AUC) when compared to the membrane trapping drug administered to mice at 5 mg / kg. There was improvement, a maximum concentration (2.5 times) measured at 15 minutes (Cmax), a two-fold decrease in clearance rate (CL), and a one-hour extension in _half- life (t _1/2 ). Table 2B shows the results of comparing a passively loaded liposome GI147211 sample, prepared with or without PG, to a free drug administered at 1 mg / kg in rats. Herein, when comparing liposome drugs with free drugs, there is a 110-140 fold increase in AUC and Cmax, a 120-140 fold decrease in clearance rate, and the same half-life. Table 2B also shows that there are further improvements and reductions in AUC and clearance rates compared to liposome GI147211 samples actively loaded for citric acid or ammonium sulfate and in one case removed with ammonium chloride after loading. . When GI147211 liposomes that are actively loaded are compared to the free drug, an increase of 800-1,200-fold in AUC, 200-fold increase in Cmax, 600-1,200-fold decrease in clearance rate, and an additional half-life of 2-7 hours are observed. It became. These findings indicate that the preferred loading method for achieving the best duration of drug with liposomes in vivo is active loading in the presence of counterions that can precipitate some drug in the inner aqueous core of the liposome.

Example 2

Liposomal Formulations of GI147211.

Phospholipids and cholesterol used herein were obtained as dry powders from Avanti Polar Lipids, Lipoid or Sigena and used without further purification.

For each liposome formulation below, a solution of a different counterion excipient was prepared and trapped in the inner aqueous core of the liposome, followed by the addition of a water-soluble camptothecin derivative to the outside of the liposome, between the inside and outside of the liposome to enhance drug loading. It was loaded for the membrane potential produced by the pH or ion concentration difference of.

First, a lipid film containing various phospholipids, including phosphatidylcholine, cholesterol and phosphatidylglycerol, by covalently sharing lipid components in an organic solvent system and then drying the solution using nitrogen gas and elevated temperature to form a film or powder. Or spray dried powder was prepared. In the range of molar ratios as listed in Table 3, different phospholipid sources (synthetic, semisynthetic, bran, soybean), chain length (14-18 carbons) and unsaturation (1-4 double bonds) were investigated. Acid, sodium or ammonium form of counterion solution (citrate, sulfate, succinate, phosphate, formate, aspartate or lactobionate) for precipitation at a pH range of 2.0 to 7.4 at a lipid concentration of 100 to 150 mg / ml With an aqueous solution containing), each lipid powder and film was hydrated. Small monolayer liposomes (<100 nm, micro-track ultrafine particles) from these mixtures were then subjected to homogenizer or sonication at 10,000 to 14,000 psi and above their respective lipid phase transition temperatures (approx. 40 to 70 ° C). Median diameter using an analyzer). Excipients not captured in the aqueous core of liposomes are generally liposomes by exchanging the buffer with 9% sucrose using dialysis, size exclusion column chromatography (Separdex G-50 resin) or ultrafiltration (100KD to 300KD molecular weight cutting). It is removed from the dispersion. Each preparation of small monolayer membrane liposomes is then actively loaded in about 10-30 minutes to the membrane potential produced when the external pH is titrated with sodium hydroxide or other suitable base in the range of 5.0-6.5. The temperature range during the drug loading phase is generally from 50 to 70 ° C. with a lipid to drug ratio of 5: 1 to 100: 1. Uncaptured drug is removed from the liposome dispersion by replacing the buffer with 9% sucrose using diafiltration, size exclusion column chromatography (Separdex G-50 resin) or ultrafiltration using 100KD to 300KD molecular weight cutting membranes. The sample was filtered at 55-65 ° C. through a 0.22 micron filter consisting of cellulose acetate or polyether sulfone. The results of constellation identification are shown in Tables 3, 4, 5, and 7 below.

Although this example describes a liposome preparation of GI147211, one skilled in the art will appreciate that other liposome preparations of camptothecin can also be prepared by the methods described.

Removal by Ammonia

In the above formulations, a gradient method was used to load camptothecin derivative GI147211 into liposomes. The composition of the gradient method involves low pH conditions of the inner aqueous space of the liposome, which may be incompletely neutralized during loading. Residual internal acid causes chemical instability (eg, lipid hydrolysis) in the liposome preparations, thereby limiting the storage period. Residual internal acidity is removed from certain liposome formulations in the following manner.

A 410 mL solution containing liposomes consisting of a HSPC: cholesterol ratio of 2: 1 and encapsulating 50 mM citric acid buffer at 58.6 mg / mL total lipid concentration was heated to 55 ° C. 60 ml of GI147211C stock solution was added to the liposome solution at a concentration of 20 mg / ml at 55 ° C. The pH of the solution was titrated from 2.5 to about 4.4 by adding about 3 ml of 2.5M NaOH. The pH was then titrated carefully to 5.8 with 1M NaOH. After heating for about 20 minutes from the first base addition, the solution was cooled to 35 ° C. or lower and ultrafiltered against a 5 L pH 6.5 dialysis solution containing 10 mM NH ₄ Cl, 9% w / w sucrose. At the end of the ultrafiltration, the solution pH was adjusted to 6.5, begged to a target concentration, and filtered through a 0.2 μm pore size filter.

The liposome GI147211 sample thus treated has a significantly lower hydrolysis rate of lipids, giving a more chemically stable state than the untreated sample. In order to enhance the shelf life of the sample as a liquid phase, it is necessary to continuously adjust the rate of lipid hydrolysis to produce an appropriately stable liposome sample. This is a general note for any liposomes loaded at pH values that differ significantly from neutral, in this case specifically for liposome GI147211 formulations.

FIG. 8 shows the results of lipid hydrolysis under accelerated conditions (25 ° C.) for liposome GI147211 samples prepared with 100 mM citric acid in hydration and loaded with a target ratio of lipid to drug at 20: 1. After drug loading, the solution was divided into a control solution and a second sample to which ammonium chloride was added. The lipid hydrolysis rate for the sample to which ammonium chloride was added showed a dramatic decrease of about 167 times compared to the control system.

The pharmacokinetics and potency of the ammonium elimination and non-elimination liposome GI147211 samples showed the same PK and efficacy characteristics (see Table 2B and Table 15). Thus, ammonium chloride has no detrimental effect on liposome preparations and provides improved chemical stability for phospholipids.

Example 3

Precipitation of GI147211 Salt.

Several excipients have been identified that can induce precipitation of GI147211 from aqueous media. Excipients include, but are not limited to, monovalent anions such as chlorides, acetates, lactobionic acid salts and formates; Divalent anions such as succinate, aspartate and sulfate; Acid, sodium or ammonium forms of trivalent anions such as citrate and phosphate. To assess the state of precipitation caused by each excipient, 5 mg / ml of GI147211 was prepared at a low pH of the solution (2 ± 0.5) and different counterions were added. After leaving GI147211 solution and counterion for 15 to 20 minutes, the precipitate was maintained by centrifugation at 3600 rpm for 10 minutes. The amount of GI147211 remaining in solution for each sample was then evaluated by analyzing the supernatant using HPLC under the following conditions: C18 ODS Hypersil column from Keystone, mobile phase 30 / 70 (v / v) acetonitrile / 0.1% acetic acid + 0.1% aqueous triethylamine buffer, UV detection at pH 3.30, 270 nm, the concentration of soluble fraction remaining in the solution differed from the original GI147211 concentration of 5 mg / ml Was taken and the amount of precipitated GI147211 was tabulated. Each value is listed in Table 6 as a percentage of the original solution concentration of 5 mg / ml. In some instances, high concentrations of counterions lead to precipitation beyond that detectable in Table 6. At the counterion concentration tested, sulfate, citrate and succinate precipitated at least 97% of the drug; Chlorides and phosphates induce drug precipitation of at least 82%; Formate and aspartate result in drug precipitation of greater than 55%; Sucrose and lactobate are less than 10% GI147211 and do not cause detectable precipitation. Precipitates consist of closed lactone rings of camptothecin derivatives when the solution pH of these tests is about 2. "A" ring-substituted water soluble camptothecin derivatives such as topotecan, irinotecan (CPT-11) and 9-aminocamptothecin can also exhibit the same degree of precipitation in the presence of the counterions described above.

Example 4

Concentration dependence of GI147211 salt precipitation using selected excipients.

To more fully investigate the concentration dependence of GI147211 precipitation, a 5 mg / ml drug solution was prepared as described in Example 3 and titrated with increasing concentrations of counterion excipient, sulfate, citrate, phosphate and chloride. After leaving GI147211 solution and counterion for 15-20 minutes, the precipitate was liberated by centrifugation at 3600 rpm for 10 minutes and the supernatant was analyzed as described in Example 3. Figure 1 details the concentration dependence of the precipitations involved. Sulfates and citrate are the most effective and preferred excipients for drug precipitation, reducing the fraction of drug remaining in solution at concentrations lower than chloride or phosphate. The loading rate of the drug varied from 13% (NaCl), 61% (phosphate) to 75% and 89% depending on the counterion for ammonium sulfate and citric acid, respectively.

In a separate investigation, after loading in the presence of 50 mM ammonium sulfate, precipitation of camptothecin GI147211 at the predetermined concentration (approximately 40 mg / ml camptothecin GI147211) inside the liposome was examined. As a result, only 0.5% of camptothecin GI147211 remained in solution, essentially indicating 99% of the drug was precipitated. Precipitation of similar drugs in the liposomes is expected. Spontaneous precipitation of the drug is also expected inside the liposomes.

Also shown in FIG. 2 is the stability score for liposome camptothecin GI147211 samples containing citrate or sulfate counterions. After 80-90 days storage at 2-8 ° C., GI147211 loss was observed to be 4% for sulfate or citrate samples, with a typical exact value of 3% for this type of HPLC analysis.

Example 5

In vitro efficacy screening and in vivo pharmacokinetics of liposome GI147211 formulations.

Liposomal formulations were prepared as described in Example 2, samples were characterized as shown in Table 7 below, and in vitro efficacy screening and in vivo function of liposome GI147211 were tested.

In vitro efficacy test

In vitro efficacy screening was performed using liposome formulations of GI147211 and GI147211. The purpose of this in vitro experiment is to demonstrate that liposome encapsulation of GI147211 does not result in reduced activity compared to non-liposomal compounds. Standard tumor cell reference cell characterization was performed to evaluate ³ H-thymidine incorporation.

About 4 hours before the experiment, tumor cells were seeded in 96-well tissue culture plates (1 × 10 ⁴ cells / well). Serial dilutions (1:10 dilution, 10 ^{5-10 -3} ng / ml, n = 8 wells / dilution) of GI147211, liposome GI147211 and TP treatment were prepared (immediately before use) and added to cells containing culture plates. It became. Positive and negative (trised) control groups were included on each assay plate. Tumor cells were then labeled with 0.2 μCi of [methyl- ³ H] thymidine and subjected to tissue culture conditions (37 ° C., 5% CO ₂ , except C6, U251, A673 and B16-F1 cells incubated for 24 hours). , 100% state humidity) for 42 hours. Cells were then lysed, harvested on glass fiber filters and unconjugated [methyl- ³ H] thymidine removed by filter washing. The filter is scintillated and cpm- ³ H / well is measured. Data (of one cell type on a cell type basis) was normalized to control thymidine incorporation and applied to a standard four-parameter nonlinear regression, with IC ₅₀ values (50% inhibitory concentration) for each treatment. ) Is measured. To test each cell line for differences in IC ₅₀ measurements between cells treated with liposomes GI147211, GI147211 and Topotecan, sequencing of variance with multiple comparisons (not adjusted for the multiplicity of the test) Statistical difference was measured by Analysis of Variance. For each treatment group (median, minimum and maximum), IC ₅₀ data is presented with the indicated importance (P <0.05). Data is presented from multiple sets of experiments involving multiple liposome preparations of GI147211.

The GI147211 and liposome GI147211 activities were also compared with those of the commercially available phase isomerase I inhibitor Topotecan. The data from this experiment (Tables 8 and 3A to 3D) suggest that the liposome preparation of GI147211 and the free GI147211 are significantly stronger than Topotecan in most of the tumor cell lines examined (P <0.05, all Occation). The data for several tumor cell lines also support the fact that the liposome preparation of GI147211 is more potent than the GI147211 free drug in some cases (P <0.05, as indicated in Table 8). The data presented in table form show results from multiple (n> 100) cytotoxicity experiments involving multiple lots of liposome GI147211 generated during formulation preparation. Thus, IC ₅₀ values are presented as median (ng / ml) with the maximum and minimum indicated. In addition, representative data showing the consistency of the schedule data are presented along with the results obtained using the preferred formulations (FIGS. 4A-4D). The data provided (FIGS. 4A-4D) support the claim that drug-free coliposomal vesicles do not cause convincing cytotoxicity, which is because the observed activity of liposome GI147211 is due to the drug and not the liposome component. These data support the fact that liposome GI147211 is an active and effective chemotherapeutic agent with increased activity compared to free GI147211.

In vivo pharmacokinetic studies

By intravenous bolus administration, pharmacokinetic studies of liposome preparations of various GI147211 have been conducted in rats and mice. Plasma samples were precipitated with acetonitrile and the supernatants of each sample were analyzed by liquid HLPC using isocratic elution and monitoring fluorescence. In a similar manner, standard and quality control samples were analyzed. Concentrations of unknown and quality control samples were determined based on interpolation using a linear fit of standard samples. For each investigation, the average plasma concentration at each time point was calculated and used for pharmacokinetic analysis. Noncompartmental analysis was performed using WinNonlim ^TM (Standand Edition, version 1.5 GI147211, Scientific Consulting, Inc.) and the following parameters were calculated: maximum extrapolated at manufacturing time Concentration (Cmax), area under the curve from production to infinity (total AUC), final state half-life (t1 / 2), clearance rate (Cl), and volume of distribution in sustained state (Vss).

Table 9 summarizes the plasma pharmacokinetic parameters measured from several of these studies. In all cases the liposome preparations showed a significant increase in total exposure compared to the free drug administered at similar doses as measured by total AUC. These results were consistent with the investigations conducted in rats and mice. The increase in total AUC for the actively loaded liposome formulations ranged from 473 to 1173 times compared to the free drug. Most of the increase in exposure is due to a decrease in volume of distribution in the sustained state for liposome preparations that approach the plasma volume. These data support the model in which the liposome preparation resides primarily in the plasma volume and becomes transparent with a final half-life of 3-8 hours depending on the formulation.

Dose dependence on plasma pharmacokinetics was investigated for two liposome preparations, NA-908-73 (sulphate counterion) and NA943-072B (citrate counterion). In both investigations the dose dependence appeared to be linear as a function of dose, as indicated by similar clearance rates over the dose range tested for a given formulation.

Citrate and sulfate formulations with a 2: 1 ratio of HSPC or DSPC: cholesterol are preferred formulations for increased efficacy data and pharmacokinetic properties. 5 shows plasma concentrations as a function of time after administration of 1 mg / kg to Sprague-Dawley rats. Investigations of liposome GI147211 preparations (NA943-072B) performed about 3 months apart indicate that pharmacokinetics remain unchanged even after liposome drugs are stored for more than 3 months at 2-8 ° C. In addition, citrate preparations made with HSPC or DSPC show very similar plasma pharmacokinetics. FIG. 6 shows plasma pharmacokinetics of the formulation (2: 1 HSPC: cholesterol, loaded with 50 mM citric acid, removed with NH ₄ Cl) compared to free drug administered at 1 mg / kg in Sprague-Dawley rats. Indicates. The AUC is about 978 as compared to the free drug.

The route of administration and corresponding plasma pharmacokinetics were examined with GI147211 free drug and liposome preparation NA-908-73 (see Table 7 for formulation characteristics) in mice. Animals were administered 1 mg / kg by intravenous (I.V), intraperitoneal (I.P) or subcutaneous administration and plasma samples were collected over 24 hours. FIG. 7 shows the plasma concentrations of free drug and liposome GI147211 as a function of time, and plasma pharmacokinetics are summarized in Table 6. Comparison of the exposure measured by AUC between the free drug and liposome preparations showed an increase of 190-500 fold in the three transport routes. The findings also support the benefits of liposome GI147211 formulations that increase plasma cycle time and that other routes of vesicular means can be used to maintain high concentrations in plasma.

Example 6

In vivo antitumor efficacy of liposome encapsulation GI147211 against free GI147211.

The antitumor efficacy of liposome preparations of GI147211 has been shown in several human tumor xenograft models. In all investigations performed, subcutaneous tumors implanted on the anterior flank of naked mice were allowed to grow to a set size of 200 ± 500 mm 3 before administration of the drug.

In vivo efficacy studies were performed to compare activity to free GI147211 for liposome combination drugs. When administered equally, liposome preparations of GI147211 have been shown to be more potent and effective at lowering tumor growth (Table 10). Xenograft models included HT29 and SW48 human colon tumor cell lines, and KB and KBV laryngeal tumors. The drug was administered intravenously at 6 or 9 mg / kg according to the weekly schedule for every 3 consecutive weeks for 3 consecutive weeks. In the SW48 xenograft study, animals received a total of two rounds of treatment (6 injections) with a 10-day recovery period between the two treatment periods. In the HT29 xenograft irradiation, 12 and 14 mg / kg dose groups were added. In this experiment, liposome GI147211 administered animals belonging to the high dose group were given the drug only two times in succession, while the free GI147211 group was all administered for three weeks. In both KB and KBV investigations, liposome GI147211 was shown to be more potent than the free drug alone. This has been explained by both the magnitude of the response and the duration. This was particularly pronounced in the multiple drug resistant (MDR ⁺ ) tumor cell line KBV, where the free drug had little effect on tumor growth, whereas liposome GI147211 showed a dose dependent inhibition of tumor growth. In two colon tumor xenograft studies, there was little difference initially, only in the SW48 study after two rounds of treatment. In the HT29 investigation, the difference in tumor response was more dramatic, with the lower side of the 1 mg / kg dose of liposome GI147211 as effective as the higher side of the 14 mg / kg dose of free GI147211. When compared at the same dose and schedule in SW48 and HT29 colon tumor models, liposome GI147211 showed 95% tumor growth inhibition effect in both models, compared to 86% and 54% produced by the free drug. A more significant difference appeared in the KB tumor model, where liposome GI147211 showed a log 10 cell killing index of 7.13, compared to 1.64 of the free drug. In addition, liposome GI147211 showed 65% tumor growth inhibition effect in the MDR tumor model KBV, while the free drug was virtually inactive. Even when administered at the same toxicity level, liposome GI147211 was still more potent than the free drug alone.

Example 7

Measurement of Treatment Index of GI147211, Topotecan and Liposomal Formulations in Two Separate Xenograft Models.

For the purpose of determining differences in treatment indices, single dose toxicity and efficacy tests of liposome preparations of GI147211, GI147211 and topotecan were performed. Tumor digraft models constructed for use in these investigations included KB larynx and ES2 ovarian tumors. All test groups consisted of 10 naked mice and the drug was delivered in a single intravenous bolus injection via the tail vein. Topotecan was administered at 6-40 mg / kg, GI147211 at 6-30 mg / kg, and liposome GI147211 at 3-40 mg / kg. Treatment index was measured by dividing LD50 by ED60 or ED80 at 27 days post dose. The results from both studies showed that liposome GI147211 showed a continuous increase in the therapeutic index in the range of 3 to 14 times greater than free GI147211 (Table 11).

Example 8

Repeated dose study of liposome GI147211 compared to free GI147211 at the same dose.

KB, KBV, and ES2 tumor xenograft models also repeatedly examined the efficacy of liposome GI147211 as compared to free GI147211. The drug was administered on days 1, 8 and 15. In all three model systems, liposome GI147211 exhibited hardened tumor growth retardation compared to free GI147211 when administered at the same toxicity level (Tables 12-14).

Example 9

Comparison of HSPC vs. DSPC Reference Liposome GI147211 Formulation and Free GI147211 in Repeated Dose Investigations in KB Tumor Transplantation Model.

Dosing studies were performed in the KB tumor xenograft model, where, after intravenous administration, formulations of two different liposomes GI147211 were compared to free GI147211. The same course of treatment was repeated on days 23 and 30 after a break of 1 and 8 days and 2 weeks according to the dosing schedule. All animals in the liposome GI147211 group were dosed four times in total, whereas the control and GI147211 groups were dosed only two times because of excess tumor growth. The results are shown in Table 15, where there was no apparent difference between the two liposome GI147211 formulations, but it clearly shows that the GI147211 group is significantly less effective as an antitumor agent. The difference between the two liposome GI147211 formulations tested was that HSPC was used in place of DSPC in batch ALM993-028 (see Table 7 for details on formulation).

The invention set forth in the claims herein relates to the specifically illustrated embodiments. However, the technology thus far is not intended to limit the invention to the illustrated embodiments, and those skilled in the art should recognize that various modifications may be made within the scope and spirit of the invention described above. The invention includes all alternatives, modifications and equivalents that may be included within the true spirit and scope of the invention as defined in the appended claims.

Claims (80)

A liposome preparation comprising at least one phospholipid and 7- (4-methylpiperazinomethylene) -10,11-ethylenedioxy-20 (S) -camptothecin (GI147211). The liposome preparation of claim 1 wherein the at least one phospholipid is phosphatidylcholine. The liposome preparation according to claim 2, wherein the phosphatidylcholine is selected from the group consisting of distearoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, soy phosphatidylcholine, bran-phosphatidylcholine, hydrogenated bran phosphatidylcholine, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. The liposome preparation of claim 3 wherein the phosphatidylcholine is hydrogenated soybean phosphatidylcholine. The liposome formulation of claim 2 wherein the liposome also comprises cholesterol. The liposome preparation of claim 4, wherein the liposome also comprises cholesterol. The liposome preparation of claim 2 wherein the liposome also comprises phosphatidylglycerol. The liposome preparation of claim 7 wherein the phosphatidylglycerol is selected from the group consisting of dimyristoyl phosphatidylglycerol, dilauryl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and distearoyl phosphatidylglycerol. The liposome preparation of claim 8 wherein the phosphatidylglycerol is disteraroyl phosphatidylglycerol. The liposome preparation of claim 6, wherein the liposome also comprises phosphatidylglycerol. The liposome preparation of claim 10 wherein the phosphatidylglycerol is selected from the group consisting of dimyristoyl phosphatidylglycerol, dilauryl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and distearoyl phosphatidylglycerol. The liposome preparation of claim 11 wherein the phosphatidylglycerol is disteraroyl phosphatidylglycerol. The liposome formulation of claim 1 wherein counterions are used to initiate or promote loading of GI147211 on liposomes. A liposome preparation according to claim 13 wherein the counterion is in the acid, sodium or ammonium form of monovalent, divalent and trivalent anions. 15. The method of claim 14, wherein the monovalent anion is selected from chloride, acetate, lactobionate or formate; Divalent anions are aspartate, succinate or sulfate; And liposome formulations wherein the trivalent anion is citrate or phosphate. A liposome preparation according to claim 5 wherein the molar ratio of phosphatidylcholine: cholesterol is 1.5: 0.5-2: 1.5. The liposome formulation of claim 16 wherein the molar ratio is 2: 1. The liposome preparation according to claim 17, wherein the liposome is 100 nm or less as a monolayer film. 19. The liposome preparation of claim 18 wherein the molar ratio of lipid: GI147211 is from 5: 1 to 100: 1. 20. The liposome preparation of claim 19 wherein the molar ratio of lipid: GI147211 is from 5: 1 to 40: 1. The liposome preparation of claim 20 wherein the molar ratio of lipid: GI147211 is 20: 1. The liposome preparation according to claim 6, wherein the molar ratio of hydrogenated soybean phosphatidylcholine: cholesterol is 1.5: 0.5-2: 1.5. The liposome preparation of claim 22 wherein the molar ratio is 2: 1. The liposome preparation according to claim 23, wherein the liposome is 100 nm or less as a monolayer film. The liposome preparation of claim 24 wherein the molar ratio of lipid: GI147211 is from 5: 1 to 100: 1. The liposome preparation of claim 25 wherein the molar ratio of lipid: GI147211 is from 5: 1 to 40: 1. The liposome preparation of claim 26 wherein the molar ratio of lipid: GI147211 is 20: 1. The liposome preparation of claim 27 wherein counterions are used to initiate or promote loading of GI147211 on liposomes. The liposome preparation according to claim 28 wherein the counterion is in the acid, sodium or ammonium form of monovalent, divalent and trivalent anions. The method of claim 29, wherein the monovalent anion is selected from chloride, acetate, lactobionate or formate; Divalent anions are aspartate, succinate or sulfate; And liposome formulations wherein the trivalent anion is citrate or phosphate. 31. A liposome preparation according to claim 30 wherein the counterion is citrate. Next, a method of preparing a liposome formulation of claim 5, comprising the steps a) to e). a) forming a lipid film or powder comprising phosphatidylcholine and cholesterol; b) hydrating the lipid film or powder with an aqueous buffer containing a counterion solution; c) applying shearing force to produce liposomes that are monolayer and less than 100 nm; d) addition of GI147211 to the aqueous medium; And e) forming a gradient between the aqueous medium inside the liposomes and the aqueous medium outside the liposomes to load GI147211 into the liposomes by the gradient. The method of claim 32, wherein the gradient is a pH gradient. 33. The method of claim 32, wherein the molar ratio of phosphatidylcholine: cholesterol is from 1.5: 0.5 to 2: 1.5. A method of inhibiting tumor growth, comprising administering a therapeutic or effective amount of the liposome preparation of claim 1 to a tumor. The method of claim 35, wherein the tumor is drug resistant or drug sensitive. The method of claim 36, wherein the tumor is derived from a cancer selected from the group consisting of ovarian cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, and laryngeal cancer. The liposomes comprise hydrogenated soybean phosphatidylcholine (HSPC) and cholesterol, the molar ratio of HSPC: cholesterol is 2: 1, the molar ratio of lipid: camptothecin is 10: 1-40: 1, and liposomes have an average size of 100 nm or less A liposome preparation comprising camptothecin encapsulated in a liposome, which is a monolayer membrane. A method for preparing a liposome formulation according to claim 38, comprising the following steps a) to e). a) forming a lipid film or powder comprising phosphatidylcholine and cholesterol; b) hydrating the lipid film or powder with an aqueous buffer containing a counterion solution; c) applying shearing force to produce liposomes that are monolayer and less than 100 nm; d) adding camptothecin to an aqueous medium; And e) forming a gradient between the aqueous medium inside the liposomes and the aqueous medium outside the liposomes to load camptothecin into the liposomes by the gradient. The method of claim 39, wherein the gradient is a pH gradient. 40. The method of claim 39, wherein the molar ratio of phosphatidylcholine: cholesterol is from 1.5: 0.5 to 2: 1.5. A method of inhibiting tumor growth, comprising administering to a tumor a therapeutic or effective amount of the agent of claim 38. The method of claim 42, wherein the tumor is drug resistant or drug sensitive. The method of claim 43, wherein the tumor is derived from a cancer selected from the group consisting of ovarian cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, and laryngeal cancer. A method for preparing a liposome preparation comprising GI147211, comprising the following steps a) to e). a) forming a lipid film or powder comprising phosphatidylcholine and cholesterol; b) hydrating the lipid film or powder with an aqueous buffer containing a counterion solution; c) applying shearing force to produce liposomes that are monolayer and less than 100 nm; d) addition of GI147211 to the aqueous medium; And e) forming a gradient between the aqueous medium inside the liposomes and the aqueous medium outside the liposomes to load GI147211 into the liposomes by the gradient. 46. The method of claim 45, wherein the molar ratio of phosphatidylcholine: cholesterol is from 1.5: 0.5 to 2: 1.5. 46. The method of claim 45, wherein the gradient is a pH gradient. 48. The method of claim 47, wherein the pH of the liposome internal aqueous medium is lower than the pH of the liposome external aqueous medium. 49. The method of claim 48, further comprising increasing the pH of the liposome internal aqueous medium by adding a membrane permeable amine to the aqueous medium external to the liposome. The method of claim 49, wherein the membrane permeable amine is selected from the group consisting of ammonium salts and alkylamines. 51. The method of claim 50, wherein the ammonium salt has a monovalent or polyvalent counterion. 52. The ammonium salt of claim 51, wherein the ammonium salt is selected from the group consisting of ammonium sulfate, ammonium hydroxide, ammonium acetate, ammonium chloride, ammonium phosphate, ammonium citrate, ammonium succinate, ammonium lactobate, ammonium carbonate, ammonium tartarate and ammonium oxalate How to be. The method of claim 52, wherein the alkylamine is selected from the group consisting of methylamine, ethylamine, diethylamine, ethylenediamine and propylamine. 46. The method of claim 45, wherein the phosphatidylcholine is selected from the group consisting of distearoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, soy phosphatidylcholine, bran phosphatidylcholine, hydrogenated bran phosphatidylcholine, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. 55. The method of claim 54, wherein the phosphatidylcholine is a liposome that is hydrogenated soybean phosphatidylcholine. The method of claim 55, wherein the liposomes also comprise phosphatidylglycerols. The method of claim 56, wherein the phosphatidylglycerol is selected from the group consisting of dimyristoyl phosphatidylglycerol, dilauryl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and distearoyl phosphatidylglycerol. 58. The method of claim 57, wherein the phosphatidylglycerol is disteraroyl phosphatidylcholine. 46. The method of claim 45, wherein the molar ratio of phosphatidylcholine: cholesterol is 2: 1. 46. The method of claim 45, wherein the molar ratio of lipid: GI147211 is 10: 1 to 40: 1. 61. The method of claim 60, wherein the molar ratio of lipid: GI147211 is 20: 1. A method for preparing a liposome preparation comprising camptothecin, comprising the following steps a) to e). a) forming a lipid film or powder comprising phosphatidylcholine and cholesterol; b) hydrating the lipid film or powder with an aqueous buffer containing a counterion solution; c) applying shearing force to produce liposomes that are monolayer and less than 100 nm; d) adding camptothecin to the aqueous buffer; And e) forming a gradient between the aqueous medium inside the liposomes and the aqueous medium outside the liposomes to load camptothecin into the liposomes by the gradient. 63. The method of claim 62, wherein the molar ratio of phosphatidylcholine: cholesterol is from 1.5: 0.5 to 2: 1.5. 63. The method of claim 62, wherein the gradient is a pH gradient. 65. The method of claim 64, wherein the pH of the liposome internal aqueous medium is lower than the pH of the liposome external aqueous medium. 66. The method of claim 65, further comprising increasing the pH of the liposome internal aqueous medium by adding a membrane permeable amine to the aqueous medium external to the liposome. 67. The method of claim 66, wherein the membrane permeable amine is selected from the group consisting of ammonium salts and alkylamines. 67. The method of claim 66, wherein the ammonium salt has a monovalent or polyvalent counterion. 69. The ammonium salt of claim 68, wherein the ammonium salt is selected from the group consisting of ammonium sulfate, ammonium hydroxide, ammonium acetate, ammonium chloride, ammonium phosphate, ammonium citrate, ammonium succinate, ammonium lactobionate, ammonium carbonate, ammonium tartarate and ammonium oxalate How to be. 68. The method of claim 67, wherein the alkylamine is selected from the group consisting of methylamine, ethylamine, diethylamine, ethylenediamine and propylamine. 63. The method of claim 62, wherein the phosphatidylcholine is selected from the group consisting of distearoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, soy phosphatidylcholine, bran phosphatidylcholine, hydrogenated bran phosphatidylcholine, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. The method of claim 71, wherein the phosphatidylcholine is hydrogenated soybean phosphatidylcholine. 73. The method of claim 72, wherein the liposomes also comprise phosphatidylglycerol. 74. The method of claim 73, wherein the phosphatidylglycerol is selected from the group consisting of dimyristoyl phosphatidylglycerol, dilauryl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and distearoyl phosphatidylglycerol. 75. The method of claim 74, wherein the phosphatidylglycerol is disteraroyl phosphatidylglycerol. 63. The method of claim 62, wherein the molar ratio of phosphatidylcholine: cholesterol is 2: 1. The method of claim 62, wherein the molar ratio of lipid: camptothecin is 10: 1-40: 1. 78. The method of claim 77, wherein the molar ratio of lipid: camptothecin is 20: 1. A method for preparing a liposome preparation comprising camptothecin, comprising the following steps a) to e). a) forming a lipid film or powder comprising phospholipids and cholesterol; b) hydrating the lipid film or powder with an aqueous buffer containing a counterion solution; c) adding camptothecin to an aqueous buffer; d) forming a gradient between the aqueous medium inside the liposomes and the aqueous medium outside the liposomes to load camptothecin into the liposomes by the gradient; And e) adding a membrane permeable amine to the external aqueous medium of the liposomes to increase the pH of the internal aqueous medium of the liposomes. A liposome preparation comprising camptothecin encapsulated in liposomes, prepared by the method of steps a) to e) below. a) forming a lipid film or powder comprising phospholipids and cholesterol; b) hydrating the lipid film or powder with an aqueous buffer containing a counterion solution; c) adding camptothecin to an aqueous buffer; d) forming a gradient between the aqueous medium inside the liposomes and the aqueous medium outside the liposomes to load camptothecin into the liposomes by the gradient; And e) adding a membrane permeable amine to the external aqueous medium of the liposomes to increase the pH of the internal aqueous medium of the liposomes.