JPH06507372A - How to administer the drug - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to administer the drug Systemic administration of drugs, i.e., delivery of drugs to the patient's blood or lymph fluids, is Necessary for pathological conditions. Systemic delivery of some drug units can be achieved by oral administration. This can be achieved by non-invasive means such as invasive methods such as intravenous, intraperitoneal, intramuscular or subcutaneous injections to avoid problems inherent in In particular, lipid particles encapsulating drugs, such as liposomes, are characterized by They are often particularly susceptible to degradation by gastric juices and therefore lipid/drug units (released (for drugs that have been administered) cannot be delivered systemically by ingestion.

Drug particles associated with lipids, such as liposomes (also known as vesicles) is used to prevent toxic agents from coming into contact with healthy, sensitive tissue in specific areas of the body. To prevent and halving of the drug in the blood and/or other organs of the body Used in drug delivery systems to target drugs to increase drug delivery. liposome is a cell-like cell consisting of one or more spherical lipid bilayers surrounding an internal aqueous space. It is a smaller particle. The drug is encapsulated within an internal aqueous space or within a lipid bilayer membrane. , it is usually a phospholipid, such as saturated or unsaturated phosphatidylcholine. pear sterols, and various other charged or neutral, Consists of natural or synthetic lipids.

In some applications, liposomes slow the release of encapsulated drugs. This helps prolong the effectiveness of the drug. For example, vesicles slowly As it undergoes metabolism, early intravenous formulations were released into the circulation by delayed release from liposomes. increased the half-life of free drug in the medium.

Some applications include local, sustained release of drugs for subsequent entry into the blood or lymphatic system. liposomes to deliver released free drug across skin or mucosal membranes This includes using the system.

However, the effects of many liposomal drug formulations are limited to blood or lymph fluids. requires the delivery of intact liposome-encapsulated drug units. For example, some repo Somosomal formulations target anti-cancer drugs against tumors and release intact liposomes in the bloodstream. Circulation of somes helps to enhance the efficacy of the formulation. Other drug-loaded liposomes is absorbed by macrophages in the body, i.e., the endothelial system, and delivered to the site of infection. However, drugs without encapsulated vesicles do not have this property. In these cases, no Local application is possible because only free drug is delivered systemically, rather than wound liposomes. administration was not the same as direct intravenous infusion.

Systemic delivery of intact liposomal drug delivery methods has so far been limited to invasive methods. As a result, the treatment of lipid-associated drugs exceeds currently available methods. Expanding therapeutic arsenals and providing methods for systemic delivery of intact vesicles by local application It was necessary to provide.

The inventors have discovered that lipid particles, in particular drugs associated with lipids, preferably encapsulating drugs, that drugs containing wound liposomes can be administered systemically to the mammalian body; , i.e. delivered internally (in the circulation, blood or lymph) by mucocutaneous administration. can be set. As used herein, the term mucocutaneous refers to vaginal and Refers to the mucosal surface of the anus, which is composed of stratified suture epithelium. The present invention follows Lipid particles with dimensions smaller than 250 nm for mammalian mucocutaneous tissue Systemic delivery of intact lipid particles to the blood or lymph of a mammal by application of a or by applying the composition to the mucocutaneous tissue of the mammal. For the production of compositions for the systemic delivery of intact lipid particles into the blood or lymph of The use of lipid particles with dimensions smaller than 250 nm at Preferably , the lipid particles encapsulate the active agent that is delivered systemically as part of the intact particle; The particles are preferably liposomes, most preferably having a diameter of 30-200 nm. It is a single lamellar vesicle (UVs).

This method removes the active agent from within the intestinal tract without exposing it to damaged gastric components. (i.e., a therapeutic or diagnostic agent). It is advantageous in that it makes it possible to After rectal or vaginal administration, liposomes enter the bloodstream or or by the lymphatic system to other parts of the body (e.g. liver, kidneys or pancreas). It will be delivered to the viscera (viscera) intact and distributed. Macrophages engulf liposomes This allows it to circulate to inflamed areas throughout the body. these Mucocutaneous, preferably rectal, administration of lipid particles can be applied to the body to treat systemic conditions. It is a non-invasive method of effectively delivering drugs to deep organs of the body.

Detailed description of the invention Lipid bilayer vesicles can optionally contain cholesterol and other sterols. Individuals with polar (hydrophilic) and non-polar (lipophilic) moieties may be It is a closed microscopic vesicle formed mainly from molecules of The hydrophilic group is phosphoric acid, glyceryl phosphate, carboxy, sulfate, amino, hydroxy, choline or other It can be a polar group. Examples of non-polar groups are alkyl, alkenyl or other lipid groups. saturated or unsaturated hydrocarbons such as

Liposomes are a subset of these bilayer vesicles and contain primarily phospholipid components. Consists of children. When exposed to water, these molecules form each layer associated in the center of the membrane. lipid ends of the molecules in the membrane, and form the respective inner and outer surfaces of the bilayer. form a bilayer membrane with opposite polar ends. Therefore, each side of the membrane has a parent Although providing an aqueous surface, the interior of the membrane consists of a lipophilic medium. These membranes contain excess In the presence of water, initially around the internal aqueous space, similar to the layers of an onion, the same Arranged in a system of membranes in close proximity to the heart. Each membrane consists of an indestructible, double layer of lipid molecules. It is a sheet. By additional energy such as homogenization, sonication or extrusion , these multilamellar vesicles (MLVs) form unilamellar vesicles I be able to. The technical aspects of liposome formation are that liposomes are biologically active As well as the fact that it is advantageous for encapsulating materials, well known.

Liposomal formulations are formulated as aqueous liposomal dispersions or in this technical field. The present invention can be applied to mucosal tissues in other known forms, e.g. suppositories. It can be used in the following embodiments. For example, drug-containing lipid particles can take the following shapes: It can also be mixed with molten Witepsol™. lipid - as a powder after initial spray drying of the drug organic phase (i.e. exposed to mucocutaneous moisture) Then, the liquid after microemulsification (as proliposomes forming single lamellar vesicles) Wi tepsOl (glycerol trilaurate) is liquid at 37°C and solid at room temperature. become. WitepsOl produces little or no irritation to the rectum and reduces body temperature. It is commonly used as an excipient for suppositories. Vaginal suppositories are the same It can be manufactured in various ways. Effective for the above treatment in all applications. The amount to be administered to a given human patient will be apparent to those skilled in the art.

Although the inventor does not wish to be bound to any particular theory, M cells located along the lateral axis are responsible for liposome uptake in this region. Conceivable. These M cells are found close to the goblet cells of the intestinal tract. this The difference between these cells is that goblet cells secrete mucin to prevent the invasion of particulates. However, on the other hand, M cells engulf particles (less than 250 nm) into the lumen of the intestinal tract, and It is specialized to deliver these to the Beyer concentrators beneath the intestinal epithelium. be. Petri concentrators are specialized cells present in high numbers throughout the subcutaneous tissue of the intestinal tract. This is the structure of the immune system. These clusters are where immune responses against foreign antigens are generated. It consists of localized accumulations of lymphocytes and macrophages that can be divided into two groups. Macrophages are encapsulated in liposomes It leaves the Bayer concentrator carrying the therapeutic agent and enters the circulation for systemic delivery.

Mucocutaneous tissue, i.e., the mucosal surfaces of the vagina and anus are composed of stratified suture epithelium It will be. These epithelia lack a stratum corneum and have a density similar to that of the epidermis. Contains Hans cells. Langerhans cells transfer particles to the outside world by invaginating the cell. It absorbs them, leaves the epidermis through the basal membrane, and moves into the lymphatic vessels. Ru. The cells eventually reach the draining lymph nodes and become resident there. This is a vagina A single lamella inserted with amphotericin when applied to mucocutaneous surfaces of the body and rectum. Another mechanism for systemic delivery of lipid-associated particles, such as liposomes It can be a summation.

A liposome-encapsulated drug that crosses the intestinal lining and treats systemic disorders in vivo. In order to determine the capacity of the infected organ, this is the intestinal tract in the example below. However, from the point of view of application, we used a model in which this organ was systemically isolated. It is necessary to. In some cases, infections that primarily reside in the kidneys (systemic fungal infections) are treated with liposome-encapsulated amphotericin B (amphotericin B) administered rectally. treated using (sin). The ability of therapeutic agents applied to the intestinal wall to reach the kidneys This model is compatible with the aforementioned lipids by intestinal delivery, since the power is not greater than systemic delivery. This provides a good example of systemic administration of associated particles. Following rectal administration, Amphoteri of Example 1 The biodistribution of syn-loaded liposomal formulations and free amphotericin is different. This suggests that drug particles associated with lipids are delivered intact to the circulation. shows.

Systemic fungal infections are common in cancer patients and other immunocompromised patients. is the main cause of mortality in individuals with .

The preferred treatment of systemic fungal infections is primarily limited to two groups of drugs: a. Amphotericin B (referred to as amphotericin), and nystatin (nystat) antifungal polyene antibiotics such as in), and ketaconazole, miconazole Primarily bacteriostatic imidazoles such as Ru and fluconazole. Unfortunately, most These drugs that are antifungal (e.g., polyenes) are also extremely harmful to the host. Due to their virulent nature, fungal infections are very often characterized by treatment, especially polyester. Antifungal antibiotics easily bind to sterol components of host cells, causing membrane destruction and results in cell permeability and lysis. Furthermore, they are particularly toxic to kidney tissue and Therefore, free drug is associated with irreversible kidney damage and even kidney failure at therapeutic dosage concentrations. are doing.

Due to the toxicity of these drugs, the lowest possible effective dosage must be given. do not have. Unfortunately, the drug is diluted in the blood and large amounts of the drug are lost to uninfected tissues. Nontoxic dosages are optimal for therapeutic (efficacy) because they are broken down, excreted, or absorbed. There isn't.

The perceived benefits of liposomal encapsulation of various therapeutic agents have led to the advancement of this technology. Researchers in the field have formulated various liposomal polyene antibiotic compositions.

For example, European Patent Publication No. 0260811 and European Patent Publication No. 031712 Specification No. 0 describes specific dimensions of gluebosomal amphoteric cells that are useful in the practice of the present invention. It describes the syn preparation. In particular, the ribosomal amphoteric acid used in the example below is smaller than 250 nm, preferably from 30 to 2500 m, most preferably It consists of single lamellar vesicles with a diameter of 30-1100n. Polyene antiseptic Bacterial antibiotics are amphipathic in nature, and amphotericin is aqueous inside liposomes. Encased in a UV bilayer rather than in space.

Liposomes with diameters smaller than 250 nm are known in the art. It can be manufactured by the method described. In short, phospholipids are dissolved in organic solvents. It is dissolved and dried to form a film or powder, which is then hydrated with an aqueous solution. profit The resulting dispersion is subjected to high shear force such as sonication, which agitates the dispersion. to form smaller vesicles. Preferably, the dispersion is European No. 01 Improved Gau Sheared in an emulsifier.

The liposomes used in the examples are iodine, which is incorporated herein by reference. Preparation according to the method described in more detail in Roppa No. 0317120 4.5 or less, preferably 1.0 to 3.0 as measured by moistened pH test paper The drug and phosphatidylglycerol are first combined in an acidified organic solvent with a pH of (preferably distearoyl-phosphatidylglycerol) was formed by forming a sex complex. The complex is amphotericin B1 or tet Another polyene such as laene, pentaene, or hexaene can be added to Amphotericin was dissolved in a chloroform-methanol solution of is formed by acidification using approximately 1 mole of acid for each mole of.

Complex formation is facilitated by briefly warming the solution to about 65°C.

However, amphotericin-phospholipid complexes can be produced in solution in small amounts of organic solvents (i.e. The drug is administered in an amount of less than 7, preferably 10 mg of drug per ml of solvent. ) contains substances such as phosphatidylcholine and cholesterol. Mixed with terol and dried to yield a lipid powder, which contains zacharide of low ionic strength. Stable in a buffered aqueous solution (by application of shear force) having a diameter smaller than 0.2μ processed into single-lamellar liposomes. In this formulation, the aqueous buffer solution p1] such that the pH of the solution is 5.5 or less, preferably 4.5 to 5.5. Must be.

In this solvent environment, amphotericin has a positive charge and phosphatidylglyceride The roll has a negative charge. Therefore, phosphatidylglycerol and amphote Forms a strong association with ricin. Liposomes formed in this manner have dimensions or Lyophilize and store for later rehydration and injection without significant changes in toxicity I can do it.

Prior to the present invention, all known liposomal or lipid-complexed polyene antibiotics were prepared. Personal use was by injection, eg intravenous administration.

Approximately 30 g of 155.7 g of distearoyl phosphatidylglycerol sodium salt Dissolved in 4.8 L of chloroform:methanol (1:1 vol/vol) at °C.

Then 892.6 g of amphotericin was added with stirring to form a yellow suspension. and then add 2.5M hydrochloric acid to obtain a clear golden colored solution with a pH of approximately 4.5. A liquid was formed. Hydrogenated soy phosphatidylcholine 392.3g and Co1 96.5 g of Nosterol was added with stirring to produce approximately 29 mg/m of amphotericin. A clear yellow solution containing 1 was obtained.

This lipid solution was spray dried to a pale yellow powder. This powder was heated to 65℃ for 77 minutes. 9% sucrose containing 10 mM sodium succinate at pH 5.5 ( (weight/volume) at a concentration of 40 mg/ml and processed into liposomes. next Improved shear forces to form small unilamellar vesicles in the Gaulin emulsifier Dynamic laser scattering by applying pressure up to 10,000 psi for 30 minutes The average liposome diameter measured by the turbulence was reduced to 31 nm. Sterile 0 ．． After 2μ filtration, amphotericin-containing liposomes were analyzed for drug content. The in-process loss of nphotericin was found to be 10.4%. Lyophilization and After reconstitution with water for injection, the liposome dispersion was heated at 65 °C for 10 min. Ta. LD of this liposome formulation in several groups of C57BL/6 mice. Ha, this There were no deaths in the groups of five animals injected at a dosage of 150 mg/kg. I found out that it was also big. This is about 2.3 mg/k in the same mouse family. LDs for free amphotericin in g. comparable to

Example 2 C57BL/6 female mice were given a lethal dose of Candida albicans (3 x lO@cells/ml) was injected intravenously.

Two days after infection, three groups of mice (5 mice/group) were treated intravenously with one of the following: It was treated accordingly.

0.75 mg/kg free amphotericin B1 or up to 0.75 mg/kg or 5.0 mg/kg (Anho UV).

Two other groups of mice (5 mice/group) were treated rectally with one of the following: : 5 mg/kg free amphotericin B or 5 mg/kg ampho UV0 One group of control mice received phosphate buffered saline intravenously. Continue treatment daily for 5 days Subsequently, two weeks after infection, mice were killed, their kidneys were removed, homogenized, and Yeast cultured in an incubator and expressed as colony forming units (CFU)/mg kidney Clearance was measured.

PBS one control 10.100 intravenous 0.75 mg/kg free antitericin 440.75 mg/kg a Anho UV 375, 0 mg/kg Anho UV 11 Rectum 5, 0 mg/kg Anho U V 2955, 0 mg/kg Free Anti Tericin 535 Intravenous administration of free amphotericin is associated with nephrotoxicity and therefore These data are for LD in C57BL/6 mice. Against unho UV based on It should be noted that the toxicity of the drug is shown to be reduced by at least 100-fold. two The results in the example of amphotericin, either in free or liposomal form, Intravenous treatment using B is more effective than rectal administration of equivalent or higher dosages of the drug. also shows that it is more effective in renal yeast clearance.

However, CFU/mg kidney was significantly lower than control mice. In mice treated rectally with SynB, there was a 34-fold decrease compared to control mice. There is a 19-fold decrease in mice treated rectally with free amphotericin B. Data is , rectal administration of both liposomal and free amphotericin B. demonstrated the negative effects of infection in the kidneys of mice systemically infected with Candida aldicans. This indicates that it can be used to reduce the burden. For drugs injected into the rectum Higher dosages or longer term treatment of mice due to yeast clearing from the kidneys further improve performance.

In this example, the amphotericin-inserted single lamellar vesicle formulation of Example 1 is It was delivered both intravenously and rectally as a rehydrated liposomal formulation. the drug is in the rectum When administering the drug, mice are first anesthetized with ketamine and acepromacine. This suppressed the transtransportive response. Then, it was modified with a thin metal rod that was swollen into a bulbous shape. Aqueous liposome formulations can be used to deliver anesthesia without irritating removal of the material using a syringe. It was delivered across the sphincter into mice that were infected.

The same group of mice was treated with a lethal dose of Candida albicans. 5RBC (4X 107 cells) was intraperitoneally injected again 2 days after infection and 10 days after infection. It was administered by injection. When the mice were killed 2 weeks after infection, the lungs were removed and homogenized. did. 6n+l lung homogenate with lung white blood cells diluted in RPMi tissue medium. by centrifugation on a density gradient consisting of 4 ml of neutrophil isolation medium layered under the were isolated from other tissue cells. White blood cells in the lungs have a high percentage of B lymphocytes, Contains some T lymphocytes and macrophages.

The number of B lymphocytes that produced anti-5RBC antibodies was determined using a modified Jerne plaque assay. It was measured using a white surface method using a cell suspension. In this analysis, anti-5RBC antibodies were Each B-lymph cell that is generated, when cultured on a molecular layer of 5RBC, absorbs the surrounding 5R Dissolve BC. The dissolved regions are transparent regions or platinum in the RBC monolayer. It appears as a mark.

Table 2 PBS one control 60 intravenous 0.75 mg/kg Free antitericin 1070.75 mg/kg Anho UV 1205, 0 mg/kg Anho UV 180 rectal 5, 0 mg/kg Anpho UV 1605, 0 mg/kg Free Antite Ricin 120 The results in Table 2 show that ricin 120, in either free or liposomal form, Both intravenous and rectal treatment of Candida-infected mice using tericin B both stimulate pancreatic B lymphocyte responses to 5RBCs more than untreated controls. .

At comparable dosages, intravenous delivery of anpho-UV significantly reduced splenic B cells compared to controls. produced a 3-fold increase in response; rectal route produced 2.6-fold greater splenic B cells than untreated group Responses were detected in live egg-infected C57BL/6 female mice with 5RBC (4X 107 cells) were injected intraperitoneally. On days 0 and 7, three groups of mice (4 Mice/group) were also treated intravenously using one of the following: PBS buffer, 0.75 mg/kg free antitericin or 0.9 mg/kg ampho Four mice in one group of UV0 were treated rectally with 5.0 mg/kg Anpho-UV. . All mice were sacrificed on day 111, at which time blood samples were obtained from each mouse. . The pancreas was removed from each group of mice, homogenized, and the white blood cell suspension was prepared in Example 3. and prepared as described above. Pancreatic leukocyte cell suspensions were prepared as previously described. Use of modified Jerne plaque analysis to differentiate B lymphocyte responses to 5RBCs analyzed. Each serum sample was analyzed for hemagglutinating antibody titer against 5RBC.

PBS one control 60 11 intravenous 0, 9 mg/kg Anho UV 440 56 Rectal 5, 0 mg/kg Anho UV 133 40” Arithmetic mean of reciprocal of antibody dilution It was expressed as

The results in Tables 2 and 3 show that liposomes and non-liposomes compared to untreated mice Both intravenous and rectal administration of amphotericin B can induce plaque-forming cells in the pancreas. against 5RBCs as measured by increased cell number and serum hemagglutinating antibody titers. has been shown to enhance B cell responses. This study further demonstrated that mammalian mucocutaneous of drug associated with lipid particles having dimensions smaller than 250 nm to skin tissue. Demonstrates systemic delivery of active agents by administration.

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Claims (10)

[Claims] 1. Lipids with dimensions smaller than 250 nm for mammalian mucocutaneous tissues Application of the particles to the mammalian blood or lymph fluid of intact lipid particles Methods including personal delivery. 2. mammalian blood or by application of the composition to the mammalian mucocutaneous tissue; 2 in the manufacture of a composition for systemic delivery of intact lipid particles to lymph fluid; Use of lipid particles with dimensions smaller than 50 nanometers. 3. 3. The method according to claim 1, wherein the lipid particles are lipid bilayer membrane vesicles. 4. Unilamellar vesicles in which the lipid bilayer vesicles have a diameter of 30-200 nm 4. The method according to claim 3. 5. 3. A method according to claim 1 or 2, wherein the lipid particles encapsulate the active agent. 6. 4. The method of claim 3, wherein the lipid bilayer vesicle encapsulates the active agent. 7. 5. The method of claim 4, wherein the lipid bilayer vesicle encapsulates the active agent. 8. 6. The method of claim 5, wherein the active drug is amphotericin B. 9. 7. The method of claim 6, wherein the active drug is amphotericin B. 10. 8. The method of claim 7, wherein the active drug is amphotericin B.