METHODS FOR THE ADMINISTRATION OF DRUGS

Abstract: Particulate lipid associated drugs, such as liposome encapsulated drugs, are systemically administered to the body of a mammal as intact lipid/drug particles by administration to mucocutaneous tissue.

Description: METHOD FOR THE ADMINISTRAΗON OF DRUGS The systemic administration of drags, that is, the delivery of a pharmaceutical agent to the blood or lymph of a patient, is necessary for certain pathological conditions. While systemic delivery of some drag units may be accomplished by non- invasive means such as oral administration, others require invasive methods such as intravenous, intraperitoneal, intramuscular or subcutaneous injection to avoid problems inherent in oral administration. In particular, lipid particles which encapsulate drags, eg., liposomes, are often particularly susceptible to degradation by gastric juices, and the lipid/drag unit (as opposed to the released drug) is thus incapable of being delivered systemically by oral ingestion. Lipid associated drug particles such as liposomes (also known as vesicles) are used in drug delivery systems to target drugs to particular areas of the body, to prevent toxic drags from coming into contact with healthy, sensitive tissues, and to increase the half-life of drags in the blood and/or other organs of the body. Liposomes are subcellular particles comprised of one or more spherical lipid bilayers which surround an internal aqueous space. Drags may be encapsulated either within the internal aqueous space or in the lipid bilayer, which is usually composed of a phospholipid such as saturated or unsaturated phosphatidylcholine, some type of sterol, and various other, charged or neutral, natural or synthetic lipids. In some applications, liposomes serve to extend the effectiveness of a drug by delaying the release of the encapulated drag. For example, early intravenous formulations extended the half-life of the free drug in circulation by the delayed release from the liposomes as the vesicles were slowly metabolized. Some applications involve the use of liposomes to provide the gradual topical release of the drag, followed by the delivery of the released free drag across a dermal or mucosal membrane to enter the blood or lymph system. However, the efficacy of many liposomal drag formulations requires the delivery of the intact liposome encapsulated drug unit to the blood or lymph. For example some liposome formulations target anticancer drags to tumors, and the circulation of the intact liposome in the bloodstream is instrumental in the increased efficacy of the formulation. Other drag containing liposomes are taken up by the macrophages in the body or the reticuloendothelial system and delivered to the site of infection, and the drug without the encapsulating vesicle does not have this characteristic. In these instances, topical application has not been the same as direct intravenous infusion since only the free drag, rather than the intact liposome, has been systemically delivered. Since the systemic delivery of intact liposomal drag delivery systems has heretofore been limited to invasive methods, it has been a desideratum to expand the therapeutic avenues of treatment for lipid associated drugs beyond the methods which are currently available and provide a method for the systemic delivery of unaltered vesicles by topical application. I have discovered that lipid particles, especially those including lipid associated drags and preferably intact liposomes encapsulating the drags, may be systemically administered to the body of a mammal, Le., delivered internally (to the circulation, either blood or lymph) by mucocutaneous administration. The term mucocutaneous, as used herein, refers to the mucosal surfaces of the vagina and the anus, which are composed of stratified squamous epithelia. The invention is thus a method comprising the systemic delivery of intact lipid particles to the blood or lymph of a mammal by the application of lipid particles having a size of less than 250 nm to the mucocutaneous tissue of the mammal, or the use of lipid particles having a size of less than 250 nm in the preparation of a composition for systemically delivering intact lipid particles to the blood or lymph of a mammal by the application of the composition to the mucocutaneous tissue of the mammal. Preferably, the lipid particles encapsulate an active agent which is systemically delivered as part of the intact particle, and the lipid particles are preferably liposomes, most preferably unilamellar vesicles (UVs) having a diameter of from 30 to 200 nm. This method is advantageous in that it can permit the placement of intact liposomes containing an active agent (ie., a therapeutic or diagnostic agent) in the intestinal tract, without exposure to damaging gastric constituents. After rectal or vaginal administration, the liposomes are transmitted intact and distributed to other parts of the body (for example to the liver, kidney or spleen) either through the bloodstream or lymphatic system. Macrophages may engulf the liposomes and enable circulation to sites of inflammation throughout the body. Mucocutaneous, preferably rectal, ad^ninistration of these lipid particles is a non-invasive method to effectively deliver drag to deep organs of the body to treat systemic conditions. Detailed Description of the Invention lipid bilayer vesicles are closed microscopic vesicles which are formed principally from individual molecules having a polar (hydrophilic) and a non-polar (lipophilic) portion, although cholesterol and other sterols may be included if desired. The hydrophilic groups can be phosphato, glycerylphosphato, carboxy, sulfato, amino, hydroxy, choline or other polar groups. Examples of non-polar groups are saturated or unsaturated hydrocarbons such as alkyl, alkenyl or other lipid groups. Liposomes are a subset of these bilayer vesicles and are comprised principally of phospholipid molecules. Upon exposure to water, these molecules form a bilayer membrane with the lipid ends of the molecules in each layer associated in the center of the membrane and the opposing polar ends forming the respective inner and outer surface of the bilayer membrane. Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium. These membranes, in the presence of excess water, can initially be arranged in a system of concentric closed membranes, in a manner which is not dissimilar to the layers of an onion, around an internal aqueous space. Each of the membranes is an unbroken, bilayer sheet of lipid molecules. With additional energy such as homogenization, sonication or extrusion, these multilamellar vesicles (MLVs) can form unilamellar vesicles. The technical aspects of liposome formation are well known in the art, as is the fact that liposomes are advantageous for encapsulating biologically active substances. Liposome preparations may be used in the manner of the invention by application to mucosal tissue as an aqueous liposomal dispersion, or in other forms which are known in the art, e.g., suppositories. For example, the drag-containing lipid particles could be mixed with melted Witepsol tm in any of the following forms: as a powder after initial spray drying of the lipid-drag organic phase (Le., as proliposomes which form unilamellar vesicles upon exposure to mucocutaneous moisture); as a liquid after microemulsification; or as a powder after drying (e.g., lyophilization) of a UV dispersion. Witepsol (glycerol trilaurate) is a liquid at 37°C and becomes a solid at room temperature. Witepsol is commonly used as a vehicle for drag suppositories since it liquefies at body temperature while producing little or no irritation of the rectum. Vaginal suppositories could be prepared similarly. In all applications, the amount to be administered to human patients which is effective for such treatment will be apparent to those of ordinary skill in the art. While I do not wish to be bound to a particular theory, it is thought that the M cells located along the intestinal microvilli of the rectum are responsible for the liposome uptake in this area. These M cells are found near the intestinal goblet cells. The difference between these cells is that the goblet cells secrete mucin to prevent particulate penetration whereas the M cells are specialized to engulf particles (250 nm or less) in the intestinal lumen and deliver them to the Peyer's patches underlying the intestinal epithelium. The Peyer's patches are specialized immune structures present in high numbers throughout the subcutaneous tissue of the intestines. These patches consist of localized accumulations of lymphocytes and macrophages in which the immune response to foreign antigens can take place. Macrophages leave the Peyer's patches, bearing the liposomal encapsulated therapeutic agent, and enter the circulation for systemic delivery. Mucocutaneous tissue, ie., the mucosal surfaces of the vagina and the anus, are composed of stratified squamous epithelia. These epithelia lack a horny layer and contain Langerhans cells in a density similar to that of the epidermis. Langerhans cells endocytose the particles, internalize them, leave the epidermis through the basement membrane and migrate into lymph channels. They eventually reach the draining lymph node where they then reside. This may be another mechanism for systemic delivery of lipid-associated particles, such as amphotericin intercalated unilamellar liposomes, when applied on the mucocutaneous surface of the vagina and rectum. To determine the ability of a liposome encapsulated drug to traverse the intestinal lining and treat systemic disorders in vivo, it is necessary to employ a model in which the infected organ is systemically separated from the point of application, which in the examples which follow is, the intestinal lining. In the examples, infections which are resident primarily in the kidney (systemic fungal infections) are treated with liposome encapsulated amphotericin B (amphotericin) which is administered rectally. Since there is no ability of the therapeutic agent applied to the intestinal wall to reach the kidney other than by systemic delivery, this model exemplifies systemic administration of such lipid associated particles by intestinal deliveiy. Following rectal administration, the biodistribution of free amphotericin and the amphotericin intercalated liposomal formulation of Example 1 appears to be different, indicating that the lipid associated drag particles are transmitted intact to the circulation. Systemic fiingal infections are a major cause of mortality in cancer patients and other immunocompromised individuals. The preferred treatment for systemic fungal infections is primarily limited to two groups of drugs: the fungicidal polyene antibiotics such as amphotericin B (referred to as amphotericin) and nystatin, and primarily fungistatic imidazoles, such as ketaconazole, miconazole and fluconazole. Unfortunately, fungal infections very often defy treatment because those drags which are the most fungicidal (e.g., the polyenes) are also extremely toxic to the host. Specifically, the polyene antifungal antibiotics readily bind to sterol components of host cells causing disruption of the membrane, cell permeability and lysis. Further, because they are particularly toxic to kidney tissue, the free drug has been associated with irreversible renal damage and even kidney failure at therapeutic dosage levels. Due to the toxicity of these drags, the lowest possible effective doses must be given. Unfortunately, because the drags are diluted in the blood, and because large amounts of the drugs are degraded, excreted or taken up by uninfected tissue, the non-toxic doses are not optimally therapeutic (efficacious). Due to the advantages which have been perceived from the liposomal encapsulation of various therapeutic agents, workers in the art have formulated a variety of liposomal polyene antibiotic compositions. For example, European patent publications EP0260811 and EP0317120 describe liposomal amphotericin formulations of a particular size, which are useful in the practice of this invention. Specifically, the liposomal amphotericin employed in the following examples comprises unilamellar vesicles having a diameter of less than 250 nm, preferably from 30 to 250 nm, and most preferably from 30 to 100 nm. Since polyene antifungal antibiotics are amphiphilic in nature, the amphotericin is encapsulated in the bilayer membrane of the UVs rather than in the inner aqueous space of the liposomes. liposomes having a diameter of less than 250 nm may be produced by methods which are known in the art. Briefly, phospholipids are dissolved in an organic solvent and dried to form a film or powder, which is then hydrated with an aqueous solution. The resultant dispersion is subjected to a high shear force, such as sonication, which agitates the dispersion to form smaller vesicles. Preferably, the dispersion is sheared in a modified Gaulin microemulsifier such as is described in EP 0190050. The liposomes employed in the examples were formed according to the method described in more detail in EP0317120, which is incorporated herein by reference, by first foπning a soluble complex between the drag and a phosphatidylglycerol (preferably distearoyl-phosphatidylglycerol) in an acidified organic solvent having a pH of 4.5 or less, preferably from 1.0 to 3.0, as measured on prewetted pH paper. The complex is formed by dissolving amphotericin B, or another polyene such as a tetraene, pentaene, or hexaene, in a 1:1 (by volume) chloroform:methanol solution, and acidifying the solution with approximately one mole of acid for each mole of amphotericin. Complex formation is facilitated by briefly warming the solution to about 65°C. The amphotericin-phospholipid complex, while in solution in a small amount of the organic solvent (Le., containing the drag in an amount of at least 7 and preferably 10 mg drag per ml of solvent), is mixed with a phosphatidylcholine and a sterol such as cholesterol and dried to yield a lipid powder which is processed (by the application of shearing force) in a low ionic strength saccharide aqueous buffer solution into stable unilamellar liposomes having a diameter of less than 0.2 . In this formulation, the aqueous buffer solution must have a pH such that the pH of the final solution is from 5.5 or less, preferably from 45 to 5.5. In this solvent environment, the amphotericin will have a positive charge and the phosphatidylglycerol will have a negative charge. Thus, the phosphatidylglycerol and the amphotericin will form a strong association. The liposomes formed in this manner can be lyophilized and stored for later rehydration and injection without significant change in size or toxicity. Prior to this invention, all known systemic uses of liposomal or lipid complexed polyene antibiotics have been by injection, e.g., intravenous administration. Example 1 Formation of Amphotericin Intercalated Unilamellar Liposomes (AmphoUVs) 155.7 g distearoylphosphatidylglycerol sodium salt was dissolved in 4.8 L of chloroform : methanol (1 : 1 v/v) at about 30°C. 92.6 g amphotericin B was then added with stirring to generate a yellow suspension and 2.5 M hydrochloric acid was then added in to generate a clear golden yellow solution with a pH of approximately 4.5. 392.3 g hydrogenated soy phosphatidylcholine and 96.5 g cholesterol were added with stirring to yield a clear yellow solution containing about 29 mg/ml amphotericin, This lipid solution was spray dried to a light yellow powder. The powder was processed into liposomes by hydration at a concentration of 40 mg/ml in 9% sucrose (w/v) containing 10 mM sodium succinate at pH 5.5 warmed to 65°C for 77 minutes. Shearing force to form small unilamellar vesicles was then provided by a modified Gaulin emulsifying device at a pressure of up to 10,000 psi for 30 minutes to reduce the mean liposome diameter to 31 nm as measured by dynamic laser light scattering. Following sterilizing 0.2 μ filtration, the amphotericin containing liposomes were analyzed for drag content and in-process loss of amphotericin was found to be 10.4%. After lyophilization and reconstitution with water for injection, the liposome dispersion was heated at 65°C for ten minutes. The LD^ of this liposome preparation in several groups of C57BL/6 mice has been found to be greater than 150 mg/kg, with no deaths in a group of five animals injected at this dose. This is compared to an LD^ for free amphotericin of about 2.3 mg/kg in the same mouse strain. Example 2 C57BL/6 female mice were injected intravenously with a lethal dose of Candida albicans (3 x 10 6 cells/ml). Two days post-infection, three groups of mice (5 mice/group) were treated intravenously with one of the following: 0.75 mg/kg free amphotericin B, or 0.75 mg/kg or 5.0 mg/kg (amphoUVs). 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 amphoUVs. One group of control mice was given intravenous phosphate buffered saline. Treatments were continued daily for five days, and 2 weeks post-infection, the mice were sacrificed, their kidneys removed, homogenized and plated to determine yeast clearance expressed as colony forming units (CFU) /mg kidney. TABLE I Treatment group CFU/mg Iddney PBS - control 10,100 Intravenous 0.75 mg/kg free amphotericin 44 0.75 mg/kg amphoUVs 37 5.0 mg/kg amphoUVs 11 Rectal 5.0 mg/kg amphoUVs 295 5.0 mg/kg free amphotericin 535 It should be noted that the intravenous administration of free amphotericin is associated with renal toxicity, and thus these data indicate at least a hundred fold decrease in toxicity for the amphoUVs based on the LD^ in C57BL/6 mice. The results in this example show that intravenous treatment with amphotericin B, either in the free form or the liposomal form, is more effective in renal yeast clearance than rectal administration of comparable or higher doses of the drag. However, the CFU/mg kidney is reduced 34-fold in mice treated rectally with liposomal amphotericin B compared to the control mice, and 19-fold in mice treated rectally with free amphotericin B compared to the control mice. The data show that rectal administration of both liposomal amphotericin B and free amphotericin B can be used to reduce the burden of infection in the kidneys of mice systemically infected with Candida albicans. Higher doses or more prolonged treatment of the mice with rectally administered drug would further improve yeast clearance from the kidneys. In this Example, the amphotericin intercalated unilamellar vesicle preparation of Example 1 was delivered as the rehydrated liposome preparation both intravenously and rectally. When the drag was administered rectally, the mice were first anesthetized with ketamine and acepromazine to inhibit peristaltic response. Using a syringe modified with a bulbed thin metal rod, the aqueous liposome preparation was then delivered past the sphincter into the anesthetized mice without stimulating ejection of the material. Example 3 The same groups of mice with a lethal dose of Candida albicans were given an intraperitoneal injection with sRBC (4 x 10 7 cells) two days post-infection and again on day 10 post-infection. When the mice were sacrificed two weeks post- infection, the spleens were removed and homogenized. The splenic white blood cells were separated from the other tissue cells by centrifugation in a density gradient composed of 4 ml of neutrophil isolation medium layered beneath 6 ml of spleen homogenate diluted in RPMI tissue culture medium. The splenic white blood cells contain a high percentage of B lymphocytes, some T lymphocytes and macrophages. The number of B lymphocytes which produced anti-sRBC antibody was determined by using the white blood cell suspension in a modified Jerne plaque assay. In this assay, each B lymphocyte which produces anti-sRBC antibodies will lyse surrounding sRBC when cultured on a monolayer of sRBC. The lysed area will appear as a clear area or a plaque in the RBC monolayer. TABLE 2 #Plaque Forming Cells Treatment Group (PFO/10 6 Cells PBS-control 60 Intravenous 0.75 mg/kg free amphotericin 107 0.75 mg/kg amphoUVs 120 5.0 mg/kg amphoUVs 180 Rectal 5.0 mg/kg amphoUVs 160 5.0 mg/kg free amphotericin 120 The results in Table 2 show that both intravenous and rectal treatment of Candida-infected mice with amphotericin B, either in the free form or the liposomal form, stimulates the splenic B lymphocyte response to sRBC more than the untreated control group. At comparable doses, intravenous delivery of amphoUVs produces a three-fold increase in splenic B cell response compared to the control group; the rectal route yields a 2.6-fold greater splenic B cell response than the untreated group. Example 4 Uninfected C57BL/6 female mice were injected intraperitoneally with sRBC (4xl0 7 cells) on day 0 and day 7 of the experiment. On days 0 and 7, three groups of mice (4 mice/group) were also treated intravenously with one of the following: PBS buffer, 0.75 mg/kg free amphotericin, or 0.9 mg/kg amphoUVs. One other group of 4 mice were treated rectally with 5.0 mg/kg amphoUVs. All mice were sacrificed on day 11 at which time a blood sample from each mouse was obtained. The spleens were removed, and homogenized and a white blood cell suspension from each group of mice prepared as described above in Example 3. As previously described, the splenic white blood cell suspension was used in the modified Jerne plaque assay to assay the B lymphocyte response to sRBC. Each serum sample was assayed for hemagglutinating antibody titer to sRBC. TABLE 3 *Expressed as the arithmetic mean of the reciprocal of the antibody dilution. The results in Tables 2 and 3 demonstrate that compared to untreated mice, both intravenous and rectal administration of liposomal and non-liposomal amphotericin B enhances the B cell response to sRBC as measured by increased numbers of splenic plaque forming cells and serum hemagglutinating antibody titer. This study further demonstrates the systemic delivery of an active agent by the administration of the agent associated with lipid particles having a size of less than 250 nm to the mucocutaneous tissue of a mammal.

Claims: I CLAIM 1. A method comprising the systemic delivery of intact lipid particles to the blood or lymph of a mammal by the application of lipid particles having a size of less than 250 nm to the mucocutaneous tissue of the mammal. 2. The use of lipid particles having a size of less than 250 nanometers in the preparation of a composition for systemically delivering intact lipid particles to the blood or lymph of a mammal by the application of the composition to the mucocutaneous tissue of the mammal. 3. The method of claim 1 or 2 in which the lipid particles are lipid bilayer vesicles. 4. The method of claim 3 in which the lipid bilayer vesicles are unilamellar vesicles having a diameter of from 30 to 200 nm. 5. The method of claim 1 or 2 in which the lipid particles encapsulate an active agent. 6. The method of claim 3 in which the lipid bilayer vesicles encapsulate an active agent. 7. The method of claim 4 in which the lipid bilayer vesicles encapsulate an active agent. 8. The method of claim 5 in which the active agent is amphotericin B. 9. The method of claim 6 in which the active agent is amphotericin B. 10. The method of claim 7 in which the active agent is amphotericin B.

Inventors: ADLER-MOORE JILL, ADLER-MOORE, JILL
Applicants: VESTAR INC, VESTAR, INC.
Application year: 1991
Main classification: A61K9
IPC codes: A61K9, A61K31