Chapter one:Site-specific drug delivery using liposomes as carriers 17 Table 1.2 Formulation Parameter Liposomal Conventional Number of Patients 29 29 Graft losses 6/11(55%) 14/16(88%) Mean duration of antifungal treatment 21.3 days 21 days Adverse reaction reports 3 in 3 patients Deaths 9 17 Survival rates Liver transplant 71.4%(0=7) 20.0%(n=5) Kidney transplant 72.7%(n=11】 62.5%(n=16) Bone marrow transplant 63.6%(n=11) 12.5%(n=8) Kidney or pancreas transplant only. L-NDDP (cis-bis-neodecanoato-trans-R,4-1,2-Daminocyclohexane plati- num),vincristine (in stealth liposome),cytochalasin B,vaginal antifungal agent uch as n nazole,stero omes from ster such as cholee terol,vitam in D,steroid h ormones and f topical and in-aerosol devices for anti-inflammatories,beclomethasone dipropionate,dexamethasone palmitate,bronchodilators,such as metapro- terenol (Metasome)and terbutaline,indomethacin,daunorubicin (Cerbu- dine),radiopharm maceutical VS-101,cisplatin(Platinol),minoxidil,calcitonin 111 nalothin,nystatin,a-t cop eol, nicotinate,vitamins A and E,tin mesoporphyrin, -tocopher rin A(aeroso formulation),apolipoprotein B,5-fluoro-2'-deoxyuridine and its pro-drug 3-5'-O-dipalmitoyl derivative,(dpFUdR)corticosteroids,14-O-palmi- toyl-hydroxyrubicin,plasmid DNA,glutathione,idarubicin,dideoxvinosine triphos hate bov somatotropin,antimonial drug m nine antimonate ma Novasome vaccines,2- Interleukin 2,imida zolidines,and dyphylline A. Highlights of current research Liposome products to deliver medication to the eye (e.g,dry eye syndrome)or gastrointestinal tract have been described. ve b carried out lipos nes and heat application to deliver anti cancer drugs.Anticancer drug-contain ing liposomes are injected into the bloodstream.At temperatures a few degrees above normal,the liposomes melt,allowing drugs to leak out.ss .Primaquin(an antimalarial agent)has been coupled to a liver cell-tar geting peptide complex that can be encapst 86.7 A new liposomal product as has been devel- oped that has a significantly stronger membrane. Multivesicular liposomes for the administration of anticancer agents have been developed.These liposomes are composed of multiple
Chapter one: Site-specific drug delivery using liposomes as carriers 17 L-NDDP (cis-bis-neodecanoato-trans-R,4–1, 2-Daminocyclohexane platinum), vincristine (in stealth liposome), cytochalasin B, vaginal antifungal agents, such as miconazole, steroidal liposomes from sterols, such as cholesterol, vitamin D, steroid hormones and fluorinated steroids, benzylpenicillin, topical and in-aerosol devices for anti-inflammatories, beclomethasone dipropionate, dexamethasone palmitate, bronchodilators, such as metaproterenol (Metasome) and terbutaline, indomethacin, daunorubicin (Cerbudine), radiopharmaceutical VS-101, cisplatin (Platinol), minoxidil, calcitonin, camptothecin,111 indium, cephalothin, nystatin, a-tocopherol, a-tocopherol nicotinate, vitamins A and E, tin mesoporphyrin, cyclosporin A (aerosol formulation), apolipoprotein B,5-fluoro-2’-deoxyuridine and its pro-drug, 3–5’-O-dipalmitoyl derivative, (dpFUdR) corticosteroids, 14-O-palmitoyl-hydroxyrubicin, plasmid DNA, glutathione, idarubicin, dideoxyinosine triphosphate, bovine somatotropin, antimonial drug meglumine antimonate for leishmaniasis, hamycin, Novasome vaccines, 2–133-Interleukin 2, imidazolidines, and dyphylline.77 A. Highlights of current research • Liposome products to deliver medication to the eye (e.g., dry eye syndrome)82,83 or gastrointestinal tract have been described.77 • Animal studies have been carried out using liposomes and heat application to deliver anticancer drugs. Anticancer drug-containing liposomes are injected into the bloodstream. At temperatures a few degrees above normal, the liposomes melt, allowing drugs to leak out.85 • Primaquin (an antimalarial agent) has been coupled to a liver cell-targeting peptide to form a complex that can be encapsulated.86,87 • A new liposomal product known as pro-liposome has been developed that has a significantly stronger membrane.82 • Multivesicular liposomes for the administration of anticancer agents have been developed. These liposomes are composed of multiple, Table 1.2 Formulation Parameter Liposomal Conventional Number of Patients 29 29 Graft lossesa 6/11 (55%) 14/16 (88%) Mean duration of antifungal treatment 21.3 days 21 days Adverse reaction reports 3 in 3 patients Deaths 9 17 Survival rates Liver transplant 71.4% (n = 7) 20.0% (n = 5) Kidney transplant 72.7% (n = 11) 62.5% (n = 16) Bone marrow transplant 63.6% (n = 11) 12.5% (n = 8) a Kidney or pancreas transplant only
18 Drug delivery systems,second edition nonconcentric,aqueous chambers,allowing more efficient drug en- trapment and improved incorporation of drugs,including cytarabine and bleomycin. Battelle has developed a process to dehydrate drug-encapsulated osomes allowing storage as a stable powder that can be econsti- tuted when re Macromolecular carriers and liposomes have been covalently cou- pled to monoclonal antibodies targeted against cardiac myosin heavy chain.Deferoxamine-modified polymers were bound tightly with Ga and oGa radioisotopes. The penetration behavior of liposomes (prepared from NAT 106) ated with s be n proteins has een investigated in vio utilizing MAbs.Within 20 minutes of topical application to young pig skin, an even distribution through all skin layers was demonstrated. Dioleoyl-N-(monoethoxy polyethyleneglycol succinyl)-phosphati- dylethanolamine(PGE-PE)(mol wt of PEG 5000),an amphipathic polymer,can be e incorporated into the liposome membrane and sig nificantly pro ong the blo ulation time of the lipc me.0 Two rat MAbs,34A and 201B,that specifically bind to a surface glycoprotein(gp112)of the pulmonary endothelial cell surface,have been coupled to unilamellar liposomes (immunoliposomes)of ap- ing that bind ing takes place during the first few passages through the lung capillary bed. ● The MAb DAL K29 against a human renal cell carcinoma associated cell surface antigen has been covalently linked to small unilamellar lipid vesicles(SUV)containing the antifolate methotrexate,with full ention of antibody TwoT surface molecules,CD4 and CD7,have beer studied as targets for the specific delivery of drugs from antibody-di- rected liposomes.The efficiency of uptake by peripheral lympho cytes,thymocytes,and two CEM sublines(CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate have be ediated inhibition of the ration of d-[3H]Urd into DNA. s compared with sim liposomes targeted to MHC-encoded HLA class I molecules,which are known to be taken up efficiently by T cells.3 Generation of cytotoxic T lymphocytes(CTL)in vitro and tumor-re- antigen recor situted lipos s has been in d were prepared from a crude butanol extract of BALBRVD leukemi cells and egg phosphatidylcholine (PC):1,2-Dimyristoylami- do-1,2-Deoxyphosphatidylcholine(DDPC)(3:2),or dimyristoylphos- phatidylcholine(DMPC):DDPC(1:4).4
18 Drug delivery systems, second edition nonconcentric, aqueous chambers, allowing more efficient drug entrapment and improved incorporation of drugs, including cytarabine and bleomycin.14 • Battelle has developed a process to dehydrate drug-encapsulated liposomes, allowing storage as a stable powder that can be reconstituted when required for drug delivery.77 • Macromolecular carriers and liposomes have been covalently coupled to monoclonal antibodies targeted against cardiac myosin heavy chain. Deferoxamine-modified polymers were bound tightly with 67Ga and 68Ga radioisotopes.88 • The penetration behavior of liposomes (prepared from NAT 106) incorporated with proteins has been investigated in vivo utilizing MAbs. Within 20 minutes of topical application to young pig skin, an even distribution through all skin layers was demonstrated.89 • Dioleoyl-N-(monoethoxy polyethyleneglycol succinyl)-phosphatidylethanolamine (PGE-PE) (mol wt of PEG = 5000), an amphipathic polymer, can be incorporated into the liposome membrane and significantly prolong the blood circulation time of the liposome.90 • Two rat MAbs, 34A and 201B, that specifically bind to a surface glycoprotein (gp112) of the pulmonary endothelial cell surface, have been coupled to unilamellar liposomes (immunoliposomes) of approximately 0.25 mm in diameter. Time-course studies reveal that 34A liposomes bind to lung antigens within 1 min after injection, indicating that binding takes place during the first few passages through the lung capillary bed.91 • The MAb DAL K29 against a human renal cell carcinoma associated cell surface antigen has been covalently linked to small unilamellar lipid vesicles (SUV) containing the antifolate methotrexate, with full retention of antibody activity.92 • Two T lymphocyte cell surface molecules, CD4 and CD7, have been studied as targets for the specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate have been evaluated by methotrexate-mediated inhibition of the incorporation of d-[3H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be taken up efficiently by T cells.93 • Generation of cytotoxic T lymphocytes (CTL) in vitro and tumor-rejection responses by sensitization of semisyngenic mice with tumor antigen reconstituted liposomes has been investigated. Liposomes were prepared from a crude butanol extract of BALBRVD leukemia cells and egg phosphatidylcholine (PC): 1,2-Dimyristoylamido-1,2-Deoxyphosphatidylcholine (DDPC) (3:2), or dimyristoylphosphatidylcholine (DMPC):DDPC (1:4).94
Chapter one:Site-specific drug delivery using liposomes as carriers 19 A new method for the elimination of mononuclear phagocytic cells from cell suspensi ons has been described.By making use of lipo some-encapsulated dichloromethylene diphosphonate,macrophages were effectively removed from spleen cell suspensions.This effect was not observed when using the free-drug or control liposomes.3 Comparative studies of the preparation of immunoliposomes with the e of two bifunctional coup and investigat 0 electron microscopy have been carried out.The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy.Antibody-mediated endocytic uptake of immunoliposomes was demonstrated.%6 in tw systems:a human malignant melanoma xenografted into nude mice and a syngeneic murine lymphoma ESb.Mp exhibiting spontaneous metastasis to the liver.Both MAbs tested were partly released from immunoliposomes within a few hours and produced a constant level of circulati A differentiation inducer,sodium butyrate (SB),encapsulated in li posomes conjugated covalently to an MAb directed to CD19 antigen has been successfully targeted to human lymphoma cell lines SKLY-18 and Ramos grown in vitro and in vivo in nude mice.Various control osomes(lacking SB or antibody,or coupled to an irrelevant anti- not effe ctive rgeters w of the limited number of umor-specin c antigens,targeting of differenti tion inducers rathe than cytotoxic drugs to tumor cells may provide a useful approach for conversion of highly malignant to less malignant tumors.3 A technique for loading certain types of drug molecules into pre formed lip mes has been describe d.The drug must be amphiphatic and exist a cha ged,protonated f .Liposome e suspen ar formed with a higher concentration of ammonium ions within the liposome than in the external aqueous phase,thus establishing a pH gradient across the liposome boundary.The amphiphatic drug mol- ecule is added to the suspension and,because of the pH gradient,is eferentially loaded in the core of the liposome targeted to the liver ave been prepared with a high content of a nonionic surfactant.This formulation was prepared by mixing soybean phosphatidylcholine,a-tocopherol,and ethoxylated hydrogenated castor oil (HCO-60)in methanol,concentrating the mixture under vacuum,and then shaking it with water.Transport to rat liver after iy administration was claimed to be ximately twice as high for liposome containing castor oil thoxyl te as for controls without surfactant. The extent to which liposomes promote the permeability of insulin through the nasal mucosa with or without pretreatment with sodium
Chapter one: Site-specific drug delivery using liposomes as carriers 19 • A new method for the elimination of mononuclear phagocytic cells from cell suspensions has been described. By making use of liposome-encapsulated dichloromethylene diphosphonate, macrophages were effectively removed from spleen cell suspensions. This effect was not observed when using the free-drug or control liposomes.95 • Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome target cell binding by cytofluorometry and electron microscopy have been carried out. The specificity of the binding of B8–24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody-mediated endocytic uptake of immunoliposomes was demonstrated.96 • The potential of small unilamellar liposomes coupled to antitumor MAbs to accumulate in solid tumor tissue has been tested in two systems: a human malignant melanoma xenografted into nude mice and a syngeneic murine lymphoma ESb.Mp exhibiting spontaneous metastasis to the liver. Both MAbs tested were partly released from immunoliposomes within a few hours and produced a constant level of circulating antibody.97 • A differentiation inducer, sodium butyrate (SB), encapsulated in liposomes conjugated covalently to an MAb directed to CD19 antigen has been successfully targeted to human lymphoma cell lines SKLY-18 and Ramos grown in vitro and in vivo in nude mice. Various control liposomes (lacking SB or antibody, or coupled to an irrelevant antibody) are not effective targeters. In view of the limited number of tumor-specific antigens, targeting of differentiation inducers rather than cytotoxic drugs to tumor cells may provide a useful approach for conversion of highly malignant to less malignant tumors.98 • A technique for loading certain types of drug molecules into preformed liposomes has been described. The drug must be amphiphatic and exist in a charged, protonated form. Liposome suspensions are formed with a higher concentration of ammonium ions within the liposome than in the external aqueous phase, thus establishing a pH gradient across the liposome boundary. The amphiphatic drug molecule is added to the suspension and, because of the pH gradient, is preferentially loaded in the core of the liposome.99 • Liposomes targeted to the liver have been prepared with a high content of a nonionic surfactant. This formulation was prepared by mixing soybean phosphatidylcholine, a-tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then shaking it with water. Transport to rat liver after IV administration was claimed to be approximately twice as high for liposomes containing castor oil ethoxylate as for controls without surfactant.100 • The extent to which liposomes promote the permeability of insulin through the nasal mucosa with or without pretreatment with sodium
Drug delivery systems,second edition glycocholate (an enhancer of nasal absorption)has been studied Results indicate that sodium glycocholate remaining in the nasal mucosa causes the lysis of liposomes that come to the surface of the nasal mucosa and the subsequent release of insulin from the lipo- some.The relatively high level of insulin in the mucosal surface caused perm eation of insulin through the mucosa .10 The release of 5(6)-carboxyfluorescein(CF)from liposomes and phos. pholipid peroxidation against time in the presence of different con- centrations of collagen,albumin,and y-globulin has been studied Results indicate that collagen decreases liposome permeability by an antioxidant effect and also by a specific interaction with phospholip ids.Col agen provides ific protec gent-induced release of CF from liposomes.Thus,a liposome-col- lagen complex may provide an improved drug delivery system. Removal of intravenously injected liposomes from the circulation has been achieved by cells of the mononuclear phagocyte system (i.e. RES)On e xposure to blood,lipo somes beco coated with plasma proteins. me of these proteins(opsonins)are thought to become determinants for subsequent recognition by mononuclear phago cytes.The review by Patel provides a critical discussion of factors that control opsonization of liposomes and their phagocytosis in vivo and in vitro.103 The distribution of 2-imidazolines in neutral dimyristoylphoaphati- dylcholine(DMPC)liposomes in negatively charged liposomes con taining dicetylphosphate (DCP)or phosphatidylserine (PS)and in positively charged liposomes containing stearylamine(STA)has been investigated.Electrophoretic mobilities of multilamellar liposomes were measured as a function of drug concentration.The results in- dicate the re ve importance of th e membr an e surface cter tics on partiti ioning behavior and the membrane transport behavior of the 2-imidazoline drugs.0 The effect of P(a cell-adhesion molecule from avian peripheral nerve myelin)on the rate of interaction of liposomes with human M21 melanoma cells has been studied.Liposome uptake by the cells was quantitated using radioactive lipids and lipo entrapped drugs under various conditions.The results suggest that the attachment of liposomes to the cell surface can increase their drug delivery poten- tial.This may be the result of triggering binding endocytic processes or a temporary permeability increase of liposome and cellular mem- brane,thereby leading to enhanced uptake.0 posome have be g two lipophilic drugs 11 a-tocopherol nicotinate(TN)and the anti-inflammatory substance L440.In the case of TN liposomes,oleoyl-hydrolyzed animal protein (OHAP)was added in order to control the vesicle size.The skin penetration ability of both drugs from liposomal gels into human stratum corneum was determined in vivo b a stripping method and
20 Drug delivery systems, second edition glycocholate (an enhancer of nasal absorption) has been studied. Results indicate that sodium glycocholate remaining in the nasal mucosa causes the lysis of liposomes that come to the surface of the nasal mucosa and the subsequent release of insulin from the liposome. The relatively high level of insulin in the mucosal surface caused permeation of insulin through the mucosa.101 • The release of 5(6)-carboxyfluorescein (CF) from liposomes and phospholipid peroxidation against time in the presence of different concentrations of collagen, albumin, and g-globulin has been studied. Results indicate that collagen decreases liposome permeability by an antioxidant effect and also by a specific interaction with phospholipids. Collagen provides nonspecific protection against the detergent-induced release of CF from liposomes. Thus, a liposome-collagen complex may provide an improved drug delivery system.102 • Removal of intravenously injected liposomes from the circulation has been achieved by cells of the mononuclear phagocyte system (i.e., RES). On exposure to blood, liposomes become coated with plasma proteins. Some of these proteins (opsonins) are thought to become determinants for subsequent recognition by mononuclear phagocytes. The review by Patel provides a critical discussion of factors that control opsonization of liposomes and their phagocytosis in vivo and in vitro. 103 • The distribution of 2-imidazolines in neutral dimyristoylphoaphatidylcholine (DMPC) liposomes in negatively charged liposomes containing dicetylphosphate (DCP) or phosphatidylserine (PS) and in positively charged liposomes containing stearylamine (STA) has been investigated. Electrophoretic mobilities of multilamellar liposomes were measured as a function of drug concentration. The results indicate the relative importance of the membrane surface characteristics on partitioning behavior and the membrane transport behavior of the 2-imidazoline drugs.104 • The effect of Po (a cell-adhesion molecule from avian peripheral nerve myelin) on the rate of interaction of liposomes with human M21 melanoma cells has been studied. Liposome uptake by the cells was quantitated using radioactive lipids and liposome entrapped drugs under various conditions. The results suggest that the attachment of liposomes to the cell surface can increase their drug delivery potential. This may be the result of triggering binding endocytic processes or a temporary permeability increase of liposome and cellular membrane, thereby leading to enhanced uptake.105 • Liposomes have been prepared containing two lipophilic drugs, dl-a-tocopherol nicotinate (TN) and the anti-inflammatory substance, L440. In the case of TN liposomes, oleoyl-hydrolyzed animal protein (OHAP) was added in order to control the vesicle size. The skin penetration ability of both drugs from liposomal gels into human stratum corneum was determined in vivo by a stripping method and
Chapter one:Site-specific drug delivery using liposomes as carriers 21 compared with conventional galenical formulations.The anti-inflam- mat ned in the ear edema absorption rates for both drugs after application of the liposomal preparations in comparison to other topical formulations.However, only a slight relationship between drug permeation into the stratum and lipo me di neter was The pulmonary delivery of omes ha erved.1s as recently bee n reviewed The technological aspects of delivering liposomes to the lungs are discussed,including the characterization of liposome-containing aerosols and the potential advantages and disadvantages of the var- ious methods that have been employed for their generation.Studies indicate that lipo can be effectively ed in the huma respiratory tract,wh n they may remain for onged periods Prolonged retention in the airways may markedly alter the pharma- cokinetics of liposome-associated materials.thereby increasing loca concentrations while decreasing levels at sites distant from the lung The future potential for such systems,including the possibilities for selective deliv hin the lung has yet to b Treatment of pulmonary diseases using the immunosuppressive drug cyclosporin A(CsA)is limited,in part,by poor penetration into the lungs following oral or intravenous administration and by the devel- opment of,hepatic,and oher toxicity following pro .CsA ol deli ivery may pr vide an aterate systemic toxicity.The total CsA dosage administered via aerosol is expected to be less than the conventional oral or intravenous dosage routes.Lipophilic CsA prepared with different phosphatidylchon liposome fo rmulations has bee nstudied in this r some ed of dioleo lylphoapha idyleth ne,1,2-Di. palmitoyls uccinylglycerol with polyethylene glyco a e pr nsitive plasma-stable and have a long circulation time in the blood.The com- plete destabilization of these liposomes might be useful for the targeted delivery of drugs such as anticancer agents. Polyethylene glycol-coated lip osome 3.5-dip entadecy hydrochlor rongly and sele bind to sub al cells via certain kinds of chondroitin sulfate proteogly cans and have an advantage for use as a specific drug delivery system.Fusogenic liposomes composed of the UV-inactivated Sendai virus effectively and directly deliver their encapsulated contents into the cytoplasm 111 ing a fusion nechanism of the Sendai virus,whereas mes are taken up y end osis 125 To enhance the efficiency of gene delivery by the introduction of molecules directly into the cells,virosomes have been developed by combining liposomes with fusogenic viral envelope proteins
Chapter one: Site-specific drug delivery using liposomes as carriers 21 compared with conventional galenical formulations. The anti-inflammatory effect of the released L440 was examined in the ear edema model in mice. The penetration tests showed significantly higher absorption rates for both drugs after application of the liposomal preparations in comparison to other topical formulations. However, only a slight relationship between drug permeation into the stratum corneum and liposome diameter was observed.106 • The pulmonary delivery of liposomes has recently been reviewed. The technological aspects of delivering liposomes to the lungs are discussed, including the characterization of liposome-containing aerosols and the potential advantages and disadvantages of the various methods that have been employed for their generation. Studies indicate that liposomes can be effectively deposited in the human respiratory tract, wherein they may remain for prolonged periods. Prolonged retention in the airways may markedly alter the pharmacokinetics of liposome-associated materials, thereby increasing local concentrations while decreasing levels at sites distant from the lung. The future potential for such systems, including the possibilities for selective drug delivery to specific cell populations within the lung, has yet to be determined.107 • Treatment of pulmonary diseases using the immunosuppressive drug cyclosporin A (CsA) is limited, in part, by poor penetration into the lungs following oral or intravenous administration and by the development of limiting renal, hepatic, and other toxicity following prolonged administration. CsA aerosol delivery may provide an alternate route of local administration that could improve treatment and reduce systemic toxicity. The total CsA dosage administered via aerosol is expected to be less than the conventional oral or intravenous dosage routes. Lipophilic CsA prepared with different phosphatidylcholine liposome formulations has been studied in this regard.108 • Liposomes composed of dioleolylphoaphatidylethanolamine, 1,2-Dipalmitoylsuccinylglycerol with polyethylene glycol are pH-sensitive, plasma-stable and have a long circulation time in the blood. The complete destabilization of these liposomes might be useful for the targeted delivery of drugs such as anticancer agents.124 • Polyethylene glycol-coated liposomes containing 3,5-dipentadecyloxybenzamide hydrochloride strongly and selectively bind to subendothelial cells via certain kinds of chondroitin sulfate proteoglycans and have an advantage for use as a specific drug delivery system. Fusogenic liposomes composed of the UV-inactivated Sendai virus effectively and directly deliver their encapsulated contents into the cytoplasm using a fusion mechanism of the Sendai virus, whereas conventional liposomes are taken up by endocytosis.125 • To enhance the efficiency of gene delivery by the introduction of molecules directly into the cells, virosomes have been developed by combining liposomes with fusogenic viral envelope proteins.126,127