Selected Examples Of Potential Medical Applications Of Liposomes

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Because of their ability to carry a wide variety of pharmaceuticals, liposomes have been studied for many different therapeutic situations. Therefore, the literature on this topic is abundant. Excellent reviews are available (e.g., Poznansky and Juliano, 1984; Gregoriadis, 1984, 1988b). We will restrict ourselves to the description of certain applications which illustrate the potential benefits of the use of liposomes in the field of drug delivery.

A. Intravenous Injection 1. MPS-Directed Drug Delivery

One approach where the characteristics of the liposomal carrier system are well matched to the intended therapeutic application is the delivery of drugs to the MPS. Because of their particulate nature, the major route of clearance of liposomes, when administered in vivo by a variety of routes, is phagocytosis by MPS cells, especially macrophages in liver and spleen. Obviously, this "natural" fate of liposomes in vivo is an advantage if one attempts to treat diseases

A) ENDOCYTOSIS OF LIPOSOMES

B) FUSION

A) ENDOCYTOSIS OF LIPOSOMES

degradation release of contents

B) FUSION

degradation release of contents

C) SURFACE ADSORPTION OF LIPOSOMES

C) SURFACE ADSORPTION OF LIPOSOMES

FIGURE 7 Various mechanisms of liposome-cell interaction. (Adapted from Pagano and Weinstein, 1987.)

involving the MPS or in cases where drug delivery to macrophages is required. For instance, when macrophages themselves become infected by foreign organisms, liposomes can be efficient, targeted drug delivery systems. Intracellular infections are caused by organisms which are capable of surviving inside the cells. In some cases, they' are capable of multiplying in the extracellular environment as well; these organisms are known as facultative intracellular parasites. Poor drug penetration into affected cells in combination with toxicity of and resistance to antiinfectious compounds often makes such intracellular infections difficult to treat.

The first reports on this approach were published independently by three different groups using liposomes as carriers of antimonial drugs like meglumine antimoniate in experimental leishmaniasis infection (Alving et al., 1978; Black et al., 1977; New et al., 1978). The Leishmania parasites are lodged inside the lysosomes of phagocytic cells, precisely the intracellular site where liposomes end up after intravenous injection. Liposomal encapsulation of the antimonial drug resulted in a more than 700-fold increase in therapeutic efficacy of this drug (Alving et al., 1978). Although this parasitic disease is infecting an estimated 100 million individuals throughout the world (Ostro, 1987), unfortunately in the near future the high therapeutic potential of this formulation is not expected to have a major impact on the treatment due to lack of current commercial interest for a mostly "third world" disease.

A highly successful use of a liposomal carrier system in infectious diseases which seems to be more promising from a commercial point of view is the therapy of systemic fungal infections with amphotericin B (AMB) incorporated into liposomes. Systemic fungal infections occur frequently in patients suffering from cancer or an immunodeficiency disorder (e.g., AIDS). For example, fungi, in particular Candida albicans, are responsible for about 20% of the lethal infections in leukemia patients. The use of AMB, a polyene antibiotic, is hindered by acute (fever, chills) and chronic toxicity to kidneys, central nervous system, and hematopoietic system (Miller and Bates, 1969). Amphotericin B has a certain preference to interact with fungal membranes due to their ergostefol content. However, it also binds to the cholesterol-containing mammalian membranes resulting in toxicity. Despite the serious side effects, limited efficacy and the development of several new antifungals, AMB is still the cornerstone of therapy for systemic fungal infections. As MPS organs such as liver, spleen, and lung are a frequent target of systemic fungal infections, encapsulation of AMB into liposomes was proposed as an approach to increase its therapeutic index.

Liposomal AMB has been shown to be effective in experimental fungal and also parasitic diseases (reviewed by Emmen and Storm, 1987; Lopez-Berestein, 1988; and Wiebe and Degregorio, 1988). Liposomal AMB was shown to be effective in the treatment of experimental histoplasmosis (Taylor et al., 1982) and cryptococcosis (Gray-bill et al., 1982). Mehta and coworkers (1984) found that, while free AMB is extremely toxic to both fungal cells and mammalian cells in vitro, the liposomal drug remains toxic to fungal cells but has little effect on mammalian cells. The AMB entrapped in multilamellar or small unilamellar liposomes of different lipid compositions was shown to be active and less toxic than conventional AMB solubilized in de-oxycholate (Fungizone) in the treatment of experimental candidiasis in mice. As a result much higher doses of AMB could be administered to the animals when given in liposomal form (Lopez-Berestein et al., 1983; Tremblay et al. , 1984; Ahrens et al., 1984). Animals treated with these high doses survived for much longer periods. In mice made neutropenic by the administration of cyclophosphamide— conditions which relate to systemic fungal infections occurring in cancer patients—AMB liposomes were more effective than AMB in the treatment of advanced candidiasis (Lopez-Berestein et al., 1984c). When injected intravenously, liposomal AMB induced higher antibiotic concentrations in liver, spleen, lungs, and kidneys in normal and in C. albicans-infected mice. All these animal studies point to.the superiority of AMB liposomes over AMB in the treatment of systemic fungal infections. However, it appears that a favorable outcome is not necessarily dependent on the entrapment of the drug into liposome structures. Recently, results were reported on an AMB colloidal dispersion (AMB-CD) being a size-controlled complex of AMB and sodium cholesteryl sulfate at a stoichiometric molar ratio of 1:1. The AMB-CD was as effective as Fungizone against species of Candida and Aspergillus. As AMB-CD was significantly less toxic than the free drug, a much higher maximum tolerated dose of AMB-CD couli be administered to rats and mice (Newman et al., 1989).

Striking results have been obtained in clinical trials by two groups of investigators using AMB liposomes in cancer patients with systemic fungal infections. One group used multilamellar liposomes made of dimiristoylphosphatidylcholine and dimiristoylphosphatidyl-glycerol (molar ratio 7:3) which were tested at the M.D. Andersor Hospital and Tumor Institute at Houston, Texas (Lopez-Berestein et al., 1985; Lopez-Berestein, 1988, 1989). The other group used sonicated liposomes made of egg phosphatidylcholine, cholesterol, and stearylamine (molar ratio 4:3:1) which were tested at the Institut J. Bordet, Brussels (Sculier et al., 1988). Clinical data from these studies indicate that two different types of liposomes, as far as structure and lipid composition are concerned, were well tolerated by cancer patients suffering from a systemic fungal infection. The liposomal product appears to be more effective and less toxic. The lack of serious toxicity at the dosage levels used is in agreement with data obtained in the treatment of experimental infections in animals by liposome-entrapped AMB. Hemolysis, kidney function deterioration, or central nervous system toxicity were not observed in the patients. Thus, upon intravenous administration, liposomes appear to be a safe carrier for AMB, a highly lipophilic compound which is not soluble in water. The true impact of liposomal AMB in the treatment of systemic mycoses and parasitic diseases can be fully evaluated once clinical trials are conducted in populations other than cancer patients and immunocompromised hosts. The mechanism behind the decreased toxicity and the enhanced activity is far from clear. Although natural targeting of liposomes to organs such as liver, spleen, and lung, which are rich in macrophages and which are a frequent target of systemic fungal infections, made the development of liposomal AMB formulations attractive, it must be noted that fungal cells are by no means exclusively located in these macrophages. Therefore, the improvement of the therapeutic index after encapsulation is not solely due to passive targeting of AMB to macrophages but seems to be related to a preference of liposomal AMB to interact with fungal cells and a degree of localization in affected areas possibly resulting from passage of AMB liposomes through damaged capillaries or from transportation inside migrating peripheral phagocytic cells (Lopez-Berestein, 1989).

A variety of other clinically important infections, such as brucellosis, listeriosis, salmonellosis, and various Mycobacterium infections, are of interest as these are often localized in organs rich in MPS cells. Liposome encapsulation has been demonstrated to improve therapeutic indices of several drugs in a number of infectious models. The natural avidity of macrophages for liposomes can also be exploited in the application of the vesicles as carriers of immunomodulators to activate these cells to an microbicidal, antiviral, or tumoricidal state. These studies were recently reviewed by Emmen and Storm (1987), Popescu et al. (1987), and Alving (1988). In addition to the treatment of "old" infectious diseases, the concept of MPS-directed drug delivery is of considerable interest for the therapy AIDS, possibly enabling control of human immunodeficiency virus replication in human macrophages.

Besides infectious diseases, liposomes offer opportunities for the treatment of other MPS-associated diseases. In metal poisonings and metal storage diseases the intracellular accumulation of metals induces intoxication. The primary sites of metal deposition are MPS cells of the liver, bone marrow, and the lungs. Chelating agents, with strong electron donor groups, have difficulties in crossing cell membranes. Clearly, liposome encapsulation can be useful to overcome this problem by allowing passive targeting to the diseased sites (Rahman, 1988).

2. MPS Avoidance Drug Delivery

In addition to MPS-directed delivery, liposomes have the potential of providing a controlled "depot" release of an encapsulated drug in the blood compartment over an extended period of time, thereby reducing toxic side effects of the drug by avoiding toxic peak concentrations of free drug in the bloodstream as will be illustrated in the section on DXR delivery via liposomes. However, it was early recognized (Poste, 1983; Poznansky and Juliano, 1984) that the rapid accumulation in the MPS system can limit the fields of application of intravenously administered drug-containing liposomes. Therefore, several groups explored the idea of avoiding uptake by the MPS (Davis and Illium, 1986; Allen and Chonn, 1987; Gabizon and Papa-hadjopoulos, 1988). Liposomes which are capable of evading the MPS would provide two important advantages. First, the liposome circulation time in the blood is increased. This would prolong the time frame available for slow drug release in the bloodstream and would also improve the prospect of directing liposomes to tissues other than liver, spleen, and lung. The other advantage may be decreased liposome loading of the MPS, thus avoiding blockade and impairment of an important host defense system (Storm et al., 1990a). One of the early approaches which have been followed to increase liposome circulation time was "blocking" of the phagocytic uptake mechanisms of the MPS by predosing with high doses of liposomes or other particles. Another approach was based on improving liposome stability in plasma (Senior, 1987). During their stay in the bloodstream liposomes can interact with serum proteins (e.g., lipoproteins and opsonins) and sometimes become destabilized, resulting in leakage of entrapped solute. In particular high-density lipoproteins (HDL) appear to be responsible for the desta-bilization. Manipulations that reduce these interactions, e.g., by rigidifying the bilayers by the inclusion of cholesterol and phospholipids yielding gel state bilayer structures, have been shown to decrease leakage of liposome contents in plasma and to prolong the residence time of liposomes in the circulation (Senior and Gregoriadis, 1982; Allen and Everest, 1983; Senior, 1987). However, although both approaches resulted in reduced clearance rates of circulating liposomes, they were not very successful in achieving substantial liposome uptake by tissues outside the MPS (Poste, 1983). Profitt et al. (1983) reported a 50% increase in the uptake of labeled liposomes in tumors of mice with a suppressed MPS system by predosing with "cold" liposomes compared to "nonsuppressed animals." However, MPS suppression seems to be of limited value in clinical practice since the MPS plays an important role in the body's defense system. Thus, it is not feasible to close it down for prolonged periods of time.

However, recent evidence from the laboratories of Allen (1989) and Papahadjopoulos (Gabizon and Papahadjopoulos, 1988) suggests that longer circulation of liposomes still may offer viable therapeutic opportunities. It was found that inclusion of monosialoganglioside (GM1), phosphatidylinositol, or sulfatides prolongs the circulation times of liposomes, particularly in combination with liposomal formulations with rigid bilayers (e.g., containing sphingomyelin, distearoyl-phosphatidylcholine, cholesterol, or other rigidifying phospholipids). Allen (1989) showed that as much as 50% of the injected dose remains in the circulation for 8 hr with up to 20% of the injected dose still circulating after 24 hr. It has been proposed that the phenomenon of prolonged circulation and reduced MPS uptake might be related to a decreased opsonization, probably obtained by mimicking important properties of the outer monolayer of red blood cells (Allen and Chonn, 1987). Clearly, such liposomes have a far greater opportunity than conventional liposomes to be distributed to non-MPS sites. A remarkable increase in tumor uptake (up to 25-fold as compared to conventional liposomes) was observed when such liposome formulations, called "stealth" liposomes because of their ability to avoid detection and uptake by the MPS, were tested in mice bearing an implanted intramuscular tumor (Gabizon and Papahadjopoulos, 1988).

The "stealth" concept may offer two other opportunities for liposome application: (1) Conventional immunoliposomes (see Sec. VI.C) have been shown to be removed rapidly from the circulation by the MPS (Peeters et al., 1987). The combination of the stealth approach for longer circulation with the attachment of antibodies or antibody fragments may provide a means of delivery of drugs to their sites of action with a high degree of specificity. This could be useful for treating leukemia, graft-vs. -host diseases, and HIV disease. (2) Extending the circulation time of relatively large liposomes (0.2-0.4 pm) offers the potential for creating an encapsulated circulating reservoir. With the benefit of the stealth coating such liposomes would avoid MPS uptake for many hours and could be engineered to release entrapped drugs as they circulate. The therapeutic availability of many drugs which are rapidly degraded or excreted in their free form may be substantially increased using this approach. Peptides often have short blood half-lives requiring large doses or multiple daily injections to be effective. Such molecules might well benefit from a long-circulating stealth microreservoir engineered to gradually release drug. This might allow dosing frequency to be reduced to only one or two injections daily. The circulating micro-reservoir concept may be preferred for the delivery of peptides over the subcutaneous or intramuscular route of administration usually used for these drugs, as the peptide would be released within the blood compartment so that distances to most target sites are much shorter thereby maximizing the likelihood of the peptide to reach its site of action.

B. Diagnosis and Therapy of Cancer

As most antitumor drugs have a narrow therapeutic window, cancer chemotherapy presents serious challenges for the design of drug delivery systems. A large number of investigations using liposomes as carriers for currently utilized antitumor drugs, such as anthra-cyclines (Gabizon, 1989; Storm, 1987; Storm et al., 1989a,b), cis-platin (Yatvin et al., 1981; Steerenberg et al., 1988), methotrexate (Kimelberg et al., 1976; Kosloski et al., 1978), and cytosine arabino-side (Kobayashi et al., 1977; Rustum et al., 1979; Mayhew et al., 1982), have been published. Additionally, other compounds not currently accepted as antitumor agents have been formulated in liposomes in order to enlarge the anticancer drug arsenal, e.g., lipophilic cisplatin analogs (Perez-Soler et al., 1986; Lautersztain et al., 1986), nocodazole, a water-insoluble, experimental drug (Soulier et al., 1986), so-called liposome-dependent drugs (referring to drugs with poor ability to enter cells and whose toxicity can be significantly enhanced by intracytoplasmic delivery) such as methotrexate-y-aspartate (Heath et al., 1985a) and 5-fluoroorotate (Heath et al., 1985b). and valinomycin, an antibiotic with ionophoric activity (Daoud and Juliano, 1986). Research on liposomal antitumor agents and the difficulties inherent to this approach have been reviewed in detail by Weinstein and Leserman (1984), Poznansky and Juliano (1984), and Gabizon (1989). In the initial literature on this subject, enthusiasm was generated primarily by the idea that liposomes might be used for specific delivery of the antitumor drug to tumor cells. However, a better understanding of the biological barriers separating the drug-liposome entity from the ultimate site of action led to the view that uptake of liposomes by nonphagocytic cells, including most tumor cells generally is questionable or, at best, of little significance (Poznansky and Juliano, 1984). Nowadays the leading rationale for liposome encapsulation of antitumor drugs is based on the expected favorable effects on the pharmacokinetic and distribution characteristics of the encapsulated drug resulting in reduced toxicity without loss of antitumor activity. To illustrate this valuable application of liposomes in anticancer therapy, we will focus below on the description of results obtained in our group on the mechanism(s) underlj/ing the therapeutic effects obtained by encapsulation of doxorubicin (DXR) and cisplatin (cDDP).

Different types of DXR- and cDDP-containing liposomes (extrusion MLV) were tested in rats bearing a solid IgM-immunocytoma on the flank. The encapsulation of DXR in liposomes resulted in a marked prolongation of the survival times (Table 1). In the animal group treated with DXR-DPPC:DPPG:chol (10:1:10) liposomes no deaths were scored 30 days after the onset of therapy; in the group treated with PC:PS:chol (10:1:4) liposomes 20% of the animals died within 30 days, while in the group treated with free DXR no long-term survivors were scored. In Fig. 8 the antitumor activity of DXR in free and in liposomal form is depicted. It can be derived that the liposome-induced enhancement of survival time was not caused by major changes in the antitumor activity as compared to free DXR. Thus, for the dose regimen used, DXR encapsulation in liposomes did not impair the antitumor activity of the drug, whereas it apparently lowered the toxicity resulting in prolonged survival and an overall increase in the therapeutic index.

Encapsulation of cDDP in liposomes did not show such favorable effects. Liposome encapsulation of cDDP decreased the antitumor effect (Fig. 9). It was demonstrated that administration of cDDP liposomes resulted in a lower incidence as well as reduced severity of focal alterations of the epithelium of the proximal tubuli compared to administration of the free drug (Steerenberg et al., 1988). However, despite this reduction in renal toxicity the therapeutic index

TABLE 1 Effect of Treatment of Solid IgM Immunocytoma Bearing Lou/M Rats with Different Formulations of DXR

Type of treatment0

Long-term survivors b (>30 days)

Saline

0/10

Free DXR

0/10

DXR-DPPC/DPPG/chol

10/10

DXR-PC/PS/chol

8/10

DXR-DSPC/DPPG/chol

5/10

aTumor-bearing rats were injected with 2 mg DXR/kg body weight i.v. daily for 5 days and on day 11. Tumor diameter at the start of the experiment was 2-3 cm.

^Number of surviving versus the total number of rats. Liposomes: DPPC/DPPG/chol: (molar ratio 10:1:10); size about 0.7 ym. PC/PS/chol: (molar ratio 10:1:4); size about 0.3 ym. DSPC / DPPG/chol: (molar ratio 10:1:10); size about 0.7 ym. Source: Crommelin et al., 1990b.

was not improved due to the loss of antitumor activity. Studies were performed in order to obtain a better understanding of the mechanism(s) underlying these therapeutic results. For DXR liposomes, the results obtained pointed to sustained release as the primary mechanism behind the beneficial effects resulting from liposome encapsulation of DXR. No direct DXR liposome accumulation in the tumor was found; DXR concentrations in the cardiac tissue were lower after administration of the free drug (Van Hoesel et al. , 1984; Storm et al. , 1989a). Interestingly, two different pathways for the sustained release of DXR were identified: DXR is released directly from liposomes being present in the blood but also indirectly from the MPS following uptake and processing by macrophages (Storm et al., 1990b). Peak concentrations of free DXR in organs which are particularly sensitive to the toxic action of the drug, like the heart, are avoided, while apparently the prolonged presence of free (i.e., nonliposomal) DXR levels in the blood can result in sufficient exposure levels for tumor cells. Comparison of liposomal DXR delivery with DXR delivery via long-term infusions in the same tumor model confirmed the indicated concept of sustained release (Storm et al., 1989b). Steerenberg et al. (1988) offered an explanation for the observed decrease in antitumor activity of cDDP after incorporation into liposomes (Fig. 9). They reported that the reduction in antitumor activity might be related to conversion of liposomally delivered cDDP by macrophages of liver and spleen in a less cytotoxic form.

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