I. Introduction

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There are many potential barriers to the effective delivery of a drug in its active form to solid tumors. Most small-molecule chemotherapeutic agents have a large volume of distribution on i.v. administration (Speth et al., 1988; Chabner and Longo, 1996). The result of this is often a narrow therapeutic index due to a high level of toxicity in healthy tissues. Through encapsulation of drugs in a macromolecular carrier, such as a liposome, the volume of distribution is significantly reduced and the concentration of drug in the tumor is increased (see IIF. Comparison of Pharmacokinetic Parameters for Different Liposomal Formulations and III. Accumulation of Liposomal Drugs in Tumors). This results in a decrease in the amount and types of nonspecific toxicities and an increase in the amount of drug that can be effectively delivered to the tumor (Papahadjopoulos and Gabizon, 1995; Gabizon and Martin, 1997; Martin, 1998). Under optimal conditions, the drug is carried within the liposomal aqueous space while in the circulation but leaks at a sufficient rate to become bioavailable on arrival at the tumor. The liposome protects the drug from metabolism and inactivation in the plasma, and due to size limitations in the transport of large molecules or carriers across healthy endothelium, the drug accumulates to a reduced extent in healthy tissues (Mayer et al., 1989; Working et al., 1994). However, discontinuities in the endothelium of the tumor vasculature have been shown to result in an increased extravasation of large carriers and, in combination with an impaired lymphatics, an increased accumulation of liposomal drug at the tumor (see III. Accumulation of Liposomal Drugs in Tumors; Huang et al., 1993; Yuan et al., 1994, 1995; Hobbs et al., 1998). All of these factors have contributed to the increased therapeutic index observed with liposomal formulations of some chemotherapeutic agents (Papahadjopoulos et al., 1991; Gabizon, 1994; Martin, 1998).

A diagram depicting both a conventional liposome (CL)³ and a sterically stabilized liposome (SSL) is shown in Fig. 1. The two types of liposomes share a lipid membrane that is relatively impermeable to both amphipathic and highly water-soluble molecules at physiological temperatures (37°C). This feature is important for the maintenance of stable liposome drug formulations, both during storage and in plasma (see VII. Stability in Plasma and Storage). Liposomes composed of a comparably more-fluid membrane are being used as a rapid-release system for doxorubicin (DOX) and are described to a limited extent in this review. A liposome also has an internal aqueous space, which can be used to entrap a variety of chemotherapeutic drugs or diagnostic dyes. We discuss in VII. Stability in Plasma and Storage how different drugs are efficiently loaded into this space. The two types of liposomes differ in the presence of the polymer coating [most commonly, polyethylene glycol (PEG)] on the surface of the SSLs but not CLs. This coating provides steric stabilization to the liposome, which is thought to limit binding of serum opsonins as well as direct interactions with cells, most importantly, of the reticuloendothelial system (RES; Allen et al., 1991, 1994; Lasic et al., 1991). The result is enhanced circulation times and increased localization in the tumor (Papahadjopoulos et al., 1991, 1995; Gabizon and Martin, 1997).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Diagram of a drug-loaded liposome both with (SSL) and without (CL) a PEG coating. The liposome contains a lipid membrane that encapsulates an internal aqueous space used to entrap chemotherapeutic drugs. DOX can be encapsulated at concentrations exceeding its aqueous solubility, forming drug crystals in the liposome interior. Alternatively, some drugs can be carried within the lipid bilayer. Further modifications of the surface through covalent attachment of targeting ligands such as Fab' fragments can result in liposomes that are specifically endocytosed by cancer cells expressing a receptor for that ligand (e.g., the HER2 receptor found on certain breast cancer tumors). The structures of the three most commonly used lipids that compose the lipid bilayer are also given. DSPC (A) or an equivalent, HSPC, is the primary phospholipid component, whereas Chol (B) is the neutral lipid component. PEG-DSPE (C) is incorporated at concentrations of 4 to 6 mol% in SSL formulations.

Steric stabilization refers to the colloidal stability (Lasic and Needham, 1995; Lasic and Papahadjopoulos, 1996) conferred on the liposome by a variety of hydrophilic polymers or hydrophilic glycolipids (Allen and Chonn, 1987; Papahadjopoulos et al., 1991; Woodle and Lasic, 1992; Allen, 1994; Torchilin et al., 1995; Zalipsky et al., 1996), the best studied of which are PEG and the ganglioside GM₁. An important finding was that SSLs also show a prolonged lifetime in the circulation (Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; Klibanov et al., 1990; Allen et al., 1991; Papahadjopoulos et al., 1991). SSLs then typically refer to any liposomes containing PEG-PE, GM₁, or another of these glycolipids or polymers that has a relatively long half-life in the general circulation. The term "conventional liposomes" has a much broader definition and refers to liposomes composed of a variety of different lipid compositions, but typically the most commonly used of these compositions are very high in phosphatidylcholine (PC) and cholesterol (Chol). The pharmacokinetics and tissue distribution of CLs depend on properties such as size, surface charge, and membrane packing. These factors are discussed in more detail in II. Pharmacokinetics and Biodistribution of Liposomes and Liposomal Drugs. However, to perform a careful comparison, we limit this discussion to formulations optimized for increased residence in the circulation, accumulation in tumors, and stability in the plasma. For SSLs, we consider liposomes containing 4 to 6 mol% PEG-DSPE, ~30 mol% Chol, and the remainder hydrogenated soy phosphatidylcholine (HSPC) or distearoylphosphatidylcholine (DSPC; Fig. 1). The size of the carrier is usually 60 to 120 nm. For CLs, the optimized formulations are composed of DSPC and Chol in either a 55:45 or 66:33 M ratio or phosphatidylcholine derived from egg yolk (eggPC)/Chol (3:2) and have a similar average size distribution.

The choice of drug for delivery via liposomes is essential to the success of this approach. Broad generalizations as to the usefulness of a certain liposome composition for the delivery of all chemotherapeutic drugs or as to the superiority of liposomal formulation for all classes of drugs is extremely dangerous considering the present limitations in liposome technology. To be effective as a carrier, a liposome must be able to efficiently balance stability in the circulation with the ability to make the drug bioavailable at the tumor. In choosing a drug, there are several criteria to consider. The drug must have sufficient activity against the chosen tumor; a drug such as DOX with a relatively broad activity against a variety of different tumor models is an ideal choice in this regard (Young et al., 1981; Doroshaw, 1996). Second, the drug must be efficiently loaded into the liposomal carrier. Ammonium sulfate and pH gradients have been used for remote loading of a variety of amphipathic basic amines, resulting in encapsulation efficiencies of ~100% (Madden et al., 1990; Lasic et al., 1992a; Haran et al., 1993; Cullis et al., 1997). Finally, the drug must be compatible with the carrier; it must be stably transported in the circulation but still released at the tumor. A wide array of different drugs have been encapsulated in liposomes for the treatment of cancer (Fig. 2; Heath et al., 1983; Papahadjopoulos et al., 1991; Allen et al., 1992; Vaage et al., 1993b; Burke and Gao, 1994; Sharma et al., 1995; Jones et al., 1997; Working, 1998). The listed examples illustrate a diversity of different classes of chemotherapeutic drugs, with distinct chemical stabilities, solubility and membrane partitioning properties, modes of action, and modes of drug resistance.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Structures of a few chemotherapeutic drugs that have been used with liposomes either in vitro or in vivo. These drugs operate via a variety of different mechanisms; some have different mechanisms of drug resistance and varying physical characteristics that make them more or less compatible for encapsulation in liposomes. At the present time, only formulations of anthracyclines (daunorubicin and DOX) have been sufficiently developed for use in the clinic, although a liposomal VCR formulation is presently under study in clinical trials.

Barenholz and coworkers (Barenholz and Cohen, 1995; Barenholz, 1998) classified these drugs into one of three classes depending on their hydrophobic properties measured as octanol-to-water partition coefficient (K_p): 1) highly hydrophilic drugs such as N-(phosphonoacetyl)-L-aspartate, 2) hydrophobic drugs such as paclitaxel, and 3) amphipathic drugs such as DOX, which represent many current chemotherapeutic agents. Liposomal formulations of highly hydrophilic drugs can be limited by the bioavailability of these drugs at the tumor site, which may be prohibitively low due to their extremely low membrane permeability and, therefore, low drug release once the carrier has reached the tumor. Drugs such as 1--D-arabinofuranosylcytosine (ara-C) or methotrexate, which are taken by tumor cells using membrane transporters (Plageman et al., 1978; Wiley et al., 1982; Westerhof et al., 1991, 1995; Antony, 1992), may be useful members of this class of drugs, assuming they can be released from the liposome in adequate quantities (Heath et al., 1983; Matthay et al., 1989; Allen et al., 1992). Future improvements in the design of carriers that are destabilized and release the drugs specifically at the tumor site may make their utilization more feasible, as discussed in VIII. Bioavailability of Encapsulated Drug. Highly hydrophobic drugs tend to associate mainly with the bilayer compartment of the liposome; this leads to lower entrapment stability due to faster redistribution of the drug to plasma components. However, liposomes may be used with this class of drugs simply as the means to formulate them for i.v. administration rather than using liposome encapsulation to achieve enhanced tumor delivery of the drugs. For example, paclitaxel has formulated into liposomes (Sharma et al., 1995, 1997) but may be equally suitable when formulated as a microemulsion (Wheeler et al., 1994). Liposomes have also used to solubilize and administer hydrophobic photosensitizers for use in photodynamic therapy (Allison et al., 1990; Reddi, 1997).

Considering the present state of liposome technology, amphipathic drugs appear to be the most suitable for liposomal carriers; these drugs include anthracyclines, such as DOX and daunorubicin, and Vinca alkaloids, such as vincristine (VCR), vinblastine, and vinorelbine (Fig. 2). With this class of drugs, it is possible to tune the drug-release rates to maintain the stability of the formulation in the plasma, yet allow the drug to be released at the tumor site. This is in large part due to the development of gradient-based loading techniques leading to stable liposomal drug formulations (Nichols and Deamer, 1976; Mayer et al., 1985; Madden et al., 1990; Haran et al., 1993). Indeed, the first liposomal oncology drugs approved for medical use in liposomal form are of the anthracyclines daunorubicin (DaunoXome; Nexstar Pharmaceuticals, Boulder, CO) and DOX [Doxil; Alza Corporation, Palo Alto, CA (CAELYX in Europe)]. DaunoXome is formulated as a CL (DSPC/Chol), whereas Doxil is an SSL formulation (hydrogenated soy PC/Chol/PEG-DSPE; Table 1). Another CL formulation (eggPC/Chol) of DOX (Harris et al., 1998), as well as formulations of other amphipathic drugs, such as VCR (Embree et al., 1998) or cisplatin (Newman et al., 1999), is in preclinical or clinical trials or under Food and Drug Administration consideration for commercial release.

In the remaining sections of this review, we attempt to show how optimization of the balance of circulation lifetimes, drug-induced toxicities, accumulation in tumors, and drug release rates from liposomes results in the most clinically effective formulations. This is accomplished through adjustments of both the pharmacological and physical properties of the liposome, including the injected dose, liposome size, presence of steric stabilization, and lipid composition of the carrier.

	II. Pharmacokinetics and Biodistribution of Liposomes and Liposomal Drug

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For free DOX, the volume of distribution has been estimated at 25 l/kg, suggesting a significant uptake by tissues (Speth et al., 1988). This large volume of distribution, when combined with the relatively rapid clearance rate from the circulation, results in low drug levels in the tumor and significant toxicity to normal tissues. Liposomes can alter both the tissue distribution and the rate of clearance of a drug by causing the drug to take on the pharmacokinetic parameters of the carrier. Pharmacokinetic parameters of the liposomes depend on physicochemical attributes of the liposomes, such as size, surface charge, membrane lipid packing, and steric stabilization, as well as on the administered dose and route of administration. The pharmacokinetics of both CLs and SSLs have been extensively reviewed (Hwang, 1987; Allen et al., 1995; Allen and Stuart, 1999).

Both slow-release CLs and SSLs have a volume of distribution for DOX not significantly different from the total blood volume (see Table 3), indicating the drug is generally confined to the systemic circulation. However, after i.v. administration, CLs have saturable, nonlinear kinetics, whereas SSLs have nonsaturable, log-linear kinetics (Hwang, 1987; Allen et al., 1995). The dose-dependent kinetics for CLs result in relatively rapid clearance rates for liposomes at low doses and complicates the calculations of clinical dosages. Clearance of CLs has been suggested by Allen et al. (1995a) to be due to both a high-affinity, low-capacity system, likely the macrophages of the RES, and a low-affinity, high-capacity system. Steric stabilization slows uptake by the high-affinity, low-capacity system, resulting in dose-independent kinetics. The potential mechanisms responsible for the reduced clearance and dose-independent pharmacokinetics of SSLs are described in more detail in IIE. Effect of Steric Stabilization on Pharmacokinetic Parameters.

Liposomes are cleared from the circulation by macrophages of the RES, in particular those of the liver and spleen (Gregoriadis, 1976; Weinstein, 1984; Senior, 1987). Opsonization by serum proteins such as the complement C3b fragment, ₂-glycoprotein I, and the Fc portion of IgG molecules is thought to play a critical role in the recognition and subsequent clearance by RES macrophages (Senior, 1987; Patel, 1992; Devine et al., 1994; Chonn et al., 1995; Devine and Marjan, 1997). The success of a liposome-based approach for drug delivery to sites other than those making up the RES is one that limits the uptake of liposomes by macrophages, either directly by preventing the interaction of liposomes with receptors on the macrophage surface or indirectly by decreasing the binding of serum opsonins. Many studies have concentrated on understanding the mechanisms responsible for regulation of these interactions. These factors are often intricately intertwined, making it impossible to construct sweeping assumptions based on any one factor.

The first aspect of a liposome that affects its disposition is size. Liposomes of a defined size are readily prepared by extrusion of lipid suspensions through filters containing pores of a similar size (Olson et al., 1979; Szoka et al., 1980). Liposomes prepared through this method are slightly larger (20-50%) than the average pore size of the filter. The general trend for liposomes of similar composition is that increasing size translates into more rapid uptake by the RES (Abra and Hunt, 1981; Hwang, 1987; Senior, 1987). However, although the trend remains the same, the clearance of liposomes is affected to differing extents depending on the composition. For example, DSPC/Chol (3:2) liposomes extruded through 400-nm filters are cleared 7.5 times as fast as liposomes extruded through 200-nm filters, which in turn are cleared 5 times as fast as small unilamellar vesicles (Senior et al., 1985). The inclusion of PEG-DSPE in the liposome composition results in clearance rates that are relatively insensitive to size in the range of 80 to 250 nm (Allen et al., 1989; Liu et al., 1992; Woodle et al., 1992). Now, a 2-fold increase in size from 100 to 200 nm results in only a 54% increase in clearance (Fig. 3; Woodle et al., 1992). A similar dependence of liposome clearance on size was observed for DSPC liposomes stabilized with small quantities of N-glutaryl-phosphatidylethanolamines (Ahl et al., 1997). These liposomes also showed an increased plasma area under the curve (AUC) compared with DSPC/Chol controls, similar to PEG-DSPE-stabilized liposomes. The authors suggested that the aggregation of nonstabilized neutral liposomes may result in an increase in the effective size and, thus, clearance from the circulation via a size-dependent mechanism. Although the dependence of liposome clearance rates on size is relatively less for these two stabilized formulations than for that with CLs, it nevertheless highlights the importance of optimization of liposome size in drug delivery systems not aimed at the RES. For neutral CLs, the window for optimal behavior is considerably narrower, and these data suggest that liposomes should be small enough (preferably <100 nm) but still maintain reasonable drug encapsulation efficiencies.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of liposome size on plasma levels of PEG-DSPE/PC/Chol (0.15:1.85:1 mol/mol/mol). Female adult Sprague-Dawley rats were injected with 67Ga-desferoxamine-loaded liposomes, and blood was drawn at prescribed time points, when 67Ga levels were determined with a gamma counter. From these data, the half-life in the circulation was determined by fitting the data to a single exponential curve (), and the 24-h time point was recorded (). The liposomes were prepared by extrusion through polycarbonate filters of defined size as described by Olson et al. (1979), and their size distribution was determined by dynamic light scattering. PC in these liposomes refers to partially hydrogenated egg PC. This figure was adapted from Woodle et al. (1992).

The administered dose can also play a significant role in the circulation lifetime of a carrier. CLs are removed from the circulation in a dose-dependent manner, indicating a saturation of the mechanisms responsible for their uptake (Gregoriadis and Senior, 1980; Abra and Hunt, 1981; Senior et al., 1985; Hwang, 1987). Circulation lifetimes typically increase as a function of increasing lipid dose. This effect is likely due to a decreased phagocytic capacity of RES macrophages after the ingestion of high lipid doses or to a saturation of plasma factors that bind to circulating liposomes and result in their opsonization. The fact that liposomes composed of high-phase transition lipids, such as SM/Chol or DSPC/Chol, can more readily saturate RES uptake may indicate that these difficult-to-metabolize lipids saturate metabolic pathways responsible for their destruction (Senior et al., 1985; Hwang, 1987). Alternatively, liposomes have been shown to bind serum proteins in a manner inversely proportional to their blood clearance rates (Chonn et al., 1992; Semple and Chonn, 1996; Semple et al., 1996), giving rise to the hypothesis that the depletion of plasma opsonins at high lipid doses results in an increase in blood circulation half-lives (T_1/2; Harashima et al., 1993; Oja et al., 1996).

RES blockade can also be achieved by delivering cytotoxic drugs such as DOX or dichloromethylene diphosphonate to RES macrophages (Bally et al., 1990b; Parr et al., 1993; Qian et al., 1994; Buiting et al., 1996). Parr et al. (1997) recently considered the effect of dose on DOX-loaded liposomes. In these experiments, the presence of DOX resulted in a ~1.5- to 2-fold increase in the plasma levels of liposomal lipid at higher doses for SSL DOX compared to CL DOX. In DOX-loaded liposomes, a 10-fold increase in plasma levels of liposomal lipid observed at lower lipid doses (<1 µmol lipid/20-22 g mouse) was reduced to a 3-fold increase at higher doses (>2 µmol lipid/mouse). It should be noted it is at these lower doses that SSL preparations are routinely used. Thus, with SSL DOX, long circulation does not necessarily come at the expense of RES toxicity. The possible implications of the use of dose escalation, and the resulting RES toxicity, simply to achieve long circulation times are described in more detail in VI. Toxicology of Liposomal Chemotherapy. This indicates that RES blockade is in part due to the drug and not solely to a saturation of plasma opsonins or inability to metabolize liposomal lipid components.

Steric stabilization with PEG-DSPE offers a unique advantage to liposome delivery in that clearance kinetics become dose independent (Allen and Hansen, 1991; Huang et al., 1992; Woodle et al., 1992). The data in Fig. 4 illustrate the relative effect of liposome dose on clearance of both an SSL formulation (SM/eggPC/Chol/PEG-DSPE, 1:1:1:0.2) and a CL formulation (eggPC/Chol, 2:1). For the SSLs, the plasma AUC increases linearly, whereas the T_1/2 remains relatively unchanged. This dose independence was recently shown to extend down to concentrations of lipid as low as 1 µmol/kg in rabbits (Utkhede and Tilcock, 1998). In stark contrast, the plasma AUC for CLs increases slowly at low doses (<2.5 µmol phospholipid/23-27 g mouse) and then increases exponentially with increasing lipid dose. A look at the circulation T_1/2 of the conventional formulation shows a leveling off of the T_1/2, indicating a saturation of the mechanism responsible for their clearance. Although the CLs used in this particular study used a fluid-phase phospholipid component, eggPC, similar pharmacokinetics have been seen with DSPC/Chol and SM/Chol liposomes (Beaumier et al., 1983; Hwang, 1987; Chow et al., 1989). In one of these studies (Beaumier et al., 1983), liposome levels in the liver were shown to saturate at the same dose where plasma clearance rates leveled off, consistent with RES saturation being responsible for increased plasma levels at high lipid doses.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Dependence of circulation T1/2 values (, ) and plasma AUCs (, ) on administered dose. Female ICR mice (three per group) were given a single bolus tail-vein injection of liposomes containing 125I-tyraminylinulin and 0.1 to 10 µmol of phospholipid. Liposomes were composed of either eggPC/Chol (2:1 mol/mol) or SM/eggPC/Chol/PEG-DSPE (1:1:1:0.2 mol/mol/mol). This figure was adapted from Allen and Hansen (1991).

The effect of liposome surface charge on liposome clearance kinetics is an increasingly misused predictive factor of circulation lifetimes. Early studies have shown that the presence of negatively charged lipids in liposomes, including phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylglycerol (PG), results in rapid uptake by the RES (Senior et al., 1985; Senior, 1987). However, this relationship between the presence of charged lipids and circulation lifetimes is extremely complex and cannot be readily explained with simple models in which the presence of an anionic lipid necessitates increased clearance from the circulation. Indeed, it now appears that each lipid must be analyzed separately and in the context of similar liposomes with respect to size, membrane packing constraints, and surface charge density.

A more careful characterization of the effect of surface charge on liposome clearance in mice was conducted using liposomes containing different anionic phospholipids (Gabizon and Papahadjopoulos, 1992). In these experiments, anionic lipids were added to fluid eggPC/Chol liposomes in a 1:10:5 ratio (anionic lipid/eggPC/Chol). Although liposomes containing PG, PA, and PS (PS > PA > PG) were cleared more rapidly than neutral liposomes, the inclusion of other anionic lipids such as the ganglioside GM₁ or phosphatidylinositol (PI) resulted in longer circulation lifetimes. Later, this second group of anionic lipids was shown to include PEG-PE conjugates (Papahadjopoulos et al., 1991; Woodle et al., 1992). The new model then divided negatively charged lipids into those with and those without a sterically shielded negative charge. Those with a sterically hindered charge were cleared more slowly, whereas those without were cleared more rapidly than neutral liposomes of a similar composition. This too may have proved to be too simple of a model.

The last statement concerning a similar composition is extremely important, and the effect of the phase transition of the lipid is intricately interrelated with the effect of charge. 1,2-dipalmitoyl-3-sn-phosphatidylglycerol (DPPG)/DSPC/Chol liposomes were previously shown to be cleared more rapidly than DSPC/Chol liposomes in mice (Fig. 5; Lasic et al., 1991; Woodle et al., 1992), and in a separate study, eggPG/DSPC/Chol liposomes were cleared more rapidly than DPPG/DSPC/Chol liposomes (Gabizon et al., 1990). However, DSPC has a gel-to-liquid crystalline phase transition (T_m) of 55°C, whereas the T_m value of DPPG is 41.1°C (Table 2; Boggs et al., 1989). Thus, the replacement of some of the DSPC with DPPG does not necessarily result in liposomes with similar permeability and membrane packing characteristics. Recently, DOX-loaded 1,2-distearoyl-3-sn-phosphatidylglycerol (DSPG)/HSPC/Chol liposomes, in which the source of PG was distearoylphosphatidylglycerol (T_m = 53.0; Table 2), were shown to have plasma levels of DOX at 24 h that were greater than twice those of HSPC/Chol liposomes (Gabizon et al., 1996). In addition, the requirement for a high phase transition anionic lipid component may also be necessary for PI, where hydrogenated soy PI is most commonly used as the source of PI in long-circulating liposomes (Gabizon and Papahadjopoulos, 1988; Gabizon et al., 1990). Thus, from these few cases, it appears that that the dependence of long circulation is more related to membrane packing and permeability considerations, and that the inclusion of high-phase transition anionic lipids into solid liposomes can actually increase circulation lifetimes.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of steric stabilization and lipid composition on plasma levels of liposomes. Extruded liposomes (70-100 nm) loaded with 67Ga-deferoxamine were injected by i.v. administration into female Swiss-Webster mice at a dose of 1 µmol of phospholipid per mouse. Blood levels of 67Ga were determined by gamma counting 24 h after injection. Lipid molar ratios were 1:10:5 except for eggPC/Chol and DSPC/Chol, both at 10:5, and PEG-DSPE/EPG/eggPC/Chol at 1:3:7:5. This figure was adapted from Woodle et al. (1992) and Lasic et al. (1991).

However, as was stated previously, all cases must be considered individually. In one study with another anionic phospholipid, PA, liposomes composed of DSPC/1,2-distearoyl-3-sn-phosphatidic acid (DSPA)/Chol (3:1:4) were cleared ~10 times faster than DSPC/Chol liposomes (1:1; Senior, 1987). DSPA has a T_m value comparable to those of DSPG and DSPC at 58°C, and so for DSPA at least, liposome charge appears to become more important than membrane packing in the determination of rates of uptake. Of course, DSPA was introduced at 25% of the total phospholipid content and has two negative charges per molecule. In the previous example, DSPG was incorporated at only 10% of the total phospholipid and has only one negative charge, consequently adding an additional layer of complexity involving surface charge density. Janoff and coworkers have indeed shown a steep dependence of blood clearance rates on the mol% of the negatively charged component N-glutaryl-dipalmitoylphosphatidylethanolamine (N-glutaryl-DPPE) in DSPC liposomes (Ahl et al., 1997). The normalized AUC in plasma was greatest at 10 mol% N-glutaryl-DPPE and rapidly declined both below and above this value. The authors suggested the small amounts of negatively charged lipids stabilize neutral liposomes against an aggregation-dependent uptake mechanism. All of these examples point to the reality that different liposomes, and even comparable liposomes with different phospholipid headgroups of similar charge, may have very different mechanisms responsible for their uptake (Daemen et al., 1997).

An even more intriguing question is raised based on these analyses. Have CLs really been optimized? Are small DSPC/Chol liposomes really the most efficient liposomal carriers in the absence of steric stabilization? At least three studies have suggested that the inclusion of small amounts (10 mol%) of certain negatively charged lipids such as DSPG or N-acylated phosphatidylethanolamines (Park et al., 1992; Gabizon et al., 1996; Ahl et al., 1997) actually increase circulation T_1/2 even further. Additional studies will be needed to elucidate the exact nature of this stabilizing effect and determine more carefully the dependence of this stabilization on the structure of the stabilizing lipid and such parameters as membrane packing. Whether these liposomes would offer any improvements over SSLs remains to be seen, but at least one study has suggested that some DOX-loaded anionic liposome formulations demonstrate an efficacy similar to that of SSL DOX (Gabizon et al., 1996).

The effect of bilayer fluidity and the relative nature of the lipid components can have a considerable impact on the clearance from the circulation of both the liposome and the associated drug. These effects can either be direct effects, such as inhibition of penetration and thus binding of serum proteins (Papahadjopoulos et al., 1973b), or indirect effects, such as stabilization of the drug formulation to reduce the rate of drug leakage (VII. Stability in Plasma and Storage). The presence of Chol probably has one of the most important roles in the maintenance of membrane bilayer stability and long circulation times in vivo (Gregoriadis and Davis, 1979; Senior and Gregoriadis, 1982; Senior, 1987). In the absence of Chol, CLs are destabilized by HDL particles (Chobanian et al., 1979; Damen et al., 1980) and upon release, their components can be readily eliminated from the circulation. For liposomes with and without Chol, clearance rates were shown to negatively correlate with increased stability in plasma (Senior and Gregoriadis, 1982). The presence of steric stabilization makes the need for Chol less apparent for empty liposomes, but for drug-loaded liposomes, Chol is necessary for maintenance of the drug in the liposomal interior. The phospholipid component also plays a prominent role in the maintenance of high plasma levels of liposomes. DSPC/Chol and SM/Chol liposomes have higher T_1/2 values in the circulation compared with more fluid liposomes containing eggPC or even 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC; Gregoriadis and Senior, 1980; Senior, 1987). This is presumably due to the decreased affinity of these liposomes for serum opsonins required for their uptake. To be most effective, the PC component must have a phase transition that is significantly above 37°C. An inspection of the gel-to-liquid-crystalline phase transitions (T_m) of a variety of different PC molecules (Table 2) shows the T_m value for eggPC is below 37°C, whereas DPPC has a T_m value of only a few degrees above (with a pretransition at 37°C). However, both DSPC and HSPC have a T_m value that is ~15-17°C higher than 37°C. Thus, at 37°C, HSPC- and DSPC-containing liposomes have a considerably more rigid membrane bilayer that resists penetration of serum opsonins than do eggPC- or DPPC-containing formulations. It is no surprise, then, that these liposomes tend to be the most stable in the circulation and display the longest circulation lifetimes.

Sphingomyelin (SM) has an added effect on circulation lifetimes. SM/Chol and SM/DSPC/Chol liposomes were both shown to have longer circulation lifetimes than DSPC/Chol (Hwang, 1987; Allen et al., 1991), indicating an additional stabilizing effect of SM. SM can form intermolecular hydrogen bonds with neighboring Chol molecules (Schmidt et al., 1977; Sankaram and Thompson, 1990), resulting in greater stability and a decreased ability of plasma proteins to insert into liposomal membranes.

The rate of elimination of a liposomal drug from the circulation is also dependent on the rate of drug leakage from the carrier. Because drugs considered for liposome encapsulation often have circulation times significantly shorter than the liposomal carrier, premature release can lead to an apparent increase in the rate of elimination of the liposomal drug from the circulation. In DOX-loaded SSLs (SSL DOX) with HSPC, DPPC, or eggPC as the phospholipid component of the formulation, a correlation was observed among the phase transition (T_m) of the phospholipid component, the stability of the formulation in in vitro plasma stability tests, and plasma levels of the drug in vivo (Fig. 6 and Table 2; Gabizon et al., 1993). Liposomes containing high-phase transition lipids formed more stable formulations, which were better able to retain their drug and showed apparent increases in drug circulation lifetimes. A similar result was seen by Mayer and coworkers using CLs and DOX (Bally et al., 1990b). A more detailed explanation of the different factors responsible for maintaining a stable formulation of different drugs in the plasma is given in VII. Stability in Plasma and Storage.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Dependence of plasma drug levels on liposome composition. SSL DOX lipid formulations containing different species of phosphatidylcholine were injected into Sabra female mice (four per group) at a dose of 10 mg/kg DOX. The molar ratio of PEG-DSPE/PC/Chol was kept constant at 0.75:9.25:8 for each formulation, whereas the species of the PC component was varied. The different PCs used were HSPC, DPPC, and eggPC. HSPC/Chol (10:8) liposomes were used as a CL control. DOX measurements were taken 24 h after drug administration. This figure was adapted from Gabizon et al. (1993).

The conclusions that can be drawn from these data differ for CLs and SSLs. For CLs, a membrane composed of Chol and high-phase transition phospholipids appears to be imperative for maintaining long circulation times and subsequent delivery of high levels of liposomes to solid tumors (see III. Accumulation of Liposomal Drugs in Tumors). SSLs are more pliable and can be used with fluid-phase lipids to obtain long circulation times and high tumor levels of liposomes. For both types of liposomes, the lipid composition of the liposome membrane is essential in maintaining a stable encapsulation of the drug while in the circulation. For most amphipathic drugs that are either weak acids or weak bases (the majority of classic chemotherapeutic agents), this is of considerable importance because these drugs will more rapidly leak from the carrier while in the circulation, unless high-phase transition lipids are used.

Original attempts to mimic the surface of red blood cells by including the sterically hindered GM₁ or PI in liposome preparations led to the development of long-circulating liposomes (Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; Gabizon et al., 1990). Later, PEG-DSPE was substituted for GM₁ or PI (Klibanov et al., 1990; Allen et al., 1991; Papahadjopoulos et al., 1991). A common misconception is that the attachment of PEG to the surface of a liposome prevents liposome uptake by the RES; rather, it simply reduces the rate of uptake. One of the most significant advantages of SSLs is the nonsaturable, log-linear pharmacokinetics, as described perviously. SSLs likely resist uptake by the high-affinity, low-capacity RES macrophages, resulting in increased circulation lifetimes (Allen et al., 1995a). Like CLs, the primary site of accumulation for SSLs is also the spleen and liver (Huang et al., 1992). However, the rate of accumulation in these tissues is considerably slower than that for CLs. Plasma levels of PEG-DSPE containing liposomes are increased 2- to 2.5-fold over DSPC/Chol (2:1) CLs and 7- to 10-fold over eggPC/Chol (2:1) liposomes in mice (Fig. 5; Lasic et al., 1991; Woodle et al., 1992). As mentioned earlier, CLs containing anionic lipids or those containing unsaturated lipid components, such as eggPC, are removed more readily from the circulation than those containing high-phase transition phospholipids (SM/Chol or DSPC/Chol). However, in the presence of PEG-DSPE, liposomes containing some charged lipids or low-phase transition phospholipids are found in plasma after 24 h at similar levels to those containing neutral high-phase transition phospholipids (Fig. 5). The presence of steric stabilization thus allows for the rate of clearance to be relatively independent of the remaining lipid composition for "empty" liposomes (Lasic et al., 1991; Woodle et al., 1992).

This is not inclusive of all phospholipid components. Both GM₁ and PEG-DSPE were unable to prevent liposomes containing PS from being cleared rapidly from the circulation (Allen et al., 1988, 1991), indicating that some membrane components may confer a very powerful propensity for a type of liposome being recognized and taken up by the RES. In addition, although the lipids composing the majority of the liposome may not have a direct effect on the removal of the liposomal carrier itself, they may have an indirect effect on clearance of the encapsulated drug. As previously described, when amphipathic drugs such as DOX are loaded into these liposomes, the rate of leakage from the liposome can become the rate-limiting step for clearance of the drug from the circulation if liposomes are not optimized to prevent leakage. The dose independence of liposome clearance, reduced recognition and clearance of liposomes by the RES, and flexibility in lipid compositions that can provide considerable advantages for SSLs that make them more desirable for an assortment of different applications.

The mechanism by which steric stabilization of liposomes increases their longevity in the circulation has been extensively discussed (Lasic et al., 1991, 1992; Needham et al., 1992a, 1999; Allen, 1994; Lasic and Martin, 1995). The basic concept of this discussion has been that a flexible-chained hydrophilic polymer or a glycolipid, such as PEG or GM₁, which occupies the space immediately adjacent to the liposome surface ("periliposomal layer"), tends to exclude other macromolecules from this space. Consequently, access and binding of blood plasma opsonins to the liposome surface are hindered, and thereby interactions of RES macrophages with such liposomes are inhibited. The exclusion of extraneous macromolecules from the periliposomal layer creates steric hindrance ("steric stabilization effect"), which is manifested as increased interbilayer repulsive forces and results in an increased interbilayer separation of PEG-decorated bilayers compared with unmodified ones (Lasic et al., 1992b; Needham et al., 1992b). Ganglioside GM₁ has less effect on the interbilayer separation compared with PEG. Measurements of streptavidin-induced agglutination rate of biotinylated SSLs demonstrated that the steric barrier decreased with decreasing PEG chain length and at higher PEG lengths (PEG M_r = 1900 and 5000) was significantly greater than that produced by GM₁ (Mori et al., 1991).

Several studies have argued that the presence of PEG or GM₁ does in fact lead to both a decreased extent and rate of binding of plasma proteins to liposomes (Senior et al., 1991; Chonn et al., 1992; Blume and Cevc, 1993; Semple and Chonn, 1996), although direct experimental evidence is not abundantly clear for PEG-DSPE-containing liposomes. In two earlier studies in which decreased protein binding or opsonic activity was shown for SSLs, the incubation of PEG-stabilized liposomes with plasma components was completed in 2 to 15 min (Allen et al., 1994; Semple and Chonn, 1996). Another study showed similar findings in vivo after a 2-min incubation but approximately equivalent levels of bound protein for a CL and a PEG-stabilized liposome formulation after 30 min (Harvie et al., 1996). Chonn et al. (1992) have previously shown a correlation of protein-binding levels in CLs and GM₁-containing liposomes to in vivo circulation lifetimes, which is consistent with the observations seen with SSLs. Thus, decreased binding of serum opsonins by GM₁-containing liposomes can result in decreased opsonin activation, as has been shown for complement factor C3 (Chonn et al., 1992), or reduced uptake by macrophages (Wassef et al., 1991; Alving and Wassef, 1992). Finally, reduced clearance may be partially due to steric hinderance for the binding of liposome-bound opsonins to their receptors on RES macrophages (Klibanov et al., 1991; Mori et al., 1991; Allen, 1994). Allen and coworkers have suggested that the elimination of SSLs may occur via the same mechanism as CLs, after a slow removal of PEG-DSPE from the membrane (Allen et al., 1991; Allen, 1994). Blume and Cevc (1993) suggested that SSLs may simply require longer to bind the opsonins necessary for uptake by the RES. The net effect of these phenomena is that by sterically hindering the approach to the liposome surface by large molecules or cells, liposomes can attain longer circulation lifetimes, allowing them greater time to accumulate in tumors.

SSL DOX has a long half-life in the circulation compared with free DOX (Table 3). Pharmacokinetic data for free DOX are best described by a biexponential fit with a rapid distribution phase and a slow terminal elimination phase. The majority of free DOX is eliminated in the initial rapid phase. With SSL DOX, a major portion of the plasma AUC is attributed to the prolonged terminal phase (Papahadjopoulos and Gabizon, 1995; Gabizon and Martin, 1997). Half-lives in rats were found to between 22 and 23.6 h (Mayhew et al., 1992; Working and Dayan, 1996), whereas those in humans approximated 45 h (Gabizon et al., 1994). For conventional formulations in humans, the terminal T_1/2 is significantly shorter: from 6.7 to 25 h for TLC D-99 and from 2.8 to 8.3 h for DaunoXome. Although the T_1/2 was found to be independent of dose for SSL DOX, it increased with increasing dose for both DaunoXome and TLC D-99. Recently, the dose-independence of SSL DOX at very high concentrations of drug, 60 mg/m², was called into question when it was reported that a decreased rate of clearance was observed at these higher doses (Martin, 1998). This may result from a drug-induced toxicity to macrophages responsible for their elimination, similar to the effect described by others with CLs (Bally et al., 1990b; Parr et al., 1997). The increased T_1/2 values of liposomal drugs relative to free drugs were also consistent with a decreased rate of clearance of the liposomal carriers. DOX is cleared 560 times slower in humans when encapsulated in SSLs, and daunorubicin is cleared 35 to 56 times slower with DaunoXome, a CL formulation, compared with the free drugs. The stable encapsulation of drug within the liposome, combined with the large size of the carrier, likely prevents filtration and removal of the drug by the kidneys.

It should be emphasized that the AUCs that are typically referred to in this discussion are based on drug that is for the most part nonbioavailable. It is entrapped inside the carrier and unable to elicit any response. Thus, the term "AUC," which is commonly used by those in the liposome field of study, for liposomal drugs may appear a little misleading to some because it does not truly represent the pool of bioavailable drug in the plasma or in the tumor. Several studies have shown for DOX that >95% of the drug remains liposome associated in the plasma (Gabizon et al., 1994; Martin, 1998). A technical limitation in the ability to accurately measure the rate at which drug is released from the carrier has also prevented us from expressing true AUC measurements for bioavailable drug in the tumor. The AUC measurements typically being referred to are of total drug, including both liposome and free drug. Depending on the lipid composition used and the rate of clearance in the particular organ or compartment being studied, these relative amounts may vary significantly.

The increased T_1/2 values of liposomal drugs translate into an increased AUC for Doxil in plasma compared with the free drug (Tables 3 and 4). In rats, the AUC for Doxil is >60 times that of free DOX, and this increase is elevated to a 368-fold increase in rabbits (Working and Dayan, 1996). In humans, the AUC is increased by 250 to 600 times in the case of Doxil over free DOX, 20 to 30 times in the case of TLC D-99, and ~60-fold in the case of DaunoXome (Table 4; Conley et al., 1993; Cowens et al., 1993; Gabizon et al., 1994). The smaller plasma AUCs for DaunoXome and TLC D-99 relative to Doxil may reflect both the shorter circulation times of the carriers, as a result of the lack of steric stabilization, and their increased rate of drug leakage. Even doses greater than three times those used for DOX were unable to provide comparable plasma levels of the relevant anthracycline (609 mg/*h/liter for 25 mg/m² DOX and 375.3 mg*h/liter for 80 mg/m² daunorubicin). For TLC D-99, plasma AUCs reached their maximum value at 50.5 ± 44.9 mg*h/liter, 10-fold lower than that for Doxil. Mayer and coworkers recently demonstrated they could obtain plasma AUCs for DOX in mice from 27 to 57% of those obtained with an SSL formulation by encapsulating DOX in DSPC/Chol liposomes and injecting them at the relatively high doses of 20 mg/kg DOX and 100 mg/kg lipid (Parr et al., 1997; Bally et al., 1998; Mayer et al., 1998). Assuming that a stable formulation can be prepared with SSLs (this is not always the case as will be seen with liposomal VCR), the data suggest that plasma drug levels, and thus the total AUC, will always be greater with SSL formulations. Although there is little debate on this particular point, there is a significant amount of debate over whether higher plasma levels necessitate a more favorable clinical outcome. This question is a complex one, and a considerable part of the remainder of this review focuses on how and whether this question can be answered.

The volume of distribution for free DOX is high in all species examined, indicating a wide tissue distribution. The small molecular size and amphipathic nature of the free drug allow it to rapidly distribute to both healthy and diseased tissues. However, when administered in liposomal form, the volume of distribution was reduced >60-fold to values approximating the plasma volume, suggesting that both DaunoXome and Doxil are restricted to the central compartment (Gabizon et al., 1993; Tables 3 and 4). The relatively large size of liposomal carriers (45-150 nm) prevents them from passing through the 2-nm pores found in the endothelium of blood vessels in most healthy tissues or even the 6-nm pores found in postcapillary venules (Seymour, 1992). In addition to the size of the carrier, the stability of the formulation can also have an effect on the volume of distribution. If drug leaks from the liposome before leaving the circulation, then the free drug can readily redistribute to healthy tissues. A comparison of Doxil or DaunoXome, both of which contain high-phase transition phospholipids (DSPC or HSPC), with TLC D-99, which contains the highly unsaturated eggPC, shows a significantly higher volume of distribution (4- to 5-fold at 25 mg/m²) for the latter (Table 4). This indicates that DOX was released more rapidly from the carrier with TLC D-99 and has distributed more extensively into normal tissues than for more stable preparations with lower leakage rates. The consequences of this are an alteration in the toxicity profile and lower tumor levels of the drug. A more thorough review of the factors that contribute to the stability of a formulation in the plasma is given in VII. Stability in Plasma and Storage.

Due in part to the size of the carrier, L-DOX has an altered tissue distribution compared with free DOX (Tables 5 and 6). Free DOX has a wide distribution, accumulating in most tissues to a significant extent. L-DOX preferentially accumulates in areas containing a discontinuous microvasculature, such as tumors, or in organs containing the macrophages of the RES, such as liver and spleen. This altered distribution reduces the concentration of drug at potential sites of toxicity, such as the heart. A comparison of the biodistribution of free drug and that encapsulated in both CLs and SSLs is given in Tables 5 and 6. When DOX levels are reported as peak levels of drug in various tissues, a significant increase in DOX is found in healthy tissues, such as kidneys, heart, and lung when administered as free DOX compared with both CL and SSL DOX (Table 5). L-DOX shows increased levels in blood, liver, spleen, and tumor (Table 5). In the liver, SSLs were found almost exclusively in Kupffer cells and rarely in the more abundant hepatocytes (Huang et al., 1992; Litzinger et al., 1994). This is consistent with the role of Kupffer cells in removing liposomes from the circulation and suggests less damage to liver tissue than if delivered to parenchymal cells. However, in addition to macrophages, other investigators have demonstrated a significant uptake of liposomes by a low-affinity, high-capacity system involving hepatocytes in a manner dependent on both the size of the liposomes and the presence of PEG-DSPE (Scherphof et al., 1994). The nature of this discrepancy is unclear but may involve problems in detection of lipo-somes in hepatocytes by some methods. From these data, it appears as though liposomes preferentially accumulate in tumor and tissues of the RES, whereas free DOX distributes more uniformly between the various tissues.

Although peak drug levels indicate L-DOX reaches healthy tissues to a reduced extent, when tissue drug levels are reported as the AUC, even in healthy tissues, tissue drug levels approach those for free DOX (Table 6). DOX delivered via CLs accumulates to a reduced extent in non-RES tissues compared with delivery by SSLs. This is most likely a result of the reduced circulation lifetimes of CLs. The AUC for tumors is still between 2.5- and 10-fold greater than that for free DOX. A more detailed comparison of the extents of accumulation in tumors is given in IIIB. Rate and Extent of Accumulation in Tumors. In one of the earliest comparisons of SSLs (GM₁) versus CLs (Huang et al., 1992), liposome levels in tumor (followed with encapsulated ⁶⁷Ga) were greater than twice those of DSPC/Chol liposomes (Table 6). The levels in spleen and liver for DSPC/Chol liposomes were 1.35 to 1.7 times those of the GM₁-containing formulation, reflecting their more rapid uptake by the RES. In the three healthy non-RES tissues measured (heart, lung, and kidneys), liposome levels were significantly greater for the SSL formulation, suggesting that longer circulation also leads to higher AUCs for liposomes in healthy tissues. The few comparative studies with L-DOX or mitoxantrone showed either insignificant differences or slightly elevated AUCs in healthy tissues for SSLs relative to the CL formulation (Table 6; Unezaki et al., 1995; Chang et al., 1997). In all cases, the total AUC was either comparable or decreased for the liposomal form compared with the free drug. Considering that the overall exposure for these tissues is approaching equivalent amounts for free and encapsulated drug, it might be reasonable to expect the level of toxicity in these tissues to be similar. However, some acute toxicities are dependent on peak levels of the drug, and liposomal drugs accumulate in these tissues at a much slower rate than the free drug. In addition, the liposomal drug is not completely bioavailable, and thus the effective concentration of the drug in these tissues is considerably reduced. In one study, a histological section of cardiac muscle showed accumulation of liposomes only within blood vessels between muscle fibers and not within the muscle itself, indicating that liposomes were unable to extravasate in the heart to areas where they may do considerable damage (Working et al., 1994). This is discussed in greater detail in VI. Toxicology of Liposomal Chemotherapy.

Anthracyclines are metabolized in human plasma to a variety of both active and inactive metabolites (Takanashi and Bachur, 1976; Fig. 7). The reduction in DOX by an aldo-ketoreductase results in the formation of the most prominent metabolite, doxorubicinol (II), in plasma, bile, and urine (Takanashi and Bachur, 1976). A two-electron reduction in DOX with subsequent elimination of the sugar results in the inactive metabolite, a 7-deoxyaglycone (V: Takanashi and Bachur, 1976; Doroshaw, 1996). In several studies, researchers looked for the presence of DOX metabolites in plasma and urine after the administration of L-DOX (Gabizon et al., 1991, 1994; Northfelt et al., 1996). Although several of the more common metabolites (doxorubicinol and glucoronide and sulfate derivatives of 4-dimethyl,7-deoxyaglycones) were observed in urine, they were at diminished levels (2.5%) compared with the administration of free DOX (11%; Gabizon et al., 1994). In two separate studies (Gabizon et al., 1994; Northfelt et al., 1996), doxorubicinol (II) was not observed in plasma at any time after the administration of SSL DOX. This is not surprising considering that the liposomal membrane protects its contents from inactivation by plasma enzymes. The importance of a stable formulation is essential in maintaining this advantage. In formulations containing unsaturated phospholipids, DOX leaks rapidly from the liposome and doxorubicinol was detected in plasma at times as short as 30 min (Gabizon et al., 1991; Embree et al., 1993). Although small amounts of DOX metabolites have been observed in tumor and tumor exudates (Gabizon et al., 1994; Siegal et al., 1995), its protection from inactivation by plasma enzymes almost certainly increases the percentage of drug that arrives in the active form at the tumor site.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Metabolism of DOX in vivo. DOX (I) can be converted to either inactive (deoxyaglycones; III or V) or active (doxorubicinol; II) metabolites in the circulation. Initially, DOX is converted to doxorubicinol (II), DOX aglycone (III), or deoxydoxorubicin aglycone (V), although the preferred pathway is for the metabolism to II. Doxorubicinol (II) is the primary metabolite found in both plasma and urine. Doxorubicinol can be further metabolized to doxorubicinol aglycone (IV) or deoxydoxorubicinol aglycone (VI), with metabolism to VI being the preferred pathway. Finally, VI can be converted to more polar metabolites such as o-sulfate or o-glucoronide derivatives. A notable advantage of liposomes is that they are able to protect their contents from metabolism and inactivation in the circulation, thus allowing higher levels of the parent compound to arrive at the tumor site. This figure was modified from Takanashi and Bachur (1976).

L-DOX is eliminated in the urine at a much slower rate than free DOX (Vaage et al., 1998). In a mouse model, free DOX was found in urine samples as readily as 15 min and up to 48 h after the administration of the drug. SSL DOX was not detected in the urine until almost 1 h and could still be detected up to 5 days after drug administration. This is consistent with a controlled-release mechanism for the liposome-encapsulated drug, where the drug is released from its carrier at a very slow rate.

An understanding of the mechanisms responsible for maintaining high circulating levels of drug in the plasma is essential to design carriers that remain in the circulation sufficiently long to have a high probability of accumulating in tumors. Nevertheless, long circulation time is only one aspect of liposomes that results in their preferential antitumor activity. If liposomes were unable to preferentially accumulate in tumors, they would be useful only as a controlled-release type of therapy. This is increasingly available through mechanical means, and thus there is minor clinical importance for the development of liposomes for this purpose. However, the fact that liposomes do accumulate preferentially in tumors allows them to be passively targeted and gives rise to substantial increases in antitumor efficacy. In III. Accumulation of Liposomal Drugs in Tumors, we review the various mechanisms responsible for the uptake of liposomes into tumors and how they may be exploited for further increasing drug delivery to tumors in the future.

	III. Accumulation of Liposomal Drugs in Tumors

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The accumulation of liposomes or large macromolecules in tumors is a result of a "leaky" microvasculature and an impaired lymphatics supporting the tumor area (Matsumura and Maeda, 1986, 1989; Huang et al., 1992; Seymour, 1992; Yuan et al., 1994; Jain, 1996). This effect is often referred to as the enhanced permeability and retention effect ("EPR phenomenon"; Matsumura and Maeda, 1986, 1989). With gold-labeled liposomes, both extravasation and transcytosis of liposomes in Kaposi's sarcoma-like dermal lesions were demonstrated (Huang et al., 1993). The principal pathway for the movement of liposomes into the tumor interstitium is via extravasation through the discontinuous endothelium of the tumor microvasculature, and transcytosis is thought to be a relatively minor pathway. Once in the tumors, nontargeted liposomes are localized in the interstitium surrounding the tumor cells (Huang et al., 1992; Yuan et al., 1994). Liposomes were not seen within tumor cells, although they were observed in resident tumor macrophages. The limited distribution of liposomes within the tumor interstitium results from a high interstitial pressure and a large interstitial space compared with normal tissues (Jain, 1989, 1990). Large tumors are more difficult to treat than small ones, in part because of the resulting incr