Advantages of liposomal delivery systems for anthracyclines

Advantages of Liposomal Delivery Systems for Anthracyclines Theresa M. Allena and Francis J. Martinb Liposomes, closed vesicular structures consisting of one or more lipid bilayers, have generated a great deal of interest as drug delivery vehicles. In particular, they have been investigated for their ability to improve the delivery of chemotherapeutic agents to tumors, in efforts to increase therapeutic efficacy and decrease toxicity to normal cells. Development of liposomal chemotherapeutic agents has, in the past, been hindered primarily by the rapid uptake of liposomes by the reticuloendothelial system. Numerous strategies that seek to either exploit or avoid this phenomenon have been used. As a result, several liposomal chemotherapeutic agents are now available in the clinic. STEALTH, a novel liposomal system coated with polyethylene glycol, avoids uptake by the reticuloendothelial system, thus improving drug delivery to the tumor while decreasing toxicity. In pegylated liposomal doxorubicin (Doxil/Caelyx [PLD]), this delivery system encapsulates doxorubicin within polyethylene glycol-coated liposomes, leading to promising new applications for a well-established drug. Liposome-encapsulated doxorubicin citrate complex (Myocet [NPLD]), another liposomal delivery system for doxorubicin, lacks the polyethylene glycol coating, resulting in much shorter circulation times than those of PLD. Daunorubicin citrate liposome (DaunoXome [DNX]) contains daunorubicin encapsulated in a smaller liposome of a different lipid composition. It has circulation times between those of PLD and NPLD. This article reviews the advantages of liposomal delivery systems in general and the divergent approaches that have been taken in developing these agents. Semin Oncol 31(Suppl 13):5-15 © 2004 Elsevier Inc. All rights reserved.

Delivery of Oncologic Drugs

D

rugs used in oncology are far from ideal, particularly in their ability to selectively accumulate in cancerous tissues or cells. Many troublesome side effects associated with distribution of the drugs to normal tissues limit the maximum dose or dose intensity that can be administered to patients. Less than ideal pharmacokinetic and biodistribution profiles are problematic, and chemical stability is also a problem with many oncologic drugs. A variety of drug delivery systems have been studied in attempts to solve some of the

aDepartment

of Pharmacology, University of Alberta School of Medicine, Edmonton, Alberta, Canada. bALZA Corporation, Mountain View, CA. Supported in part by the Canadian Institutes for Health Research, the National Cancer Institute of Canada, and Ortho-Biotech Products, L.P (T.M.A.). Dr Allen is a consultant to Tibotec Therapeutics, Division of Ortho Biotech Products, L.P. Address reprint requests to Theresa M. Allen, PhD, 9-31 MSB Department of Pharmacology, University of Alberta School of Medicine Edmonton, AB, T6G 2H7, Canada.

0093-7754/04/$-see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2004.08.001

problems associated with the lack of selectivity and stability of oncologic drugs. Liposomal drug delivery systems meet most, if not all, of the criteria of an ideal drug delivery system for oncologic agents (Table 1). Of the several types of drug carrier systems researched to date, liposomal carriers are the most advanced, with several liposomal formulations of anticancer drugs already on the market or in the late stages of clinical trials.1 Not all oncologic drugs, however, are appropriate for liposomal delivery, as they may lack some of the biologic and chemical characteristics that would make them suitable (Table 2).

Liposomes as Drug Delivery Vehicles Liposomes, which were first described as “bimolecular leaflets” in the early 1960s,2 are closed vesicular structures that consist of one or more lipid bilayers. They may have a unilamellar structure, ranging from 20 to 1,000 nm (internal volume, 0.5 to 11 ␮L/mg lipid), or an onion-like structure, with an equal number of lipid bilayers and aqueous compartments, ranging from 400 to 3,500 nm (internal volume, 4 ␮L/mg lipid).3,4 Because their size and permeability are easily 5

6 Table 1 Characteristics of an Ideal Anticancer Drug Delivery System Localized drug delivery to tumors Decreased toxicity to normal tissues Intrinsic lack of toxicity High ratio of drug to carrier (ie, high concentration of encapsulated contents) Efficient loading mechanism for drug (to avoid drug waste) Versatility (to accommodate a wide variety of drugs with different solubilities) Appropriate release kinetics to release the drug at tumor cells Protection of drug from premature degradation Capable of preventing or overcoming multidrug resistance Easy to store and administer

altered, liposomes can be tailored to a variety of specific uses. For example, water-soluble molecules can be enclosed in the aqueous compartment(s), lipid-soluble molecules can be associated with liposomes through partitioning into the lipid bilayers, and peptides or small proteins can be adsorbed to the interface between the lipid bilayer surface and adjacent water.4 In vivo, conventional liposomal formulations (ie, those that lack a polyethylene glycol [PEG] coating) are taken up primarily by phagocytic cells in the blood and by cells of the reticuloendothelial system (RES) in the liver (specifically Kupffer’s cells),5 spleen, and bone marrow.4,6 Plasma proteins, immunoglobulins, and complement bind to the surface of the liposomes while they are circulating. These attached proteins then serve as opsonins and trigger the rapid RES uptake.7 Very large liposomes are also taken up by the lung as they become trapped in the alveolar capillaries.4 There is little extravasation of liposomes from blood into tissues fed by continuous, nonfenestrated capillaries, such as muscle, skeletal, connective, central nervous system, cardiac, gastrointestinal, gonadal, skin, and subcutaneous tissue, as well as into serous and mucous membranes and exocrine and endocrine glands.4 Liposomes have attracted considerable interest as drug delivery systems, particularly for cancer chemotherapeutics, because they can significantly increase the concentrations of drug delivered to diseased tissues while, at the same time, they significantly reduce the amount of drug delivered to normal tissues.8 By changing drug pharmacokinetics and biodistribution (eg, by reducing the peak plasma drug levels) liposomal delivery systems can reduce serious drug toxicities, such as cardiotoxicity associated with doxorubicin or renal toxicity associated with amphotericin B, thereby increasing the therapeutic index of the drugs.9-11 Liposome administration is perceived to be low risk because the lipids used are usually extracted from natural sources, such as egg yolks or soybean oil, that have been safely used as emulsifying agents in lipid emulsions for human parenteral administration. However, the use of liposomes has its limitations. Drug delivery to tissues with continuous, nonfenestrated capillar-

T.M. Allen and F.J. Martin ies has been difficult.4,6 Because of rapid uptake by the RES, the half-lives of conventional liposomal preparations are measured in minutes rather than hours or days.12,13 Although large unilamellar liposomes have a large carrying capacity for drugs within their aqueous interior, they have short circulation half-lives relative to smaller liposomes.3 The premature breakdown of liposomes mediated by various components of plasma or by uptake by blood monocytes may cause premature release of the encapsulated drug into the bloodstream rather than into target tissues.6

RES-Targeting Liposomal Formulations The first liposomes to be formulated, often termed classical or conventional liposomes, were simple mixtures of cholesterol and phospholipids (eg, phosphatidylcholine). It was recognized early on that classical liposomes naturally targeted the RES, which led to the development of liposomal formulations of drugs for which RES delivery was desirable (Fig 1). Delivery of immune modulators to RES cells to stimulate cellmediated or humoral immune responses against cancer cells could take advantage of this natural homing ability to RES cells. For most anticancer agents, however, uptake of classical liposomes loaded with cytotoxic drugs by RES cells is to be avoided because RES function could be compromised, thus adversely affecting patient outcome.6,14 Three approaches to developing RES-targeting liposomal antitumor products have emerged. The first incorporates acidic lipids (cardiolipin and egg phosphatidylglycerol) into the lipid bilayer. As a result, weakly basic drugs such as doxorubicin, which is positively charged at physiologic pH, form “ion pairs” with the acidic components of the membrane. This results in stable in vitro formulations suitable for long-term storage in freeze-dried form.15 Liposomal versions of doxorubicin (LED; NeoPharm, Lake Forest, IL) and mitoxantrone (LEM ETU; NeoPharm) formulated with this approach are in phase I/II trials.16-22 In the second approach, drug is encapsulated in the aqueous compartment using a pH gradient. The pH gradient beTable 2 Characteristics of Anticancer Drugs Suitable for a Liposomal Carrier System Intermediate-to-high potency Inherent instability (rapid degradation) Hydrophilicity (low octanol:water partition coefficient [log Poct < 1.7]) Weak acids or bases of intermediate solubility (1.7 > log Poct < 5)* Very poor solubility (log Poct > 5) Large volume of distribution (leading to high distribution to normal tissues) Rapid rate of plasma clearance Poor distribution to solid tumors (poor selective tumor toxicity) Given by sustained infusion (sustained-release properties of liposomes mimic infusion) *Drugs with “intermediate solubility” are suitable for a liposomal carrier system if they possess titratable acid or basic groups that change their water solubility when protonated or deprotonated.

Advantages of liposomal delivery systems

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Figure 1 Family tree illustrating the relationship between formulation strategy and the development of liposomal anthracycline products.15 RES, reticuloendothelial system. *Not marketed in the United States.

tween the exterior and interior of the liposome causes the drug to migrate into and become entrapped in the liposome interior.23-25 A nonpegylated product based on this technology (Myocet, liposome encapsulated doxorubicin citrate complex [NPLD]; Medeus Pharma, Stevenage, Herts UK) is approved, in combination with cyclophosphamide, for treatment of metastatic breast cancer in Europe, but not in the United States. The use of NPLD results in reductions in the dose-limiting cardiotoxicity and the gastrointestinal toxicity of conventional doxorubicin (Fig 2, Table 3).25 An NDA has been filed for liposomal vincristine (Onco TCS; INEX Pharmaceuticals, Vancouver, Canada), which is also based on the pH-loading technology, and two others are in clinical development: (1) liposomal topotecan (Topotecan TCS; INEX Pharmaceuticals) and (2) liposomal lurtotecan (OSI 211; OSI Pharmaceuticals, Melville, PA).26-29 In the third approach, water-insoluble drugs such as taxanes are mixed with matrix lipids (ie, those that form the bulk

of the liposome structure). For example, liposomal paclitaxel (LEP ETU; NeoPharm) and liposomal SN38 (LE-SN38; NeoPharm) are formulated directly into the liposome membrane in this way and are in clinical trials.30-32 RES-targeting liposomes have also been developed in other fields. AmBisome (Gilead Pharmaceuticals, Inc, San Dimas, CA), a liposomal formulation of amphotericin B, is licensed for treatment of fungal infections.33 Amphotericin B is intercalated into the liposomal membrane as an integral part of the liposome structure. This product allows dose escalation with acceptable tolerability and results in reduced renal toxicity compared with conventional amphotericin.3

RES-Avoiding Liposomal Formulations Scientists recognized the RES as the primary obstacle to sitespecific drug delivery and sought ways to formulate lipo-

Figure 2 Structural differences between conventional liposomes and pegylated liposomes. (A) Conventional liposome. (B) Pegylated liposomal doxorubicin.

T.M. Allen and F.J. Martin

8 Table 3 Key Characteristics of Liposomal Anthracyclines Characteristic RES Relationship Diameter Lipid composition

Pegylated Active drug Administration

Half-life Safety v conventional anthracycline

Approved indications

PLD (Doxil)50 Avoiding 80 to 100 nm Soybean-derived phosphatidylcholine (hydrogenated), cholesterol, N(carbonyl-methoxy-polyethylene glycol 2000)-1,2-distearoyl-snglycero-3-phospho-ethanolamine sodium salt Yes Doxorubicin Liquid suspension to be diluted in 250 mL of 5% dextrose before administration ⬃50 to 80 hr92 Reduced GI toxicity, cardiotoxicity, and alopecia. Slight reduction in bone marrow toxicity. Hand-foot syndrome and stomatitis are doselimiting.9,94-98 United States: second-line treatment of advanced HIV-associated Kaposi’s sarcoma, advanced ovarian carcinoma refractory to paclitaxel and platinum. EU and Canada: also approved as monotherapy for metastatic breast cancer

DNX (DaunoXome)39

NPLD (Myocet)25,52

Avoiding 45 nm Distearoylphosphatidylcholine, cholesterol

Targeting 150 to 180 nm Egg yolk-derived phosphatidylcholine, cholesterol

No Daunorubicin Liquid suspension to be diluted 1:1 with 5% dextrose before administration 2 to 4 hr Reduced cardiotoxicity, alopecia. Myelosuppression is dose limiting.

No Doxorubicin 3-vial kit. Drug loaded into liposomes (requires 30 to 40 minutes) in pharmacy just before administration24,90,91 10 to 15 minutes93 Reduced GI toxicity and cardiotoxicity. Slight reduction in alopecia. Myelosuppression is dose limiting.90,99-101

First-line therapy for advanced HIVassociated Kaposi’s sarcoma

Not approved in United States. Approved in Europe for metastatic breast cancer.

Abbreviations: EU, European Union; GI, gastrointestinal; HIV, human immunodeficiency virus; RES, reticuloendothelial system.

somes that would avoid opsonization and have longer plasma half-lives.15,34 Several approaches emerged to develop RESavoiding liposomes, including identification of liposomal characteristics that would lead to a longer half-life, blockade of uptake into the RES, and addition of coatings to shield the liposomal surface from interactions with other molecules and cells. Size, surface charge, and degree of unsaturation of the matrix lipids critically influence liposomal circulation times in vivo. In one approach to avoiding RES uptake, highly sonicated or homogenized liposomes of small diameters (⬍50 nm) and bearing a net neutral charge, including lipids with high phase-transition temperatures and cholesterol, were found to circulate for several hours. Prolonged circulation time was related to delayed uptake of liposomes into the RES.4,35,36 Some evidence of tumor targeting by these liposomes exists,37 but the use of such liposomes is limited by low aqueous-entrapped volumes and particle instability caused by the lack of charge stabilization.38 To date, DaunoXome (daunorubicin citrate liposome injection [DNX]; Gilead Pharmaceuticals, Inc) is the only pure lipid (distearoylphosphatidylcholine and cholesterol in a 2:1 ratio) product to emerge from this approach (Fig 1, Table 3).15 It has a mean diameter of 45 nm and an initial (alpha) clearance half-life of approximately 4 hours.39 Another approach takes advantage of RES saturation.

Once the saturation point of liposomal uptake by the RES has been reached, liposomes that remain in the blood have increased circulation times. Efforts have been made to block the RES by dosing with empty liposomes before administration of liposome-encapsulated drugs.40 However, while this approach may result in some increased uptake of liposomes by other tissues,37 it carries risks associated with long-term loss of RES function.14,38 The third approach to formulation of RES-avoiding liposomes involves coating the surface of the liposome with inert materials that hinder interaction between blood components (opsonins) and the liposome surface, thereby camouflaging the liposome from host defense systems in the RES.15,34 This concept was modeled after erythrocytes, which use a carbohydrate coating to evade immune detection.14,15 The first demonstration that this approach produced longer circulation times was made in 1987 using monoganglioside GM1, which was derived from bovine brain tissue.41 Subsequently, phosphatidylinositol, which is extracted from soybeans and is therefore more readily accessible, was also found to increase circulation time.42 The next major advance in the use of surface coating was the development of polymer-coated liposomes (Fig 1).43-45 Liposomes coated in PEG have terminal half-lives of 12 to 30 hours in animal models and 21 to 54 hours in humans.10,43,46 This prolonged circulation is a result of increased surface

Advantages of liposomal delivery systems hydrophilicity, hindrance of negative charges at the liposome surface, and decreased opsonization and RES uptake.5,13,34,38 It is noteworthy that both the rate and extent of uptake of liposomes and their entrapped drugs by the RES are decreased by the PEG coating, whereas other methods of prolonging liposome circulation merely delay the rate, but not the extent, of uptake.13 Such PEG-coated liposomes have been named “STEALTH” (Alza Corporation, Mountain View, CA) because of their ability to evade detection by the immune system. Other polymers (eg, poly[acryl amide], poly[vinyl pyrrolidone], poly[acryloyl morpholine], poly[2-oxazoline]) also have been shown to prolong the circulation of liposomes and limit accumulation in the liver.34,47 Although none have shown greater success than pegylated liposomes, circulation times similar to those achieved with PEG have been observed in animal models using certain concentrations of poly(2-oxazoline) and poly(acryloyl morpholine).47-49 PEG has been used rather than alternatives because it is relatively inexpensive and widely used in the pharmaceutical industry in a variety of applications. Overall, the strategy of RES avoidance through use of polymer coatings offers the most versatility in lipid composition and particle size.38

Pegylated Liposomal Doxorubicin Doxil (doxorubicin HCl liposome injection [PLD]; distributed in the United States by Tibotec Therapeutics, a division of Ortho Biotech Products, L.P., Bridgewater, NJ; Caelyx is distributed outside the United States by Schering-Plough, Kenilworth, NJ) consists of doxorubicin encapsulated in a pegylated liposome composed of hydrogenated soy phosphatidylcholine, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (mPEG-DSPE), and cholesterol.50 Doxorubicin is loaded into liposomes to a very high concentration under the influence of an ammonium sulfate gradient and is suspended in a nonelectrolyte solution.51 Each liposome contains 10,000 to 15,000 molecules of doxorubicin. More than 90% of the drug is encapsulated within the liposomes.50 The liposomes are 80 to 100 nm in diameter, with linear segments of surface-grafted PEG extending in a brush formation about 5 to 6 nm from the surface.13 By comparison, NPLD is approximately 160 nm in diameter,52 although both pegylated and classical liposomes may be formulated in a variety of sizes.13 The PEG coating on pegylated liposomes is the unique feature that distinguishes them from conventional liposomes (Fig 2). In addition to preventing opsonization by plasma proteins, leading to reduced RES uptake of liposomes, PEG provides a steric stabilization effect that results in reduced adhesion to cells, blood vessel walls, and other surfaces. The steric stabilization effect leads to the remarkable colloidal stability reported for pegylated liposome suspensions. Pegylated liposomal doxorubicin, for example, does not aggregate or settle out of suspension over a 2-year period. Sterically stabilized liposomes circulate for many hours, and, if they are small enough, pass through capillaries that have increased permeabilities, such as capillaries in diseased

9 tissues (eg, solid tumors) or in the liver and certain other organs. Evidence suggests that such long-circulating liposomes eventually distribute as intact particles to normal tissues as well, perhaps by a process of transcytosis and/or extravasation at the level of postcapillary venules. The liposomes are slowly removed from circulation by a combination of RES uptake and extravasation in normal tissues.

Effect of Encapsulation on Pharmacokinetics and Biodistribution In PLD, the rate of release of doxorubicin from the carrier is slow (the release half-life is several days). Because the liposomal drug stays associated with the carrier, it has pharmacokinetics similar to that of the carrier, which results in significantly altered plasma pharmacokinetics for PLD when compared with conventional doxorubicin. This includes significantly higher area under the plasma-concentration-time curve (AUC), increased distribution half-life, lower rate of clearance, smaller volume of distribution, and 15- to 40-fold higher peak concentrations.15,46,50,53 Tissue (eg, tumor) concentrations of doxorubicin are also significantly higher, and remain higher for significantly longer periods of time following delivery via pegylated liposomes, compared with unencapsulated drug.46 Alterations in the pharmacokinetics and biodistribution of doxorubicin, when delivered in pegylated liposomes, reduce cardiotoxicity and the myelotoxicity and increase drug efficacy.46

Tumor-Targeting Mechanism Because the release rate of doxorubicin from PLD is slow, unlike conventional (nonpegylated) liposomes, nearly all (93% to 99%) of an administered doxorubicin dose remains encapsulated in the pegylated liposome while in plasma.15 Importantly, the liposomes are generally too large to extravasate through the continuous or fenestrated capillaries found in most normal tissues outside of the RES, so there is little exposure of these tissues to the toxicities associated with conventional doxorubicin.54 Tumor angiogenesis results in discontinuous capillaries with 100-nm to 800-nm gaps between capillary endothelial cells and at vessel termini.54,55 These vessels have wide interendothelial junctions and a large number of fenestrae, transendothelial channels formed by vesicles, and discontinuous or absent basement membranes.5,46,54-57 Thus, tumor vessels are inherently permeable or “leaky,” allowing extravasation of liposomes from the tumor vessels into the interstitial space between tumor cells (Fig 3).5,46,55,56 Because of the stability of pegylated liposomes in plasma, nearly the entire doxorubicin dose that reaches tumors is delivered in encapsulated form. It has not yet been possible to determine the time when peak levels of released (ie, bioavailable) drug occur in human tumor tissue, but peak concentrations of liposomal drugs in implanted rodent tumors are observed 2 to 3 days after liposome administration,

T.M. Allen and F.J. Martin

10

Figure 3 Proposed mechanism for PLD accumulation in tumors. (1) Liposomes containing doxorubicin circulate for 2 to 3 weeks after injection. During this period, virtually all doxorubicin remains encapsulated. (2) Intact liposomes extravasate through defects/gaps present in newly sprouting vessels and enter the tissue compartment, lodging in the tumor interstitium near the vessel. (3) Drug molecules are released from the extravasated liposomes. (4) Free drug molecules penetrate deeply into the tumor and enter tumor cells. (5) Doxorubicin molecules bind to nucleic acids and kill tumor cells.15

and significant levels can be measured in tumors for more than 1 week.58 Thus, tumor targeting with pegylated liposomes occurs because of their physical characteristics. They circulate in the blood with little degradation until they escape

into tissues, and they can escape from the vasculature only in areas with leaky blood vessels. Passive targeting of pegylated liposomes to solid tumors has been studied in humans administered radiolabeled lipo-

Figure 4 Distribution of radiolabeled liposomes in a patient with Kaposi’s sarcoma (KS) of the upper trunk and lower left extremity. Pegylated liposomes loaded with 111In-DTPA were injected intravenously at time 0 in an AIDS-KS patient and serial whole-body gamma scintigrams were recorded at 4, 24, and 72 hours and 7 days after injection.59

Advantages of liposomal delivery systems

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Table 4 Mean ⴞ SD Doxorubicin Concentrations (␮g Doxorubicin/g Tissue) in a KS Lesion and Adjacent Normal Skin Following a Single Dose of PLD (Doxil)10 48 hours

96 hours

Location

10 mg/m2 (n ⴝ 8)

20 mg/m2 (n ⴝ 7)

10 mg/m2 (n ⴝ 4)

20 mg/m2 (n ⴝ 4)

Normal skin KS lesion Ratio

0.36 ⴞ 0.31 5.0 ⴞ 7.8 9.6

0.84 ⴞ 0.52 13.2 ⴞ 8.5 20.1

0.95 ⴞ 1.00 11.7 ⴞ 16.6 20.4

0.27 ⴞ 0.11 2.9 ⴞ 1.4 13.1

NOTE: Although 24 patients were enrolled, the 48-hour biopsy data could not be interpreted for one patient after the 20-mg/m2 dose. Specimens were carefully blotted to remove blood and minimize contamination by extracellular doxorubicin in the assay.

somes. Whole-body gamma imaging (Fig 4) confirms the selective accumulation of pegylated liposomes in a variety of solid tumors.59,60 The patient in Fig 4 had Kaposi’s sarcoma (KS) with multiple and deep skin lesions over the upper body and left lower extremity. At 4 and 24 hours following injection of the radiolabeled liposomes, most of the radioactivity was in the vasculature, reflecting the long circulating nature of pegylated liposomes. Gradually, however, the radioactivity became concentrated in the KS lesions. Very little uptake was seen in normal tissues except in the liver where there was some uptake in the hepatic RES. Biopsy studies in patients with KS also showed the targeting potential of pegylated liposomes.10 Twenty-four patients consented to biopsies of both normal skin and a KS lesion on the skin at 48 and 96 hours after an infusion of either 10 or 20 mg/m2 of PLD (Table 4).10 Regardless of the time point or dose, there was significantly more doxorubicin in the KS lesion than in normal skin. At 48 hours after the 20 mg/m2 dose, tissue doxorubicin concentrations were, on average, 19 times higher in the KS lesions than in normal skin. However, in individual patients this ratio of tumor to skin concentration ranged from 3:1 to 53:1 (Fig 5).15 With varying assumptions about the relative vascularity of the two types of tissues,

it was estimated that the ratio of intracellular drug in KS lesions to intracellular drug in normal skin for the 11 patients treated with 20 mg/m2 could be as little as 1:1 or as high as 22:1.50 Once the liposomes are released from the blood vessels at the site of the tumor, doxorubicin is thought to be released slowly from liposomes by leakage down the drug’s concentration gradient, or on degradation of the liposomes by enzymes such as phospholipases, by inflammatory cells, or by phagocytic cells.15,46 The release of ammonium sulfate (also present in the liposome) helps to catalyze the drug leakage.61 Hence, cytotoxicity to the tumor cells is caused in large part by uptake of released drug present in the tumor interstitial fluid. The long circulation half-lives of the liposomes, along with the slow release of contents and uptake of released drug, lead to a longer time to peak drug concentration and prolonged duration of tumor exposure relative to free doxorubicin. Clinical data support these preclinical observations. The concentration of total doxorubicin (encapsulated drug plus released drug) in KS lesions 72 hours after a single injection is significantly higher when administered in pegylated liposomes rather than as conventional doxorubicin (Fig 6).10 On

Figure 5 Biopsy results from seven patients in the study described in Table 4. Doxorubicin levels in normal skin and KS lesions 48 hours after a single injection of PLD 20 mg/m2. The largest observed difference in tissue levels of doxorubicin after conventional doxorubicin or PLD treatment was 1:53 in patient no. 2.15

T.M. Allen and F.J. Martin

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Figure 6 Doxorubicin levels in KS lesions 72 hours after a single 15-minute injection of PLD or conventional doxorubicin in doses of 10, 20, or 40 mg/m2. Each cohort consisted of three patients. Doxorubicin levels at 72 hours were 5.2 to 11.4 times higher after PLD administration compared with conventional doxorubicin administration.10

average, the difference in tissue levels was 10-fold; this was true regardless of the administered dose of conventional doxorubicin or PLD. The Cmax ratios of plasma doxorubicin with PLD versus conventional doxorubicin administration ranged from 13.4 to 20.2. This suggests that, even at time points earlier than 72 hours, the maximum total doxorubicin levels in the KS lesions were higher for PLD than for conventional doxorubicin treatment.10 In a subsequent study, the daily average maximum plasma doxorubicin concentration (Cmax,avg) and dose intensity were significant predictors of differences in tumor response among AIDS patients who had KS.62 The Cmax,avg was most predictive of a partial response. Fifty-one percent of patients who received PLD at a dose of 20 mg/m2 every 3 weeks achieved the Cmax,avg that maximized the probability of a partial response. These observations are consistent with those made in preclinical models where the AUC (area under the curve or drug concentration multiplied by the length of time during which drug could be measured) consistently favored the pegylated liposome formulation.58

Influence of Pegylated Liposome Formulation on Resistance Testing In vitro drug sensitivity assays and drug resistance tests such as the Extreme Drug Resistance assay (Oncotech, Tustin CA) are being used increasingly to evaluate the potential activity of chemotherapeutic agents. However, contradictory results are often obtained when different tests are used to evaluate the same tumors and same drugs.63 Furthermore, in vitro resistance to a single agent, using this assay, predicted treatment responses to combination therapy that included the agent in some studies,64 but not in others.65 One potential contributor to the inconclu-

sive results with the Extreme Drug Resistance assay is that it is designed to identify highly resistant agents, so results should not be interpreted to provide estimates of relative activity between agents with low or moderate resistance. Several issues specific to pegylated liposome-encapsulated agents may interfere with sensitivity and resistance assays, leading to underestimation of clinical activity against a particular tumor. As discussed in the article by Vail et al (elsewhere in this issue),66 PLD has shown activity in the treatment of tumors resistant to conventional doxorubicin. However, in vitro cytotoxicity measurements (which are similar to sensitivity assays) for these tumors may result in greater IC50 values for PLD than for conventional doxorubicin,67 in part because the cytotoxicity of the conventional drug is overestimated in culture because no redistribution of the drug takes place, unlike the in vivo situation. When PLD is added to in vitro cell cultures, the liposomes are not taken up by the cells, and most of the administered concentration of PLD remains encapsulated and is not bioavailable, whereas in vivo, the pegylated liposomes enter the tumor intact and drug release is subsequently provoked by many factors that may require several weeks. In vitro sensitivity and resistance tests do not reproduce these conditions, nor is the timing of drug release from pegylated liposomes reproduced. Therefore, when sensitivity and resistance assays show activity of conventional doxorubicin for a particular tumor, it is reasonable to predict that PLD will also be active, but evidence of resistance to conventional doxorubicin or PLD with these tests does not rule out activity of PLD in vivo. The results of resistance testing obtained before first-line therapy should not be used to guide treatment decisions for second-line or third-line therapy because resistance patterns may change after exposure to first-line therapy. Direct tests of

Advantages of liposomal delivery systems PLD with the Extreme Drug Resistance assay just before therapy still has limited clinical utility because of how the tests are performed. As described above, enhanced drug delivery and retention of agents encapsulated in pegylated liposomes are dependent on in vivo tumor properties such as neovascularization, but these properties are absent with in vitro testing. In addition, many resistance tests use samples of chemotherapeutic agents that are frozen and thawed as many times as needed, but repeated freezing of pegylated liposomes affects their integrity. Clinically, toxicity and treatment convenience are often at least as important to patients as relative levels of resistance to chemotherapy, particularly among patients with treatmentrefractory disease that is likely to progress regardless of the chemotherapeutic agent that is used. Based on its comprehensive review of the available evidence, a working group from the American Society of Clinical Oncology concluded that oncologists should make chemotherapy treatment recommendations on the basis of published reports of clinical trials and a patient’s health status and treatment preferences.68 The working group also concluded that the use of chemotherapy sensitivity and resistance assays to select chemotherapeutic agents for individual patients is not recommended outside of the clinical trial setting. Thus, sensitivity and resistance assays appear to have little clinical utility, particularly for the rational use of pegylated liposomeencapsulated liposomal products.

Future Uses of Pegylated Liposomal Encapsulation Within the next decade, the pegylated liposome delivery system is likely to have applications beyond the uses of conventional doxorubicin. Other chemotherapy drugs, such as the camptothecin analogues, are possible candidates for delivery via pegylated liposomes. Other possible uses of pegylated liposomes include: ● Encapsulation of agents that target monocytes/macrophages to treat intracellular bacterial/parasitic infections (eg, leishmaniasis, Mycobacterium avium, M. tuberculosis)69-71; ● Encapsulation of anti-inflammatory agents and anti-infectives that target sites exhibiting increased vascular permeability72-79; ● Targeted delivery of small molecules (eg, antibiotics, antisense oligonucleotides, DNA plasmids for gene therapy)80,81; ● Targeting to cell-surface receptors on specific tumor cell types using ligands chemically attached to the liposome surface, such as growth factors (vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor), vitamins (folate), anti-integrins that bind RGD motifs (␣v␤3), and monoclonal antibodies (or fragments thereof)82-87; ● Linkage with anti-angiogenesis agents to cut off tumor blood supply and halt growth.88,89

13

A Comparison of Liposomal Anthracyclines Three liposomal formulations of an anthracycline are now available in the clinic. They differ in the primary target (RESavoiding or RES-targeting), the size of the liposome, the lipid composition, the rate of drug release, the presence or absence of pegylated liposome technology, and the active drug encapsulated (Table 3)9,24,25,39,50,52,90-101. These differences in the nature of the liposomal product have practical effects on how the drug is administered and its half-life in the plasma. As a result, the toxicities associated with the three drugs are different, as are the approved indications. Despite the fact that PLD, NPLD, and DNX are all liposomal-encapsulated anthracyclines, it is not possible to extrapolate preclinical and clinical evidence from one product to another within this class. Recognition of differences among the liposomal formulations of these products enhances understanding of their unique pharmacokinetic, pharmacodynamic, and clinical characteristics.

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