Pegylated Liposomal Doxorubicin: Scientific Rationale and Preclinical Pharmacology
Pegylated Liposomal Doxorubicin: Scientific Rationale and Preclinical Pharmacology
Soon after Bangham and Horne first described liposomes in the mid- 1960s as closed vesicular structures able to envelop water-soluble molecules, pharmacologists recognized their potential value in drug delivery. The rationale was simpleuse liposomes as a safe vehicle for delivering drugs more specifically to sites of disease while limiting exposure of normal tissues. The envelopment of cytotoxic antitumor agents was of particular interest because these drugs generally have a narrow therapeutic window, ie, dose-limiting side effects limit their therapeutic utility.
Among the dozens of liposome-encapsulated antitumor agents studied in animal models, the anthracycline antibiotics, in particular, doxorubicin and daunorubicin, emerged as benefiting substantially from liposome encapsulation. Animals were able to tolerate greater doses of a variety of formulations of liposome-encapsulated doxorubicin (Doxil) with antitumor activity, in general, being maintained. Although this safety advantage was considered meaningful, attempts to improve antitumor activity by actually targeting encapsulated drugs to tumors were frustrated by rapid removal of the liposomes from blood by elements of the mononuclear phagocyte system, fixed macrophages residing in liver, spleen, lung, and bone marrow. It is believed that the binding of plasma proteins (lipoproteins, immunoglobulins, complement) to the liposome surface triggers this rapid macrophage uptake.
Despite the lack of true targeting, internalization of liposome-encapsulated anthracyclines by mononuclear phagocyte system cells was found to diminish exposure of certain tissues to the toxic effects of such drugs. For example, doxorubicin-related nausea/vomiting and cardiomyopathy are believed to be related to the drugs peak levels in plasma. In theory, liposome encapsulation results in sequestration of the majority of an injected dose in the mononuclear phagocyte system, thus attenuating the initial plasma levels of free drug and improving safety. The drug is eventually released from mononuclear phagocyte system cells and is distributed to peripheral tissues in free (ie, unencapsulated) form. If this theory is true, the pharmacokinetic pattern would mimic that of doxorubicin administered as a prolonged infusion, a regimen known to reduce drug-related side effects. Indeed, it has been shown that administration of liposome-encapsulated doxorubicin reduces the drugs acute and chronic toxicities in preclinical animal models. Moreover, results from animal models indicate that doxorubicin delivered in this fashion retains its activity against systemic tumors.
The pharmacokinetics and safety of various clinical formulations of liposomal doxorubicin have been reported in the scientific literature.[2,6-24] Clinical pharmacokinetic measurements confirm that conventional liposome formulations are cleared rapidly from plasma. These data also suggest that a considerable amount of encapsulated doxorubicin is released into plasma prior to mononuclear phagocyte system uptake.[10,21]
Armed with the knowledge that mononuclear phagocyte system uptake can provide favorable safety advantages for encapsulated doxorubicin, formulation scientists began to optimize liposome carriers for this purpose. As shown in Figure 1, the first major branch of the liposome anthracycline family tree was represented by these Mononuclear Phagocyte System Targeted formulations. Two alternative formulation approaches (subbranches) soon emerged.
The first approach relied upon acidic lipids incorporated into the liposome bilayer, such as cardiolipin (CL) and egg phosphatidyl glycerol (EPG), to bind doxorubicin, which is positively charged at physiological pH, to the membrane itself.[8,12] Formation of such ion-pairs between the drug and an acidic membrane component provided a stable association resulting in robust formulations that were stable in vitro and could be freeze-dried for long-term storage.
The second approach, represented by TLC D-99, used true encapsulation of doxorubicin into the aqueous compartment of the liposome and employed a clever technique to circumvent the problem of leakage. In this case doxorubicin is loaded into the liposomes (in a hospital pharmacy) immediately prior to administration by adding an aqueous solution of doxorubicin (at neutral pH 7.0) to liposomes containing a low pH internal buffer (pH 4.0). The pH gradient thus formed across the liposome membrane leads to movement of doxorubicin into the liposomes. Once inside, the low pH environment traps the drug, preventing it from leaking out as long as the pH gradient is maintained.
The ion-pair formulations have been tested clinically but have not progressed beyond phase I/II studies. TLC-D99 is in advanced phase III trials in metastatic breast cancer.
Recognizing that mononuclear phagocyte system uptake represented the main obstacle to targeting, another branch of liposome scientists attempted to formulate liposomes that could resist binding/interaction with plasma proteins (opsonization), thus prolonging their blood residence times and targeting potential. Early work suggested that a modest degree of mononuclear phagocyte system avoiding activity could be obtained by formulations composed of high phase transition lipids and cholesterol. Size was also a critical parameter: the smaller the liposome, the longer it circulated. This pure lipid subbranch arrived at two formulations of small diameter (approximately 50 nm), one composed of DSPC/cholesterol and the other of sphingomyelin/cholesterol, both of which showed relatively slow mononuclear phagocyte system clearance. DaunoXome, a DSPC/cholesterol formulation of daunorubicin, is the only product to emerge from this pure lipid approach. DaunoXome is approved in the US and Europe for the treatment of AIDS-related Kaposis sarcoma.
Not satisfied with the plasma longevity achieved by the pure lipid formulations, another branch of liposome scientists explored the possibility of coating the surface of liposomes with inert materials designed to camouflage the liposome from the bodys host defense systems. The biological paradigm for this surface modified subbranch was the erythrocyte, a cell which is coated with a dense layer of carbohydrate groups, and which manages to evade immune system detection and to circulate for several months before being removed by the same type of cell responsible for removing liposomes. The first breakthrough came in 1987 when a glycolipid (the brain tissue-derived ganglioside GM1) was identified which, when incorporated within the lipid matrix, allowed liposomes to circulate for many hours in the blood stream. A second glycolipid, phosphatidylinositol, was also found to impart long plasma residence times to liposomes and, because it was extracted from soy beans not brain tissue, was believed to be a more pharmaceutically acceptable excipient.
A major advance in the surface modified subbranch was the development of polymer-coated liposomes. Polyethylene glycol modification had been used for many years to prolong the half-lives of biological proteins (such as enzymes and growth factors) and to reduce their immunogenicity. Several groups of liposome scientists reported in the early 1990s that pegylated-coated liposomes circulated for remarkably long times after intravenous administration. Half-lives on the order of 24 hours were seen in mice and rats and over 30 hours in dogs. The term stealth was applied to these liposomes because of their ability to evade interception by the immune system in much the same way as the stealth bomber was able to evade radar. Pegylated liposomal doxorubicin (PEG-LD) (Doxil) is the first product to emerge from the surface modified liposome subbranch. It too is approved in the US and Europe for treatment of Kaposis sarcoma.
The efficacy of PEG-LD has been evaluated in a variety of different tumor models, including several human xenograft models.[30-36] In every model examined, PEG-LD was more effective than the same dose of free doxorubicin in inhibiting or halting tumor growth, in effecting cures and/or in prolonging survival times of the tumor-bearing animals. In most cases, all three endpoints were improved by PEG-LD, and in no case was PEG-LD less effective than doxorubicin. Not only was PEG-LD more active in both solid and dispersed tumors, it was also found to be more effective than doxorubicin in preventing spontaneous metastases from two different intramammary implanted tumors in mice. These findings also are supported by studies performed with PEG-LD in several murine tumor models and a human xenograft model (Figure 2).
The efficacy of free doxorubicin in these animal models was generally limited by its toxicity at high doses. Therefore, the ability to use PEG-LD at higher doses offers a therapeutic advantage. In addition, pharmacokinetic and tissue distribution studies suggest that the greater persistence, particularly in tumor tissue, achieved with PEG-LD compared with conventional doxorubicin also contributes a therapeutic advantage.
Furthermore, PEG-LD was found to be significantly more effective than conventional (non-Stealth) liposomal doxorubicin, demonstrating the impact of the long-circulating Stealth liposome. Based on the results of these nonclinical studies, PEG-LD appears to be an effective agent for the treatment of both solid and dispersed tumors.