1. Field of the Invention
The present invention relates to a method for controlling loading of active agents into liposomes. More particularly, the present invention relates to methods for modulating loading of active agents into multivesicular liposomes.
2. Description of Related Art
Optimal treatment with many drugs requires that the drug level be maintained at a specified level for a prolonged period of time. For example, optimal anti-cancer treatment with cell cycle-specific antimetabolites requires maintenance of a cytotoxic drug level for a prolonged period of time. Cytarabine is a highly schedule-dependent anti-cancer drug. Because this drug kills cells only when they are synthesizing DNA, prolonged exposure at therapeutic concentration of the drug is required for optimal therapeutic effect. The therapeutic effectiveness of such agents is often further complicated by the fact that the half-life after an intravenous or subcutaneous dose may be as short as a few hours. To achieve optimal therapeutic effect against cancer cells with a cell cycle phase-specific drug like cytarabine, there are two major requirements: first, the cancer cells must be exposed to a high concentration of the drug without doing irreversible significant harm to the host; and second, the tumor must be exposed to the drug for a prolonged period of time to maximize the number of cancer cells that are contacted during DNA synthesis, the susceptible portion of the cycle of cell proliferation. This kind of treatment regimen requires a high drug load in a slow release formulation.
Certain other types of drugs are so toxic that it is important to maintain a low level of the drug over an extended period of time. For instance, amikacin is an aminoglycoside antibiotic with clinically significant activity against strains of both gram negative and gram positive bacteria. Under existing therapeutic procedures, the drug is normally administered by intravenous or intramuscular routes on a once or twice a day schedule. The most commonly used clinical dose is 15 mg/Kg/day, which is equivalent to a maximum recommended daily dose of 1 g per day. However, administration of the drug by spaced injections results in systemic exposure to the patients, and depending on the drug, increases risk of toxic side effects. Consequently, a local depot slow-release preparation for treatment of infections such as those confined to a local region of soft tissue or bone would be advantageous in increasing local tissue levels of the drug, compared with therapeutic systemic doses, while reducing or avoiding the systemic toxicity of the free drug. If the drug is highly toxic or the treatment regimen requires a low therapeutic dose, a relatively low drug load in a slow release formulation is beneficial.
One approach which has been used to provide controlled release compositions for drug delivery is liposome encapsulation. Among the main types of liposomes, multivesicular liposomes (Kim, et al., Biochim. Biophys. Acta; 728:339-348, 1983), are uniquely different from unilamellar liposomes (Huang, Biochemistry; 8:334-352, 1969; Kim, et al., Biochim. Biophys. Acta; 646:1-10, 1981), multilamellar liposomes (Bangham, et al., J. Mol. Bio., 13:238-252, 1965), and stable plurilamellar liposomes (U.S. Pat. No. 4,522,803). In contrast to unilamellar liposomes, multivesicular liposomes contain multiple aqueous chambers. In contrast to multilamellar liposomes, the multiple aqueous chambers of multivesicular liposomes are non-concentric.
The prior art also describes methods for producing multivesicular liposomes (Kim, et al., Biochim. Biophys. Acta, 728:339-348, 1983). However, the encapsulation efficiency of some small molecules, such as cytosine arabinoside, also known as cytarabine or Ara-C, proved relatively low, and the release rate of encapsulated molecules in biological fluids was faster than is therapeutically desirable. EP 0 280 503 B1 discloses a method developed for controlling the release rate of encapsulated molecules from multivesicular liposomes wherein a hydrochloride is introduced into the encapsulation process to control the rate of release in biological fluids (of the active agent. Further research, disclosed in WO 95/13796, has shown that the release rate of agents from multivesicular liposomes in human plasma can be controlled by introduction of a non-hydrochloride acid into the aqueous solution in which the agent is dissolved prior to forming the multivesicular liposome
U.S. Pat. No. 5,077,056 discloses studies that show the rate of release of the encapsulated biological agent from liposomes into an aqueous environment can be modulated by introducing protonophores or ionophores into liposomes to create a membrane potential. In addition, a method is known (U.S. Pat. No. 5,186,941) for controlling the release rate of drugs from vesicle compositions wherein the liposomes containing a therapeutic agent encapsulated are suspended in a solution containing sufficient solute to provide an osmolarity substantially isotonic with respect to that of the solution within the vesicles, and hypertonic with respect to physiological saline. In multivesicular liposomes, it is also known (WO 96/08253) to control the rate of release of active agents by introducing an osmotic spacer into the aqueous solution in which the active agent is dissolved prior to formation of the multivesicular liposomes.
In addition to the biologically active agent and acids or osmotic spacers intended to control the rate of release of the biologically active agent from the liposomes, it is common practice to coencapsulate compounds that are intended to serve any of a number of helper functions. For instance, certain biologically active compounds retain activity only when kept at a particular pH. Thus acids or buffers are often necessarily encapsulated in addition to the active agent to control the pH of the drug environment. In other cases, a counterion is incorporated to enhance solubility of a biologically active agent that has a low solubility.
These methods for producing liposome formulations with slow release characteristics have sometimes proven incompatible with the goal of producing liposomes containing a high load of active agent with good encapsulation efficiency so that little of the expensive active agent is wasted by failure to capture it within the liposomes.
Thus the need exists for new methods for producing liposomes, for instance multivesicular liposomes (MVLs), that allow for control of drug loading, either high or low, while maintaining desirable slow release of the active agent into storage and biological fluids. Of particular interest is the development of high-load, controlled release formulations for peptides and proteins. A need also exists for new methods of achieving these goals without sacrifice of high encapsulation efficiency to avoid the waste of expensive active agents, such as drug and therapeutic proteins.