Formulation of Hydrophobic Drugs Into Cochleate Delivery Vehicles: A Simplified Protocol & Bioral? Formulation Kit

Formulation of Hydrophobic Drugs Into Cochleate Delivery Vehicles: A Simplified Protocol & Bioral? Formulation Kit
By: David Delmarre, PhD; Ruying Lu; Nadine Tatton, PhD; Sara Krause-Elsmore, PhD; Susan Gould-Fogerite, PhD; and Raphael J. Mannino, PhD
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Drug Delivery Technology
Cochleate delivery vehicles represent a new technology platform for oral and systemic delivery of drugs. A novel lipid-based system, they represent a unique technology platform suitable for the oral and systemic administration of a wide variety of molecules with important therapeutic biological activities, including drugs, genes, and vaccine antigens. Cochleate formulation technology is particularly applicable to macromolecules as well as small molecule drugs that are hydrophobic, positively charged, negatively charged, and that possess poor oral bioavailability. Proof-of-principle studies for cochleate-mediated oral delivery of macromolecules as well as small molecule drugs is being carried out in appropriate animal models with well established, clinically important drugs, which currently can only be effectively delivered by injection. The leading formulation is Amphotericin B cochleates, a potent antifungal agent.
STRUCTURE & PROPERTIES OF COCHLEATES
Cochleate delivery vehicles are stable phospholipid-cation precipitates composed of simple, naturally occurring materials (ie, phosphatidylserine and calcium). They have a unique multilayered structure consisting of alternating layers of calcium and phospholipid in large, continuous, solid, lipid bilayer sheets rolled up in a spiral or stacked, with little or no internal aqueous space (Figure 1). This structure provides protection from degradation for associated ?encochleated? molecules. Divalent cation concentrations in vivo in serum and mucosal secretions are such that the cochleate structure is maintained. Hence, the majority of cochleate-associated molecules are present in the inner layers of a solid, non-aqueous, stable, impermeable structure.
STABILITY OF COCHLEATE FORMULATIONS
Encochleation (Figure 2) protects and provides increased stability for associated molecules. Because the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact, even though the outer layers of the cochleate may be exposed to harsh environmental conditions or enzymes. The interior of cochleates is essentially free of water and resistant to penetration by oxygen, two components most responsible for the decomposition and degradation of drugs, leading to reduced shelf-life. Thus, encochleation of drugs imparts extensive shelf-life stability. For example, the encochleation efficiency, the percentage of supercoiled versus relaxed plasmid, and immunogenicity as DNA vaccines (a property requiring in vivo intracellular delivery and gene expression) is equivalent to fresh preparations for more than 1 year. Cochleates may be lyophilized to a powder and stored at room temperature or 4?C. Lyophilized cochleates can be reconstituted with liquid prior to in vitro use or in vivo administration. Lyophilization has no adverse effects on cochleate morphology or functions.
SAFETY/BIOCOMPATIBILITY OF THE COCHLEATE DELIVERY VEHICLES
Cochleates are made from safe, simple, well-defined, naturally occurring substances, generally phosphatidylserine (PS) and calcium. Phosphatidylserine is a natural component of all biological membranes and is most concentrated in the brain. The phospholipids used can be produced synthetically, or prepared from natural sources. Soy PS is inexpensive, available in large quantities and suitable for use in humans. Clinical studies by other investigators (more than 30 have been published) to evaluate the potential of PS as a nutrient supplement indicate that PS is very safe and may play a role in the support of mental functions in the aging brain. Unlike many cationic lipids, cochleates (which are composed of anionic lipids) are non-inflammatory and biodegradable. We have evaluated the in vivo tolerance of mice to multiple administrations of cochleates by various routes, including intravenous, intraperitoneal, intranasal and oral. Multiple administrations of high doses of cochleate formulations to the same animal show no toxicity and do not result in either the development of an immune response to the cochleate matrix or to any side effects relating to the cochleate vehicle.
DELIVERY OF BIOACTIVE MOLECULES
Many naturally occurring membrane fusion events involve the interaction of calcium with negatively charged phospholipids (generally PS and phosphatidylglycerol). Calcium-induced perturbations of membranes containing negatively charged lipids and the subsequent membrane fusion events are important mechanisms in many natural membrane fusion processes. Therefore, cochleates can be envisioned as membrane fusion intermediates.
Our current working hypothesis is that as the calcium-rich, highly ordered membrane of a cochleate first comes into close approximation to a natural membrane, a perturbation and reordering of the cell membrane is induced, resulting in a fusion event between the outer layer of the cochleate and the cell membrane. This fusion results in the delivery of a small amount of the encochleated material into the cytoplasm of the target cell. The cochleate could then break free of the cell and be available for another fusion event, either with this or another cell. Alternatively, particularly with active phagocytic cells, the cochleate may be taken up by endocytosis and fuse from within the endocytic vesicle. Cochleates made with trace amounts of fluorescent lipids have been shown to bind and gradually transfer lipids to the plasma membrane and interior membranes of white blood cells in vitro.
EXAMPLES OF COCHLEATE-MEDIATED DRUG DELIVERY
Cochleates for the Delivery of OTC Antimicrobial Agents
Using Amphotericin B (AmB) as a model, cochleates have been shown to be highly effective at mediating the oral delivery of drugs that are currently only available in injectable formulations. Oral administration of AmB-cochleates has been shown to be as effective as equivalent, injectable doses of the leading AmB formulation (Fungizone) in mouse models of systemic candidiasis, cryptococcal meningitis, and aspergillosis. In addition, AmB-cochleates demonstrate increased stability and substantially lower toxicity than currently existing products.
These finding have been published in peer reviewed journals.1-4 BioDelivery Sciences International (BDSI) is currently moving forward with the scale-up GMP manufacturing of AmB cochleate formulations and anticipates filing an IND for the clinical trials of this product in 2004.
Cochleates for the Delivery of OTC Anti-inflammatory Agents
BDSI is currently investigating the potential for using cochleate delivery vehicles to formulate and effectively deliver anti-inflammatory agents, including aspirin, ibuprofen, naproxen, acetaminophen, and COX-2 inhibitors. BDSI scientists have prepared formulations of various NSAIDS in cochleates.
In Vitro Studies: In tissue-culture studies using the established macrophage-derived cell line J774, nano-encapsulate aspirin and acetaminophen cochleate formulations were shown to be 5- to10-fold more efficient than free drug at inhibiting nitric oxide synthase, one of the enzymes used by macrophage during the inflammatory response.
In Vivo Studies: The ability of cochleates to protect the gastrointestinal tract from aspirin toxicity and to enhance the anti-inflammatory efficacy of aspirin was evaluated in vivo using a standard carrageenan rat model for acute inflammation performed by an independent contract laboratory. Standard (large) aspirin cochleates and nano (small) aspirin cochleate formulations were prepared and tested.
In these studies, cochleate formulations showed substantially reduced gastric irritation in comparison to aspirin controls. In addition, aspirin cochleates were highly efficacious against the carrageenan model in the rat paw within 3 hours when administered orally. Impressively, less than one-third the dose of encochleated aspirin is 50% to 75% more effective than free drug. In summary:
Cochleates can be used to formulate a variety of biologically active molecules.
Protocols have been developed to control and stabilize particle size.
Particle size can influence and improve biological activity.
Cochleates mediate and enhance effective oral bioavailability.
Formulation into cochleates can reduce toxicity.
Cochleates for the Delivery of OTC Anti-inflammatory Agents
BDSI is currently investigating the potential for using cochleate delivery vehicles to formulate and effectively deliver anti-inflammatory agents, including aspirin, ibuprofen, naproxen, acetaminophen, and COX-2 inhibitors. BDSI scientists have prepared formulations of various NSAIDS in cochleates.
In Vitro Studies: In tissue-culture studies using the established macrophage-derived cell line J774, nano-encapsulate aspirin and acetaminophen cochleate formulations were shown to be 5- to10-fold more efficient than free drug at inhibiting nitric oxide synthase, one of the enzymes used by macrophage during the inflammatory response.
In Vivo Studies: The ability of cochleates to protect the gastrointestinal tract from aspirin toxicity and to enhance the anti-inflammatory efficacy of aspirin was evaluated in vivo using a standard carrageenan rat model for acute inflammation performed by an independent contract laboratory. Standard (large) aspirin cochleates and nano (small) aspirin cochleate formulations were prepared and tested.
In these studies, cochleate formulations showed substantially reduced gastric irritation in comparison to aspirin controls. In addition, aspirin cochleates were highly efficacious against the carrageenan model in the rat paw within 3 hours when administered orally. Impressively, less than one-third the dose of encochleated asp