The present invention relates to the preparation of carriers for lipophilic materials in general. More specifically it relates to the formation of an improved carrier for these compounds which disperses in the presence of the aqueous contents of the gastro-intestinal tract (GI) to form drug-carrying lipid aggregates. The invention is particularly suitable for oral applications but can be readily adapted for other uses. The invention especially relates to novel phospholipid-cyclosporin formulations having improved bio-availability, increased efficacy and reduced toxicity and to a process of manufacture of such formulations.
Cyclosporins are fungal metabolites. They are hydrophobic neutral cyclic peptides and have essentially similar chemical and physical properties. Cyclosporin A (CyA) is representative and is the best known example. It is widely used in organ transplants to prevent rejection and as an immunosupressive agent in the treatment of systemic and local autoimmune disorders in which T cells play a major role. CyA has also been used to treat chronic conditions such as rheumatoid arthritis, asthma and non-malignant skin disorders. Derivatives of CyA are also known to prevent multi-drug resistance from developing during treatment with cytotoxic drugs.
The clinical use of CyA in oral and intravenous dosage forms to prevent organ rejection was approved by the FDA in 1983. It has dramatically improved long-term survival rates in transplant patients. Most patients, however, still need to be maintained on life-long CyA therapy. This is normally provided in an oral form but may involve intravenous injection when it is necessary to obtain an adequate blood concentration quickly or oral therapy proves ineffective. Unfortunately, there are two major problems associated with oral therapy. Firstly, since the drug is lipophilic, its absorption from the GI tract is variable and incomplete, and bioavailability can range from 6% to 60%. This results in variable or inadequate blood concentrations which can bring about graft rejection and failure Secondly, use of CyA is associated with nephrotoxicity. Impairment in kidney function is dose-related and increases with prolonged exposure, again emphasising the importance of controllable and predictable bioavailability.
There are few therapeutic compounds that have received more extensive and exhaustive pharmacodynamic and pharmacokinetic examination than CyA. Investigations have shown that CyA has a narrow therapeutic index and that drug absorption takes place across an absorption window located along the upper part of the small intestine. Little absorption takes place in the stomach or colon.
The first CyA oral formulation introduced into clinical use (Sandimmune) comprised a solution of CyA dissolved in a solvent system of olive oil and ethanol (Patentschrift (Switz.) CH 64I 356, Feb. 29, 1984, Appl. 79/1949. Feb. 27, 1979). The oil was emulsified in water using a polyethoxylated oleic glyceride surfactant to give a coarse O/W emulsion. This system was found to be inherently thermodynamically unstable. It is markedly affected by external conditions such as pH, temperature, diluting medium surrounding medium As a result, drug tended to precipitate out of solution, and thus not be absorbed. The release of CyA from the oil-droplets and its subsequent absorption was also found to be highly dependent on the prevailing conditions in the GI tract e.g. composition of food and presence of bile and pancreatic enzymes. This formulation thus gave erratic inter- and intra-patient bioavailability.
Although these problems were widely recognised, Sandimmune was relied upon exclusively by transplant patients for a number of years. It is only recently that a new oral formulation of CyA called Neoral with improved pharmacokinetics has been introduced to address these problems. This formulation was introduced as a xe2x80x98high-technologyxe2x80x99 microemulsion system in which the CyA is dissolved in a solvent consisting of a mixed lipophilic (corn oil mono-, di- and triglycerides) and hydrophilic (propylene glycol) solvent stabilised by an appropriate amount of a powerful surfactant, polyoxyl-40 hydrogenated castor oil (Kovarik et al, J. Pharm Sciences, 83, 444 (1994), and Hall, Inpharm, 10 December p 13 (1994)). This new formulation is reported to have self-emulsifying properties and immediately forms a transparent microemulsion in aqueous fluids. The CyA is dissolved in colloidal oil droplets (10-100 nm diameter) stabilised by the surfactant and can be diluted without precipitation, having similar properties to a real aqueous solution.
Neoral is at present the only known oral formulation generally available that gives consistent absorption, independent of bile and food. Clearly, in view of the number of patients world-wide who need to be on long-term CyA maintainance and their individual circumstances, it would be most desirable for there to be a comparable bioequivalent formulation that does not rely on the presence of potentially harmful synthetic surfactants.
A number of alternative approaches to the solubilisation of CyA and the development of formulations that avoid the dual problems of variable bioavailabilty and incomplete absorption from the GI tract have been described in the prior art.
Polyvinyl pyrollidone (PVP) with molecular weights of 40,000 and 17,000, have been used as solubilising agent to carry the drug (Yonish-Rouach et al Journal of Immunological Methods 135, 147-153 (1990)). It was demonstrated that CyA can be solubillsed and retain its activity (in vitro) in aqueous solutions of PVP. However, no evidence that the formulation would work in vivo was presented.
Co-administration of d-alphatocopheryl polyethylene glycol succinate (TPGS) which can form micelles has been reported to lead to an improvement of CyA absorption in children after liver transplantation (Sokol et al., The Lancet 338, 212-215, (1991)).
In order to counter the poor solubility of CyA, Guzman et al., have immobdilsed the drug in nanoparticles of polymeric nanomatrix composed of either isobutyl-2-cyanoacrylate monomer or poly-E-caprolactone, in the presence of Pluronic F-68 (Journal of Pharmaceutical Sciences 82, 498-502 (1993)). However, the drug-free nanoparticles also exhibited immunosupressive activity suggesting that they are unlikely to be a suitable vector for carrying CyA.
The enhancement of the intestinal absorption of a cyclosporine derivative (used as a model for CyA) by using milk fat globule membrane (MFGM) as an emulsifier of lipophilic cyclopeptides has been reported (Biol. Pharm. Bull. 17, 1526-1528(1994)).
In cases, where it is necessary to administer CyA intraveneously, it is normally formulated in an injectable form using a solvent consisting of ethanol and Cremophor EL, a tri-ricinoleate ester of ethoxylated castor oil. This solubiliser frequently gives rise to anaphylatic reactions and is itself known to cause nephrotoxicity exacerbating problems associated with the inherent renal toxicity of CyA.
A well-recognised approach to the formulation of lipophilic drugs is liposome encapsulation in which the drug is intercalated into the lipid bilayer(s) of the liposome. Compositions, methods of preparation, applications, advantages and disadvantages of liposomes have all been extensively reported, and there are more than 30 publications describing liposomal entrapment of CyA mainly for intravenous and systemic use.
From purely pharmaceutical considerations, there is general consensus that liposome entrapment significantly reduces nephrotoxicity. However, there is less certainty about whether the reduced nephrotoxicity reported with intravenous liposomal formulations is in fact due to altered pharmacokinetics of liposome encapsulated CyA or the non-specific, physical binding of the drug to other lipids present in the system. Some reports claim that CyA pharmacolknetics depend on such factors as liposome charge, size and composition. Fahr (Pharmaceutical Research, 12, 1189-1198 (1995)), however, dismisses this idea and cites evidence suggesting that high lipid doses tend to bind CyA in blood, thereby minimising the amount of drug available in sensitive organs like the kidney.
Apart from factors influencing the inherent nephrotoxicity of CyA, the three key factors in determining the suitability of carriers for CyA for oral and systemic use are: that the vector system should be non-toxicrirritant, it should have high entrapment levels and it should be stable.
Membrane lipids are present in all living cells and represent a significant component of our diet and thus their use present no toxicity problems. There are, however. problems regarding entrapment levels and stability, The charge, nature of the headgroups, and the saturation of the hydrocarbon chains have all been shown to influence the level of entrapment of CyA in liposomes. There is, however, consensus amongst those engaged in lposome work that the lipid:CyA molar ratio at equilibrium is about 20:1 for egg phosphatidylcholine. This should, however, be considered as a lower limit as in our own experience, unless the lipid:drug ratio is substantially greater than 20:1, the bound CyA in the liposome membrane will diffuse out into the surrounding aqueous medium and will precipitate out as untrapped CyA crystals on standing.
This problem is not fully recognised and many of the earlier studies, particularly those in which drug entrapment is measured by the analysis of liposomal pellets obtained by ultracentrifugation and no account is taken of the proportion of non-entrapped drug, tend to cite unrealistically high entrapment values. This is of importance as it is well known in formulation work that free CyA crystals are not absorbed from the GI tract resulting in poor bioavailability. In the case of intravenous injection, the formation of CyA crystals must be avoided at all costs. In practice, it is this crystallisation process that is the main reason why many liposome formulations perform so badly and do not proceed beyond animal testing.
EP 0 697 214 A1, describes aqueous compositions, with liposomes having a siz less than 100 nm, prepared by homogenising a specific mixture of a phosphatidylcholine, phosphatidyl glycerol and cyclosporin in a mole ratio of from 25:3:1 to 17:3:1. The claims for particle size and drug entrapment would appear to render the compositions suitable for intravenous administration of CyA.
PCT Publication No: 90/00389 discloses a method for the preparation of freeze-dried compositions of CyA in liposomes. The liposomes are intended to be reconstituted immediately before use in an attempt to solve the problems of stability and crystal formation. It discloses lipid:drug ratios in the region of 20:1.
EP 0 355 095 describes a pharmacological agent-lipid solution preparation comprising a lipophilic pharmacological agent, which may be CyA, a desalted charged lipid and an aqueous-miscible lipid solvent such that uponr introduction into an aqueous medium a suspension of lipid associated with the pharmacological agent is formed. As such it is clearly an example of the prior art pro-liposome compositions containing charged lipids, disclosed in the earlier EP 0 158 441.
Even if the formulations described in both the above disclosures have successfully managed to overcome these problems, they would still be exceedingly expensive to produce because of the lipids used, particularly at the high lipid/drug ratios involved, and the relatively complex production processes involved.
In general, technical problems relating to entrapment and stability combined with high production costs have, to date, limited the wider use of liposomes as carriers for drugs. Only amphotericin and doxorubicin are presently in clinical use. These products are for lifethreatening conditions and the quantities used are relatively small to justify the high costs of the lpids and the complex manufacturing processes involved.
Apart from their use in liposomes, there is some report in the prior art describing the use of phospholipids for improving the dissolution of oil-soluble compounds or improving their absorption from the GI tract.
The preparation of solid lipid-drug co-precipitates using diacyl phospholipids to increase the dissolution behaviour of poorly water-soluble drug solvates, and the possibility of modifying drug release from such dispersions by incorporation of small amounts of polymers, has been described (J. Pharm. Sci. 81, 282-286 (1992)). The amount of phospholipid employed, was much lower than the amount of drug and these preparations involved the incorporation of lipid in the crystalline structure of the drug solvate.
PCT/US86/00637 discloses the use of non-esterified fatty acids and monoglycerides in molar ratios between 1:2 and 2:1 together with up to 30 mole percent of a monacyl lipid, lyso-phosphatidylchouine, to form lipid particles which show improved oral absorption when used as carriers for various lipophilic compounds.
Vehicles described as circulating micro-reservoirs, suitable for delivering xenobiotics are disclosed in U.S. Pat. No. 4,298,594. The compositions consist of diacyl phospholipids together with sufficient cholesterol esters to render them more hydrophobic. They are claimed to give improved in vitro and in vivo stability to lipophilic drugs as well as enhanced oral absorption.
U.S. Pat. No. 5,009,956 discloses a method of stabilising small unilamellar vesicles (SUVs) having an outer and an inner layer, comprising between 15-32.5 mol percent of a monoacyl phospholipid in the outer layer of the singlebilayer membrane. It is claimed that sonication of a mixture of diacyl and monoacyl lipids in the proportions stated, for a period of time, is necessary in order to equilibrate the mixture of lipids and obtain maximal stabilisation. There is no suggestion that the SUVs described can be used to solubilise large amounts of lipophilic compounds through molecular association.
An object of the present invention is to provide a bulk lipid carrier, particularly for lipophilic compounds, that is safe, efficient and effective. The existing carriers for lipophilic compounds are systems based on combinations of hydrophilic and lipophilic solvents and ethoxylated chemical surfactants. Although the carrying capacity may be adequate, some compositions can be potentially harmful, particularly if administered in large amounts over a prolonged period.
Given the many benefits of phospholipids, it would be highly desirable to find a means to exploit their unique carrier potential without the practical limitations of presently available systems. The work reported in the prior art points to the need for an efficient, effective and non-toxic carrier for lipophilic compounds. Such needs are not unique to the cyclosporins. There are many biologically active compounds where optimum bioavailibilty cannot be expressed because of poor solubility. For example, in some of the new antifungal and cytotoxic compounds, activity is often linked with lipophilicity. Many lipophilic drug candidates do not progress to further clinical evaluation because of the inability to formulate a suitable dosage form that would allow the potential benefits of the compound to be assessed. Therefore, a non-toxic carrier that transports lipophilic compounds in molecular dispersion would be of significant benefit.
In one aspect, the present invention employs the solubilisation of lipophilic drugs such as CyA in mixtures of diacyl lipids, for example pliosphatidyl choline (PC) and monoacyl lipids, for example mono-acyl phosphatidyl choline (MAPC).
The reasons for the use of such mixtures is three-fold. Firstly, we find that such mixtures are capable of solubilising much higher amounts of CyA than diacyl lipids alone. The reasons for this are not clear but may reflect an association due to steric factors and/or membrane topography. Secondly, the presence of the monoacyl lipid appears to enhance the dispesability of these mixtures in aqueous media. Thirdly, the bioavailability of CyA (or other lipophilic compound) is greatly improved. The reasons for this are again not fully dear but are probably related to the fact that PC and fat-soluble compounds such as CyA are absorbed in the same region of the gastro-intestinal tract.
The absorption, transport and phanmacokinetics of phospholipids are well-known. Over 90% of the diacyl lipid phosphatidylcholine (PC) entering the GI tract is absorbed from the upper region of the intestinal lumen where fat-soluble substances are also absorbed. Almost all of this PC is first hydrolysed to form monoacyl lipid. This, together with bile salts, monoacylglycerols and free fatty acids, then form mixed micelles within the lumen which are taken up by intestinal epitheleal cells. Fat-soluble materials such as CyA tend to partition into such micelles and be co-transported across the mucosal membrane. Whilst it is not suggested that the presence of phospholipids employed in the invention actively transport the associated compounds per se across intestinal mucosa, it is likely that absorption of lipids and lipid-soluble compounds take place in parallel. The increased presence of PC and MAPC are likely to improve the bioavailability of CyA.
In sharp contrast to the synthetic ethoxylated surfactants used in earlier formulations, PC and MAPC are endogenous compounds naturally present together in the intestinal mucosa and their presence is likely to be helpful rather than harmful. The mechanism of uptake of CyA from the micelles formed by such detergents is not known but their strong detergency could potentially damage and alter permeability of the mucosa. This may, of course, be one reason why ethoxylated surfactants are used as carriers to promote improved absorption
Following transport into the epitheleal cells, the CyA enters the blood-stream where it probably partitions into the lipid components of the high and low density lipoproteins and the membranes of erythrocytes and other cells as hypothesised by Fahr (supra) in the case of direct intravenous injection.
A surprising discovery in this invention is the high solubilising capacity of the lipid mixture when MAPC is present, and the improvement in bioavailability. Furthermore, the physical characteristic of the composition can be a soft wax that can be extruded or a plastic wax that can be broken down into granules or spheronised and presented in unit dosage form. Alternatively, the composition may be presented as a fluid preparation by adding suitable non aqueous hydrophilic or lipophilic media for filling into soft gelatin capsules. The composition may also be dispersed in aqueous media to form aqueous dispersions just before use. With careful control of the phospholipid mixture and processing, the invention could in certain circumstances be suitable for parenteral use after dilution. These unique features also enable it to have other novel uses, such as in inhalation and topical delivery.
In another aspect, the invention provides a lipid carrier composition based on the use of monoacyl phospholpids, preferably in combination with diacyl lipids to solubilise water insoluble, lipophilic compounds and thereby improve their bioavaitablity. The physical characteristics can range from fluid compositions to amorphous wax-like compositions. However, the drug-carrying lipid aggregates formed on dilution with water or other aqueous fluids are organlised lipid aggregates that can be lposomes, mixed micelles or micelles. It should be understood that the type of drug-associated lipid particle(s) obtained is not critical, as long as they have the capacity to cary the lipophilic compound in molecular association and obtain improved bioavailablity. In some instances, where an oil or a lipophilic component is also present, stabilised oil globules may be seen in the heterogeneous suspension at equilibrium.
Embodiments of the invention may overcome two major disadvantages in using liposomes as carriers, namely, physical instability of the vesicles and low entrapment. Unlike liposome preparations, no external aqueous medium is necessary and therefore stability and microbial contamination should not be an issue. Furthermore, expensive and energy intensive equipment is not required to produce liposomes with well defined characteristics. Absence of intensive shearing forces involved in some methods of preparing liposome suspensions avoids the loss of entrapped compound. Furthermore, large scale production is easily undertaken. Although most compounds can be carried in the invention to obtain improved bioavailability, it is particularly suitable for solubilising water-insoluble lipophilic compounds particularly fungal metabolites (e.g. cyclosporiny, and anti-fungal and cytotoxic agents. It may also be useful to deliver peptides and proteins and nucleic acids associated in the form of lipid complexes.
A further unexpected feature of the compositions described is that they will readily disperse into discrete microscopic/colloidal lipid aggregates in the presence of an aqueous fluid, even at room temperature, with minimum agitation. The lipid aggregates obtained on dilution are uniform and mostly in the region of 100 rim when the ratio of diacyl to monoacyl lipid is less than approximately 1:1. Lipophilic compounds remain in association within the aggregates. Depending on the combination of diacyl- to monoacyl lipids and their configuration, the aggregates may be vesicular or non-vesicular. They may be bilayer in form, complexes of bilayers and micetles, or totally micellar. Given the appropriate lipid mixture, the size of the lipid aggregates is unaffected on dispersion in aqueous fluid between the physiological pH range i e. 2 to 8. The monoacyl components both promote solubilisation in the lipid mixture and also aid dispersion into small aggregates in the presence of aqueous medium. Bile salts and other emulsifiers are not essential for release of the compound for absorption in the GI tract as the compound is largely in molecular dispersion in a partially digested lipid mixture. However, as a bonus, dispersion into lipid aggregates may be fuirther improved in the presence of emulsifiers such as bile salts particularly at 37xc2x0 C.
The present invention can be used to carry different types of compounds for all kind of applications, but it is particularly suitable for carrying lipophitic compounds, especially for oral administration. By way of example, and not by way of limitation the compounds being carried may be CyA and miconazole, an antifiungal compound. In addition to these two examnples of lipophilic compounds, a further example of a highly water insoluble lipophilic compound, astaxanthine is given to demonstrate the utility of the invention in non-pharnaceutical applications. Astaxanthine is widely used in aquaculture to confer pigment to fish, but large amounts have to be given because of poor bioavailability.
It must be understood that these formulations are not limited to the examples shown. Many biologically active compounds eg, peptides, proteins, vaccines, DNA, steroids, hormones, vitamins, anti-arrythinc compounds, etc and other lipophilic compounds can be incorporated in the composition, by selecting the appropriate quantity of lipids, ratio of diayl to monoacyl fractions and cognisant of physical properties of the lipid, such as charge, chain length, degree of saturation and phase transition temperature. The lipid carrier may also be formulated as a fluid composition with appropriate hydrophilic or lipophilic media. Liquid compositions may be more convenient to administer because they can be diluted prior to use or filled into soft gelatine capsules. Solvents used in processing and the presence of residual hydrophilic medium left in the bulk lipid carrier should also be taken into account, as they could affect the association of the compound and bioavailability.
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