Title:  Nano-emulsion of 5-aminolevulinic acid

United States Patent:  6,559,183

Issued:  May 6, 2003

Inventors:  Schmid; Hans W. (Zug, CH); Burmeister; Gerd (Oberarth, CH)

Assignee:  ASAT AG Applied Science & Technology (Zug, CH)

Appl. No.:  831564

Filed:  July 26, 2001

PCT Filed:  November 12, 1999

PCT NO:  PCT/EP99/08711

PCT PUB.NO.:  WO00/28971

PCT PUB. Date: May 25, 2000

The present invention relates to a composition comprising a nano-emulsion that contains 5-aminolevulinic acid as well as a carrier in an aqueous phase. This invention also relates to a pharmaceutical preparation containing this composition. The nano-emulsions of this type can be used in photodynamic therapy as well as in the photodiagnostic detection of proliferatives cells.

Description of the Invention

The present invention relates to nanoemulsions which contain 5-aminolevulinic acid or its derivatives, precursors or metabolites.

Photodynamic therapy is a novel and promising method for treating various premalignant and malignant diseases which are connected to cell proliferation. The principle of photodynamic therapy is based on introducing what is termed the photosensitizer into the tumor tissue and using irradiation with light of a suitable wavelength to convert this photosensitizer into a cytotoxically active compound which in the end destroys the cells. The selectivity of this method is based on the sensitizer being concentrated to a greater extent in rapidly proliferating tumor cells than in normal tissue. Irradiation with light in a locally restricted manner can then be used to specifically activate the sensitizer which is present in the tumor cells, thereby destroying the cancer cells while to a large extent sparing the healthy tissue.

Until now, an intravenously administered mixture of hematoporphyrin derivatives has in the main been used as the photosensitizer. Despite the encouraging clinical successes which have been achieved in connection with a number of different types of cancer, these hematoporphyrin derivatives nevertheless suffer from a variety of disadvantages. In the first place, relatively high concentrations of the active compound appear in normal tissue due to the low degree of tumor selectivity and the fact that the active compound is only slowly eliminated from the body. Undesirable photochemical reactions therefore take place in healthy tissue in connection with the irradiation. In the second place, this treatment results in a general sensitivity to light such that the patient is not allowed to expose himself to daylight for a period of some four weeks.

In certain cases, it is possible, particularly in connection with dermatological and gynecological applications, to bring about a reduction in the high concentration of active compound in normal tissue, and therefore in the undesirable side-effects, by developing topically applicable active compound formulations in place of the known systemic formulations. Attempts are also being made to reduce the sensitivity to light by using photosensitizer precursors which are photochemically inactive and are only converted into a photosensitizer within the target cell.

5-Aminolevulinic acid is an endogenous substance which is synthesized from glycine and succinyl-CoA. In heme biosynthesis, the extremely photoactive protoporphyrin IX is formed from 5-aminolevulinic acid (5-ALA) in several rapidly proceeding reactions steps, and is then converted into heme in a slow reaction. If the heme concentration is too high, a natural control mechanism inhibits both the endogenous synthesis of 5-aminolevulinic acid and the breakdown of protoporphyrin IX.

This control mechanism is circumvented by exogenously administering synthetically prepared 5-aminolevulinic acid, thereby giving rise to an increased production of protoporphyrin IX. Since the breakdown of protoporphyrin IX is still inhibited by the natural control mechanism, this compound becomes concentrated in the cells. When irradiated with light, protoporphyrin IX is able to enter into a photochemical oxidation reaction and consequently acts as a photosensitizer. When the sensitizer molecule absorbs a quantum of light, it is first of all transferred into an electronically excited state (singlet state), which is relatively short-lived, and either releases its excess energy once again within a few nanoseconds by emitting a fluorescence photon or else passes over into a relatively long-lived triplet state. Energy from this triplet state can be transferred to oxygen molecules which are present in the cell. The singlet oxygen which is formed in this connection has a cytotoxic effect, in particular on proliferating cells, since it reacts with cell components, for example the cell membrane and the mitochondria, or triggers the formation of cell-damaging free radicals. Furthermore, irradiation of the photosensitizer gives rise to a characteristic fluorescence radiation which can be used for detection reactions, for example for detecting proliferating cells.

A number of investigations using topically applicable 5-aminolevulinic acid compositions are known from the prior art. While these investigations have the feature in common that the 5-aminolevulinic acid employed is in the form of an oil-in-water emulsion, differences exist with regard to other parameters, such as period of penetration, period of treatment, type of light employed and the dose of light applied.

B. Thiele et al. (H+G, Volume 69, No. 3, pages 161-164 (1994)) describe investigations which involve using 20% .delta.-aminolevulinic acid in the form of an oil-in-water emulsion, with a penetration period of from 5 to 6 h, and subsequently irradiating with an argon ion-pumped dye laser (emission maximum 630 nm) giving a cumulative total dose of from 50 to 100 J/cm².

Wolf et al. (Journal of the American Academy of Dermatology Vol. 28, pages 17 to 21, 1993) describe investigations which involve using 20% 5-aminolevulinic acid in the form of an oil-in-water emulsion, with a penetration period of 4, 6 or 8 h, and irradiating with unfiltered light or red light, giving a light dose of from 30 J/cm² to 100 J/cm².

Although the investigations disclosed in the prior art clearly demonstrate the promising potential of photodynamic therapy using 5-aminolevulinic acid, oil-in-water emulsions which are so far known suffer from a number of disadvantages.

Thus, M. Novo Rodriguez et al. (SPIE, Vol. 2371, pages 204-209) showed that, in the high concentrations which are required for a clinical application, aminolevulinic acid is unstable in aqueous solutions in the neutral to basic pH range. In the time period of 25 h investigated, satisfactory results are only obtained at a pH of 5.01, and a concentration of 3% and a pH of 5 are specified as the optimal conditions for aqueous solutions of 5-aminolevulinic acid. However, for clinical use, it will in general also be necessary to provide compositions in a higher concentration range; furthermore, to be used commercially, the 5-ALA solutions have to be stable for a period which is of the order of weeks or months.

V. von Arx et al. (J. Pharm. Pharmacol. 49: 652-656, 1997) describe investigations relating to the topical application of 5-aminolevulinic acid in a variety of gels. This publication states that the best formulation for maintaining the stability of 5-aminolevulinic acid is a combination with Novion AA-1, a polyacrylic acid, at a pH <6.

Another disadvantage of the known oil-in-water emulsions is that the depth to which the photosensitizer penetrates into the damaged tissue is not optimal. As a result, the diseased tissue is in many cases only accessible to the photodynamic therapy in its superficial layers even though the depth to which the light employed for activating the photosensitizer penetrates would also enable more deeply lying layers to be treated.

The object of the present invention was therefore to make available 5-aminolevulinic acid-comprising compositions in which the disadvantages known from the prior art are at least partially eliminated and which, in particular, possess adequate stability and exhibit an improved ability to penetrate into tissue.

This object is achieved by a composition which is characterized in that it contains a nanoemulsion which comprises a substance selected from 5-aminolevulinic acid, or a derivative, a precursor and/or a metabolite thereof, and a carrier in an aqueous phase.

It was observed, surprisingly, that the stability of 5-aminolevulinic acid can be substantially increased when the acid is formulated into a nanoemulsion. While the reasons for this are not known, it appears that a microenvironment created by nanosomes has a particularly favorable effect on the stability of the 5-aminolevulinic acid.

It has furthermore been shown, surprisingly, that very high tissue penetration depths can be achieved with the nanoemulsions according to the invention, resulting in more deeply lying diseases, or diseases with higher layer thicknesses, also becoming accessible to treatment. The greater penetration depths were particularly surprising because it had previously been assumed that, due to its small size, 5-aminolevulinic acid would in any case be readily able to penetrate through a damaged epidermis which is present, for example, over inflammations, precancerous stages and tumors.

A third surprising advantage is that, when packed into nanosomes in accordance with the invention, 5-aminolevulinic acid is evidently taken up very efficiently by the cells. This firstly improves targeting; secondly, it means that the penetration period, i.e. the time between applying the composition and irradiating the diseased tissue with light, can be reduced, with this representing a distinct relief for the patient.

According to the invention, the nanoemulsion comprises an active substance which is selected from 5-aminolevulinic acid or a derivative, a precursor and/or a metabolite thereof. "Derivative" is to be understood as being, in particular, salts, complexes and addition compounds. "Precursor" and "metabolite" are in this connection to be understood as being those substances which are converted in a cell into protoporphyrin IX. Particular preference is given to the active substance being 5-aminolevulinic acid or one of its derivatives. The carrier can be any carrier as long as it is able to form the nanoemulsion in an aqueous phase. The carrier preferably comprises an oil phase, i.e. a material which is immiscible with water, for example lipids, and an emulsifier. Physiologically harmless carrier substances are expediently used.

The size of the emulsified particles in the nanoemulsion (nanosomes) is on average .ltoreq.200 nm, e.g. from 10 to 200 nm. The particle size which is in each case optimal depends on other parameters such as the viscosity of the composition. For example, good results were obtained with a gel having a viscosity of 5 mPas at an average particle diameter of about 110 nm, and also for a lotion having a viscosity of 1.6 mPas at an average particle diameter of about 20 nm.

Suitable carrier systems, which are stable over a long period of time, which do not contain any high concentrations of surfactants and cosurfactants, and which are free from toxic emulsifier complexes, are disclosed, for example, in U.S. Pat. No. 5,152,923. These nanoemulsions comprise a glycerophosphatide, such as a lecithin or a cephalin, as the emulsifier and physiologically tolerated lipids, e.g. triglycerides, such as vegetable or animal oils, for example groundnut oil, soybean oil, etc., as the oil phase. The emulsifier/oil weight ratio is from 0.05 to 0.4:1.

Examples of emulsifiers which have already been employed successfully in practice in 5-aminolevulinic acid nanoemulsions are egg lecithin, soybean lecithin and phosphatidyl choline. An example of an approved lipid is Miglyol 812.

The proportion of active substance, for example 5-aminolevulinic acid, in the composition essentially depends on the application which is envisaged. In general, from about 1 to 25% by weight, based on the total weight of the composition, are present. However, it is also possible to use higher or lower doses. A proportion of from 5 to 15% by weight, in particular of about 10% by weight, has proved to be suitable for applications in connection with photodynamic therapy.

The composition can additionally comprise adjuvants and/or additives, in particular those substances which are customary in cosmetics or pharmacy. Examples of such substances are buffers, stabilizers, additional emulsifiers, thickeners, etc.

In a particularly preferred embodiment, the composition according to the invention is a gel which, based on the total weight of the composition, comprises from 1 to 25% by weight, preferably from 5 to 15% by weight, of active substance, from 40 to 60% by weight, preferably from 45 to 55% by weight, of carrier and from 0 to 10% by weight, preferably from 1 to 5% by weight, of adjuvants, with the remainder being water.

According to another particularly preferred embodiment, the composition according to the invention is a lotion which, based on the total weight of the composition, comprises from 1 to 25% by weight, preferably from 5 to 15% by weight, of active substance, from 10 to 30% by weight, preferably from 15 to 25% by weight, of carrier and from 10 to 30% by weight, preferably from 15 to 25% by weight, of adjuvants, with the remainder being water.

As mentioned at the outset, the 5-aminolevulinic acid composition according to the invention exhibits a surprisingly high degree of stability on storage, with the proportion of active substance in the composition having a pH of between 1.5 and 3 preferably being reduced, after one year of storage at room temperature, by not more than 5% and, particularly preferably, by not more than 4%. After one year of storage at 5^oC., the proportion of active substance is preferably reduced by not more than 3% and particularly preferably by not more than 2.5%.

The present invention also relates to the composition according to the invention which is in the form of a pharmaceutical preparation. In this case, the composition is free of constituents which are not pharmaceutically acceptable and preferably free of constituents which, for example, provoke irritation. In addition to the carrier substances which have already been mentioned, the pharmaceutical preparation can also comprise further adjuvants and/or additives which are acceptable and preferably well tolerated.

The pharmaceutical preparation can be present in a form which is suitable for systemic administration, such as an injectable liquid. However, for dermatological and gynecological applications, the the preparation is preferably in a form which is suitable for topical administration. The preparation possesses properties, e.g. viscosity and rheology, which are favorable for the administration form which is in each case required in order to ensure that, after the preparation has been administered, the nanosomes loaded with 5-aminolevulinic acid penetrate to an adequate extent into the target tissue. These viscosity and rheology properties can be adjusted by adding thickeners such as polyethylene glycol stearyl ethers, polyethylene glycol stearates and/or polysaccharides such as polysaccharide B-1459, for example.

The present invention also relates to a process for producing the composition or the pharmaceutical preparation according to the invention. In this process, the constituents of the carrier material are initially introduced in an aqueous phase and the mixture is converted into a nanoemulsion by homogenizing thoroughly. It is possible, for example, to use commercially available high pressure homogenizers for this purpose. The 5-aminolevulinic acid, and any additives which may be present, can be added before and/or after the homogenization. After the nanoemulsion has been prepared, it is then possible to add other adjuvants and additives whose presence was not desirable during the homogenization.

Preference is given to excluding air while carrying out the process, for example by means of applying a vacuum and/or a protective gas atmosphere. In addition, it is preferred to implement the process while excluding light. The process is carried out at a temperature at which the desired nanoemulsion can be formed and the constituents, in particular the active substance, is adequately stable. In general, it has been found that a temperature range of from about 5 to 45^oC. is suitable. However, adjuvants and/or additives which are, for example, first of all mixed, and homogenized where appropriate, in a separate mixture, and only after that added to the composition, can be processed at higher temperatures, for example up to about 80^oC. For a pharmaceutical application, care is taken to ensure that the resulting product is sterile, for example by employing sterile starting materials and maintaining sterile process conditions and/or by inserting a sterilization step after the preparation.

An important area of use for the compositions according to the invention is in the field of photodynamic therapy, with particular preference being given to applying the nanoemulsion topically. The nanoemulsion according to the invention can be used in association with all diseases whose control comprises inhibiting the proliferation of, or destroying, cells or tissues by photoactivating a sensitizer which is formed from 5-aminolevulinic acid. The diseases include, in particular, those which are associated with an increase in cell proliferation since, in this case, the photosensitizer is concentrated to a particularly high degree by the increased cell metabolism in diseased cells.

The compositions according to the invention are consequently suitable for treating tumor diseases such as basal cell carcinoma, squamous cell carcinoma, Bowen's disease, solar keratosis, condylomata acuminata (CIN), epithelial neoplasia of the vulva (VIN), and nodose and subcutaneous cancer diseases. Psoriasis is an example of a nontumorous disease.

The treatment is effected, for example, by topically applying a nanoemulsion which contains the active substance, e.g. 5-aminolevulinic acid, and then incubating in order to allow an adequate quantity of the 5-aminolevulinic acid to penetrate into the tissue which is being treated. During the incubation, irradiation of the treated area with light is preferably avoided, for example by covering it, in order to prevent any undesirable premature activation. After the incubation period, which is generally from about 1 to 8 h and usually about 4 h, has expired, the tissue is irradiated with an adequate dose of radiation using a light source. Suitable light sources include lamps which emit white light and also monochromatic light sources, such as a laser, in particular an argon dye laser which emits at about 630 nm. The radiation doses are normally in a range of from about 20 J/cm² to several hundred J/cm² per application.

Another area for using the nanoemulsions according to the invention relates to detecting the presence of proliferating cells in a sample, for example a tissue sample. The detection is based on selectively concentrating a photosensitizer, which is produced by metabolism of the active substance, in proliferating cells as compared with normal cells. Preference is given to the active substance being 5-aminolevulinic acid and the photosensitizer being protoporphyrin IX. The extent to which the photosensitizes has been concentrated can be determined by means of photodiagnostic methods, for example by irradiating with light having a wavelength of 405 nm and measuring the fluorescence radiation generated by the photosensitizer. The nanoemulsions according to the invention are particularly suitable for being used in tumor diagnosis.

The invention furthermore relates to the use of the nanoemulsion according to the invention for producing a drug for photodynamic therapy.

Finally, the invention relates to a kit which comprises a nanoemulsion according to the invention, which is suitable for being applied topically, and one or more auxiliary substances. Examples of these auxiliary substances are a covering material, such as a plastic film which is applied to the site being treated, after the nanoemulsion has been applied, in order to prevent premature activation by light, and means for attaching the covering material or else means for applying the nanoemulsion to the site being treated.

Claim 1 of 21 Claims

What is claimed is:

1. A composition comprising a nanoemulsion which comprises an active substance which can be converted into protoporphyrin IX, the active substance being selected from the group consisting of at least one of 5-aminolevulinic acid, a salt compound thereof, a complex compound thereof, an addition compound thereof, precursors thereof, and metabolites thereof, and (b) a carrier in an aqueous phase, with the carrier being formed from at least one lipid and from at least one emulsifier comprising soybean lecithin.
 

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