RU2379026C2 - Composition for fluorescent diagnostics and photodynamic therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and concerns pharmacological composition for fluorescent diagnostics (FD) and photodynamic therapy (PDT) with application of prosensibiliser -5-aminolevulinic acid (ALA) hydrochloride. Composition for fluorescent diagnostics and photodynamic therapy includes 5-aminolevulinic acid hydrochloride as active substance, gel-forming polymer-hydroxyethylcellulose and water. Composition additionally contains plasticiser and organic additive. Composition can also include surface-active substance - block-copolymer of ethylene oxide and propylene oxide in amount 2.5-5.0 % wt, as well as preservative in amount 0.05-0.3 % wt. Claimed composition can be efficiently applied for FD and PDT.

EFFECT: invention insures creation of stable in storing gel composition, which contains ALA as active component in amount not less than 5% and water gel as base.

6 cl, 27 ex, 4 tbl, 1 dwg

Description

The invention relates to medicine, and more specifically relates to a pharmacological composition for fluorescence diagnostics (PD) and photodynamic therapy (PDT) using a sensitizer - 5-aminolevulinic acid hydrochloride (ALA).

The PDT method is based on the use of photosensitizers, which have the ability to selectively accumulate in pathological, in particular oncological, tissues and, when exposed to laser radiation in the presence of oxygen, cause photochemical reactions leading to the selective destruction of tumor cells.

The introduction of ALA into the body induces an increased level of fluorescent protoporphyrin IX (PpIX) in it, the first porphyrin in the biosynthesis chain of all natural porphyrins. Due to the lack of the ferrochelatase enzyme in the cancer cells, the PpIX content in them, 3-4 hours after the introduction of ALA into the body, is several times higher compared to healthy tissue. As a result, by illuminating the tissue with light at a wavelength of 630 nm, the presence of blue light can determine the presence of cancerous tissue.

Currently, the most well-developed diagnostic methods using fluorescence of PpIX malignant tumors of the bronchi, digestive tract, bladder and superficial skin tumors. In the latter case, ALA is most often used in the form of solutions. However, for external use, more viscous outlet forms are much more convenient. The literature describes a number of ALA-containing formulations for external use, which can be considered as analogues of the present invention, various ointments, creams, lotions. Conventionally, all these compounds can be divided into those containing water and non-aqueous. The disadvantages of the latter, in particular liposomal, are the complexity of the composition and manufacture [Casas A., Fukuda N., DiVenosa G., Del AM, Batlle C. - The influence of the vehicle on the synthesis of porphyrins after topical application of 5-aminolaevulinic acid . Implications in cutaneous photodynamic sensitization - Br. J. Dermat. 2000, 143, 564-572], [Pierre M.B.R., Tedesco A.C., Marchetti J.M., Bentley M.V.L.B. - Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study http://www.biomedcentral.com/1471-5945/1/5 BMC Dermatology 2001, 1: 5], as well as low storage stability. So, the content of ALA in liposome form after a month of storage is 30-60% of the initial level [Auner BG, Petzenhauser E., Valenta C. - Influence of 6-ketocholestanol on skin permeation of 5-aminolevulinic acid and evaluation of chemical stability - J . Pharm. Sci. 2004, 93, 2780-2787], in non-aqueous gel form after 200 days of storage at 5 ° C. — less than 45% [McCarron R.A., Donnelly R.F., Andrews G.P., Woolfson A.D. - Stability of 5-aminolevulinic acid in novel non-aqueous gel and patch-type systems intended for topical application - J. Pharm. Sci. (2005) Vol. 94, No.8, pages 1756-1771].

The known ALA compositions in the form of aqueous lotions [US Patent 6,559,183 (2003) "Nano-emulsion of 5-aminolevulinic acid"] and creams [US Patent 6,034,267 (2000)], US Patent 7,247,655 (2007)], but their stability is also low. Examples are cream and lotion, in which after a month of storage at 4 ° C, no more than 50% and 75% of the initial ALA content remains, respectively [Auner BG, Petzenhauser E., Valenta C. - Influence of 6-ketocholestanol on skin permeation of 5-aminolevulinic acid and evaluation of chemical stability - J. Pharm. Sci. 2004, 93, 2780-2787].

The gel forms of ALA containing water as a component of the medium are closer to our composition in composition. Known biphasic gel, prepared on the basis of water and glycerol monooleate in a ratio of 30: 70 by weight [Turchiello F., Vena F.C.B., Maillard Ph., Souza C.S., Bentley M.V.L.B., Tedesco A.C. - Cubic phase gel as a drug delivery system for topical application of 5-ALA, its ester derivatives and m-THPC in photodynamic therapy (PDT) - Journal of Photochemistry and Photobiology B: Biology 2003, 70, 1-6]. The main disadvantages of this composition is the extremely low content of active substance - only 0.2%. This limits the use of such a composition in PDT, since it is shown that the minimum ALA content should not be less than 5% [Asta Juzeniene, Petras Juzenas, Vladimir Iani Topical Application of 5-Aminolevulinic Acid and its Methylester, Hexylester and Octylester Derivatives: Considerations for Dosimetry in Mouse Skin Model. - Photochemistry and Photobiology, 2002, 76 (3): 329-334]. Another disadvantage is the low storage stability (over 30 days, the amount of ALA decreased by ~ 8%), in addition, the process of obtaining a composition based on a gel of water - glycerol monooleate is quite complicated and takes at least 24 hours.

The gel forms of water-based ALA are closest in composition to our composition. An alginate-based aqueous gel was selected as a prototype of the invention as a gelling polymer in a buffer solution providing a pH of composition 5-6 [US Patent 6,860,879 (2005)]. The main disadvantage of this composition is that it is prepared ex-tempore by mixing a solid mixture of ALA and alginate with a buffer solution.

The present invention is the creation of a storage stable gel composition containing ALA as an active component in an amount of not less than 5% and an aqueous gel as a base.

To solve this problem, a composition is proposed that includes, as an active substance, 5-aminolevulinic acid hydrochloride, a gelling polymer - hydroxyethyl cellulose, a plasticizer, an organic additive and water in the following ratio of components (wt.%)

5-aminolevulinic acid hydrochloride 5.0-20.0 hydroxyethyl cellulose 1.15-1.65 plasticizer 3.0-5.0 organic supplement 5.0-20.0 water rest

The problem is also solved by the fact that polyethylene glycol or polypropylene glycol is used as a plasticizer.

The problem is also solved by the fact that propylene glycol and / or ethylene glycol and / or diethylene glycol and / or glycerol and / or dimethyl sulfoxide are used as an organic additive.

The problem is also solved by the fact that the composition further includes, as a surfactant, a block copolymer of ethylene and propylene oxide in an amount of 2.5-5 wt.%.

The problem is also solved by the fact that the composition additionally contains a preservative in an amount of 0.05-0.3 wt.%.

The problem is also solved by the fact that, as a preservative, the composition contains 4-hydroxybenzoic acid methyl or propyl ester or N- (3,3bis (hydroxymethylmethyl) -2,5-dioxo-4-imidazolidinyl) -N, N'-bis (hydroxymethyl) urea or mixtures thereof

The gelling polymer hydroxyethyl cellulose (HEC) can be used of various types, differing in the degree of polymerization, for example, with a molecular weight of 7.2 × 10 ⁵ or 1.3 × 10 ⁶ , while the required rheological properties of the composition (viscosity, plasticity) are controlled by the amount of HEC used.

Polyethylene glycol (PEG) 300-1500 or polypropylene glycol (PPG) 250-1050 contribute to the production of a plastic gel with high adhesive properties.

Organic additives are added in order to increase the penetration rate of the active substance through the upper layers of the skin.

A nonionic surfactant, which is a block copolymer of ethylene and propylene oxide (Proxanol 268), helps to increase the wettability of the upper layer of the skin.

Preservatives are administered to prevent the development of microorganisms in the composition during prolonged storage.

As for the technology for preparing the proposed gel compositions, it is as simple as possible and actually boils down to the sequential mixing of the components, which takes no more than 3 hours.

Specific activity i.e. the ability to induce the formation of PpIX was evaluated by fluorescence of induced PpIX in healthy rabbit skin and mouse tumors. The distribution of PpIX induced by local application of ALA gel in the tissues of mice with Ehrlich tumor is summarized in Table 1.

From the data in Table 1, it follows that ALA in the gel composition penetrates well through the stratum corneum and is absorbed into the blood, as evidenced by the presence of specific PpIX fluorescence in the internal organs and areas of the skin and muscles that are distant from the application zone. Moreover, the amount of PpIX induced in the tumor increases with increasing duration of application and ALA concentration in the gel. Figure 1 shows the time dependence of the fluorescence intensity of the induced PpIX when applying a gel with 20% ALA content in different parts of the Ehrlich tumor (1 - adjacent to the skin, 2 - at a depth of 2-3 mm, 3 - at a depth of 4-6 mm) , and in Fig.2 the same dependence during the application for 4 hours of a gel with a different content of ALA.

The use of a gel composition with an ALA content of 5-20% for PDT of S-37 sarcoma on male F1 hybrid mice showed that the proposed composition is an effective tool for PDT and PD of oncological diseases.

Table 1 the cloth Fluorescence intensity, rel. units before application after 4 hours Tumor 1.6 ± 0.2 7.0 ± 1.0 Epidermal skin 1.0 ± 0.003 4.3 ± 0.3 Dermis skin 1.0 ± 0.003 3.5 ± 0.1 Ear skin 1.1 ± 0.1 2.6 ± 0.3 Muscle 1.6 ± 0.4 2.7 ± 0.9 Liver 2.2 ± 0.1 8.0 ± 2.6 Gall bladder 1.6 ± 0.1 28.2 ± 10.5 Kidney 1.6 ± 0.2 4.9 ± 0.4

The invention is illustrated by the above examples, but is not limited to.

Examples 1-18. Preparation of ALK Gel Samples

Samples of ALA gel were prepared in a four-necked round-bottom flask with a capacity of 50 cm ³ equipped with a magnetic stirrer with heating, a thermometer, and a reflux condenser in an argon atmosphere, which was bubbled during the entire preparation process. At the end of the process, ALA gel is packaged in dark glass bottles in a special box in an argon atmosphere.

If a block copolymer of ethylene and propylene oxide was used in the composition, Method A was used; otherwise, Method B.

Method A. Preparation of ALA gel samples containing a block copolymer of ethylene and propylene oxide (proxanol 268)

To the calculated amount of water sufficient to prepare 25 g of ALA gel, the SCE is added at room temperature with stirring, the temperature is raised to 50 ° C and stirred at this temperature for 40 minutes. After the mass has cooled to room temperature, crushed proxanol 268 is added and mixed. A pre-prepared mixture of propylene glycol, PEG, and, if necessary, a preservative (methyl paraben, propyl paraben and diazolidinyl urea or a mixture thereof) are added to the obtained transparent homogeneous mass and mixed for another 40 minutes. Then make ALA, mix until completely dissolved, then mix for another 15 minutes and pack.

Method B. Preparation of ALA gel samples not containing a block copolymer of ethylene and propylene oxide

To the calculated amount of water sufficient to prepare 25 g of ALA gel, at room temperature, with stirring, add a pre-prepared suspension of the calculated amounts of HEC, if necessary, a preservative, an organic additive and a plasticizer. The temperature was raised to 50 ° C and stirred at this temperature for 40 minutes. Then the mass is allowed to cool to room temperature, 5 g of ALA is added to the resulting transparent gel, stirred for another 15 minutes and packaged.

The compositions of the ALA gel samples prepared according to methods A and B are shown in Table 2.

All the compositions shown in Table 2 turned out to be stable during the year.

Example 19-22. Assessment of the local irritating effect of ALA gel

The local irritating effect of the studied ALA gel samples was evaluated macroscopically by the presence of edema, hyperemia, peeling, ulceration, and other pathological manifestations of the skin in the gel exposure zone.

A gel composition containing 5-20% ALA (composition according to examples 1-4) was applied to previously epilated areas of the back of a mouse or rat or to the inner surface of the forearm of volunteers, covered with a plastic film and fixed with a plaster. The application time was 4 hours.

Visual inspection of the areas subjected to the application showed that this composition with multiple (daily for 5 days) cutaneous application did not have a local irritant effect.

Examples 23-25. Healthy Skin Gel Testing

The studies were performed on chinchilla rabbits, females weighing 2.5-3.5 kg. A gel sample (composition according to examples 1, 2, 4) was applied to previously epilated areas of the skin of the back of a rabbit, covered with a plastic film and fixed with a plaster. The application time of the drug was 4 hours. Previously, animals were sedated by intravenous administration of Relanium (10-30 mg) or droperidol (5 mg).

Induced fluorescence in healthy skin was evaluated by local fluorescence spectroscopy with excitation by radiation from a solid-state laser with frequency doubling (532 nm). In mathematical processing, the integrated fluorescence intensity in the range of 620-650 nm was normalized to the integrated tissue autofluorescence intensity in the range of 555-585 nm [Sokolov VV, Filonenko EV, Telegin L.V. et al. Quantum Electronics, 2002, No. 11, 963-969], the obtained value was designated as D _f . Fluorescence was recorded by the contact method at the Spectr-Cluster diagnostic unit (Cluster LLC, Moscow); to obtain a reliable result, 5 to 10 skin spectra were measured.

The fluorescence intensity of protoporphyrin IX in the area of application of the gel to the skin exceeded that for intact areas of rabbit skin by at least two times. Table 3 shows the normalized fluorescence intensity (D _f ) of induced protoporphyrin IX in healthy rabbit skin after 4 hours of ALA gel application. From the above data it follows that in the range of ALA concentrations of 5-20%, the diagnostic contrast does not decrease.

Table 3 Example No. Composition D _f 23 as in example 1 2.5 ± 0.5 24 as in example 2 2.4 ± 0.3 25 as in example 4 2.3 ± 0.2

Examples 26, 27. Carrying out photodynamic therapy

Studies were conducted on mice hybrids F1 males with sarcoma S-37. The tumor strain S-37 was maintained in vivo in the ascites variant. For experiments, ascitic fluid at a dilution of 1: 3 with an isotonic solution was inoculated subcutaneously on the outer surface of the thigh in an amount of 0.05 cm ³ / animal. Treatment began on the 6th day after inoculation of the tumor material. The size of the tumors on the day of treatment was (4-5) × (4-5) mm ² .

A gel containing 10% or 20% ALA (composition according to examples 2, 4) was applied to the skin at the rate of 60 mg of active substance per mouse; PDT was performed after 4 hours of application.

An LED source with a radiation maximum at 530 ± 15 nm was used as a source of optical radiation. The distance between the end of the emitter and the tumor was 2-3 mm. The device was set to maximum power (power density ~ 30 mW / cm ² ) and irradiated for 1 h. Thus, the light dose was ~ 100 J / cm ² .

The criterion for evaluating the antitumor effect was inhibition of tumor growth (SRW,%), which was calculated by the formula

where V _counter and V _experience - tumor volume in the control and experimental groups, respectively.

Data on the antitumor efficacy of PDT with a gel composition containing ALA are presented in Table 4.

Table 4 presents the data on the antitumor efficacy of PDT after 4-hour application of ALA gel in mice with S-37 sarcoma. As can be seen from the data in Table 4, PDT with a gel composition containing 20% ALA (4-hour application) led to SRW by 71-50%, while the effect remained at a level of ≥50% for 11-14 days after treatment .

Table 4 Experience number Composition Dose mg / cm ² Exposure parameters SRW per day after treatment,% Power density, mW / cm ² The energy density, j / cm ² 7 eleven fourteen eighteen twenty 26 as in example 4 40 thirty 54 71 55 51 35 29th 27 as in example 2 40 52 fifty 44 eighteen 3

Thus, a gel-stable composition was created during storage, containing ALA as an active component in an amount of 5-20%, to induce the formation of protoporphyrin IX in fluorescence diagnostics and photodynamic therapy.

The proposed composition is an effective tool for PDT and PD of cancer.

Claims (6)

1. Composition for fluorescence diagnostics and photodynamic therapy, comprising as active substance 5-aminolevulinic acid hydrochloride, a gelling polymer and water, characterized in that it contains hydroxyethyl cellulose as a gelling polymer, and further comprises a plasticizer and an organic additive in the following ratio of components :
5-aminolevulinic acid hydrochloride 5.0-20.0% hydroxyethyl cellulose 1.15-1.65% plasticizer 3.0-5.0% organic supplement 5.0-20.0% water rest 2. The composition according to claim 1, characterized in that as a plasticizer it contains polyethylene glycol or polypropylene glycol. 3. The composition according to claim 1, characterized in that as an organic additive it contains propylene glycol or ethylene glycol, or diethylene glycol, or glycerin and / or dimethyl sulfoxide. 4. The composition according to claim 1, characterized in that it further comprises a surfactant block copolymer of ethylene oxide and propylene oxide in an amount of 2.5-5 wt.%. 5. The composition according to claim 1, characterized in that it further comprises a preservative in an amount of 0.05-0.3 wt.%. 6. The composition according to claim 5, characterized in that it contains 4-hydroxybenzoic acid methyl or propyl ester or N- (1,3-bis (hydroxymethyl) -2,5-dioxo-4-imidazolidinyl) -N as a preservative , N'-bis (hydroxymethyl) -urea or mixtures thereof.

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