MXPA06002220A - Non-polar photosensitizer formulations for photodynamic therapy - Google Patents

Abstract

A pharmaceutical liposomal formulation for photodynamic therapy comprising a non-polar porphyrin photosensitizer and one or more phospholipids, which are stable in storage without requiring freeze-drying is described. The liposomal formulation provides therapeutically effective amounts of the photosensitizer for intravenous administration. The phospholipids may be modified by pegylation, i.e. they contain poly ethylene glycol as an integral part of the phospholipids. The formed liposomes contain the non-polar photosensitizer within the membrane and are useful for the combined targeting of a nonpolar photosensitizer and a second polar substance. When a formulation includes the presence of monosaccharides or polyalcohols, it can be efficiently freeze-dried preserving the size of the liposomal vehicles and the content of a therapeutically effective amount of the photosensitizing agent. The invention also relates to the liposome composition formed upon reconstitution with an aqueous vehicle. The freeze-dried formulation upon reconstitution with a suitable aqueous vehicle forms liposomes that are also useful for intravenous administration.

Description

NON-POLAR PHOTOSENSIBILIZER FORMULATIONS FOR PHOTODYNAMIC THERAPY BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the preparation of liposomal formulations containing Temoporfin or other non-polar photosensitizers and their use in therapy, particularly using intravenous injection. 2. Information of the Declaration of Description Liposomes are artificial vesicles composed of concentric lipid bilayers separated by water compartments and have been extensively investigated as drug delivery vehicles. Due to their structure, chemical composition and colloidal size, all of which can be well controlled by preparation methods, the liposomes show several properties that may be useful in various applications. The most important properties are of colloidal size, better said distributions of uniform particle size in the range of 20 nm to 10 μm, and special surface and membrane characteristics. Liposomes are used as vehicles for drugs and antigens because they can serve several different purposes (Store &Crommelin, Pharmaceutical Science &Technology Today, 1, 19-31, 1998). Encapsulated liposomal drugs are inaccessible to metabolizing enzymes. Conversely, body components (such as erythrocytes or tissues at the injection site) are not directly exposed to the total dose of the drug. The duration of action of the drug can be prolonged by liposomes due to a slower release of the drug in the body. Liposomes that have a potential for direction, that is, objective options change the distribution of the drug over the body. The cells use the mechanism of endocytosis or phagocytosis to take the liposomes in the cytosol. In addition, liposomes can protect a drug against degradation (ie, metabolic degradation). Although they are sometimes successful, liposomes have limitations. Liposomes not only supply the drug to damaged tissue, but also rapidly enter the liver, spleen, kidneys and reticuloendothelial systems, and spill drugs during circulation (Harris &Chess, Nature, March 2003, 2, 214-221). Pegylation is an alternative method to overcome these deficiencies. First, pegylation maintains drug levels within the therapeutic window for longer periods of time and provides the drug as a long circulation portion that gradually degrades into smaller, more active, and / or easier-to-clarify fragments. Second, it allows microparticles or large macromolecules containing long-circulating drug to accumulate slowly in pathological sites with affected vasculature or receptor expression and improves or reinforces the drug supply in those areas. Third, it can help achieve a better objective effect for those target drugs and drug vehicles that are supposed to reach pathological areas with decreased blood flow or with a low concentration of a target antigen. Pegylation benefits typically result in increased stability (temperature, pH, solvent, etc.), significantly reduced immunogenicity and antigenicity, resistance to proteases, maintenance of catalytic activity, and improvements in solubility, among others. characteristics, and an increased liquid stability of the product and aggregation induced by reduced agitation. Liposome membranes containing bilayer compatible species such as lipids linked by poly (ethylene glycol) (PEG-lipids) or gangllosldoses are being used to prepare stealth liposomes (Papahadjopoulos et al., PNAS, 88, 1460-4, 1991). ). Stealth liposomes have a relatively long half-life in blood circulation and exhibit altered biodistribution in vivo. Vaage I went to. (Int. J. of Cancer 51, 942-8, 1992) prepared doxorubicin stealth liposomes and used them to treat well established and newly implanted primary mouse growth carcinomas, and to inhibit the development of spontaneous metastases from intra-mammary tumor implants. They concluded that the long circulation time of the secretion liposomes of doxorubicin formulation accounts for its superior therapeutic effectiveness. The presence of MPEG-derived (pegylated) lipids in the bilayer membrane of sterically stabilized liposomes effectively provides a spherical barrier against interactions with plasma proteins and cell surface receptors that are responsible for the rapid intravascular rupture / destabilization and RES elimination observed after of the iv administration of conventional liposomes. As a result, the pegylated liposomes have a prolonged circulation half-life, and the pharmacokinetics of any encapsulated agent are altered to conform to those of the liposomal vehicle instead of those of the trapped drug (Stewart et al., J. Clin. Oncol. , 683-691, 1998). Because the mechanism of localization of pegylated liposome tumor is by means of extravasation through blood vessels punctured in the tumor (Northfelt et al., J. Clin. Oncol. 16, 2445-2451, 1998; Muggia eí al. , J. Clin. Oncol. 15, 987-993, 1997), it is likely that prolonged circulation favors accumulation in the tumor by increasing the total number of steps made by the pegylated liposomes through the tumor vasculature. Photodynamic therapy (PDT) is one of the most promising new techniques that is explored for use in a variety of medical applications and is known as a well-recognized treatment for tumor destruction ("Pharmaceutical development and medical applications of porfirin-type macrocycles" ", TD Mody, J. Porfirins Phtalocyanines, 4, 362-367 2000). Another important application of PDT is the treatment of infectious diseases due to pathogenic microorganisms including dermal, dental, suppurative, respiratory, gastroenteric, genital and other infections. A constant problem in the treatment of infectious disease is the lack of specificity of the agents used for the treatment of disease, which results in the patient generating a new group of malignancies of the therapy. The use of PDT for the treatment of various types of disease is limited due to the inherent characteristics of photosensitizers. These include its high cost, extended retention in the host organism, substantial skin photo toxicity, previous toxicity, low solubility in physiological solutions (which reduces its utility for intravascular administration as it may cause thromboembolic accidents) and low target effectiveness. These disadvantages lead to the administration of extremely high doses of a photosensitizer, which dramatically increase the possibility of accumulation of the photosensitizer in undamaged tissues and the accompanying risk of affecting undamaged sites. One of the prospective methods to increase the specificity of photosensitizers and the effectiveness of PDT is a conjugation of a photosensitizer with a ligand-vector, which binds specifically to receptors on the surface of a target cell. A number of natural and synthetic molecules recognized by the target cells can be used as such vectors. This procedure is now used in the design of new generations of photosensitizers for the treatment of tumors ("photosensitizers based on Porfirin for use in photodynamic therapy" ED Sternberg, D. Dolphin, C. Brueckner, Tetrahedron, 54, 4151 -4202 1 998 ). Another method to increase tumor selectivity by targeting photosensitizers to tumor cells is to use liposomes, for example, liposomes conjugated with transferrin (Derycke &De Witte, Int. J. Oncology 20, 1 81-187, 2002) . Because unconjugated liposomes are often easily recognized and eliminated by the reticuloendothelial system, PEG-yellated liposomes (Woodle &Basic, Sterically stabilized liposomes, Biochim Biophys Acta 11 13, 171-1 99, 1992; al., Enhanced anticancer therapy mediated by specialized liposomes, J Pharmacol 49, 972-975, 1 997). Since the application of photodynamic therapy in the treatment of cancer and other diseases is growing rapidly, there is also a greater demand for new photosensitizer formulations. These new formulations of photosensitizers need to be more stable, easy to process and handle. In addition, the non-polar photosensitizers, especially more hydrophobic, should be able to target the tissue in a selective and efficient manner.

OBJECTIVES AND BRIEF DESCRIPTION OF THE INVENTION It is an object of the present invention to provide an improved photosensitizer formulation for use in photodynamic therapy (PDT). Another object of the present invention is to incorporate non-polar photosensitizers in a liposomal membrane thereby allowing polar and non-polar substances to be transported using the same vehicle. A further object of the present invention is to preserve the structure and size of liposomal constructs with incorporated non-polar photosensitizer during lyophilization processes by adding monosaccharides or polyalcohols as cryoprotectants. Still another object of the present invention is to provide a photosensitizer formulation with improved pharmacokinetic properties. Still another object of the present invention is to improve the transport of non-polar photosensitizers through the cell membrane and thereby increase the efficiency of PDT. The present invention includes a pharmaceutical liposomal formulation for photodynamic therapy comprising a non-polar porphyrin photosensitizer and one or more phospholipids, which are storage stable without requiring lyophilization. The liposomal formulation provides therapeutically effective amounts of the photosensitizer for intravenous administration. Phospholipids can be modified by pegylation, that is, they contain polyethylene glycol as an integral part of the phospholipids. The formed liposomes contain the non-polar photosensitizer within the membrane and are useful for the combined purpose of non-polar photosensitizer and a second polar substance. When a formulation includes the presence of monosaccharides or polyalcohols, it can be lyophilized efficiently while retaining the size of the liposomal vehicles and the content of a therapeutically effective amount of the photosensitizing agent. The invention also relates to the liposome composition formed on reconstitution with an aqueous vehicle. The lyophilized formulation on reconstitution with a suitable aqueous vehicle forms liposomes which are also useful for intravenous administration. The foregoing, and other objects, features and advantages of the present invention will become apparent from the following description read together with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a liposomal formulated mTHPC gel filtration curve. Both liquid components and mTHPC show the same distribution over all fractions collected. Figure 2 shows the measurement of light-induced fluorescence (LIF) of Coló 26 tumors with intact skin, nu / nu Swiss mice 0. 5 mg / kg, with intravenous injection of mTHPC, product A and B giving tumor accumulation as a post-injection time function.

Figure 3 shows the effect PDT 6 hr after intravenous injection of product A, B and mTHPC. Mice were injected intraperitoneally (i.p.) (0.25 ml) with Evans blue (1%) immediately after PDT treatment and sacrificed after 24 h.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES A pharmaceutical liposomal formulation for photodynamic therapy comprising a non-polar porphyrin photosensitizer and one or more phospholipids, which are storage stable without requiring lyophilization, is described below. The liposomal formulation provides therapeutically effective amounts of the photosensitizer for intravenous administration. Phospholipids can be modified by pegylation, that is, they contain polyethylene glycol as an integral part of the phospholipids. The formed liposomes contain the non-polar photosensitizer within the membrane and are useful for the combined purpose of a photosensitizer and a second polar substance. The formulation can be preserved by a lyophilization procedure using a monosaccharide or polyalcohol, but does not need to have a reasonably stable half-life. The process cycle retains the size of the liposomal vehicle as well as the membrane content of the photosensitizing compound. Non-polar photosensitizers in this invention include the known porphyrin-based compounds. In particular photosensitizers, which are advantageously employed in the practice of this invention, they are based on the porphyrin macrocycle photosensitizers, which include deuteroporphyrin, etioporphyrin, protoporphyrin, hematoporphyrin, pheoforbide and derivatives thereof, especially di- and tetra-hydroporphyrins. , which have maximum absorption of light in the range of 640-780 nanometers. The phospholipids may or may not be modified by pegylation (containing polyethylene glycol as an integral part). The formed liposomes contain the non-polar photosensitizer within the membrane and are useful for the combined purpose of a non-polar photosensitizer and a second polar substance. Formulations of photosensitizers are useful for targeting the non-polar photosensitizer molecule to unwanted cells or tissues or other undesirable materials and, after irradiation with a suitable light source, damaging the target. The photosensitizer formulations are also useful for monitoring unwanted cells or tissues or other undesirable materials by using fluorescence imaging methods without or with limited photochemical activation of the photosensitizer. Especially, the liposomal formulation of the invention is useful for transporting non-polar photosensitizers. The non-polar substances are integrated into the membrane of the vehicles, thereby creating a structure that opens more easily, releasing the photosensitizer for action directly towards the cell membrane. This mechanism sups the photosensitizer directly to the cell membrane system, a preferred place of action. In this way, the photosensitizer that is effectively activated by illumination with a suitable external light source, can irreversibly damage unwanted cells, tissues or other structures. Conventionally constructed liposomal formulations are used to transport various compounds trapped in the luminal part of the vehicle. The invention focuses on the combination of two different transport compartments within a liposome allowing a combined transport of polar and non-polar substances. In this aspect, by residing the photosensitizer within the membrane, the photosensitizing agents are efficiently targeted for their site of action but the luminal part within the liposome particle remains free for the inclusion of other substances, including drugs that can have a beneficial effect on therapy. In addition, the combination of monosaccharides such as glucose and the photosensitizer bound to the phospholipids is a stable tool for preserving the size of the liposomes during lyophilization and rehydration using a physiological common carbohydrate in place of dsaccharides. This versatile concept allows the addition of substances, which will have a beneficial effect on therapy. The combination of two or more substances together with a membrane-bound photosensitizer within a vehicle will allow one to directly or indirectly influence the oxygen content of the target cells thereby influencing the efficacy of PhotoDynamic therapy. For example, such effects can be achieved by adding inhibitors of enzyme activities as inhibitors of mammalian thioredoxin reductase (TrxR). In this way, only effective after reaching the cytoplasm of the target cells, the inhibitor will block the path of Trx / TrxR that normally acts as an antioxidant.

Example 1 Preparation of Liposomes Containing m-THPC mTHPC (Temoporfin) was synthesized as described in Patents No. 4, 992,257 and 5, 162,519, incorporated herein by reference. The liposomes were prepared according to the following general procedure: The non-polar photosensitizer, ascorbic palmitate and the phospholipids are dissolved in chloroform / methanol. The solution is then dried under vacuum using a rotary evaporator until the chloroform / methanol mixture is no longer detectable by gas chromatography. The water for injection is added to rehydrate the lipid film at a temperature of 50 ° C for at least 2 hours. The mixture is thus passed through a homogenizing filter system using a final pore size of 100 nanometer. Optionally, the rehydration water is complemented with monosaccharides or polyalcohols. The filtrate is collected, filled into bottles and optional lyophilized. The lyophilized composition is reconstituted with water for injection before administration. Using the foregoing procedure, several different preparations of the liposomal formulation were prepared as follows: Example 1a Ingredient Quantity% w / v mTHPC 0.05 to 0.15 Dipalmitoyl Phosphatidyl Choline 0.5 to 2.0 Dipalmitoii Phosphatidyl Glycerol 0.05 to 0.2 Distearoyl Phosphatidyl Ethanolamine Pegylated 0.05 to 0.2 Ascorbic Palmitate 0.002 to 0.004 Water For Injection as required to achieve the desired concentrations above.

Example 1b Ingredient Quantity% w / v mTHPC 0.05 to 0.15 Dipalmitoyl Phosphatidyl Choline 0.5 to 2.0 Dipalmitoyl Phosphatidyl Glycerol 0.05 to 0.2 Ascorbic Palmitate 0.002 to 0.004 Water For Injection as required to achieve the desired concentrations above.

Example 1c Ingredient Quantity% w / v mTHPC 0.05 to 0.15 Dipalmitoyl Phosphatidyl Choline 0.5 to 2.0 Dipalmitoyl Phosphatidyl Glycerol 0.05 to 0.2 Glucose 2.0 to 12.0 Ascorbic Palmitate 0.002 to 0.004 Water For Injection as required to achieve the desired concentrations above. It was found that all worked well in their use according to the present invention.

Example 2 Chemical and Physical Stability of Liposomal m-THPC The physical stability of the liposomal formulations was measured by monitoring the particle size distribution by photon correlation spectroscopy.

Stability of liposomal mTHPC Storage Conditions Distribution of Average Particle Size (nm) Initial 166 23 ° C - 1 Month 177 23 ° C - 4 Months 167 Example 3 Location of mTHPC within the liposomal bilayer of the formulation The gel filtration of the liposomal formulation was performed on Sephadex G50 columns. As shown in Figure 1, the lipids and mTHPC show the same distribution over all the fractions indicating a physical interaction in both components, ie integration of mTHPC in the membrane bilayer. The distribution does not change markedly after destroying the roof structure by ultrasonication, as shown in Tables 1 and 2.

Example 4 Pharmacokinetic properties in Coló 26 mice, a non-metastasizing mouse colorectal tumor mouse strain, syngeneic to Balb / C mice, was used. Cells were maintained as a monolayer culture in the middle of the Roswell Memorial Park Institute (RPMI-1640) supplemented with 10% heat-inactivated fetal goat serum, 1% streptomycin-penicillin and 200 mM L-glutamine a 37 ° in 95% air and 5% CO2. The athymic female mice of six weeks (Swiss, nu / nu) were inoculated subcutaneously in the right hind paw with 2 x 10 6 of Colo26 cells. Two weeks later, as the tumor reached a diameter of 5-7 mm, the formulations of m-THPC (0.5 mg / kg) were injected intravenously. Non-invasive LIF measurements have been performed at three different sites: tumor with underlying skin, symmetric normal tissues with underlying skin on the left hind paw, and elevated skin at different times after administration of the drug. At the selected time points the mice were sacrificed and fluorescence signals from normal and tumor tissues were measured in direct contact. Figure 2 shows the accumulation of mTHPC, product A and B in the tumor as a function of post-injection time measured in four mice. The non-invasive measurements showed the tendency to the best accumulation of mTHPC in the tumor compared to normal tissues with a better ratio (1.3) to 24h. Invasive measurements made in direct contact with the tumor and the muscle at 24 hrs. and 48 hrs. of post-injection gave the proportions of 2.7 and 3.0 respectively. The measurements for product A have been made in a mouse at 0.5 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h post-dose. Product A has shown similar pharmacokinetic behavior as mTHPC. Compared to mTHPC the highest fluorescence intensity in the tumor tissue was reached already after 4 hours after the injection. The measurements for Product B were made in a mouse at 0.5 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h and 72 post-dose.

The highest fluorescence intensity in the tumor was measured 8 hours after the i.v. injection. Compared to mTHPC, product B is showing completely different pharmacokinetic properties. The concentration of Product B was increased in the tumor much faster (16 h) than mTHPC.

Example 5 Antitumor Activity of Liposomal m-THPC Col6 26, a non-metastasizing mouse colorectal tumor mouse strain, syngeneic to Balb / C mice, was used. Cells were maintained as a monolayer culture in the middle of the Roswell Memorial Park Institute (RPMI-1640) supplemented with 10% heat-inactivated fetal goat serum, 1% streptomycin-penicillin and 200 mM L-glutamine a 37 ° in 95% air and 5% CO2. The athymic female mice of six weeks (Swiss, nu / nu) were inoculated subcutaneously in the right hind paw with 2 x 10 6 of Colo26 cells. Two to three weeks later, as the tumor reached a diameter of 10-13 mm, the formulations of m-THPC (0.5 mg / kg) were injected intravenously. Unless indicated otherwise, 3 mice per group per product were used per each post-PDT. to. Photodynamic Treatment The drug-light (DLI) intervals of 0.5 h, 4 h, 6 h, 72 h were used for products such as mTHPC, product A (liposomes and m-THPC) and product B (PEG-liposomes liposomes) and m- THPC). Each animal was anesthetized by injection i.m. of ketamine (12.5 mg / ml), then photoradiated at 652 nm with 10 J / cm2 at 100 mW / cm2 using a light-emitting diode laser. b. Evaluation of PDT effect To evaluate necrosis of tumor tissue after PDT treatment, the Evans Blue dye method was used. The mice were injected i.p. (0.25 ml) with Evans blue (1%) immediately after PDT treatment. Twenty-four hours later the mice were sacrificed with an overdose of halothane, the tumors were excised and cut longitudinally. As exemplified in Figure 3, photos of whole tumors and pieces of tumor had been taken. Unstained necrotic areas were considered tumor damage: i. Drug-light interval (DLI) = 0.5 h, necrosis was not observed irrespective of the products used. ii. Drug-light interval (DLI) = 4 h. Three mice treated with mTHP-PDT demonstrated partial tumor necrosis. Two mice treated with product A gave partial tumor necrosis, while the third mouse tumor remained intact. Three mice treated with the B-PDT product demonstrated tumor necrosis: in two mice the distinct tumor necrosis was observed, while in one mouse the tumor necrosis was partial. iii. Drug-light interval (DLI) = 6 h; six mice were used for product A. Different necrosis was noted in the tumors of two mice treated with product A and four others remained intact. For product B, a different tumor necrosis was observed in all mice. Two tumors treated with mTHPC-PDT showed different necrosis, one tumor was free of necrosis. The mTHPC-PDT tumors that showed necrosis were confirmed by ocular observations. Having described the preferred embodiments of the invention with reference to the accompanying drawings, it will be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be made herein by those skilled in the art without departing from the scope and spirit of the invention as defined in the appended claims.

Claims (16)

1. CLAIMS 1. A pharmaceutical liposomal composition for photodynamic therapy comprising a liposomal bilayer which consists substantially of phospholipids, and a therapeutically effective amount of a non-polar photosensitizer. The liposomal formulation according to claim 1, wherein said phospholipids are selected from the group consisting of dipalmitoyl phosphatidyl choline, dipalmitoyl phosphatidyl glycerol, phospholipids linked by poly (ethylene glycol) and combinations of these three materials. 3. The liposomal formulation according to claim 1, wherein said photosensitizer is a porphyrin macrocycle photosensitizer. 4. The liposomal formulation according to claim 1, wherein said porphyry macrocilin photosensitizer is selected from the group consisting of deuteroporphyrin, etioporphyrin, protoporphyrin, hematoporphyrin, pheoforbide and its di- and tetrahydroporphyrin derivatives. The liposomal formulation according to claim 1, which has been lyophilized, further comprising one or more monosaccharides or polyalcohols, and wherein the lyophilized formulation, in the addition of a suitable aqueous vehicle, forms liposomes containing a therapeutically acceptable amount of the non-polar photosensitizer within the liposomal bilayer. 6. The liposomal formulation according to claim 5, wherein said monosaccharide is selected from the group consisting of glucose and fructose. 7. The liposomal formulation according to claim 5, wherein said polyalcohol is selected from the group consisting of inositol and mannitol. The liposomal formulation according to claim 5, wherein the concentration ratio of monosaccharide to phospholipid is between 1: 2 and 1: 12. 9. The liposomal formulation according to claim 5, wherein the concentration ratio of polyol to phospholipid is between 1: 2 and 1: 12. 10. The liposomal formulation according to claim 5, reconstituted with an aqueous fluid for pharmaceutical administration. eleven . The liposomal formulation according to claim 1, wherein the therapeutically effective concentration of the photosensitizer is from 0.0001 to 0.15 percent w / v. 12. The liposomal formulation according to claim 5, wherein the therapeutically effective concentration of the photosensitizer is from 0.0001 to 0.1 5 percent w / v. The liposomal formulation according to claim 1 further comprising a component selected from the group consisting of butylated hydroxytoluene, ascorbic palmitate, and combinations of these two. The liposomal formulation according to claim 5, further comprising a component selected from the group consisting of butylated hydroxytoluene, ascorbic palmitate, and combinations of these two. 15. The liposomal formulation according to claim 1, wherein the formulation further comprises at least one additional pharmaceutically active substance, especially polar, suitable for having some beneficial effect in a preselected therapy. 16. The liposomal formulation according to claim 5, wherein the formulation further comprises at least one additional pharmaceutically active substance, especially polar, suitable for having some beneficial effect in a preselected therapy.