SUBSTANCE: photosensitising agents obtained by reducing a double bond in porphyrinic macrocycle of sulphonated mesotetraphenylporphyrine, preferably disulphonated mesotetraphenylporphyrine, such as TPPS2a. Resulting sulphonated mesotetraphenylchlorines are compounds of formula (I) , (where X stands for -SO3Н; each of n, p, q and r independently stands for 0 or 1; and sum of n, p, q and r is an integer from 1 to 4, preferably at least 2, in particular, 2 or 4), isomers or isomeric mixture. Compounds in accordance with the said invention and pharmaceutically suitable salts thereof have a high extinction coefficient in the region of 630 to 680 nm.
EFFECT: compounds are widely used as photosensitising agents for photochemical internalisation of molecules and photodynamic therapy.
25 cl, 8 ex, 8 dwg
The present invention relates to new photosensitizing agents and their use in photochemical internalization of molecules and in photodynamic therapy.
Of the techniques known to a large number of photosensitizing agents. When exposed to light, they can become toxic or can release toxic substances such as singlet oxygen and other radical oxidants that can damage cellular substance or biomolecules, including cell membranes and cellular structures, and similar damage to the cells or membranes may ultimately kill the cell. Specified cytotoxic effect used in the treatment of various abnormalities or disorders, including diseases related to tumors. Such treatment known as photodynamic therapy (PDT) and includes the introduction of photosensitizing (fotohimioterapiei) agents in the affected area of the body, followed by irradiation with activating light in order to activate the photosensitizing agents and translate them into a cytotoxic form, when exposed to cells die or their ability to reproduce is reduced.
Recently, photodynamic effect was proposed as a means for introducing molecules that do not otherwise have the ability to p is to unicati through the membrane, in the cytosol of the cell so that it does not necessarily lead to the destruction or death of the cell. In said method, known as "photochemical internalization, or PHI, the molecule that must be internalized or deliver, is injected into the cell in combination with a photosensitizing agent. Effects on cell light with a suitable wavelength activates the photosensitizing compound, which, in turn, causes the destruction of intracellular cell membrane and the subsequent release of molecules into the cytosol.
Photosensitizing agents may exert its effect through different mechanisms, either directly or indirectly. For example, some photosensitizers can be activated by light directly to become toxic, while others generate toxic particles, for example, oxidizing agents such as singlet oxygen or formed under the action of oxygen free radicals, which have an extremely strong destructive effect against cellular matter and biomolecules such as lipids, proteins and nucleic acids.
Known photosensitizing agents include, for example, psoralens, porphyrins, chlorins and phthalocyanines. Porphyrin photosensitizers have a direct impact by generating toxicus is their oxygen particles, and they are considered as the most suitable candidates for use in PDT. Porphyrins are found in nature as precursors in the synthesis of heme. In particular, heme is formed when iron (Fe2+) is included in fetoprotein IX (PpIX) under the action of the enzyme ferrochelatase. PpIX is a very powerful photosensitizer, whereas heme does not show photosensitizing properties.
A large number of photosensitizers based on porphyrin or porphyrin compounds known from the art and described in the literature. Examples of such agents include Photofrin®, which was recently permitted as a photosensitizer in the treatment of some types of cancer. However, because Photofrin®must be administered parenterally (e.g. intravenously), this causes the disadvantage consists in the fact that this method leads to prolonged photosensitivity of the skin, which may persist for several weeks. Because Photofrin®consists of large porphyrin oligomers, he also poorly penetrates the skin when it is applied topically. Similar problems arise with other photosensitizers based on porphyrin, such as the so-called "hematoporphyrin derivatives" (HpD), which is also described for use with the cancer chemotherapy.
Recently for use as photosensitizing agents have been investigated sulfonated mesotetraphenylporphyrin (TPPSns), such as desulfuromonas letterfinlay TPPS2aand TPPS2oand tetrachlorophenyl letterfinlay TPPS4and it has been shown that they have a number of important advantages in comparison with HpD and Photofrin®. In particular, they have a high value of the ratio of the tumor : normal tissue, and therefore are of interest for use in photochemotherapy (Peng et al., Cancer Lett. 36: 1-10, 1987; Eversen et al., Photodynamic therapy of tumors and other diseases (Ed. G.Jori and C.Perria), p.215-219, Libreria Prongetto Publ., Padova; and Winkelman, Cancer Res. 22: 589-596, 1962). Recently it was also discovered that TPPS2asuitable for use as a photosensitizer for photochemical internalization of macromolecules (Berg et al., Cancer Res. 59: 1180-1183, 1999: Högset et al., Hum. Gene Ther. 11: 869-880, 2000: and Selbo et al., Int. J. Cancer 87: 853-859, 2000).
However, the main disadvantage of using TPPS2afor clinical purposes is the low absorption of red light. In fact, a major limitation in the clinical use of all known porphyrin derivatives is that the larger the wavelength of light to which they are sensitive, has a relatively low absorption in the range of about 620-630 nm. When the wavelength of the light is able to penetrate into the living tissue only for a short distance, and therefore cannot reach the superficial cells or tissues, such as cancerous cells. It is therefore desirable to find an alternative tetrapyrrole compounds, which still would have preferred properties of known photosensitizers based on the porphyrin, but, in addition, would have a higher absorption of light with a longer wavelength, which is better penetrates the tissues.
The aim of the present invention is solving the above problem and, in particular, the aim of the invention are agents that have a high photosensitizing effects compared with those derived porphyrin, which are known from the art.
It was found that the recovery of one of the double bonds in the porphyrin macrocycle of sulfated mesotetraphenylporphyrin (for example, desulfuromonas of mesotetraphenylporphyrin) leads to compounds that unexpectedly exhibit photosensitizing properties. In particular, such compounds have compared to the corresponding porphyrins improved spectral characteristics and have been shown to have unexpectedly high extinction coefficient at exhibiting a red light, for example, in the range from 630 to 680 nm. Thus, these compounds are considered as the most appropriate to use the Oia is not only the usual methods of photodynamic therapy, but also in the methods of photochemical internalization of macromolecules.
One aspect of the present invention is a photosensitizing agent, which is a sulfonated letterfinlay, for example desulfuromonas letterfinlay, or its pharmaceutically acceptable salt. In such compounds, at least one of the four phenyl rings typically contains 1, 2 or 3, preferably 1, alphagroup. Preferred compounds in accordance with the invention are such compounds in which two phenyl rings contain one sulfogrupp in each of the rings.
Another aspect of the present invention is a photosensitizing agent, which can be obtained by reduction of one double bond in the porphyrin macrocycle of sulfated mesotetraphenylporphyrin, in particular agent, which can be obtained by reduction of the double bond in the porphyrin macrocycle desulfuromonas of mesotetraphenylporphyrin, such as TPPS2a. Pharmaceutically acceptable salts of such compounds are another aspect of the present invention.
Examples of photosensitizing agents according to the invention include compounds of formula I:
(where X means-SO3H;
n, p, q and r each independently represents 0 and the 1, the amount of n, p, q and r is an integer from 1 to 4, preferably at least 2, for example 2 or 4) and their pharmaceutically acceptable salts.
Isomeric forms of the compounds of the formula I, for example, such compounds in which a restored double bond is located in any of the last three pyrrole rings, the authors also considered as part of the present invention. Any mixture of isomers, comprising at least one compound of formula I or its isomer, is considered as a part of the present invention. Examples of isomers of compounds of formula I, which are most suitable for use in the invention include compounds of formulas Ia-Ic:
(where the values of X, n, p, q and r above).
For compounds of formulas I, Ia, Ib and Ic, in which any of the n, p, q and r is equal to 1, it indicates the presence of only sulfopropyl that is attached to the phenyl group at any position of the ring (for example, ortho-, meta - or para-). In those cases, when there is more than one sulfopropyl, these sulfopropyl can be in the same or in different positions of each phenyl ring. Preferably these groups are in the same positions of the ring, more preferably in meta - or para-position. If any of the n, p, q or r is 0, t is this means the absence of any substituents in the ring; an example is unsubstituted phenyl.
Especially preferred compounds of formulas I, Ia, Ib or Ic are such compounds in which the sum of n, p, q and r is equal to 2. Most preferred are such compounds in which the substituted phenyl rings are located adjacent to each other, in particular adjacent to the reduced pyrrole ring, for example, such compounds of the formula I in which n=0, p=0, q=1 and r=1. Alternative preferred compounds of formulas I, Ia, Ib or Ic include compounds in which the sum of n, p, q and r is equal to 2, and the substituted phenyl rings are located opposite each other, for example the compounds of formula I in which n=0, p=1, q=0 and r=1.
Independently, in each phenyl ring alphagraph X can be in ortho-, meta - or para-position. Preferably it is in the meta - or para-position, most preferably in the para-position. Preferred compounds of the present invention include compounds of formula II:
Isomeric forms of the compounds of formula II, such that restored the double bond is located in one of the three remaining pyrrole rings, are also part of the invention. Part of the present invention is any mixture of isomers, comprising at least one compound of the formula II and its isomers.
Especially preferred compound of formula II is a compound in which the considered two substituted phenyl ring adjacent to the restored double bonds.
Compounds of the present invention can be obtained using standard methods well known from the art. Most conveniently these connections to obtain recovery of the corresponding porphyrin.
Thus, another aspect of the present invention is a method for producing compounds according to the invention, with the specified method includes at least one of the following stages:
(a) restoration of sulfonated tetraphenylporphyrin or chelate complex with iron, such as the restoration of tetraphenylporphyrin formula III:
or chelate complex with iron (where the values of X, n, p, q and r are indicated previously);
(b) separation, if necessary, the mixture of compounds obtained in stage (a), using well-known methods of separation; and
(c) conversion of the compound obtained in stage (a) or stage (b), for example the compounds of formula I, its pharmaceutically acceptable salt.
Derived porphyrin, which is used as the source of the product at the stage of (a), available on the market or can be derived from SIP is utilized methods known from the art. Sulfonated porphyrins, including TPPS2a(desulfuromonas tetraphenylporphin), for example, can be obtained from Porphyrin Products (Logan, Utah, USA).
Chemical transformation derived porphyrin of formula III to the corresponding chlorin at the stage (a) can chemically be done a few different ways, for example using p-toluensulfonate as a predecessor of diimide when recovering the free base porphyrin (see, e.g., Whitlock et al., J. Am. Chem. Soc. 91: 7485-7489, 1969). Otherwise obtaining the required chlorine can be accomplished by restoring the appropriate gelatoria, for example, sodium in boiling isoamyl alcohol (see Eisner, J. Chem. Soc. 3461-3469, 1957; Eisner et al., J. Chem. Soc. 733-739, 1957).
Most preferred for producing compounds of the present invention is a photochemical restoration of porphyrins, optionally in the presence of a tertiary amine base. For example, the free base porphyrin can photochemically to recover the chlorine in the presence of amines, in particular tertiary amine (see, for example, Harel et al., Photochem. Photobiol. 23: 337-341, 1976).
The restoration of the porphyrin molecules in the presence of diamide due to the recovery of two double bonds in the porphyrin macrocycle can result in bacteriochlorin. Why is the way of photovoltaikanlage using amine, in particular mainly tertiary amine, is preferred for use in obtaining the desired derivative of chlorin of the present invention. Examples of suitable tertiary amines for use in the specified way include triethylamine (tea), N-organic, tri-n-butylamine, trioctylamine etc., the recovery is usually carried out in the presence of visible light (i.e. spend fotovosstanovlenie).
Photochemical restoration of the porphyrin can be performed in a solvent or mixture of solvents, such as benzene, pyridine, dimethyl sulfoxide and tppi a temperature in the range from 10 to 30°C, preferably at room temperature (for example, from 20 to 25°With, in particular, on 22°).
Fotovosstanovlenie can be carried out using light in the visible range, for example, light with a wavelength in the range from 400 to 640 nm, preferably from 500 to 640 nm, for example, approximately 545±15 nm. The irradiation is usually carried out at a rate of 1 to 50 W/m2for example, approximately 15 W/m2during the time from 1 to 90 minutes, for example from 5 to 40 minutes the Formation of any unwanted bacteriochlorin can be reduced by reducing the time during which the porphyrin macrocycle is exposed to light. Methods of irradiation of porphyrins, for example, using La is p or lasers are well known from the technical field. Especially suitable for this purpose xenon lamp high pressure or odnovolova lamp, such as lamp, which delivers the company Philips. The reaction is usually carried out under anaerobic conditions, for example, a mixture of the original product and solvent can be saturate with nitrogen before exposed to light, and by choice during irradiation with light.
The compound of the present invention may be in the form of mixtures of isomers and can be used in this form. If necessary, the compounds of the present invention, for example the compounds of formula I, can be divided into their isomers due to the difference of their physical/chemical properties using methods well known in the art, for example by chromatography and/or fractional crystallization.
As indicated above, the compounds of the present invention may take the form of pharmaceutically acceptable salts. These salts are basically acid additive salts with physiologically acceptable organic or inorganic acids. Suitable acids are, for example, hydrochloric, Hydrobromic, sulfuric, phosphoric, acetic, lactic, citric, tartaric, succinic, maleic, fumaric and ascorbic acid. Hydrophobic salts are convenient to obtain, for example, deposition. Suitable salts include, for example, acetate, brough the ID, chloride, citrate, hydrochloride, maleate, mesilate, nitrate, phosphate, sulfate, tartrate, oleate, stearate, tosylate, as well as salts of calcium, meglumine, potassium and sodium. Methods of obtaining salts are well known from the technical field.
As indicated previously, the compounds of the present invention and their salts have valuable pharmacological properties, namely: exhibit photosensitizing properties that make them useful for photochemical internalization of macromolecules and as fotohimioterapiei agents.
Thus, another aspect of the present invention is a pharmaceutical composition comprising the above photosensitizing agent, in particular a compound of formula I or its pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier or excipient.
Another aspect of the invention is the above photosensitizing agent, in particular a compound of formula I or its pharmaceutically acceptable salt, for use as a medicine, for example as a photosensitizing agent used in the method of photochemical internalization, photochemotherapy or diagnosis.
Another aspect of the present invention is the use of the above photosensitizing agent, in particular, of the value of the formula I or its pharmaceutically acceptable salt, to obtain a therapeutic agent that is used in the method of photochemical internalization, photochemotherapy or diagnosis, for example in the treatment of disorders or abnormalities of external or internal surfaces of the body, sensitive to fotohimioterapiei impact.
The term "internalization"as used in the text of this application, refers to the delivery of molecules into the cytosol and involves the step of release of the molecules from the intracellular or associated with the cell membranes into the cytosol of cells. The term "cell"used in the text of this application, includes all eukaryotic cells (including cells of insects and fungal cells). Thus, representatives of the "cells" include all types of mammalian cells and memleketim, plant cells, insect cells, cells, fungi and protozoa.
Methods of introduction of molecules into the cytosol of living cells useful for the management and study of biological processes. Of particular interest are methods in which cells after internalization remain viable and/or funktsionalnostej. The use of photosensitizing agents for introduction into the cytosol of cells, molecules, which are otherwise unable to penetrate the membrane, so that it may not necessarily lead to the destruction or death of a significant number of cells, was proposed which, for example, in applications WO 96/07432 or WO 00/54802. In said method, the molecule that you want to internalizing, and a photosensitizing agent is injected into the cell simultaneously or sequentially, after which the photosensitizing agent and the molecule is subjected to endocytosis or otherwise carry out translocation of cytoplasmic bullock, complementary mechanism or other restricted intracellular membranes of the cell. The molecule that must be subjected to translocation in the cell inside the cell, and a photosensitizing agent is injected into the cell together or sequentially, and they are accumulated by the cell in intracellular compartments. The molecule that you want to internalizing in the cell, is released under the action of a cell light with a suitable wavelength that activates the photosensitizing agent, which, in turn, leads to the destruction of intracellular membranes of cells and subsequent release of molecules inside cytosole. This method, in which the cell is exposed to light in order to release interest in the molecule of intracellular cell in which it is contained, the effect of the photosensitizing agent called "photochemical internalization" or PHI.
Thus, another aspect of the present invention is a method of introducing a molecule (i.e. deliver the molecules in the cytosol of cells as in vitro, and in vivo, with the specified method comprises contacting the specified cell with the above photosensitizing agent, in particular a compound of formula I or its pharmaceutically acceptable salt, contacting the specified cells with a molecule that must be entered, and the irradiation of the specified cell with light of a wavelength effective to activate the photosensitizing agent, such as light with a wavelength in the range of 300-800 nm.
The exact time of introduction of the molecules, which need to deliver (i.e. deliver molecules), and the photosensitizing agent and the time of exposure, in order to achieve the above effects should take into account various factors, including the type of the affected cells, the type of delivered molecules, surrounded by cells, and to consider whether the way in vitro or in vivo, is introduced directly into the target tissue or carry out the application on the peripheral area. If you take all these factors into account, the appropriate time can easily be determined by experts in the field of technology. Typically, the delivered molecule and a photosensitizing agent is injected into the cells before irradiation. For example, you can enter them both simultaneously and separately for the period from 1 to 72 hours prior to irradiation, preferably from 4 to 48 hours, in particular from 4 to hours before irradiation.
In some cases deliver the molecule is administered simultaneously with a photosensitizing agent. Thus, another aspect of the present invention is a pharmaceutical composition comprising the above photosensitizing agent together with the delivered molecule. Additionally may contain pharmaceutically acceptable carrier or excipient.
Another aspect of the present invention is a pharmaceutical composition comprising the above photosensitizing agent together with the delivered molecule for use in therapy, in particular in cancer therapy or gene therapy.
Another aspect of the present invention is the use of the above photosensitizing agent and/or deliver molecules for the preparation of drugs for use in therapy, in particular cancer therapy or gene therapy, in which the specified photosensitizing agent or specified to be delivered molecule in contact (either separately, either simultaneously or sequentially) with cells or tissues of the patient, and these cells or tissue is irradiated with light of a wavelength effective to activate the specified photosensitizing agent. Treatments, including these methods are another aspect of this image is the shadow.
Photosensitizing agent according to the invention can be thus used to transfer or transfection any molecule into the cytosol of living cells as in vitro (i.e. in culture) or in vivo. It can be used not only for delivery of molecules (or parts or fragments of molecules) into the cell, but in some cases, and for prezentowania or their expression on the cell surface. So, after transportation and release deliver molecules into the cytosol of cells, if the cells are specialized cells, such as cells, presenting antigen, molecule or its fragment can be transferred to the cell surface, where it can be presented outside the cell, i.e. the cell surface. This method is particularly useful for vaccination when the vaccine components, i.e. antigens and immunogenic can be entered in the cell for prezentowania on the surface of the cell in order to cause, facilitate or enhance the immune response. Further details regarding the usefulness of molecules capable of expression on the cell surface, described in the application WO 00/54802.
Deliver molecules that can be introduced into the cytosol of cells using photosensitizing agents of the present invention include molecules that barely penetrate the cell membrane. Also, the above-mentioned agents can facilitate delivery into the cytosol and increase the activity of molecules that are only partially able to penetrate the cell membrane or the membrane of intracellular vesicles. Deliver molecules can be organic compounds, proteins or protein fragments, such as, for example, peptides, antibodies or antigens or fragments thereof. Another class of delivered molecules that can be entered using the agents according to the present invention are cytotoxic drugs, such as protein toxins or cytotoxic compounds. Molecules, which may be of clinical interest for the treatment of cancer, but its application is limited due to low digestibility or due to the absence of absorption the cytosol, by using the above method can be introduced into the cytosol and targeted to specific cells. An example of such molecules is gelonin.
Depending on the type of delivered molecules of the above methods can be used to treat various disorders, such as rheumatoid arthritis, atherosclerosis and cardiovascular diseases, viral and other infections, psoriasis, solar keratosis, wounds, fractures, warts and inherited genetic disorders such as cystic fibrosis syndrome G is line and the Louis-Bar syndrome.
Another class of suitable deliver molecules are nucleic acids. Nucleic acids can be used in the form of genes encoding, for example, therapeutic proteins, desensitizing RNA molecules, ribozymes, DNA aptamers and triplex-forming oligonucleotides. Otherwise nucleic acids can be used in the form of non-coding molecules, such as, for example, synthetic antisense DNA or RNA, ribozymes, aptamers, triplex-forming oligonucleotides, peptide nucleic acids (PNAs), a transcription factor DNA-"traps" or chimeric oligonucleotides to correct specific mutations in a patient. Where possible, the nucleic acid molecules can be in the form of a complete gene or fragments of nucleic acids, optional built-in vector molecule, in particular the vector plasmid. The last form is particularly suitable in the case when the delivered molecule intend to use in a method of gene therapy, in which genes therapeutically delivered to cells of the patient. It can be used in the treatment of many diseases, such as cancer, cardiovascular disease, viral infection and monogenic disorders such as cystic fibrosis.
Optionally, the photosensitizing agent or delivered mo is Akula, you want to enter into the cell, separately or photosensitizing agent and deliver the molecule together may be attached, connected or associated with the molecules media, targeting molecules or vectors that facilitate or accelerate the uptake of the photosensitizing agent or delivered molecules or can target or deliver these items to a specific cell type, tissue or intracellular cell. Examples of systems-carriers include polylysine and other polycation, dextran sulfate, various cationogenic lipids, liposomes, reconstructed latisevicius lysis (LDL) particles or sterically stabilized liposomes. Such carriers usually can improve the pharmacokinetics and increase the uptake by cells of delivered molecules and/or photosensitizing agent, and may also direct the delivered molecule and/or the photosensitizing agent in the intracellular cell, which is particularly useful to achieve photochemical internalization, but usually fail to deliver targeted molecule and/or photosensitizing agent in a specific cell (in particular, cancer cells) or tissue. However, to achieve the specified specific or selective targeting carrier molecules delivered molecule and/or the photosensitizing agent can be linked, attached to or associated with specific target molecules, which contribute to the absorption of delivered molecules to specific cells in the desired cells or tissues. Such targeting molecules can also be delivered direct molecule in the intracellular cell, which is particularly useful for the implementation of photochemical internalization.
You can use a large number of target molecules, in particular it is possible to use molecules as described in Curiel, D.T. (1999), Ann. New York Acad. Sci. 886, 158-171; Bilbao, G. et al. (1998) Gene Therapy of Cancer (Walden et al., eds., Plenum Press, New York), Peng K.W. and Russell S.J. (1999), Curr. Opin. Biotechnol. 10, 454-457, Wickham TJ (2000), Gene Ther. 7, 110-114.
The molecules of the carrier and/or target molecules can be linked, connected or associated with the delivered molecule with a photosensitizing agent, or both together, can be used the same or different molecules media and target molecules. Such targeting molecules or carriers or can also be used to direct the delivered molecule in specific intracellular cell, which is particularly useful for the implementation of the, for example in a complementary mechanism or endosome.
As indicated earlier, the photosensitizing agent according to the present invention can be used in photodynamic therapy, in particular of fotoh the of treatment or diagnosis. These methods are well described in the patent and scientific literature, for example, in applications WO 96/28412 and WO 98/30242.
In the case where the photosensitizers used for PDT, anomalies and disorders that can be treated include any malignant, pre-malignant and non-malignant abnormalities or disorders, sensitive to the action of photochemotherapy, in particular tumor or other neoplasm, skin disorders such as psoriasis or senile keratosis and acne, abrasions on the skin, and other diseases or infections, in particular bacterial, viral or fungal infections, for example herpes virus infection.
Internal or external body surface, which can be treated using compounds of the present invention, include the skin and other epithelial and serosal surfaces, including, for example, the mucous membranes, the lining of the organs, in particular, respiratory, gastrointestinal and genitale-urinary tracts, and glands, the ducts of which can go on these surfaces (in particular, liver, hair follicles with sebaceous glands, mammary glands, salivary glands, and seminal vesicles). In addition to the skin, these surfaces include, for example, the lining of the vagina, endometrium and urothelia. These surfaces may also include a cavity formed in the body p is after excision sick or cancerous tissue, in particular, cavities in the brain after excision of tumors, such as gliomas.
Thus, examples of surfaces include: (i) the skin and conjunctiva; (ii) the lining of the mouth, pharynx, intestine, stomach, intestines and intestinal appendages, rectum and anal canal; (iii) the lining of the nasal passages, nasal sinuses, nasopharynx, trachea, bronchi and bronchioles; (iv) the lining of the ureters, bladder and urethra; (v) the lining of the vagina, cervix and uterus; (vi) the parietal and visceral pleura; (vii) the lining of the abdominal and pelvic cavity and the lining surfaces of bodies, which inside these cavities; (viii) Dura and meningi; (ix) any tumors in solid tissues, which can be made available for photoactivating light, in particular, as at the time of surgery, and through the optical waveguide is placed inside the needle.
The composition according to the invention can be prepared in a known manner using one or more physiologically acceptable carriers or excipients in accordance with methods well known in the art. The type of composition and types of carriers or excipients, dosage, etc. can be selected in the usual manner in accordance with the choice and required by the introduction, the goal of treatment etc. Dosage can so is e to define in the usual way, and they can depend on the type of delivered molecules (where available), the goals of treatment, the patient's age, method of drug administration, etc.
The composition can be entered locally, orally or systemically. For use in PDT preferred compositions for local application, and these include gels, creams, suspensions, sprays, lotions, medicinal ointments, sticks, Soaps, powders, vaginal suppositories, aerosols, drops, solutions, and any other conventional pharmaceutical forms known from the field of technology. Local application available on the site can be achieved using methods known from the technical field, in particular using catheters and other suitable systems drug delivery.
Alternatively, compositions can be prepared in a form adapted for oral or parenteral administration, for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Thus, alternative pharmaceutical forms include plain or coated tablets, capsules, suspensions and solutions containing the active component choice together with one or more conventional inert carriers and/or diluents.
The concentration of the present invention compounds in the composition depends on the intended use is isawanya connection the type of composition, the method of introduction of the composition, condition, treatment is performed, and the patient and may be varied or adjusted in accordance with the selection made. However, in the General case, for use in PDT the range of concentration of the photosensitizer may be from 0.01 to 50 wt.%, in particular, from 0.05 to 20 wt.%, for example, 1-10 wt.%. When used for the implementation of photochemical internalization is important that the concentration of the photosensitizing agent was such that after the agent got in the cage, in particular appeared to be inside or was associated with one of the cells inside the cell was activated by irradiation, there was a destruction of one or more cell structures, in particular one or more intracellular cell undergoes lysis, or destroyed. For example, photosensitizing agents can be applied with concentration from 10 to 50 µg/ml For in vitro use, the range can be extended significantly, in particular 0.05-500 µg/ml For use in vivo in the treatment of people photosensitizing agent may be used in amounts in the range of 0.05-20 mg per 1 kg of body weight when designating systemically or with a concentration of 0.1-20% in solvent for local application. When using the considered compounds for the implementation of photochemical Intern the implementation time interaction of cells with the photosensitizing agent in the incubator (i.e. the "probe") can vary from several minutes to several hours, e.g. up to 48 hours or more. The incubation time should be such that the photosensitizing agent to soak in the appropriate cells. After processing the cells in the incubator along with the photosensitizing agent can optionally be followed by processing them in the incubator does not contain photosensitizing agent, and then the cells are light and/or type of the delivered molecule.
Identify appropriate doses of the target molecules for use in the present invention is a routine for a person skilled in the art. In cases where the delivered molecule is a protein or peptide, for use in vitro deliver molecules typically used in doses less than 5 mg/ml (in particular, 0.1 to 5 mg/ml), and to apply the in vivo delivery of the molecule is usually used in doses less than 5 mg/kg (in particular, 0.1 to 5 mg/kg). In cases where the delivered molecule is a nucleic acid, for use in vitro approximate dose of delivered molecules is approximately 0.1 to 50 μg of nucleic acid in 104cells, and for use in vivo is approximately 10-6-1 g nucleic acid per injection for human.
After administration of the compounds or compositions according to the present is obreteniyu (in particular, on the surface of the body) on the treated surface are light in order to achieve the desired effect, in particular photochemical internalization or fotohimioterapiei effect. Stage irradiation with light to activate a photosensitizing agent can be performed according to techniques and procedures well known from the technical field. Suitable light sources capable of emitting light in the desired wavelength range and with the right intensity, well known from the art. The time during which the surface of the body or cells exposed to light in accordance with the method of the present invention, may vary. For example, it turns out that the implementation of the efficiency of internalization deliver molecules into the cytosol increases with time of exposure light. In General, the duration of the phase of the exposure light is from several minutes to several hours, particularly preferably within 60 minutes, for example, from 1 to 30 min, in particular from 0.5 to 3 min, or from 1 to 5 min, or from 1 to 10 min, in particular from 3 to 7 min, and preferably approximately 3 min, for example, from 2.5 to 3.5 minutes approximate dose of light can be chosen specialist and depends on the amount of the photosensitizer accumulated in the cells-mission the x or tissues. The dose usually ranges from 40 to 200 j/cm2for example, 100 j/cm2when the integrated flux density of less than 200 mW/cm2. The irradiation of light with a wavelength in the range of 500-750 nm, in particular in the range from 550 to 700 nm, particularly suitable for in vivo use of the present invention methods.
Another aspect of the present invention is a method fotohimioterapiei treatment of disorders or abnormalities of external or internal surfaces of the body, when this method is applied on the affected surface of the photosensitizing agent of the present invention, in particular the compounds of formula I or its pharmaceutically acceptable salts, and the exposure specified surface light, preferably light with a wavelength in the range of 300-800 nm, for example, 500-700 nm.
Methods of exposure to various body surfaces, in particular, lamps or lasers are well known from the art (see, for example, Van den Bergh, Chemistry in Britain, May 1986 p.430-439). For inaccessible areas this can usually be achieved using optical fibers.
Compounds according to the invention can be mixed or administered together with other photosensitizing agents, e.g., ALA or Photofrin®or with other active ingredients that can enhance photochemiluminescence impacts is satisfied. For example, can include agents capable of forming chelate compounds such as aminopolycarboxylate acid (in particular, etc), in order to enhance the accumulation of Pp and, thus, strengthen the photosensitizing effect. Agents capable of forming chelate compounds can generally be used with a concentration of from 0.05 to 20 wt.%, in particular, from 0.1 to 10 wt.%.
In order to enhance fotohimioterapiei impact may also be used by agents that facilitate the penetration through the skin, in particular diallylsulfide, such as dimethylsulfoxide (DMSO). Agents that facilitate the penetration through the skin, can usually be used with a concentration in the range from 0.2 to 50 wt.%, in particular, approximately 10 wt.%.
Depending on the condition, the treatment is carried out, and composition type connection, which is used in the present invention may be administered in conjunction with other agents choice, for example, be in the form of joint compositions or they may be administered sequentially or separately. In fact, in many cases, especially beneficial photochemiluminescence effect can be achieved by using pre-treatment agent, facilitating penetration through the skin, which is carried out on a special stage before the introduction of the compounds used is s according to the present invention.
Another aspect of the present invention, therefore, is the product, including the above photosensitizing agent, in particular a compound of formula I or its pharmaceutically acceptable salt together with at least one agent that facilitates penetration through the skin, and optionally with one or more agents capable of forming a chelate compound, as a combined preparation for simultaneous, separate or sequential use for the treatment of disorders or abnormalities of external or internal surfaces of the body that are sensitive to the effects of photochemotherapy.
Another aspect of the invention is a kit for use in photochemotherapy of disorders or abnormalities of external or internal surfaces of the body, including:
a) a first container containing the present invention photosensitizing agent, in particular a compound of formula I or its pharmaceutically acceptable salt,
b) a second container containing at least one agent that facilitates penetration through the skin; and by choice
c) one or more agents capable of forming chelate compounds, which are contained either within the specified first container or the second container.
Will be understood that therapeutic method that uses described in esteem the invention compounds inevitably causes fluorescence of disorders or abnormalities, the treatment is carried out. Apart from the fact that the intensity of this fluorescence can be used to remove abnormal cells, localization of the indicated fluorescence can be used to visualize the size, extent and location of abnormalities or disorders.
In this way identified or confirmed in the study place abnormality or disorder can then be treated with an alternative therapeutic method, in particular surgical or chemical treatment, or therapeutic method according to the present invention by continuous amplification of fluorescence or further use in the compounds of the present invention. Will become clear that for imaging diagnostic methods may require lower levels of fluorescence than those used in therapeutic treatment. So, in General, suitable concentrations range from 0.2 to 30%, in particular 1-5% (wt./wt.). Places, methods and techniques were discussed earlier in connection with therapeutic use and is also applicable for diagnostic use, as described in the present invention.
Compounds of the present invention can also use the methods of in vitro diagnostics, for example, studies of cells contained in the fluid taken from the body. Stronger fluorescence associated with abnormal tissues may be a useful indicator of abnormalities or disorders. The method is highly sensitive and can be used for early detection of abnormalities or disorders, for example, carcinoma of the bladder or lungs by studying cells, respectively, in samples of urine or sputum. Other useful fluids from the body, which can be used in diagnosis, in addition to urine and sputum specimens include blood, semen, tears, cerebrospinal fluid, etc. Can be investigated samples or preparations of tissues such as biopsy samples of tissue or samples of bone marrow. Thus, the present invention extends to the use of compounds according to the invention or their salts to diagnose when conducting photochemotherapy in accordance with the above methods, products, and kits for carrying out the specified diagnostic.
Another aspect of the invention relates to a method of in vitro diagnosis of abnormalities or disorders consisting in the study of a sample of fluid or tissue taken from the patient, with the specified method comprises at least the following stages:
i) mixing the specified taken from the body of the Jew is awns or tissue with a photosensitizing agent, the present invention, in particular a compound of formula I or its pharmaceutically acceptable salt,
(ii) the exposure of this mixture with light, for example light with a wavelength in the range of 300-800 nm,
(iii) determining the level of fluorescence
(iv) comparing the level of fluorescence from the control values.
The present invention is illustrated in more detail in the following examples, which do not limit the invention, and reference is made to the accompanying drawings.
The figure 1 shows the absorption spectrum of TPPS2ain the presence of triethylamine (tea) in benzene before and after exposure of monochromatic light for various time intervals.
Figure 2 is a graph showing the influence of the exposure light at the maximum optical density peak TPPS2ain the band 646-655 nm. The solution TPPS2ain benzene in the presence of triethylamine exhibit monochromatic light and measure the optical density.
The figure 3 shows the HPLC chromatogram of the derivatives obtained by irradiation of TPPS2alight. TPPS2ain the presence of triethylamine in benzene exhibit light and analyze the formation of fluorescent derivatives by HPLC with reversed phase. Solution exhibit light and draw samples for measurements.
On the igure 4 shows the HPLC chromatogram TPCS 2aand cell extracts of V79 cells treated with TPCS2a. The V79 cells treated with 1 μg/ml TPCS2aformed after 10 min of exposure light (a) in accordance with figure 3. TPCS2aextracted from cells after 18 h of incubation in the incubator and assessed by HPLC (b). The retention times of the peaks are indicated on the figure.
Figure 5 shows a fluorescence micrograph of V79 cells treated with TPPS2aand TPCS2a. Cells are grown in an incubator with (a) 1 µg/ml TPPS2aor (b) 1 µg/ml TPCS2awithin 18 hours, followed by keeping the exposure to red light in the incubator for 1 hour in not containing the sensitizer environment.
Figure 6 shows curves of the response signal from the integrated flux density to inhibit the activity β-AGA in V79 cells treated with 1 mg/ml TPCS2awithin 18 hours, followed by keeping the exposure to red light in the incubator for 1 hour in not containing the sensitizer environment.
The figure 7 shows curves of the response signal from the dose for V79 cells grown with TPPS2aand TPCS2aand exposed (a) red (b) blue light. Cells grown with 1 or 2 μg/ml TPPS2aor 1 μg/ml for 18 hours followed by curing to eksponirovanie the light in the incubator for 1 hour in not containing the sensitizer environment.
The figure 8 shows protein synthesis in V79 cells after the combined effects of light and gelonida. Cells treated with 1 μg/ml TPCS2ain the absence (filled circles) or in the presence (empty circles) of 1 µg/ml gelonin and exhibiting a red light. Strip at intervals on this and the previous figures represent the standard deviation calculated as a result of three identical experiments.
Materials and methods
The photosensitizer TPPS2areceive from Porphyrin Products (Logan, Utah, USA). The original solution (2 mg/ml) TPPS2adissolved in DMSO, which is obtained from Sigma (St. Louis, Missouri, USA). Gelonin bought from Sigma. The original solution of the toxin (2 mg/ml) obtained by dissolving the powder gelonin in phosphate buffered saline solution with a pH of 8.5 and stored until use at a temperature of minus 20°C.
p-Nitrophenyl-N-acetyl-D-glucosamine purchase from Sigma (St. Louis, Missouri, USA).
Getting disulfonate of tetraphenylboron - TPCS2a
Disulfonate of tetraphenylboron (TPCS2a) get from TPPS2afully in accordance with the methodology described Harel et a. (Photochem. Photobiol. 23: 337-341, 1976).
Prepare a mixture of 950 µl of benzene/triethylamine (tea) (18:7), 32 μl of dimethyl sulfoxide (DMSO) and 18 μl of TPPS2a(from a solution with conc what Tracia 1 mg/ml in DMSO) and saturate it with nitrogen for 5 min in a cell with a capacity of 1 ml The mixture exhibiting a light xenon lamp high pressure power 500 W, which is equipped with a monochromator with a diffraction grating, Baush and Lomb. Cell exhibiting a light with a wavelength of 545±15 nm and a density of 15 W/m2. The integrated flux density is controlled by a photo detector UDT 11A, equipped 223 radiometric filter. Absorption spectra are regularly measured with a spectrophotometer Perkin-Elmer Lambda 15 UV/VIS. The optical path is 1 see
To obtain TPCS2amore (study of cells), the mixture is prepared in a coated polymer film flask, which is constantly rinsed with nitrogen during exposure light. The magnitude of the optical path is maintained at the level not more than 1 see the Mixture is irradiated with lamp block TL'/03 and gently shake during exposure. After irradiation, the mixture is subjected to freeze-drying and dissolved in DMSO.
Using cells fixed line V79 (ATCC CCL-93), isolated from lung fibroblasts Chinese hamster. Cells grown in minimal maintenance medium containing 10% serum fetal cow (FCS from Gibco, Paisley, UK), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco), at 37 ° °in the incubator, which is rinsed with a mixture of 5% carbon dioxide in air. the entrances during the week subcultured twice.
The formation of the label by using the photosensitizer
Cells were seeded in flasks of 25 cm2(Nuclon, Denmark) with minimal support medium containing 10% serum fetal cow, and leave for 4-5 hours at a temperature of 37°for a more complete fixation with the substrate. The cells are then washed three times with medium and treated for 18 h with 1 μg/ml TPPS2aor TPCS2ain a medium containing serum. Next, cells are washed three times not containing the photosensitizer environment and before exposure light was incubated for 1 hour. Then exhibiting a red light (Phillips TL 20 W/09), which is passed through the filter Cinemoid 35, or blue light (Appl. Photophysics, Nood. 3026, London). The integrated flux density reaching the cells is for red light bulbs and lamp blue light of 1.35 mW/cm2and 1.5 mW/cm2respectively.
The survival of cells is determined by test kolonialapologie, as described in Berg et al. (Photochem. Photobiol. 53: 203-210, 1991). 1500 cells were seeded in 25 cm2plastic flasks with tissue culture and treated with photosensitizer and irradiated with light, as described previously. After photochemical treatment of V79 cells leave for 5 days at a temperature of 37°in containing serum medium with the culture that formed colonies. The cells are then precipitated with ethanol, ptcr who're asked methylene blue and counted the number of colonies. Inhibition of protein synthesis analyze the number of inclusions in protein [3H]-leucine, which is determined after 24 hours after exposure with light, as described in Llorente et al. (FEBS Lett. 431: 200-204, 1998).
Porphyrins are extracted from the cells by scrapings cells in acidified methanol (5 µl of concentrated HCl in 10 ml of methanol), as described by Berg et al. (Br. J. Cancer 74: 688-697, 1996). Cellular debris granularit, and the liquid above the precipitate is separated. Porphyrins concentrate equalising through extracts nitrogen until then, until volume is reduced to approximately 150-200 µl, and granularit additionally released protein. 100 µl of the liquid above the sediment is mixed with 235 ál of 10 mm solution of Na2PO4to bring the pH to approximately 10,5 using 5M KOH solution and immediately used for analysis by HPLC. This methodology allows us to quantitatively extracted porphyrins from cells. The starting solutions of photosensitizers diluted directly in the starting buffer solution.
The HPLC system consists of a pump (Spectra Physics 8800), a column of reversed phase (Supelcosil LC-18-T (4,6×250 ml), Supelco, S.A., Gland, Switzerland), fluorescence detector (LDC Fluoromonitor III) and integrator (Spectra Physics Data-jet)connected to the computer. Solvent a was a mixture of methanol and water (30:70 by volume)containing 1.5 mm phosphate, pH whom D. which lead to 7.0. Solvent B is a mixture of methanol and water (95:5 by volume)containing 1.5 mm phosphate, pH of which was adjusted to 7.5. Establish a 30-minute linear gradient between 40% and 20% solvent A, followed by 5-minute linear gradient to 100% solvent B. the Fluorescence is determined by the excitation in the region of wavelengths 330-400 nm. The scattered light is separated from the fluorescence by using a cut-off filter, which transmits only light with a wavelength of 410 nm.
To conduct microscopic studies using cell 28 cm2(Falcon 3002, Becton Dickinson, Plymouth, UK). Cells are washed once with phosphate buffered saline and on top of layer of phosphate buffered saline carefully place the top glass. The cells are examined with a microscope Zeiss Axioplan (Zeiss, Oberkochen, Germany), equipped with epifluorescence. For excitation using a mercury lamp HBO/100 W. the Cells and the fluorescence of the cells studied using a cooled CCD (charge coupled device) camera (TE2, Astromed, Cambridge, UK). The camera is controlled by a computer, which is used for digital image processing and information storage. The microscope was equipped with a filter with a bandwidth of excitation in the range of 390-440 nm, 470 nm of dichroic is a beam splitter and a 610 nm long-wavelength filter.
Photochemical inactivation of the enzyme is complementary mechanism β-AGA determined by the method specified in Beaufay et al. (J. Cell Biol. 61: 188-200, 1974). The method is based on the formation of p-NITROPHENOL from the substrate p-nitrophenyl-N-acetyl-D-glucosamine), which can be determined spectrophotometrically at 420 nm. Cells after exposure to light immediately separated and prepare for carrying out enzymatic analysis.
Photochemical restoration TPPS2a
In accordance with the method described in Harel et al. (Photochem. Photobiol. 23: 337-341, 1976), TPPS2airradiated light in a solution of triethylamine (tea) in benzene saturated with nitrogen. The solution is irradiated with light with a wavelength of 545 nm in a cuvette, which is described earlier in the section "Materials and methods".
The figure 1 shows the change in the absorption spectrum upon irradiation with light and the spectrum of the final product, which is typical of the chlorines. Peak I-line Q-bands is shifted to 646 nm to 655 nm, and its maximum intensity is increased 5.8 times (figure 2). Somesthesia points are observed at 593 nm, and at 505 nm, 518 nm and 577 nm. The absorption bands after irradiation are typical of chlorines.
To increase the number of chlorines to examine the cells to irradiation of light is subjected to bógreater volumes of solutions TPPS2awho receive, as described earlier in the section "Mat is the rials and methods". The time scale to increase absorption 655 nm similar to that described previously and shown in figure 2. The process of the formation of chlorine is monitored by HPLC, as shown in figure 3, which shows that it forms several isomers of chlorine. Solutions which are irradiated with light for 10 minutes, then used for treatment of cells in culture. Approximately 23% of the product after 10 min of irradiation has the same retention time, and that TPPS2ahowever , the peak has an absorption spectrum typical of chlorine.
Photobiological research TPCS2a
For the biological study of photochemical effects of chlorine, TPCS2ause lung fibroblast cell line V79 Chinese hamster. Cells are grown overnight with chlorine, the photosensitizer is extracted from cells and extracts examined by HPLC. As shown in figure 4, the shape of the peaks in the fluorescence of extracts of cells similar to the one that has the mother liquor. For carrying out chromatography using a column of C-18 reversed-phase, in which, as a rule, the retention time increases with the hydrophobicity of the compounds. It was found that the absorption of chlorine cells increases with increasing hydrophobicity of the isomers.
As was previously established, the photosensitizer TPPS2a localized in endocytic vesicles of cells grown together with the specified connection (Berg et al. Photochem. Photobiol. 52: 481-487, 1990). It was shown that localization TPCS2ainside the cell is similar to the localization of TPPS2athat indicates that TPCS2aalso located in endocytic vesicles (see figure 5). This is later confirmed photochemical inaktivirovanie of the enzyme to the lysosomes β-N-acetylglucosaminidase (see figure 6). Thus, by comparing the pattern of intracellular fluorescence localized in the lysosomes of the photosensitizer TPPS2aindirectly by fluorescence microscopy and directly by measuring the photochemical inactivation of the enzyme to the lysosomes β-AGA was shown that TPCS2alocalized in endocytic vesicles V79 cells.
The advantage of using chlorine instead of porphyrin when conducting photodynamic therapy is a greater extinction coefficient in the region of the waves of the red region of the spectrum. It was reliably shown by comparing the exposure of cells treated with TPPS2aand TPCS2ablue and red light (see figure 7). From figure 7b shows that cells treated with TPPS2aor TPCS2awere equally sensitive to blue light, while cells treated with TPCS2awere approximately 6 times more Chu is Stateline to the action of red light, than cells treated with porphyrin TPPS2a(figure 7a).
Intramolecular localization TPCS2ain endocytic vesicles and, therefore, its possible use for photochemical internalization of macromolecules is assessed through the internalization of inactivating ribosomes type I protein toxin gelonin. It was shown that Galanin by itself or in combination with the light it has low toxicity (Berg et al. Cancer Res. 59: 1180-1183, 1999). Protein synthesis in cells treated for 18 h with 1 mg/ml Galanina, is reduced by approximately 10%. However, as shown in figure 8, TPCS2aand light significantly enhances the cytotoxicity gelonin that is determined by protein synthesis after 24 hours after exposure to light. Note a marked decrease (20%) of protein synthesis caused by the action of the TPCS2athat is not observed in the study of clonogenicity. The results show that Galanin internalized in cells during the photochemical process using TPCS2a.
Discussion of results
The present study has shown that desulfuromonas tetraphenylporphin can be restored to its chlorin form by photochemical recovery in anaerobic conditions in the presence of triethylamine. Photochemical reduction causes a 5.8-fold increase in the extinction coefficient of the I-line is the Q-bands due to the formation of multiple isomers of chlorine. Comparison with the original porphyrin, TPPS2ashows that the photosensitizing ability of chlorine TPCS2ain V79 cells equally effective for sensitization of cells to photoinactivation blue light and 6 times more efficient for the red light.
By fluorescence microscopy was indirectly shown that TPCS2alocalized in endocytic vesicles V79 cells. This was confirmed by direct measurement of photochemical inactivation of the enzyme to the lysosomes β-AGA. It was found that the rate of inactivation β-AGA in comparison with cell survival is similar to the one that was previously installed for TPPS2a(Berg et al., Int. J. Cancer 59: 814-822, 1994), indicating the same pattern of distribution of these compounds between the membranes endocytic vesicles and cavities.
For example, photochemical internalization gelonin it was also shown that TPCS2acan be used as a photosensitizer for photochemical internalization of macromolecules.
Additional example composition 1.
Preparation of pharmaceutical composition containing a photosensitizing agent, such as TPSC2αand at least one pharmaceutically acceptable carrier or excipient.
When grinding with the pestle in the mortar mix 500 mg TPSC2αwith 10 g basis Unguentum Merck. who received the cream is placed in a closed container of suitable volume. A cream containing 5% TPSC2αready to the local application.
Additional example composition 2.
Preparation of pharmaceutical compositions containing the photosensitizing agent, the molecules of the carrier, and at least one pharmaceutically acceptable carrier or excipient.
In a container for autoclaving put the hitch 100 mg TPSC2αand autoclave for 20 minutes at 121°C. Sterile Cremophor EL (BASF production) is heated to 60-70°and 0.4 ml of Cremophor with stirring under sterile conditions added to autoclaved TPSC2α. The mixture is stirred for approximately 5 minutes at 60-70°C, after which it slowly add the required amount of (3.6 ml) of sterile water for injection, pre-heated to 60-70°administered 0.3 ml of a solution of bleomycin (45000 units/ml), obtaining the composition for intravenous injection containing 25 mg/ml TPSC2αin 10% Cremophor EL.
An additional example of composition 3.
Preparation of a combined preparation containing photosensitizing agent, and the agent promoting the percutaneous introduction, or chelating agent.
20 mg TPSC2αdissolved in 1 ml DMSO. Water is added to the desired concentration, but the rate of not more than 40% (vol.). The resulting solution was placed in sealable containers. The solution is ready for months the nogo application.
An additional example of the composition 4.
The manufacture of the set consisting of the first container containing a photosensitizing agent, and a second container containing an agent that facilitate percutaneous introduction.
1 ml of a Solution containing 20 mg/ml TPSC2αmethanol is subjected to drying by freezing in the container 1. The container seal. In the container 2 is placed 1 mg DMSO, the container 2 is pressurized. For local use the contents of the container 2 is introduced into the container 1, seal and shake until dissolved. The mix is ready for local use. To obtain the required lower concentrations in the container, add water at the rate of not more than 40% (vol.) and mix by shaking.
Additional example applications 1.
The cream obtained in accordance with other example compositions 1, is applied to the area affected by skin cancer. After 3 hours, covered with a cream area is irradiated with a diode laser at a wavelength of 652 nm and power density of the light flux 90 W/cm2within 1-10 minutes, depending on therapeutic indications, taking into account the type and thickness of the tumor.
An additional example of the application 2.
The composition obtained in accordance with an additional example of the composition 2, intravenous patient with oral cancer at the rate of 1 mg/kg of body weight. Che is ez 72 hours cancerous tumor is irradiated with a diode laser at a wavelength of 652 nm and power density of the light flux 90 W/cm 2providing specific dose of 20 j/cm2.
1. Photosensitizing agent, which contains sulfated letterfinlay formula (I):
where X represents-SO3H;
n, p, q and r each independently represents 0 or 1; and the sum of n, p, q and r is an integer from 1 to 4, preferably at least 2, in particular 2 or 4; its isomer or pharmaceutically acceptable salt of any of these compounds.
2. Photosensitizing agent according to claim 1, in which the two phenyl rings present in the specified chlorine, each contain one sulfogrupp.
3. Photosensitizing agent according to claim 1, in which the sum of n, p, q and r is 2 and each group X is in the same position of the ring in each of the phenyl rings.
4. Photosensitizing agent according to claim 3, in which the indicated position of the ring is a meta - or para-position.
5. Photosensitizing agent according to any one of claims 1, 3 or 4, in which the sum of n, p, q and r is equal to 2, and the substituted phenyl ring is located next to the restored pyrrole ring.
6. Photosensitizing agent according to claim 1, wherein said chlorine is a compound of formula (II):
isomer or pharmaceutically acceptable salt of any of the above link is.
7. Photosensitizing agent, which can be obtained by reduction of one double bond in the porphyrin macrocycle of sulfated mesotetraphenylporphyrin, or its pharmaceutically acceptable salt.
8. Photosensitizing agent according to claim 7, wherein said porphyrin macrocycle is desulfuromonas masterfishermen.
9. Photosensitizing agent of claim 8, wherein said porphyrin is TPPS2a(disulfonato of tetraphenylporphine).
10. A method of obtaining a photosensitizing agent according to any one of claims 1 to 9, which includes at least one of the following stages:
(a) recovery of sulfated mesotetraphenylporphyrin or chelate complex of iron,
(b) separation, if necessary, the mixture of compounds obtained in stage (a); and
(c) conversion of the compound obtained in stage (a) or stage (b), its pharmaceutically acceptable salt.
11. Pharmaceutical composition for use in the method of photochemical internalization, photochemical therapy or diagnosis, including photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier or excipient.
12. Photosensitizing agent according to any one of claims 1 to 9, or it is pharmaceutically acceptable salt to obtain a therapeutic agent, which is used in the method of photochemical internalization, photochemotherapy or diagnosis.
13. The application of a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt to obtain a therapeutic agent that is used in the method of photochemical internalization, photochemotherapy or diagnosis.
14. The method of introducing deliver molecules into the cytosol of cells, the method includes:
(a) contacting the specified cells with a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt;
(b) contacting the specified cell with the specified deliverable molecule and
(c) irradiation of the specified cell with light of a wavelength effective to activate the photosensitizing agent.
15. Pharmaceutical composition for use in the method of photochemical internalization, photochemical therapy or diagnosis, including photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt, delivered molecule and at least one pharmaceutically acceptable carrier or excipient.
16. The pharmaceutical composition according to item 15, in which the delivered molecule selected from the group comprising organic compounds, proteins, fragments of proteins and nucleic acids.
17. The pharmaceutical composition according to § 15 for obtaining a therapeutic agent that is used in the method of photochemical internalization, photochemotherapy or diagnosis.
18. The pharmaceutical composition according to item 16 for receiving a therapeutic agent that is used in the method of photochemical internalization, photochemotherapy or diagnosis.
19. The application of a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salts and deliver molecules, in particular of the delivered molecule according to item 16, for the preparation of drugs for use in therapy, in particular cancer therapy or gene therapy, in which the specified photosensitizing agent and deliver specified molecule in contact (either separately, either simultaneously or sequentially) with cells or tissues of the patient, and these cells or tissue is irradiated with light of a wavelength effective to activate the specified photosensitizing agent.
20. The application of claim 19 for the treatment of rheumatoid arthritis, atherosclerosis, viral infections, psoriasis, solar keratosis, wounds, fractures, warts, cystic degeneration syndrome gorlina and Louis-Bar syndrome.
21. The application of a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt and dostavlyaemykh, which is chosen from the group comprising a gene encoding a therapeutic protein, desensitized molecule DNA or RNA, ribozyme, aptamer, triplecore oligonucleotide, peptide nucleic acid (PNA), a transcription factor DNA-"traps" and chimeric oligonucleotide to obtain medications for use in a method of gene therapy, in particular in a method of treatment of cancer, cardiovascular diseases, viral infections or monogenic disorders such as cystic fibrosis.
22. The pharmaceutical composition according to any one of p-18, in which the specified photosensitizing agent and/or specified to be delivered molecule attached, connected or associated with the molecule-carrier, the target molecule or vector.
23. The use according to any one of p-21, where specified photosensitizing agent and/or specified to be delivered molecule attached, connected or associated with the molecule-carrier, the target molecule or vector, for the treatment of any malignant, pre-malignant and non-malignant abnormalities or disorders, sensitive to the action of photochemotherapy, in particular tumors or other growths, skin disorders such as psoriasis or senile keratosis and acne, abrasions on the skin, and other diseases or infections, in particular bacterial, viral is whether fungal infections, for example, herpes virus infections.
24. The application of a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salts for the preparation of medicaments for use in the treatment of any malignant, pre-malignant and non-malignant abnormalities or disorders, sensitive to the action of photochemotherapy, in particular tumors or other growths, skin disorders such as psoriasis or senile keratosis and acne, abrasions on the skin, and other diseases or infections, in particular bacterial, viral or fungal infections, for example herpes virus infections.
25. The way fotohimioterapiei treatment of malignant, pre-malignant and non-malignant disorders or abnormalities of external or internal surfaces of the body are sensitive to photochemotherapy, which includes:
(a) applying to the surface of the affected photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt and
(b) the specified irradiation surface of the light with wavelength in the range of 300-800 nm.
26. The product, including photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt together with at least one agent that facilitates penetration through the skin, and/or with one or more agents, with osobami to form chelate compounds, in the form of a combined preparation for simultaneous, separate or sequential use in the treatment of disorders or abnormalities of external or internal surfaces of the body that are sensitive to the effects of photochemotherapy.
27. Set for use in photochemotherapy of disorders or abnormalities of external or internal surfaces of the body, including:
(a) a first container containing a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt;
(b) a second container containing at least one agent that facilitates penetration through the skin; and by choice
C) one or more agents capable of forming chelate compounds, which are contained either within the specified first container or the second container.
28. The method of in vitro diagnosis of malignant, pre-malignant and non-malignant disorders or abnormalities of external or internal surfaces of the body that are sensitive to the effects of photochemotherapy, which consists in the study of a sample of fluid or tissue taken from the patient, which includes:
i) mixing the specified taken from a body fluid or tissue with a photosensitizing agent according to any one of claims 1 to 9, or its pharmaceutically acceptable salt;
(ii) exposing the above-mentioned mixture of light is;
(iii) determining the level of fluorescence
(iv) comparing the level of fluorescence from the control values.
FIELD: molecular biology, biotechnology, medicine, pharmaceutical industry.
SUBSTANCE: invention proposes RNA molecule able to target-specific interference of RNA and represents a double-stranded RNA molecule of size 19-22 nucleotides and having 3'-bulge consisting of 1-5 nucleotides This RNA is prepared by combining two chains of RNA and used for preparing a pharmaceutical composition. After incorporation into the multicellular eucaryotic organism this RNA molecule provides promotion to target-specific interference of RNA and results to decreasing level of expression of the gene-target or to knockout of the gene-target. Method for promotion of target-specific interference of RNA using this RNA molecule is used in assay and modulation of gene function. Cell containing endogenous nucleic acid-target, RNA molecule able for target-specific RNA interference and exogenous nucleic acid-target is used in analytical procedures. Using the invention provides silence of the gene-target mediated by target-specific interference of RNA.
EFFECT: valuable biological properties of RNA.
40 cl, 24 dwg, 3 ex
FIELD: molecular biology.
SUBSTANCE: disclosed is molecular transfecting complex (MTC) comprising of plasmide DNA carrying necessary genetic information, polyethyleneimine, modifies with acylhydrazine groups and being capable to form ion complexes with plasmide DNA, ligand molecules for receptor-mediated MTC addressing to certain cell types and providing prolonged MTC circulation in blood. Ligand molecule contains one or more aldehyde groups, whereby such molecules may be attached after DNA-acylhydrazidopolyethyleneimine complex formation. Invention is useful in medicine for gene therapy.
EFFECT: complexes with prolonged storage life.
5 cl, 8 dwg, 10 ex
SUBSTANCE: claimed method includes injection of vector construct pX-RSVhGH encoding human growth hormone or pX-Ins, encoding human insulin into parenchyma of 8-9-week rabbit males. Transformation effectiveness of gene constructs is determined by immunohystochemical kit Novocastra (Sigma, USA).
EFFECT: method for genetic transformation of rabbit testicle stem cells with increased effectiveness.
3 dwg, 1 ex
FIELD: detection and measuring of at least one analyte in a sample.
SUBSTANCE: the invention is dealt with a method of detection and measurement of one or more analytes for analysis in a sample. The method provides for binding of one or more analytes for analysis in a sample with light-diffusing particles; illumination of any of such particles bound with the analytes by a light under conditions, which allow to have a diffuse light from the particles and at which the light diffused from one or more particles can be detected by a human eye at magnification of no less than in 500 times and without an electronic amplifier. In the capacity of a measure for detecting of presence of such analytes the method also provides for detection of the light diffused by any of such particles under certain conditions.
EFFECT: much easy in realization, more responsive and more universal method of detection of analytes.
55 cl, 48 dwg, 26 tbl, 34 ex
FIELD: chemistry of coordination compounds, chemical technology, medicine.
SUBSTANCE: invention relates to an improved method for synthesis of metallic complexes of chlorophyll (A) derivatives with transient metal ions (Ni2+, Zn2+, Co2+, Cu2+). Method involves boiling the parent ligand with transient metal salt followed by isolation of the end product. Method involves using methylpyropheophorbid (a) or chlorin e6 13-N-methylamide-15,17-dimethylester or 13(2)-hydroxymethlpheiphorbid (a) as a ligand, and acetyl acetonate of the corresponding metal is used as transient metal salt, and boiling is carried out in equimolar amount of reagents for 2-3 h. By alternative variant the method involves boiling the parent ligand with transient metal salt followed by isolation of the end product wherein methylpyropheophorbid (a) or chlorin e6 13-N-methylamide-15,17-dimethyl ester, or 13(2)-hydroxymethylpheophorbid (a) is used as a ligand. Acetyl acetonate of the corresponding metal is used as transient metal salt in 10-fold excess. Acetyl acetonate is added to the reaction mixture by two equal portions followed by boiling for 1-2 min after each addition. Method provides high yield and without using large excess of metal salts. Invention can be used in synthesis of antitumor and antiviral preparations used in medicine.
EFFECT: improved method of synthesis, valuable medicinal properties of complexes.
3 cl, 1 dwg, 8 ex
FIELD: medicine, radiation therapy.
SUBSTANCE: the present innovation refers to radiosensitizers that contain as an active component halogenated derivatives of borated porphyrines that contain a great number of carboranic cells which are selectively accumulated in neoplasms' tissues in the irradiated volume and could be applied in such type of cancer therapy that include but are not restricted with boron-neutron-capturing therapy and photodynamic therapy. The present innovation , also, deals with applying these radiosensitizers for visualization of the tumor and treating the cancer.
EFFECT: higher efficiency.
35 cl, 2 dwg, 8 ex, 7 tbl
FIELD: organic chemistry.
SUBSTANCE: claimed method includes interaction of 1,3-diaminopropane with paraformaldehyde at room temperature for 10-20 min under stirring and recrystallization of condensation product from ethanol or heptane.
EFFECT: method of high yield.
1 dwg, 2 ex
FIELD: organic chemistry.
SUBSTANCE: invention relates to a method for synthesis of e6 chlorine derivatives with two or three amino-groups. The proposed method provides to synthesize chlorines comprising 2 and 3 amino-groups without using activating agent and with high yields that are similar or exceed yields of analogous compounds prepared by using activating agents. In using the proposed methods di- and triaminochlorines can be synthesized selectively from methylpheophorbid (a) directly without isolation of intermediate compounds. Method for synthesis of aminochlorines involves interaction of ester groups of chlorine e6 with ethylenediamine and isolation of the end product wherein pure ethylenediamine is used, chlorine e6 13-N-(2-aminoethylamide)-15,17-dimethyl ester is synthesized in situ from methylpheophorbid (a) and 50-200-fold mole excess of ethylenediamine in chloroform medium at room temperature 15-25°C for 1.5-2.5 h followed by evaporation of chloroform in rotor evaporator under reduced pressure 20-300 mm mercury column and at bath temperature 50°C, not above. Before isolation the reaction mixture is kept in dark at room temperature and after keeping the mixture for 20 h e6 chlorine diamine is isolated as the end product that comprises two amino-groups, and after keeping the mixture for 70 h e6 chlorine triamide is isolated comprising tree amino-groups. The proposed method of synthesis chlorines can be used in synthesis of antitumor and antiviral preparations in medicinal aims.
EFFECT: improved method of synthesis.
FIELD: chemistry of metalloorganic compounds, chemical technology.
SUBSTANCE: invention relates to an improved method for synthesis of octa-4,5-carboxyphthalocyanine cobalt sodium salt, or 2,3,9,10,16,17,23,24-octacarboxylic acid of phthalocyanine cobalt (terephthal) of the formula (I) . Terephthal is a synthetic preparation used in catalytic ("dark") therapy of cancer based on generation of oxygen reactive species in tumor directly by chemical manner and in combination with ascorbic acid being without using the physical effect. Method for preparing octa-4,5-carboxyphthalocyanine cobalt sodium salt involves melting pyromellitic acid dianhydride with cobalt salt in the presence of urea followed by alkaline hydrolysis of prepared octa-4,5-carboxyphthalcyanine cobalt tetraimide. Salt formed after hydrolysis is purified from impurities, in particularly, from oligomeric compounds by column chromatography method on aluminum oxide, following precipitation of octacarboxylic acid, its, its washing out, concentrating and purifying from residual inorganic salts by washing out with distilled water and by neutralization with sodium hydroxide aqueous solution also, treatment with apyrogenic activated carbon, filtration and drying the end substance. Purification of octa-4,5-carboxyphthalocyanine cobalt from residual inorganic salt is carried out preferably by electrodialysis method after its partial neutralization to pH 5.2-5.5 at current density 0.15-0.25 A/dm2, temperature 20-35°C and the concentration 1.5-3.0% followed by complete neutralization to pH 8.7, treatment of obtained octacarboxy-PcCo salt solution with activated carbon, filtration and drying filtrate in a spray drier. Proposed method provides preparing octa-4,5-carbocyphthalocyanine cobalt salt of high purity degree and free of oligomeric compounds and residual chlorides.
EFFECT: improved method of synthesis.
FIELD: chemistry of metalloorganic compounds.
SUBSTANCE: invention describes mixtures of metallocenylphthalocyanines prepared by reaction of mixture containing two phthalocyanines of the formulae (I) and (II) wherein M1 means Cu, Zn, Ni, Pd, Pt, Mn, Co, VO, MnO, TiO or H2; X means Cl, Br; Y1, Y2 and R15 have values given in claim 1 of the invention with the metallocene derivative in the presence of a catalyst. Also, invention describes oligomeric metallocenylphthalocyanines, a method for their synthesis and the information optical carrier comprising a mixture into a recording layer by any claim 1 or 2, or compound by claims 3-10.
EFFECT: improved method of synthesis.
11 cl, 8 tbl, 22 ex
FIELD: chemical technology.
SUBSTANCE: invention relates to a method for synthesis of metal-free phthalocyanine having important and broad practical using. Invention describes a method for synthesis of metal-free phthalocyanine by high-temperature condensation of 3-nitro-5-tert.-butylphthalodinitrile in the presence of lead oxide taken in the amount 0.25-0.27 g, ammonium chloride taken in the amount 0.3-0.33 g and ammonium molybdate taken in the amount 0.02-0.025/1 g of 3-nitro-5-tert.-butylphthalodinitrile at temperature 240-260°C. Invention provides preparing metal-free tetra-(3-nitro-5-tert.-butyl)phthalocyanine with the yield 5% by the simple a single-step technology.
EFFECT: improved method of synthesis.
FIELD: chemistry, medicine.
SUBSTANCE: invention relates to hemin-peptide of general formula I , wherein R1 is ArgTrpHisArgLeuLysGlu(OMe)OH; R2 is -OH; Y is Cl; Me is Fe, or pharmaceutically acceptable salts thereof having virulicidal and anti-viral activity, including activity against herpes virus and HIV, and capability for destroying of λ fag, herpes and HIV DNA. Hemin-peptide fragment also is disclosed.
EFFECT: new anti-viral agent.
2 cl, 5 tbl, 5 ex
SUBSTANCE: invention relates to using a gadolinium complex of [1-(4-perfluorooctylsulfonyl)piperazine]amide of 6-N-[1,4,7-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecane-10-N-(pentanoyl-3-aza-4-oxo-5-methyl-5-yl)]-2-N-[1-O-α-D-carbonylmethylmannopyranose]-L-lysine as a contrasting substance used in method of the magnetic-resonance tomography in visualization of patches.
EFFECT: enhanced precision of diagnosis.
3 tbl, 136 ex
FIELD: printing inks.
SUBSTANCE: invention relates to materials generating charge, in particular to printing inks and toners for protective marking and applying protective markers in order to reveal falsifications and counterfeits. Use of counterfeit protection technology is described involving printing ink composition containing charge-generation substance and a medium, wherein charge-generation substance has maximum absorption in near IR region within a range of 700-1500 nm and in visible region within a range of 400-700 nm, this substance being compound selected from a type of polymorphous modification of X form of metal-free phthalocyanine, type of polymorphous modification of Y form and phase I and II forms of titanyloxyphthalocyanine, polymorphous modification of phase II form of vanadyloxyphthalocyanine, and polymorphous modification of phase V form of hydroxygalliumphthalocyanine and methoxygalliumphthalocyanine. Printing ink composition is applied onto an article or substrate using known printing procedure. Methods of establishing authenticity of an article or substrate are also described, which consist in determining characteristic absorption of marker in near IR region.
EFFECT: enabled effective protection of articles or substrates when using printing inks containing, for example, blue or green dyes, whose absorption bands can be shifted to infrared region and partially mask absorption properties of marker.
20 cl, 4 tbl, 6 ex
FIELD: medicine, oncology.
SUBSTANCE: method includes the consecutive stages: (a) administration of at least one dose of anti-angiogenic cyclo-(arginine-glycine-asparagine acid)-containing pentapeptide (pentapeptide cRGD), such as cyclo-(Arg-Gly-Asp-D-Phe-[N-Me]-Val); (b) administration of anti-tumor effective amount of radio immunotherapeutic agent(RIT) not later than in 1 hour following administration of pentapeptide cRGD at stage (a); and (c) administration of at least two additional doses of pentapeptide cRGD, where the first additional dose is administered within 2 days after RIT and each additional dose of pentapeptide cRGD is administered with intervals between doses not more than 2 days.
EFFECT: invention provides the synergic effect in regard to apoptosis of tumor cells and endothelial cells of tumor vessels.
30 cl, 6 dwg, 2 tbl