2 , 3 -DIHYDROPORPHINE DERIVATIVES , PROCESSES FOR THEIR PREPARATION, AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
Technical field
The present invention relates to compounds of formula 3 or salts thereof, processes for the preparation of compounds of formula 3 or salts thereof, pharmaceutical compositions comprising compounds of formula 3 or salts thereof, the use of compounds of formula 3 or salts thereof as photo therapeutic or photodiagnostic agents, and methods of treatment using compounds of formula 3 or salts thereof.
Background art
Photodynamic therapy (PDT) is a known treatment that uses light to destroy, for example, cancer tissue. Cytoluminescent therapy (CLT) is a form of photodynamic therapy. In both photodynamic therapy and cytoluminescent therapy, a photosensitizer is administered to a patient, generally orally or intravenously. The photosensitizer collects selectively in cancer tissue and, when exposed to light, becomes activated, releasing a highly energized, free radical form of oxygen known as singlet oxygen. Singlet oxygen destroys cancer cells from the inside out, while leaving normal tissues largely unaffected. The administered photosensitizer can be exposed to light and activated internally using fibre-optic catheters or endoscopes inserted into the body to bring the light directly to the seat of the tumour or externally using light of higher wavelengths, which allows a greater depth of penetration into the body.
Most known photosensitizers have mayor drawbacks, for example, they may be difficult to prepare and purify, or they may only accumulate slowly in tumours. For example, Russian patent RU-2183956 discloses photosensitizers based on a mixture of alkali metal salts, chlorine-e6, ρurρurine-5 and purpurine-18, which is obtained by extracting Spirulina biomass. However, the photosensitizers disclosed in RU- 2183956 have a low selectivity for tumour tissues, a high toxicity to normal organs and tissues, and a low therapeutic photoactivity in tumour cells. Moreover, they are
chemically and photochemically unstable, but are only slowly metabolised and cleared from normal tissues.
It is therefore an object of the present invention to provide photosensitizers with certain desired physical, chemical, photophysical and biological properties, such as high selectivity for tumour tissue, optimum speed of accumulation in tumour tissue, rapid clearance from normal tissue, slow clearance from tumour tissue, high photodynamic activity, low tendency to induce photosensitivity, low cytotoxicity towards normal tissue, homogeneity and chemical stability of medicinal forms during storage, and ease of preparation and purification of industrial quantities.
The inventors of the present invention have investigated the compound of formula 1, 18-carboxy-20-(carboxymethyl)-8-ethenyl-l 3-ethyl-2,3-dihydro-3,7,12,17- tetramethyl-21H,23H-porphine-2-propanoic acid, which is also known as phytochlorin or chlorine-e6, and derivatives and metal complexes thereof.
The inventors of the present invention have further developed a process for the preparation of derivatives and metal complexes of chlorine-e6, which is simple and effective, and provides the derivatives and metal complexes without residual toxic reagents.
Summary of the invention
A first aspect of the present invention is a compound of formula 3
or a salt thereof, wherein M is a metal atom in the M(II) oxidation state, a metal halide or a metal oxide, where the metal is Ca, Ti, N, Νb, Ct, Mo, Mn, Tc, Ru, Co, Rh, Νi, Pd, Pt, Ag, Au, Zn, Cd, Hg, Al, Ga, In, Ge, Pb or a lanthanide, or M is SiR
2 where R is a C,-C
8 saturated or unsaturated alkyl group, each R
1, R
2, R
3, R
4, R
5, R
6, R
7, R
8, R
9, R
10, R", R
12, R
13 and R
14 is independently hydrogen, (CH^-CHO, (CH
2)
n-CO
2R
15 or a C C
6 saturated or unsaturated alkyl group optionally substituted with one or more of -OH and -ΝH
2, n is 0, 1, 2 or 3, and each R
15 is independently hydrogen, lithium, sodium, potassium, magnesium, calcium, a - saturated or unsaturated alkyl group optionally substituted with one or more of -OH and -NH
2, or a naturally occurring amino acid.
The metal halide may be a metal fluoride, chloride, bromide, iodide or a mixture thereof. Preferably M is Zn, Cd, Ca, Mn, Au or Co. More preferably M is Zn.
Preferably the compound is immobilized on a protein, a polypeptide, a polymer or activated charcoal.
A second aspect of the present invention is a compound of formula 3
or a salt thereof, wherein M is a metal atom in the M(II) oxidation state, a metal halide, a metal oxide or a silicon with two axial substituents, each
R
10, R
n, R
12, R
13 and R
u is independently hydrogen, (CH^-CHO, (CH
2)
n-CO
2R
15 or a C C
6 saturated or unsaturated alkyl group optionally substituted with one or more of -OH and -NH
2, n is 0, 1, 2 or 3, each R
15 is independently hydrogen, lithium, sodium, potassium, magnesium, calcium, a C,-C
6 saturated or unsaturated alkyl group optionally substituted with one or more of -OH and -NH
2, or a naturally occurring amino acid, and wherein the compound is immobilized on a protein, a polypeptide, a polymer or activated charcoal.
The metal halide may be a metal fluoride, chloride, bromide, iodide or a mixture thereof. Preferably M is Mg, Ca, Ti, V, Nb, Cr, Mo, Mn, Tc, Fe, Ru, Co, Rh, Ni,
Pd, Pt, Cu, Ag, Au, Zn, Cd, Hg, Al, Ga, In, Ge, Sn, Pb, a lanthanide or SiR2 where
R is a C,-C8 saturated or unsaturated alkyl group. More preferably M is Mg, Ca, Ti, V, Nb, Cr, Mo, Mn, Tc, Fe, Ru, Co, Rh, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, Hg, Al, Ga,
In, Ge, Sn, Pb or a lanthanide. Even more preferably M is Zn, Cu, Cd, Ca, Mn, Au or Co. Even more preferably M is Zn.
The compound of the first aspect of the present invention is preferably immobilized on a protein, a polypeptide, a polymer or activated charcoal. The compound of the second aspect of the present invention is immobilized on a protein, a polypeptide, a polymer or activated charcoal. Either way, preferably the compound is immobilized in monomer form. Preferably the protein is serum humane albumin (SHA) or bovine serum albumin (BSA), more preferably serum humane albumin (SHA). Preferably the polypeptide is a low molecular weight polypeptide, more preferably polylysine or polyasparagine. Preferably the polymer is polyvinylpyrrolidone (PVP).
For the purposes of this invention, a "salt" of a compound of the present invention is formed between a carboxylic acid functionality of a compound of the present invention and a suitable cation. Suitable cations include, but are not limited to lithium, sodium, potassium, magnesium, calcium and ammonium. Preferably the salt is a pharmaceutically acceptable salt. The salt may be a mono-, di- or tri-salt. Preferably the salt is a mono- or di-lithium, sodium, potassium, magnesium, calcium or ammonium salt. More preferably the salt is a mono- or di-sodium salt.
Preferably each R
1, R
2, R
3, R\ R
5, R
6, R
7, R
8, R
9, R
10, R
u, R
12, R
13 and R'
4 is independently hydrogen, methyl, ethyl, propyl, allyl, CO
2H, CH
2CO
2H or
Preferably R
1 and R
3 are hydrogen. Preferably R
s, R
8 and R
n are hydrogen.
Preferably R,s is hydrogen, sodium, a C,-C3 saturated or unsaturated alkyl group or a naturally occurring amino acid, such as aspartic acid or lysine.
The compound of formula 3 has two chiral centres, 1* and 2*, and can therefore exist in the form of four stereoisomers. The present invention embraces all of these stereoisomers and mixtures thereof. Mixtures of the stereoisomers can be resolved by conventional methods, for example, chiral chromatography, fractional recrystallisation, derivatisation to form diastereomers and subsequent resolution, and resolution using enzymes. Alternatively, the compound of formula 3 can be prepared directly in substantially enantiomerically pure form by enantioselective or stereoselective synthesis.
The compound of formula 3 preferably comprises at least 95% of one enantiomer, preferably at least 98% of one enantiomer, and more preferably at least 99% of one enantiomer. Preferably the compound of formula 3 is substantially enantiomerically pure, which is defined for the purposes of the present invention as meaning that the compound of formula 3 comprises at least 99% of one enantiomer.
Preferably R1 and R3 are hydrogen, and R1 is in the down-configuration and R3 is in the up-configuration in formula 3 as shown. More preferably R1 and R3 are hydrogen, R2 is (CH^COaH, R4 is CO2H, and chiral centres 1* and 2* are in the (S)-configuration.
In the most preferred embodiment, the compound of the present invention is of formula 2
A third aspect of the present invention is a process for the preparation of a compound of formula 3 or a salt thereof, comprising the step of mixing a compound of formula 4
or a salt thereof with a metal compound in an aqueous solution having a pH ≥ 9 to yield the compound of formula 3 or the salt thereof.
An immobihzer can be added to the compound of formula 3 upon formation. Alternatively an immobihzer can be added to the compound of formula 4 prior to the mixing with the metal compound. Preferably the immobihzer is added to an aqueous solution having a pH ≥ 9.
The third aspect of the present invention further provides a process for the preparation of a compound of formula 3 or a salt thereof, comprising the steps of (i) mixing a compound of formula 4
or a salt thereof with an immobihzer in an aqueous solution having a pH ≥ 9 to yield an immobilized compound 4, and (ii) adding a metal compound to the immobilized compound 4 to yield an immobilized compound of formula 3 or the salt thereof.
Preferably the immobihzer is a protein, a polypeptide, a polymer or activated charcoal. Preferably the protein is serum humane albumin (SHA) or bovine serum albumin (BSA), more preferably serum humane albumin (SHA). Preferably the polypeptide is a low molecular weight polypeptide, more preferably polylysine or polyasparagine. Preferably the polymer is polyvinylpyrrolidone (PVP). Preferably the immobihzer immobilizes the compound of formula 3 in monomer form.
Preferably the compound of formula 4 and the metal compound are mixed in a ratio of about 1:1. Preferably the compound of formula 4, the metal compound and the immobihzer are mixed in a ratio of about 1:1:1.
Preferably the metal compound is an organometallic compound. Preferably the metal compound is a carboxylic acid metal salt. Preferably the metal compound is a Zn compound, such as zinc acetate. Alternatively the metal compound may be a Cd or Cu compound, such as cadmium acetate or copper acetate.
Preferably the aqueous solution is provided with a pH ≥ 9 by the addition of ammonia. Preferably the aqueous solution is provided with a pH of from 9 to 10.
Preferably the step of mixing the immobilized or non-immobilized compound of formula 4 with the metal compound is carried out at a temperature of from 10°C to 100°C. Preferably the step of mixing the compound of formula 3 or 4 with the immobihzer is carried out at a temperature of from 10°C to 100°C. More preferably the steps are carried out at a temperature of from 15°C to 40°C, even more preferably at a temperature of from 18°C to 20°C.
A fourth aspect of the present invention is a pharmaceutical composition comprising a compound of formula 3 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
Preferably the pharmaceutical composition is in a form suitable for oral, parental (including intravenous, subcutaneous, intramuscular, intradermal, intratracheal, intraperitoneal, intraarticular, intraabdominal, intracranial and epidural), transdermal, airway (aerosol), rectal, vaginal or topical (including buccal, mucosal and sublingual) administration, most preferably in a form suitable for oral or parental administration.
For oral administration, the pharmaceutical composition is preferably provided in the form of a tablet, capsule, hard or soft gelatine capsule, caplet, troche or lozenge, as a powder or granules, or as an aqueous solution, suspension or dispersion. Moreover, the pharmaceutical composition is preferably in a form suitable for providing 0.01 to 10 mg/kg/day of a compound of formula 3 or a salt thereof, more preferably 0.1 to 5 mg/kg/day, even more preferably about 2 mg/kg/day.
Alternatively, the pharmaceutical composition is in a form suitable for parental, in particular intravenous, administration, in which case the pharmaceutical composition is preferably an aqueous solution or suspension having a pH of from 6 to 8.5.
Preferably the pharmaceutical composition is suitable for use in the photodynamic therapy or cytoluminescent therapy of a human or animal disease. Preferably the human or animal disease is characterised by begin or malignant cellular hyperprohferation or by areas of neovascularisation. More preferably the human or animal disease is a benign or malignant tumour.
Preferably the pharmaceutical composition is suitable for the treatment of atherosclerosis, multiple sclerosis, diabetes, a benign or malignant tumour, arthritis, rheumatoid arthritis, a fungal, viral, chlamydial, bacterial, nanobacterial or parasitic
infectious disease, HIV, hepatitis, herpes simplex, herpes zoster, psoriasis, a cardiovascular disease, or a dermatological condition.
A fifth aspect of the present invention is the use of a compound of formula 3 or a salt thereof for the manufacture of a phototherapeutic agent for the use in photodynamic therapy or cytoluminescent therapy. Preferably the phototherapeutic agent is used for the treatment of a disease characterised by begin or mahgnant cellular hyperprohferation or by areas of neovascularisation. More preferably the phototherapeutic agent is used for the treatment of a benign or mahgnant tumour.
A sixth aspect of the present invention is the use of a compound of formula 3 or a salt thereof for the manufacture of a medicament for the treatment of atherosclerosis, multiple sclerosis, diabetes, a benign or mahgnant tumour, arthritis, rheumatoid arthritis, a fungal, viral, chlamydial, bacterial, nanobacterial or parasitic infectious disease, HIV, hepatitis, herpes simplex, herpes zoster, psoriasis, a cardiovascular disease, or a dermatological condition.
A seventh aspect of the present invention is the use of a compound of formula 3 or a salt thereof for the manufacture of a photodiagnostic agent for the identification of an area that is affected by begin or mahgnant cellular hyperprohferation or by neovascularisation. Preferably the area is a begin or mahgnant tumour.
An eighth aspect of the present invention is a method of photodynamic therapy or cytoluminescent therapy of a human or animal disease, comprising administering a therapeutically effective amount of a compound of formula 3 or a salt thereof to a human or animal in need thereof and subjecting the human or animal to irradiation or sound. Preferably the human or animal disease is characterised by begin or mahgnant cellular hyperprohferation or by areas of neovascularisation. More preferably the human or animal disease is a benign or mahgnant tumour. The precise wavelength of the irradiation or sound used depends on the compound administered to the human or animal. However, generally the irradiation is electromagnetic radiation with a wavelength in the range of from 500nm to lOOOn ,
preferably from 600nm to 900nm, more preferably from 620nm to 820nm, even more preferably from 630nm to 710nm.
A ninth aspect of the present invention is a method of treating atherosclerosis, multiple sclerosis, diabetes, a benign or mahgnant tumour, arthritis, rheumatoid arthritis, a fungal, viral, chlamydial, bacterial, nanobacterial or parasitic infectious disease, HIV, hepatitis, herpes simplex, herpes zoster, psoriasis, a cardiovascular disease, or a dermatological condition, comprising administering a therapeutically effective amount of a compound of formula 3 or a salt thereof to a human or animal in need thereof. Optionally the human or animal is further subjected to irradiation or sound. The precise wavelength of the irradiation or sound used depends on the compound administered to the human or animal. However, generally the irradiation is electromagnetic radiation with a wavelength in the range of from 500nm to lOOOnm, preferably from 600nm to 900nm, more preferably from 620nm to 820nm, even more preferably from 630nm to 710nm.
Brief description of the drawings
Figure 1 shows the absorption spectra of (1) chlorine-e6 (λmιx = 656nm), (2) Zn- chlorine-e6 complex (λm._ = 632nm), and (3) Zn-chlorine-e6 complex immobilized on SHA (λmax = 636nm), all in water.
Figure 2 shows the absorption spectrum of chlorine-e6 (λmax = 402, 502 and 656nm) in water.
Figure 3 shows the absorption spectra of (1) chlorine-e6 (λmax = 656nm), (2) chlorine-e6 immobilized on SHA (λmax = 662nm), and (3) Zn-chlorine-e6 complex immobihzed on SHA (λmax = 636nm), all in water.
Figure 4 shows the absorption spectra of (1) chlorine-e6 (λma3[ = 656nm), (2) Zn- chlorine-eό complex (λma!t = 632nm), and (3) Zn-chlorine-e6 complex immobihzed on PVP (λm„ = 638nm), all in water.
Figure 5 shows the absorption spectra of (1) chlorine-e6 ( max = 656nm), (2) chlorine-e6 immobihzed on PVP (λmax = 662nm), and (3) Zn-chlorine-e6 complex immobihzed on PVP (λmax = 638nm), all in water.
Figures 6 to 8 show the absorption spectra of Zn-chlorine-e6 complex (λmax = 414 and 634nm), Zn-chlorine-e6 complex immobihzed on SHA (λraax = 418 and 636nm), and Zn-chlorine-e6 complex immobihzed on PVP (λmax = 416 and 638nm), all in water, respectively.
Figures 9 and 10 show the fluorescence spectrum (λmax = 643nm) and the fluorescence stimulation spectrum (λmax = 412 and 607nm) of Zn-chlorine-e6 complex in water respectively.
Figures 11 and 12 show the fluorescence spectrum (λmax = 645nm) and the fluorescence stimulation spectrum (λmax = 446 and 673nm) of Zn-chlorine-e6 complex immobihzed on SHA in water respectively.
Figures 13 and 14 show the fluorescence spectrum (λmax = 645nm) and the fluorescence stimulation spectrum (λmax = 429 and 727nm) of Zn-chlorine-e6 complex immobihzed on PVP in water respectively.
Figures 15 and 16 show the fluorescence spectrum (λmax = 645nm) and the fluorescence stimulation spectrum (λmax = 418 and 641nm) of a biological sample taken from the hquid above the sediment of an ascite tumour taken from an experimental animal (mouse), which had previously been injected intraabdominally with a preparation comprising Zn-chlorine-e6 complex immobihzed on SHA.
Figure 17 shows the absorption spectra of (1) chlorine-e6 (λmax = 656nm), (2) chlorine-e6 immobihzed on PVP (λmax = 662nm), and (3) Cd-chlorine-e6 complex immobihzed on PVP (λmax = 646nm), all in water.
Figure 18 shows the absorption spectrum of Cd-chlorine-e6 complex immobilized on PVP (λmax = 424 and 646nm) in water.
Figure 19 shows the absorption spectra of (1) chlorine-e6 (λmax = 656nm), (2) chlorine-e6 immobihzed on PNP (λmax = 662nm), and (3) Cu-chlorine-e6 complex immobihzed on PNP (λmax = 636nm), all in water.
Figure 20 shows the absorption spectrum of Cu-chlorine-e6 complex immobilized on PVP (λmax = 410, 505 and 636nm) in water.
Figure 21 shows the results of pharmacokinetic distribution studies. The pharmacokinetic distribution of Zn-chlorine-e6 complex immobihzed on SHA over 30 hours in organs, tissues, biological liquids and tumours (embryocarcinoma) was studied.
Detailed description of the invention
The present invention provides two routes to compounds of formula 3.
The first route (see Examples 1 and 2 below) comprises the step of mixing a compound of formula 4, also called chlorine-e6, which is commercially available, with a metal compound in an aqueous solution having a pH ≥ 9 to yield the compound of formula 3. The compound of formula 3 may be immobihzed in monomer form on an immobihzer, such as a protein, a polypeptide, a polymer or activated charcoal, by adding the immobihzer to the compound of formula 3 upon formation.
More specifically, chlorine-e6 is dissolved in an aqueous solution with a pH ≥ 9. A pH ≥ 9 can be achieved, for example, by adding ammonia to an aqueous solution. Then an about equimolar quantity of a metal compound, for example zinc acetate, is added to the reaction mixture. When mixing the solution at about room temperature, chIorine-e6 and the metal ion form a complex. The progress and completion of the complex-formation reaction can be monitored with a spectrophotometer.
On completion of the complex-formation reaction, an about equimolar quantity of an immobihzer such as a protein, a polypeptide, a polymer or activated charcoal, for example serum humane albumin (SHA) or polyvinylpyrrolidone (PNP), is added to the reaction mixture. The solution is mixed at about room temperature until the compound of formula 3 is immobihzed on the immobihzer. The progress and completion of the immobilization reaction can be monitored with the help of a spectrophotometer.
The second route (see Examples 3 to 6 below) comprises the steps of (i) mixing a compound of formula 4 with an immobihzer in an aqueous solution having a pH ≥ 9 to yield an immobihzed compound 4, and (ii) adding a metal compound to the immobihzed compound 4 to yield an immobihzed compound of formula 3. Preferably the compound of formula 4 is immobihzed in monomer form on a protein, a polypeptide, a polymer or activated charcoal. The progress and completion of the immobilization and the complex-formation reaction can be monitored with a spectrophotometer.
Thus a water-soluble immobihzer, for example serum humane albumin (SHA) or polyvinylpyrrolidone (PVP), is added to the reaction mixture in an about equimolar quantity relative to chlorine-e6, either before (route 2) or after (route 1) carrying out the complex-formation reaction.
The fact that compounds of formula 3 can be immobihzed in monomolecular form on the immobihzer is surprising, since monomeric compounds of formula 3 are not particularly stable in aqueous solution. The quantity of the immobihzer required is defined by the number of sites on the molecule to be immobihzed, which is one for compounds of formula 3.
Without wishing to be bound by theory, it is beheved that it is the monomer form of the compounds of formula 3, which is the photoactive form, which may be useful as a phototherapeutic or photodiagnostic agent. However, compounds of formula 3, which have not been immobilized, have a tendency to form aggregates (dimers, trimers and oligo ers of unknown structure) with unpredictable physical, chemical,
photophysical and biological properties, in particular when the compounds of formula 3 are subjected to pHs lower than 9. For example, aggregates of Zn- chlorine-e6 are chemically very stable and attempts to disaggregate the Zn-chlorine- e6 aggregates, for example, by increasing pH, heating, using polar solvents, etc. have failed. Thus the aggregation process is difficult, if not impossible, to reverse. The present invention solves this problem by immobilizing the compounds of formula 3 in monomeric form prior to any aggregation occurring.
The compounds of formula 3 are photosensitizers and therefore useful in pharmaceutical compositions and medicaments for the use in photodynamic therapy. Moreover the photosensitizers of formula 3 can be used as photodiagnostic agents for the identification of areas that are affected by begin or mahgnant cellular hyperprohferation or by neovascularisation.
The pharmaceutical composition or medicament employed in the present invention can be administered by oral, parental (including intravenous, subcutaneous, intramuscular, intradermal, intratracheal, intraperitoneal, intraarticular, intraabdominal, intracranial and epidural), transdermal, airway (aerosol), rectal, vaginal or topical (including buccal, mucosal and subhngual) administration.
For oral administration, the compounds of the invention will generally be provided in the form of tablets, capsules, hard or soft gelatine capsules, caplets, troches or lozenges, as a powder or granules, or as an aqueous solution, suspension or dispersion.
Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose. Corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatine. The lubricating agent, if present, may be magnesium stearate, stearic acid or talc. If
desired, the tablets may be coated with a material, such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Capsules for oral use include hard gelatine capsules in which the active ingredient is mixed with a sohd diluent, and soft gelatine capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil. /- AA — ^^^ _^
Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
For parenteral use, the compounds of the present invention will generally be provided in a sterile aqueous solution or suspension, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride or glucose. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p- hydroxybenzoate. The compounds of the invention may also be presented as liposome formulations.
For topical and transdermal administration, the compounds of the invention will generally be provided in the form of ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters or patches.
Suitable suspensions and solutions can be used in inhalers for airway (aerosol) administration.
In general, a suitable dose will be in the range of 0.01 to 10 mg per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 5 mg per kilogram
body weight per day, more preferably about 2 mg per kilogram body weight per day. The desired dose is preferably presented once a day, but may be dosed as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 1 to 1500 mg, preferably 10 to 1000 mg, and most preferably 20 to 500 mg of active ingredient per unit dosage form.
The invention will now be described with reference to the following examples. It will be appreciated that what follows is by way of example only and that modifications to detail may be made whilst still falling within the scope of the invention.
Synthetic experimental details
Example 1
Ammonia was added to water until the pH of the solution was not less than 9. Then chlorine-e6 (1.0g) was dissolved in the aqueous solution. An equimolar quantity of zinc acetate (0.22g) was added and the reaction mixture was stirred for 15 minutes at about 20°C to achieve the complex-formation reaction. The progress and completion of the reaction was monitored with the help of a spectrophotometer. On completion of the complex- formation reaction, serum humane albumin (SHA) (71g) was added to the reaction mixture as an immobihzer. On completion of the immobilization reaction, which was monitored with a spectrophotometer, the product of the reaction, Zn-chlorine-e6 complex immobihzed on SHA, was purified by dialysis.
Figure 1 shows the long-wave region of the visible absorption spectra of (1) the starting material chlorine-e6 (λmax = 656nm), (2) Zn-chlorine-e6 complex (λmax = 632nm), and (3) Zn-chlorine-e6 complex immobihzed on SHA (λmax = 636nm), all in water.
As can be seen in Figure 1, the formation of the Zn-chlorine-e6 complex is accompanied by a 24nm short-wave shift of the long-wave absorption peak, and the immobihzation of Zn-chlorine-e6 on protein causes a 4nm long-wave shift. Such shifts of the long- ave peak are typical for both complex-formation with metal and immobilization on protein and prove the completeness and purity of the reactions. Moreover, the characteristic absorption peak of chlorine-e6 of medium intensity at λmax = 502nm practically disappears for Zn-chlorine-e6, and instead a weak peak at λmax = 514nm appears, which also demonstrates the completeness and purity of the reaction.
For comparison, Figure 2 shows the visible absorption spectrum of the starting material chlorine-e6 in water down to 350nm. The maxima of the main absorption peaks are at λmax = 402, 502 and 656nm.
Example 2
The synthesis of immobihzed Zn-chlorine-e6 was carried out as described in Example 1, except that as immobihzer polyvinylpyrrolidone (PVP) (62g) was used instead of SHA.
As can be seen in Figure 4, the spectral picture of the visible absorption spectra of (1) the starting material chlorine-e6 (λmax = 656nm), (2) Zn-chlorine-e6 complex (λmax = 632nm), and (3) Zn-chlorine-e6 complex immobilized on PVP (λmax = 638nm) are practically identical to the ones depicted in Figure 1. One observes a significant 24nm short-wave shift of the long-wave peak upon metal complex formation and a small 6nm long-wave shift upon immobihzation on polymer PVP. The medium intensity peak of chlorine-e6 at λmax = 502nm practically disappears, when forming the Zn-chlorine-e6 complex. All of these changes prove the completeness of the reactions and the purity and homogeneity of the products obtained.
Example 3
Ammonia was added to water until the pH of the solution was not less than 9. Then chlorine-e6 (l-Og) was dissolved in the aqueous solution. An equimolar quantity of SHA (71 g) was added and the reaction mixture was stirred for 17 minutes at about 20°C to immobilize chlorine-e6 on SHA. Then an equimolar quantity of zinc acetate (0.22g) was added and the reaction mixture was stirred at room temperature to complex Zn into the chlorine-e6, which was monitored with a spectrophotometer. The product of the reaction, Zn-chlorine-e6 complex immobilized on SHA, was purified by dialysis.
Figure 3 shows the long-wave region of the visible absorption spectra of (1) the starting material chlorine-e6 (λmax = 656nm), (2) chlorine-e6 immobihzed on SHA (λmax = 662nm), and (3) Zn-chlorine-e6 complex immobihzed on SHA (λmax = 636nm). Unhke the first method of synthesis (see Example 1), when forming chlorine-e6 immobihzed on protein, first a 6nm long-wave shift of the absorption peak occurs, and then a 26nm short-wave shift, when forming Zn-chlorine-e6 immobihzed on SHA. Such shifts of the absorption peak agree with the properties of the synthesized products and prove the completeness of the reactions and the purity of the products obtained. Moreover, the medium intensity peak of chlorine- e6 (λmax = 502nm) is observed in the spectra of chlorine-e6 as well as of chlorine-e6 immobihzed on protein, but then it disappears in the spectrum of Zn-chlorine-e6 complex immobihzed on protein and gets transformed into a peak at λmax = 514nm.
Example 4
The synthesis of immobihzed Zn-chlorine-e6 was carried out as described in
Example 3, except that as immobihzer polyvinylpyrrolidone (PVP) (62g) was used instead of SHA.
Figure 5 shows the long-wave region of the visible absorption spectra of (1) the starting material chlorine-e6 (λmax = 656nm), (2) chlorine-e6 immobilized on PVP (λmiX = 662nm), and (3) Zn-chlorine-e6 complex immobihzed on PVP (λma- = 638nm). As in Example 3, when immobilising chlorine-e6 on PVP, a 6nm longwave shift of the absorption peak takes place, and then after introduction of Zn
ions into chlorine-e6 and formation of the Zn-chlorine-e6 complex immobihzed on PVP, a 24nm short-wave shift of the absorption peak occurs. These results demonstrate the completeness of the reactions and the purity of the products obtained. They are also evidenced by the behaviour of the medium intensity peak of chlorine-e6 at λmax = 502nm, which is present in the spectra of chlorine-e6 as well as of chlorine-e6 immobihzed on PVP, but disappears in the spectrum of Zn- chlorine-e6 complex immobihzed on PVP.
The fact that the spectra of the products, synthesised by the two different routes discussed above (route 1 : Examples 1 and 2, route 2: Examples 3 and 4), are identical proves that the conclusions drawn in the final paragraphs of Examples 1 to 4 are correct.
Discussion of further spectra
Figures 6 to 8, with a spectral range of 350-700nm, show visible absorption spectra of Zn-chlorine-e6 complex, Zn-chlorine-e6 complex immobilized on SHA and Zn- chlorine-e6 complex immobihzed on PVP, all in water, respectively. The absorption spectra have main absorption peaks at λmax = 414 and 634nm for Zn-chlorine-e6 complex, λmax = 418 and 636nm for Zn-chlorine-e6 complex immobilized on SHA, and λmax = 416 and 638nm for Zn-chlorine-e6 complex immobihzed on PVP. The conclusions, drawn from these absorption spectra regarding the purity and stability of the monomeric products, were confirmed at every stage of the synthesis with the help of the highly sensitive analytical method of fluorescence spectroscopy (see Figures 9 to 14, discussed below).
Figures 9 and 10 show the fluorescence spectrum and the fluorescence stimulation spectrum of Zn-chlorine-e6 complex in water respectively. The monomeric Zn- chlorine-e6 complex has a characteristic fluorescence spectrum with λmax = 643nm, and a fluorescence stimulation spectrum with main peaks at λmax = 412 and 607nm, i.e. analogous to the peaks observed in the absorption spectrum. This shows that the fluorescence belongs to the monomeric Zn-chlorine-e6 complex and the fluorescence data prove the high purity and homogeneity of the studied product.
Figures 11 and 12 show the fluorescence spectrum and the fluorescence stimulation spectrum of Zn-chlorine-e6 complex immobihzed on SHA in water respectively. The fluorescence spectrum is similar to the fluorescence spectrum of Zn-chlorine- e6 complex in water, though slightly shifted into the red region (λmax = 645nm) and with peaks of a smaller half-width, which demonstrates the great structural similarity between the centres of Zn-chlorine-e6 complex and Zn-chlorine-e6 complex immobilized on SHA observed in these spectra. The fluorescence stimulation spectrum of Zn-chlorine-e6 complex immobihzed on SHA, shown in Figure 12, is very similar to its absorption spectrum shown in Figure 7 and shows two main peaks at λmax = 446 and 673nm with a smaller half-width and a more regular shape compared to the peaks in the absorption spectrum. This proves that the fluorescence belongs to monomeric Zn-chlorine-e6 complex immobihzed on SHA and that the studied product has a high homogeneity and purity.
Figures 13 and 14 show the fluorescence spectrum and the fluorescence stimulation spectrum of Zn-chlorine-e6 complex immobihzed on PVP in water respectively. The shape of the fluorescence spectrum is very similar to the fluorescence spectra discussed above and has a peak at λmax = 645nm as in the spectrum of Zn-chlorine- e6 complex immobihzed on SHA. The fluorescence stimulation spectrum has main peaks at λmax = 429 and 727nm, which agrees with its absorption spectrum and shows that the fluorescence belongs to Zn-chlorine-e6 complex immobihzed on PVP and that the product is highly pure.
Figures 15 and 16 show the fluorescence spectrum and the fluorescence stimulation spectrum of a biological sample taken from the liquid above the sediment of an ascite tumour taken from an experimental animal (mouse), which had previously been injected intraabdominally with a preparation comprising Zn-chlorine-e6 complex immobihzed on SHA. As can be seen by comparing the spectra of the biological sample shown in Figures 15 and 16 with the corresponding spectra of the models shown in Figures 9 to 14, the peaks in the spectra of the biological sample occur at similar λmax (fluorescence spectrum in Figure 15: λmax = 645nm; fluorescence stimulation spectrum in Figure 16: λmax = 418 and 641nm) and have a
similar peak shape and peak intensity ratio as the peaks in the spectra of the models. This means that the preparation injected into the experimental animal did not undergo substantial structural changes and comprises Zn-chlorine-e6 with a high structural homogeneity of the absorbing and fluorescent centre as was observed for Zn-chlorine-e6 complex immobihzed on SHA.
Example 5
Cd-chlorine-e6 complex immobihzed on PVP was synthesized in a similar way to Zn-chlorine-e6 complex immobihzed on PVP (see Example 4). Figure 17 shows the long-wave part of the visible absorption spectra of (1) the starting material chlorine- e6 (λ maχ = 656nm), (2) chlorine-e6 immobihzed on PNP (λmax = 662nm), and (3) Cd- chlorine-e6 complex immobihzed on PVP (λmax = 646nm). Figure 18 shows the absorption spectrum in the range of 350-750nm of the monomer form of Cd- chlorine-e6 complex immobihzed on PVP in water. As can be seen in Figure 18, the spectrum of Cd-chlorine-e6 complex immobihzed on PVP in monomer form has two main peaks at λmax = 424 and 646nm respectively.
Example 6
Cd-chlorine-e6 complex immobihzed on PVP was synthesized in a similar way to Zn-chlorine-e6 complex immobihzed on PVP (see Example 4). Figure 19 shows the long-wave part of the visible absorption spectra of (1) the starting material chlorine- e6 (λmax = 656nm), (2) chlorine-e6 immobilized on PVP (λmax = 662nm), and (3) Cu- chlorine-e6 complex immobihzed on PVP (λmax = 636nm). Figure 20 shows the absorption spectrum in the range of 350-750nm of the monomer form of Cu- chlorine-e6 complex immobihzed on PVP in water. As can be seen in Figure 20, the absorption spectrum of Cu-chlorine-e6 complex immobihzed on PVP in monomer form differs from the monomer spectra of Zn-chlorine-e6 immobilized complex and Cd-chlorine-e6 immobihzed complex. The absorption spectrum of Cu-chlorine-e6 complex immobihzed on PVP in monomer form has three main peaks at λmils = 410, 505 and 636nm respectively.
Preclinical pharmacokinetic studies
A remarkable and important feature of the immobihzed monomer Zn-chlorine-e6 complex is the possibility of preparing a stable form of the monomeric Zn-chlorine- e6 complex at a pH of from 6 to 8.5, which is required for injection usage. Zn- chlorine-e6 complex preparations suitable for injection may be prepared by acidifying the reaction medium after completion of the synthesis. Pharmaceutically acceptable additives, which do not interfere with the structural stability of the Zn- chlorine-e6 complex and the homogeneity of the preparation, may be added to such preparations suitable for injection.
The pharmacokinetic distribution of Zn-chlorine-e6 complex immobihzed on SHA over 30 hours in organs, tissues, biological liquids and tumours (embryocarcinoma) was studied. Female mice of the line Balb/c weighing 20-21 g were used as experimental animals. The pharmacokinetic studies were carried out using a Perkin- El er spectrofluorimeter on homogetates of organs and tumours, taken after the intraabdominal injection of Zn-chIorine-e6 complex immobihzed on SHA at a dose of 25 g/kg weight. The results of these pharmacokinetic distribution studies are depicted in Figure 21 and summarised in Table 1 below.
A. Intraabdominal injection of Zn-chlorine-e6 complex immobihzed on SHA at a dose of 25 mg/kg weight was well endured by the animals without any signs of toxicity and did not affect their behavioural reaction, both immediately and 30 hours after the injection.
B. The immobihzed complex was rapidly absorbed from the abdominal cavity into the blood and was deposited in the hver during the first hours after injection. Its content in the hver tissues was 10-14 times higher than its level in the blood.
C. A significant quantity of the immobihzed complex was also accumulated in the kidneys in the first 12 hours after injection (only 2-2.5 times less than in the hver), however, the immobilized complex was practically absent from the urine. During the next 18 hours, the immobihzed complex was washed out intensely from the kidney tissue into the blood. The kidneys' secretion function was not affected during the whole observation period.
D. The maximum concentrations of the immobihzed complex in the hver were found during the first 8 hours after injection. During the next 24 hours, the surplus of the immobihzed complex was discharged intensely into the small intestines. The dynamics of the distribution curves of the liver and small intestines correlate precisely with one another. It may be sufficient to inject 5-10 times smaller doses of the immobihzed complex in order to achieve maximum concentrations in the tumour.
E. 5-8 times less of the immobihzed complex accumulated in the spleen and the lungs compared to the hver or tumour, and 24 hours after injection the spleen and lungs had phone readings.
F. Skin and muscle tissue both had practically the same accumulation dynamics, the only difference being that the immobihzed complex content in the muscle was
1.5 times higher in the first 15 hours than the immobihzed complex content in the skin.
G. The accumulation of the immobihzed complex in tumour increased progressively from the moment of injection and reached its maximum 15 hours after injection. The maximum concentration plateau (12-20 hours) was found to be much longer than after chlorine-e6 injection, and after an insignificant fall by the end of the first 24 hours, a second increase of immobihzed complex concentration up to the maximum readings of the concentration plateau was observed between 24 and 30 hours. A "scissors" effect (the immobihzed complex concentration in the tumour is increasing, while the immobihzed complex concentration in the hver is decreasing) was observed twice for the hver and tumour, once 12 hours after injection and once, even more pronounced, 24 hours after injection.
To summarise, after the absorption of the immobihzed complex in the abdominal cavity, redistribution from the blood into the organs and washing out of the immobihzed complex surplus by the hver during the first 24 hours, the immobihzed complex accumulated in the tumour tissue in a concentration of 2.5 times greater than in the hver and 6 times greater than in the skin, muscle and other parenchymal organs. In comparison with the pharmacokinetics of the dimer form, the monomer form demonstrated much greater tumour selectivity and stability of the chemical structure in tissues.
The spectroscopic data (see Figures 1 to 20) and pharmacokinetic data (see Figure 21) discussed above show that the immobihzed preparations preserve the monomeric structure, purity and chemical stability of the porphyrin nucleus of the Zn-chlorine-e6 complex.
Definition of acute toxicity parameters:
To define parameters LD]0 and LD50 three preparation doses were chosen (100, 125 and 150 mg/kg weight) for single intraabdominal injection. Chlorine-e6 readings
were taken as a prototype, where LD,0 is 119 mg/kg weight and LD50 is 160 mg/kg weight.
After injection of Zn-chlorine-e6 complex immobihzed on SHA in the above stated doses, a first reaction to the injection was observed only with the third animal group (150 mg/kg weight), because the preparation was injected in 3ml physiological solution, which caused temporal animal stillness due to abdominal swelling. After the absorption of the surplus hquid, however, these animals did not differ from the animals in the other two groups in their behavioural reactions (moving activity, defence reflex, food reflex, coat condition).
During the following 72 hours, signs of acute toxicity (slow reaction, hollow sides, diarrhoea, defence and food reflex absence) did not appear. The animals were kept under observation for a further fortnight.
Further tests were carried out similarly with intraabdominal injections of 175, 200 and 225 mg/kg weight, as well as 300, 350 and 450 mg/kg weight. None of these concentrations proved toxic.
It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope of the invention, which is defined by the following claims.