HK1160848B - Process for preparing chlorins and their pharmaceutical uses - Google Patents

Description

Method for preparing chlorins and their medical use

Technical Field

The present invention relates to methods of preparation, properties, pharmaceutical compositions and therapeutic uses of sulfonated (sulfonated) chlorins and bacteriochlorins designed for photodynamic therapy (PDT) of hyperproliferative (hyperproliferative) tissues such as tumors, hyperproliferative blood vessels and other disorders or abnormalities responsive to photodynamic therapy. In particular, the present invention relates to a novel method enabling large-scale chemical synthesis of stable chlorins and bacteriochlorins, characterized in that no solvents or bases are used. In another embodiment, pharmaceutical compositions and methods for therapeutic use for systemic administration are provided. In another embodiment, pharmaceutical compositions for therapeutic use and methods of treatment for topical administration are also provided. Also provided are methods of detecting hyperproliferative tissue, such as tumors, using photodynamic methods or MRI.

I. Background of the invention

I.a. prior art

When injected into organisms, various tetrapyrrole macrocycles such as purpurins, chlorins, bacteriochlorins, phthalocyanines, benzo chlorins have shown the following capabilities: preferentially concentrated in hyperproliferative tissue, absorbs light and responds to light to form an active state. These macrocycles then exhibit cytotoxic effects on the cells or other tissues in which they are concentrated when irradiated at the appropriate wavelength. In addition, these compounds also cause energy to be emitted from the tissue, which can be used to detect their location.

In PDT, the patient is injected with a photosensitizer (usually about 0.1 to about 10mg/kg body weight) which shows some selectivity for photodamage to tumor tissue, followed by a period of time after which visible or near infrared light (about 50-200J/cm)²) The tumor region is irradiated. The photosensitizer absorbs light and fluoresces, either to react with substrate molecules through an electron or hydrogen transfer reaction (type I process), or to transfer its energy to ground-state molecular oxygen to produce singlet oxygen O which attacks the tissue₂(¹Δ_g) (type II process). The major contributor to type I processes is the formation of superoxide (O) by electron transfer from the photosensitizer of the electrical stimulation₂ ^-). Evidence suggests that type II photooxygenation processes are more favorable than type I processes in cells [1, 2]However, it is also believed that PDT response is increased when superoxide is also produced [3]. At the time of detection, fluorescence is determined when exposed to light of the desired wavelength, which requires less energy than when used for treatment. Adequate treatment generally requires the formation of high yields of singlet oxygen in the tissue, which may have a synergistic effect with concomitantly formed superoxide or other reactive oxygen species.

Properties of the best sensitizers (sensitizers) for PDT treatment include: (i) the synthesis is simple, effective and economical; (ii) stable, pure and long storage time; (iii) soluble in a biocompatible solvent or vehicle (vehicle); (iv) high extinction coefficient in the "light therapy window" (600-900 nm); (v) singlet molecular oxygen sensitization and/or superoxide generation with high quantum yield; (vi) low or no dark toxicity; (vii) selectively accumulate and remain in tumor tissue for a longer time; (viii) low skin photosensitization upon systemic administration; (ix) the photobleaching is controllable; (x) It is easy to metabolize or excrete after treatment. Sensitizers are only precursors to cytotoxic substances, particularly singlet oxygen and other reactive oxygen species such as superoxide ion. The direct precursor of singlet oxygen and the usual direct precursor of superoxide are triplet sensitizers. Therefore, high quantum yields of singlet oxygen require at least three sensitizer triplet state properties: (i) nearly uniform quantum yield, (ii) electron energy at least 20kJ/mol higher than singlet oxygen (94kJ/mol), and (iii) long lifetime (hundreds of microseconds). The addition of specific mediators can enhance accumulation and retention in tumor tissue, but the intrinsic property of the sensitizer that is relevant for these purposes is the hydrophilicity/lipophilicity of the sensitizer, and the ability to meet these properties to achieve the desired target is the most popular property.

The lower limit of the phototherapeutic window is determined by the presence of heme proteins, which account for the majority of the light absorption in the visible region of the tissue. Below 550nm, the penetration of light in tissue decreases rapidly. However, the penetration at 550-630nm is significantly enhanced, and the penetration is multiplied by 700 nm. Tissue penetration is then enhanced by 10% as the wavelength is shifted to 800 nm. The upper limit of the phototherapeutic window is determined by the infrared radiation absorption of water and the energy requirement for efficient energy transfer to oxygen. Indeed, diffusion-controlled triplet energy transfer from sensitizer to molecular oxygen requires that the triplet energy of sensitizer is at least 115 kJ/mol. Furthermore, the singlet-triplet energy split of the tetrapyrrole macrocycle is about 40kJ/mol [4], which requires that the sensitizer must have a singlet energy of more than 150 kJ/mol. Given that the Stokes shift of these sensitizers is generally small, they should only absorb light below 800 nm. The conclusion is thus: the ideal sensitizer must strongly absorb light having a wavelength of about 750 nm. The strong absorption of bacteriochlorins at this wavelength makes them ideal candidates for PDT sensitizers. Chlorins are also suitable candidates for PDT in applications where light penetration is not a critical factor.

Photosensitive elementIs a kind of bloodPorphyrin derivative [ 5]]It is the most widely used photosensitizer and has been approved for the treatment of various solid tumors [6]. Hematoporphyrin derivative (HpD) is prepared by mixing hematoporphyrin with glacial acetic acid and sulfuric acid, followed by hydrolysis and precipitation under acidic conditions. Lipson et al [ 7]]The method is described in part. The HpD thus prepared is composed of a complex mixture of porphyrins (porphyrin). When the HpD is separated into its two major fractions by gel filtration using Sephadex LH-20, the higher molecular weight fraction is called the photosensitizerIt is a more potent PDT agent [8]. Photosensitive elementThe recommended dose for the human being is 1-2mg/kg body weight. Photosensitive elementThe main components of (A) are dimers and higher oligomers linked by ether, ester, and possibly carbon-carbon bonds [ 9]]。

Photosensitive elementHas several desirable properties including good efficacy, water solubility, reasonable singlet oxygen yield, and ease of manufacture. However, the light-sensitive elementIt also has some disadvantageous properties: (i) it is a porphyrin dimer and higher oligomers connected by ether, ester and/or carbon-carbon bonds; (ii) it shows skin phototoxicity in patients 4-6 weeks after administration; (iii) the difficulty of light penetration through tissue limits the photosensitizers due to their relatively weak absorption in the red region (630nm)In current clinical applications of PDT for destruction of cancerous tissue located less than 4mm of the light source used in the treatment. Thus, there is a need for more effective, chemically pure compositionsLess phototoxic, more concentrated sensitizers which absorb light more strongly and in the infrared region.

It is known that chemical reduction of one of the tetrapyrrole rings, which is equivalent to the conversion of a porphyrin to a chlorin, results in a further shift of the longest wavelength absorption band to the red region with an enhanced extinction coefficient. These properties were studied in a second generation of PDT photosensitizers under the trade name Foscan5, 10, 15, 20-tetrakis (3-hydroxyphenyl) chlorin (m-THPC) is one of the most effective second generation photosensitizers [10]. Further reduction of the opposite pyrrole ring, equivalent to the conversion of chlorins to bacteriochlorins, leads to a shift of the absorption band into the infrared region with an additional enhancement of the extinction coefficient. However, until recently, it was generally accepted that bacteriochlorins were very unstable compounds [10 ]]Studies on PDT sensitizers have focused on chlorins [11]. It was later shown that it was indeed possible to synthesize stable bacteriochlorin [12 ]]. However, this is not fully recognised in the scientific literature, where it is required that the synthesis of stable bacteriochlorins using this route is limited to the preparation of bacteriochlorins with inert functional groups [13 ]]. Bacteriochlorins with other functional groups have been prepared (see PCT/EP2005/012212, WO/2006/053707).

Significant interest in bacteriochlorins as PDT sensitizers and reports of photosensitizers that some naturally occurring bacteriochlorins are effective in vitro and in vivo [14, 15], inspiring much effort to synthesize bacteriochlorins. Synthetic bacteriochlorins have been prepared as follows: derivatization of the corresponding porphyrins via 1, 3-dipolar cycloaddition [20] by ortho-dihydroxylation with osmium tetroxide [16], intramolecular cyclization [17], Diels-Alder reaction using porphyrins as dienophiles [18] or Diels-Alder reaction using vinyl porphyrins in which the porphyrin is a diene [19], can also be by self-condensation of dehydromethylidene dipyrrole (dipyrrin) -acetal derivatives [21 ]. Furthermore, Whitlock's classical method of preparing bacteriochlorins studied decades ago, involves the reduction of the 7, 8-17, 18-pyrrolylporphyrin site with diimide [22 ]. This is the method used by Bonnet for the synthesis of Foscan and 5, 10, 15, 20-tetrakis (3-hydroxyphenyl) bacteriochlorin [23 ]. Meanwhile, very extensive studies on the synthesis of bacteriochlorin derivatives have resulted in numerous patents based on the above-mentioned methods (see, for example, US2007/7,166,719; US2003/6,624,187; US2003/6,569,846; US2002/6,376,483; US1999/5,864,035; US1998/5,831,088; WO 90/12573; WO 94/00118; WO 95/32206; WO 96/13504; WO 97/32885; US2006/194,960).

Some newly synthesized bacteriochlorins have negligible dark toxicity and high tumor selectivity, are partially soluble in water, and have significant absorption bands in the range of 700nm-800 nm. However, there are some drawbacks, namely: (i) complex and expensive synthesis, including very difficult purification; (ii) limited water solubility, which in the case of systemic application can lead to dissolution in organic solvents, creating an additional chemical burden on the organism, or in combination with a vehicle, increasing the cost of the treatment; (iii) chemical instability, particularly in the presence of light; (iv) the singlet oxygen quantum yield is low or unknown. An interesting representative of this third generation photosensitizer is palladium-bacterial pheophorbide (bacteriopheophorbide), now known as TookadIt has been approved for phase III clinical studies. TookadIs derived from bacteriochlorophyll, and as with most natural bacteriochlorophylls, is particularly sensitive to oxygen, which results in rapid oxidation to the chlorin state, which has a maximum absorption at 660nm or below. Furthermore, if the laser is used to stimulate bacteriochlorin in vivo, the oxidation will lead to the formation of new chromophores which absorb outside the laser window, which will reduce the photosensitizing effect. Photochemical degradation of this family of compounds was measured in TX-100/PBS with 778nm (13mW) illumination, showing that 90% of the compounds irreversibly disappeared within 5 minutes (4J) with concomitant growth of chlorin band at 660nm [ 3J ]]。

PDT has been extensively tested for the treatment of skin diseases, i.e. actinic keratosis, squamous cell carcinoma, Bowen's disease (intraepithelial squamous cell carcinoma), basal cell carcinoma, but there is little information on malignant melanoma [24 ]. When visible light is used, the high pigmentation of melanoma tissue limits the effectiveness of PDT because melanin reduces the penetration of light with wavelengths less than 700 nm. There have also been reports of the use of topical PDT in non-neoplastic diseases such as psoriasis. Earlier studies used hematoporphyrin derivatives [25] and meso-tetraphenylporphyrin sulfonic acid tetrasodium salt [26] in liquid formulations containing transdermal penetration enhancers. Typically, however, when HpD or other porphyrins are applied topically in a (liquid, gel, cream, emulsion, etc.) formulation (containing a vehicle that enhances the diffusion of HpD or other porphyrins through tissue), the penetration enhancer will have a tendency to become porphyrin retentive as normal tissue fluids dilute. In these cases, the porphyrins no longer diffuse through the tissue (even remaining in solution). Thus, topical application of porphyrins is often associated with a loss of specificity for malignant tissue, and persistent photosensitization of normal tissue in the vicinity of the site of application occurs due to local concentration of porphyrins.

To overcome these problems, it has been proposed: without the topical application of porphyrins, it is advantageous to use the following agents: it is not a photosensitizer per se, but it induces the synthesis of endogenous porphyrins in vivo, namely protoporphyrin-IX (PpIX) [27 ]]. 5-amino-4-oxopentanoic acid, also known as 5-aminopentanoic acid (or ALA), is known to be a biological precursor of protoporphyrin IX. Excess ALA leads to a biological accumulation of PpIX, which is a true photosensitizer. Thus, by applying ALA topically to a skin tumour and then exposing the tumour to light after a few hours, a beneficial phototherapeutic effect can be obtained (see, e.g., WO 91/01727). Since the skin covering basal cell carcinoma and squamous cell carcinoma is more easily penetrated by ALA than healthy skin, and since PpIX biosynthesis is more efficient in skin tumors, it was found that topical application of ALA results in a selective increase in PpIX production in tumors. This has been the basis for many dermatological preparations of ALA or some of its derivatives, which have been approved and used clinicallyMost notably LevulanAnd Metvix

However, while the use of ALA represents a significant advance in the art, photodynamic therapy with ALA is not entirely satisfactory. In ALA-PDT, patients repeatedly reported the development of pain [28 ]. ALA is a prodrug, and the efficiency of drug production varies with biosynthesis in a subject. Only a very limited amount of PpIX may be biosynthesized by the cell. It is also unstable in pharmaceutical formulations. It does not penetrate all tumors and other tissues with sufficient efficiency to treat a wide range of tumors or other diseases. It is preferred that the wavelength of the light-sensitizing light is about 635nm, while it shows that only 1-10% of the incident red light (600-700nm) can pass through a 1cm thick section of human tissue. There is therefore a need for improved photodynamic therapeutic agents for topical application.

Summary of the invention

According to a first aspect, the present invention provides a process for the preparation of a chlorin or bacteriochlorin derivative having the formula:

formula (I)

Wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X¹，X²，X³，X⁴，X⁵，X⁶，X⁷，X⁸each independently selected from halogen (F, C)l, Br) and hydrogen atoms, with the proviso that all X²，X⁴，X⁶And X⁸Or all of X¹，X³，X⁵And X⁷All are halogen, or all X are halogen;

R₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈independently selected from H, -OH and-SO₂R, wherein each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms;

R₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈independently selected from H, -OH and-SO₂R, wherein each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms;

y is fluorine or hydrogen;

comprises the following steps:

(i) solid state reduction of the corresponding substituted porphyrin to the chlorin derivative or bacteriochlorin derivative using a hydrazide in the absence of a solvent and optionally in the absence of a base; wherein the corresponding substituted porphyrin has the formula:

formula (II).

Thus, the compound of formula (I) may be a chlorin derivative having the formula:

formula (V).

Alternatively, the compound of formula (I) may be a bacteriochlorin derivative having the formula

Formula (VI).

Suitably, X²，X⁴，X⁶，X⁸Each independently selected from halogen (F, Cl, Br).

Suitably, R₅，R₆，R₇，R₈Is H.

Suitably, Y is H.

Suitably, R₁，R₂，R₃，R₄is-SO₂R, wherein each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms.

In another aspect, X²，X⁴，X⁶，X⁸Each independently selected from halogen (F, Cl, Br);

R₅，R₆，R₇，R₈is H;

y is H; and

R₁，R₂，R₃，R₄is-SO₂R, wherein each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms.

In another aspect, the present invention provides a method for preparing a chlorin or bacteriochlorin derivative having the formula:

formula (III)

Wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X²selected from halogen (F, Cl, Br), X¹Selected from hydrogen or halogen (F, Cl, Br); and

r' is-SO₂R, wherein each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms,

comprises the following steps:

(i) solid state reduction of the corresponding substituted porphyrin to the chlorin derivative or bacteriochlorin derivative using a hydrazide in the absence of a solvent and optionally in the absence of a base; wherein the corresponding substituted porphyrin has the formula:

formula (IV).

Thus, the compound of formula (III) may be a chlorin derivative having the formula:

formula (VII).

Alternatively, the compound of formula (III) may be a bacteriochlorin derivative having the formula

Formula (VIII).

In another aspect, R' is-SO₂R, wherein R is-Cl for the corresponding substituted porphyrin of formula (IV); and the method comprises the following further steps:

(ii) reacting chlorin or bacteriochlorin derivatives with amines H-NHRⁿOr H-NR₂ ⁿ(ii) a Amino acid or wherein RⁿAlcohols H-OR being alkyl of 1 to 12 carbon atomsⁿCombining;

to provide a chlorin or bacteriochlorin derivative, wherein R' is-SO₂R, wherein R is-amino acid, -ORⁿ、-NHRⁿor-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms.

In another aspect, the present invention provides a pharmaceutical composition comprising:

(a) a chlorin or bacteriochlorin derivative having the formula:

formula (III)

Or a pharmaceutically acceptable derivative of the composition thereof,

wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X²selected from halogen (F, Cl, Br), X¹Selected from hydrogen or halogen (F, Cl, Br), and R' is-SO₂R；

Each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms,

wherein the chlorin or bacteriochlorin derivative is effective in photodynamic therapy for ameliorating the symptoms of a hyperproliferative disease; and

(b) a surface penetration enhancer.

In another aspect, the present invention provides the use of a chlorin or bacteriochlorin derivative, or a pharmaceutically acceptable composition derivative thereof, for detecting hyperproliferative tissue;

wherein the chlorin or bacteriochlorin derivative has the formula:

formula (III)

Wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X²selected from halogen (F, Cl, Br), X¹Selected from hydrogen or halogen (F, Cl, Br), and R' is-SO₂R；

Each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms.

In another aspect of the invention, there is provided a method of detecting the presence of hyperproliferative tissue in a subject, comprising:

(i) administering to said subject a diagnostically sufficient amount of a chlorin or bacteriochlorin derivative having the formula

Formula (III)

Wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X²selected from halogen (F, Cl, Br), X¹Selected from hydrogen or halogen (F, Cl, Br), and R' is-SO₂R；

Each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms,

or a pharmaceutically acceptable derivative of the composition thereof,

(ii) allowing sufficient time for the chlorin or bacteriochlorin derivative to bind to the target site and for any chlorin or bacteriochlorin derivative that does not preferentially bind to the target tissue to be cleared from non-target tissues, and

(iii) imaging the compound in the patient.

The imaging step may be accomplished by generating an MRI image of at least a portion of the patient's body.

Alternatively, the step of visualizing may be accomplished by exposing the compound to light of sufficient energy to cause the compound to fluoresce.

In another aspect of the invention, there is provided a pharmaceutically acceptable composition for use in the treatment of skin cancer or a skin disease selected from actinic keratosis, squamous cell carcinoma, bowen's disease, basal cell carcinoma, psoriasis, acne vulgaris and rosacea;

wherein the composition comprises:

(i) a chlorin or bacteriochlorin derivative having the formula:

formula (III)

Wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X²selected from halogen (F, Cl, Br), X¹Selected from hydrogen or halogen (F, Cl, Br), and R' is-SO₂R；

Each R is independently selected from-Cl, -OH, -amino acid, -ORⁿ、-NHRⁿand-NR₂ ⁿWherein R isⁿIs an alkyl group of 1 to 12 carbon atoms;

and

(ii) a pharmaceutically acceptable vehicle for intradermal or transdermal delivery of the compound, wherein the vehicle comprises a surface penetration enhancer that transiently penetrates the skin and facilitates penetration of the compound through various skin layers;

wherein

(a) Administering the composition to a subject;

(b) allowing sufficient time for the chlorin or bacteriochlorin derivative to preferentially concentrate near the target site of dermatological treatment; and

(c) illuminating the target to obtain the desired response to the skin cancer or skin disease.

Process for preparing derivatives

Suitably, the hydrazine is p-toluenesulfonyl hydrazide, 4-chlorobenzenesulfonic hydrazide (4-chlorobenzenesulfonic hydrazide), 4' -oxybis (benzenesulfonyl) hydrazide, benzenesulfonyl hydrazide, 4-methoxybenzenesulfonyl hydrazide or benzoyloxy hydrazide (benzoic hydrazide).

Solid state reactions require the use of a temperature above the melting point of one of the reactants to partially dissolve or disperse the other reactant or reactants into the melted reactant. For solid state reactions between the hydrazide and the porphyrin derivative, the solid state reaction is suitably carried out above the melting point of the hydrazide.

Suitably, the reduction step is carried out at a temperature of at least 70 ℃. Suitably, the reduction step is carried out at a temperature of at least 100 ℃. In another aspect, the reducing step is carried out at a temperature of 70-200 ℃. Suitably, the reduction step is carried out for at least 5 minutes.

Suitably, the reduction step is carried out under vacuum or an inert atmosphere.

Pharmaceutical composition

Suitably, the pharmaceutical composition comprises at least 0.01 wt% chlorin or bacteriochlorin derivative, or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. Suitably, the pharmaceutical composition comprises from 0.01% to 30% by weight of chlorin or bacteriochlorin derivative, or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. Suitably, the pharmaceutical composition comprises from 0.01% to 10% by weight of chlorin or bacteriochlorin derivative, or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. Suitably, the pharmaceutical composition comprises from 0.1% to 1% by weight of chlorins or bacteriochlorins or pharmaceutically acceptable salt derivatives thereof, based on the total weight of the composition.

When a surface penetration enhancer is present in the pharmaceutical composition, the composition suitably comprises from 0.05 to 10% by weight of the surface penetration enhancer, based on the total weight of the composition. Suitably, the composition comprises from 0.1 to 10 wt% of a surface penetration enhancer. Suitably, the surface penetration enhancer may be selected from dimethyl sulphoxide and other dialkyl sulphoxides, N-methyl formamide, dimethyl acetamide, glycols, various pyrrolidone derivatives and various 1-substituted azacycloalkane (cycloalkakan) -2-ones.

Suitable glycols may be selected from polyethylene glycol, polypropylene glycol 425, propylene glycol and propylene glycol monolaurate.

Suitable pyrrolidone derivatives may be selected from N-dodecylpyrrolidine-3, 5-dione, N-dodecylpyrrolidine-2-thione, N-dodecyl-2-pyrrolidone, N- (2-hydroxyethyl) -2-pyrrolidone, N-cyclohexyl-2-pyrrolidone, 1-butyl-3-dodecyl-2-pyrrolidone, 1, 5-dimethyl-2-pyrrolidone, 1-ethyl-2-pyrrolidone, 1-hexyl-4-methyloxycarbonyl-2-pyrrolidone, 1-hexyl-2-pyrrolidone, 1- (2-hydroxyethyl) pyrrolidone, 3-hydroxy-N-methyl-2-pyrrolidone, 1-lauryl-4-methyloxycarbonyl-2-pyrrolidone, and N-methyl-2-pyrrolidone.

Suitable 1-substituted azacycloalkane-2-ones include 1-dodecylazacycloheptan-2-one, hereinafter referred to as AzoneDisclosed in U.S. patent nos. 4,562,075, 4,405,616, 4,326,893 and 3,989,816.

Detection of hyperproliferative tissue

When chlorins and bacteriochlorin derivatives or pharmaceutically acceptable salts thereof are used for detecting hyperproliferative tissue, suitably, the hyperproliferative tissue may be selected from vascular endothelial tissue, neovascular tissue present in the eye, abnormal vascular walls of tumors, solid tumors, tumors of the head, tumors of the neck, tumors of the eye, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lung, non-solid tumors, malignant tumors of one of hematopoietic and lymphoid tissues, lesions of the vascular system, diseased bone marrow, and diseased cells in which the disease is one of an autoimmune and inflammatory disease.

Treatment of hyperproliferative diseases

In another aspect, the present invention provides the use of a chlorin or bacteriochlorin derivative, or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament for the treatment of a hyperproliferative disease.

When the chlorin or bacteriochlorin derivative or a pharmaceutically acceptable salt thereof is used for the treatment of a hyperproliferative disease, suitably said hyperproliferative disease is selected from cancer (cancer) or carcinoma (carcinomas), myeloma, psoriasis, macular degeneration. Suitable examples are stomach cancer, intestinal cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcoma, liver cancer, bladder cancer, maxillary cancer, cholangiocarcinoma, head and neck cancer, tongue cancer, brain tumor, skin cancer, malignant goiter, prostate cancer, colorectal cancer, parotid cancer, hodgkin's disease, multiple myeloma, kidney cancer, leukemia and malignant lymphomas.

Suitably, the treatments comprise irradiating the chlorin or bacteriochlorin derivative or a pharmaceutically acceptable composition derivative thereof with light having a wavelength matching the absorption band of the chlorin or bacteriochlorin derivative. Suitably, the wavelength of light is 600-800 nm. Suitably, when chlorins are used, the wavelength of light is 630-. Suitably, when bacteriochlorin is used, the wavelength of light is 720-780 nm.

Suitably, the light dose is 1 to 250J/cm². In some aspects, suitably, the light dose is less than 50J/cm²Less than 20J/cm²Less than 10J/cm²。

Suitably, the dose of chlorin or bacteriochlorin derivative, or a pharmaceutically acceptable salt thereof, is 0.01mg-200mg/kg body weight per day. Suitably, the dose is from 0.01mg to 100mg per kg body weight per day.

The present invention has been made in view of the above-mentioned prior art. The synthesis described in WO2006/053707(PCT/EP2005/012212) comprises only three essentially quantitative steps: (i) functionalizing a halogenated tetraphenylporphyrin via chlorosulfonylation of a benzene ring; (ii) synthesizing an amphiphilic compound by reacting chlorosulfonic acid group with nucleophilic substance water, namely amines or alcohols; iii) reducing the tetrapyrrolic macrocycle with a hydrazide derivative in the presence of an inorganic or organic non-nucleophilic base. Then, as shown in fig. 2 of patent WO2006/053707, the synthesis of halosulfonated bacteriochlorins by this method is accompanied by contamination of the analog chlorin, and purification requires very difficult operations. It is an object of the present invention to provide an economical, environmentally friendly, large scale synthesis of pure, stable and functionalized tetraphenylchlorins and tetraphenylchlorins wherein the compounds have electron withdrawing groups in the ortho position of the phenyl ring. It is also an object of the present invention to provide chemical and therapeutic properties of said chlorins and bacteriochlorins, methods of treatment and pharmaceutical compositions comprising these molecules, as well as evidence of their effectiveness in PDT.

Halogenated sulphonated bacteriochlorins have unique properties that make them preferred photosensitizers for PDT:

1) the presence of halogen atoms in the ortho position to the phenyl group serves three functions. First, they produce a controlled "heavy atom effect" that enhances the yield of sensitizer triplet states without compromising the triplet survival time and the ability to efficiently transfer electron energy to molecular oxygen [29 ]. Secondly, they stabilize the reduced state of the tetrapyrrole macrocycle by electronic and steric effects. Again, they accelerate the rate constant of energy transfer to molecular oxygen through charge transfer interactions, resulting in the production of large quantities of singlet oxygen, superoxide and other reactive oxygen species.

2) The presence of a sulfonic acid group in the meta position of the phenyl group serves two functions. First, a means to modulate the hydrophilicity/lipophilicity of the sensitizer is provided, since the very hydrophobic sensitizer appears to be less phototoxic, probably due to low water solubility and less ability to reconfigure from plasma membrane to other intracellular compartments, whereas the very hydrophilic dye may be concentrated mainly in the tumor stroma and have a lower PDT efficacy [30 ]. Secondly, sulfonic acid groups, especially larger or longer substituents, when attached thereto provide additional steric protection for the dye's bacteriochlorin nucleus against oxidation.

3) The simultaneous presence of the ortho halogen atom of the phenyl group and the meta sulfonic acid group performs an additional function. Molecular modeling and experimental data indicate that when limited rotation of a single bond exists at the meso position of a 5, 10, 15, 20-tetraphenylporphyrin having an asymmetric benzene ring, geometric isomers (called atropisomers) result from different positions of ortho and/or meta substituents relative to the porphyrin plane [31 ]]. The atropisomers have significantly different polarities and extinction coefficients of the longest wavelength absorption bands, which can differ by nearly an order of magnitude. In particular, alpha with 4 sulfonamide substituents on the same side of the porphyrin plane₄The isomer has the highest extinction coefficient and is the most amphiphilic of these atropisomers.

The widespread use of sulfonated chlorins and bacteriochlorins in PDT requires an economical and environmentally friendly synthetic method that can be implemented on an industrial scale. The central object of the present invention is to provide a new process for the preparation of these compounds based solely on the precursors porphyrin and hydrazide, wherein the hydrazide is added in the solid state, heated above the melting point in a sealed reaction vessel, in the absence of oxygen and base, and after a period of time the desired product is obtained.

It is an object of the present invention to provide a PDT method for topical application of said sulfonated chlorins or bacteriochlorins with a suitable vehicle. Vehicles for topical administration of photosensitizers may take various forms, including liquid solutions, gels, creams, emulsions, ointments and the like. Typically, the formulation of these vehicles includes at least one surface penetration enhancer. Unlike the common general knowledge in the art [32] that drugs with molecular weights above 500 daltons do not penetrate skin well, we provide formulations for effective intradermal delivery of the sulfonated chlorins or bacteriochlorins to skin diseases, where the molecules reach molecular weights slightly above 1 kD.

The present invention relates to compounds for the treatment and detection of hyperproliferative tissues, such as tumors, using photodynamic methods. The compounds of the present invention may also be used to treat dermatological diseases such as psoriasis, acne vulgaris and rosacea; gynecological diseases such as dysfunctional uterine bleeding; urological disorders such as human condyloma acuminata virus; cardiovascular diseases such as restenosis and atherosclerotic plaques; photodynamic destruction of bacteria or viruses; removing hair and caring skin; the immune response is transplanted after the organ or tissue is transplanted.

Finally, it is another object of the present invention to provide a method for diagnosing hyperproliferative tissue using halosulfonated chlorins or bacteriochlorins. Provided that these compounds preferentially accumulate in such tissues, the other property for diagnostic purposes is the clear ability to detect very small amounts of these compounds. These compounds may have very different absorption bands in the red and infrared regions, where the tissue is most permeable. Selective stimulation of these compounds produces different fluorescence at wavelengths not emitted by the biomolecules. Very sensitive equipment can be used to detect fluorescence and sub-nanomolar amounts of halosulfonated chlorins or bacteriochlorins can be determined in biological media. There is no limitation on the irradiation source for light diagnosis and light therapy, but a laser beam is preferable because intense light can be selectively used in a desired wavelength range. It is essential that the light be of sufficient intensity to cause the compound to emit fluorescent light for diagnostic purposes and to exhibit a cell killing effect for therapeutic purposes. Furthermore, when halogenated sulphonated chlorins or bacteriochlorins are used, fluorine-MRI (magnetic resonance imaging) can detect the accumulation of these compounds in small areas of the body and track the metabolic products formed which are cleared from the body.

Detailed description of the invention

Definition of II.A

As used herein, "hyperproliferative diseases" refer to diseases that have in common the underlying pathology of cell hyperproliferation resulting from unregulated or abnormal cell growth, and including uncontrolled angiogenesis. Examples of hyperproliferative diseases include, but are not limited to, carcinoma or carcinoma, myeloma, psoriasis, macular degeneration.

As used herein, "hyperproliferative tissue" refers to tissue that is out of control and includes tumors and unconstrained vascular growth such as that found in age-related macular degeneration.

As used herein, "tumor" means a neoplasm, including benign and malignant tumors. The term specifically includes malignancies, which may be solid tumors or non-solid tumors (e.g., leukemias). Examples of tumors include stomach cancer, intestinal cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcoma, liver cancer, bladder cancer, maxillary cancer, cholangiocarcinoma, head and neck cancer, tongue cancer, brain tumor, skin cancer, malignant goiter, prostate cancer, colorectal cancer, parotid cancer, hodgkin's disease, multiple myeloma, kidney cancer, leukemia, and malignant lymphoma.

As used herein, "infectious agent" refers to an invading microorganism or parasite. As used herein, "microorganism" refers to viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi, and similar microorganisms, and "parasite" refers to an infectious, generally tiny or very small multicellular invertebrate, or an egg or juvenile form thereof, that is susceptible to antibody-induced clearance or lysis or phagocytic destruction.

As used herein, "pharmaceutical formulation" or "drug" refers to a compound or composition that induces a desired therapeutic or prophylactic effect when properly administered to a subject. It includes, but is not limited to, photosensitizers that absorb light and use it as a drug or to activate other compounds that then act as drugs.

As used herein, a "pharmaceutically acceptable composition derivative" refers to a composition in which a photosensitizer is associated with a biologically active group, i.e., any group that selectively promotes binding, accumulation, or clearance in a particular biological environment. Examples known in the art include substituents generated by sugars, amino acid derivatives, oligonucleotides or ligands specific for receptors (steroid hormones, growth factors, neurotransmitters or antibodies). It also includes salts of photosensitizers.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, excipients used in the formulation of tablets, pills, capsules, creams, solutions, suspensions or emulsions. It is well known in the art how to formulate such pharmaceutical compositions.

As used herein, "surface penetration enhancers" refers to compounds or compositions that enhance or promote the transport of drugs across barriers such as the skin or other tissues, including dimethylsulfoxide and other dialkylsulfoxides, dimethylformamide, dimethylacetamide, glycols, various pyrrolidone derivatives, AzoneOr any other skin penetration assisting agent described in the literature, or mixtures thereof.

As used herein, "irradiation" refers to exposure of a subject to the electromagnetic spectrum of all frequencies. Preferably, the wavelength of illumination is selected to match one or more wavelengths at which the drug absorbs light.

As used herein, "Luzitin" refers to any sulfonated tetraphenylchlorin or tetraphenylchlorin with an electron withdrawing group in the ortho position to the phenyl group, and the following abbreviations refer to the following specific compounds which are non-limiting examples of such molecules:

-Luzitin-Cl-c is 5, 10, 15, 20-tetrakis (2-chloro-5-sulfonylphenyl) chlorin,

Luzitin-FMet-c is 5, 10, 15, 20-tetrakis (2-fluoro-5-N-methylsulfamoylphenyl) chlorin,

Luzitin-F is 5, 10, 15, 20-tetrakis (2-fluoro-5-sulfonylphenyl) bacteriochlorin,

Luzitin-Cl is 5, 10, 15, 20-tetrakis (2-chloro-5-sulfonylphenyl) bacteriochlorin,

-Luzitin-Cl₂is 5, 10, 15, 20-tetra (2, 6-dichloro-3-sulfonylphenyl) bacteriochlorin,

Luzitin-FMet is 5, 10, 15, 20-tetrakis (2-fluoro-5-N-methylsulfamoylphenyl) bacteriochlorin,

-Luzitin-F₂met is 5, 10, 15, 20-tetrakis (2, 6-fluoro-3-N-methylsulfamoylphenyl) bacteriochlorin,

-Luzitin-Cl₂et is 5, 10, 15, 20-tetrakis (2, 6-dichloro-3-N-ethylsulfamoylphenyl) bacteriochlorin,

-Luzitin-Cl₂hep is 5, 10, 15, 20-tetrakis (2, 6-dichloro-3-N-heptylsulfamoylphenyl) bacteriochlorin,

-Luzitin-FMet₂is 5, 10, 15, 20-tetra (2-fluoro-5-N, N-dimethyl sulfamoyl phenyl) bacteriochlorin,

the following abbreviations are also used herein:

-Cl₂PhB is 5, 10, 15, 20-tetrakis (2, 6-dichlorophenyl) bacteriochlorin

-BMPO is 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide

-DMPO is 5-dimethyl-1-pyrroline-N-oxide

-DMSO is dimethyl sulfoxide

II.B. precursor Compounds

Pyrrole and the desired halophenyl aldehyde were mixed in an acetic acid/nitrobenzene mixture at 120 ℃ to synthesize 5, 10, 15, 20-tetrakis (halo-phenyl) porphyrin and 5, 10, 15, 20-tetrakis (2-cyanophenyl) porphyrin, 5, 10, 15, 20-tetrakis (2-trifluoromethylphenyl) porphyrin, 5, 10, 15, 20-tetrakis (2-nitrophenyl) porphyrin and 5, 10, 15, 20-tetrakis (2-carboxymethylphenyl) porphyrin [33] by the nitrobenzene method. After cooling, the pure porphyrin is directly crystallized from the reaction medium. All the characteristic data (NMR, FAB and microanalysis) are in good agreement with the porphyrins mentioned before.

Method according to the previous study [34, 35]]To effect chlorosulfonation of the porphyrin. The desired porphyrin (200mg) and chlorosulfonic acid (10mL, 150mmol) were stirred at a temperature of 50-250 deg.C for 1-3 hours. After the stated period of time, dichloromethane (200mL) was added to the solution. Continuous water extraction was carried out with stirring until neutralization. The dichloromethane solution was then washed with sodium bicarbonate and anhydrous Na₂SO₄And (5) drying. Purification by column chromatography on silica gel using dichloromethane as eluent gave the desired chlorosulfonated porphyrin as purple crystals.

Hydrolysis of the above chlorosulfonated porphyrin was performed, comprising suspending 100mg of the desired compound in distilled water (120mL) and refluxing for 12 hours. The resulting solution was concentrated by rotary evaporation and the resulting solid was dried at 120 ℃. The sulfoporphyrin derivative was obtained in quantitative yield. Their NMR, FAB and microanalysis characteristics correspond well to literature data [34, 35 ].

II.C. plant

The absorption spectra were recorded on a Shimadzu UV-2100 spectrophotometer or with a Carry 50Biospectrophotometer (Varian, Mulgrave, USA). The fluorescence spectra were measured with a Spex Fluorolog 3 spectrophotometer, calibrated with a wavelength dependent system (RCAC31034 photomultiier) or with a Perkinelmer LS 50 fluorescence spectrometer. Transient absorption Spectra were measured using an Applied Photophysics LKS 60 nanosecond laser flash photolysis dynamic spectrometer using third harmonic excitation of a Spectra-Physics Quanta Ray GCR 130-01Nd/YAG laser, a Hamamatsu 1P28 photomultiplier tube, and a Hewlett-Packard Infinium oscilloscope (1 GS/s). Flash photolysis measurements were performed in the presence of air and argon saturated solution. Photoacoustic calorimetry uses the same Nd/YAG laser, a homemade front photoacoustic generation tube [36] with a 2.25MHz Panamerics sensor (model 5676) and a Tektronix DSA 601 transient recorder. Phosphorescence of singlet oxygen at room temperature is measured at 1270nm [37] after laser excitation of the aerated solution at 355nm using a suitably applied photophysiometer, cooled to 193K in a liquid nitrogen chamber (Products for Research model PC176TSCE005) using a Hamamatsu R5509-42 photomultiplier. The emission of singlet oxygen at 1270nm can also be detected with a liquid nitrogen cooled germanium detector (North Coast) coupled with a Technix Digitizingscope (TDS 520B) after excitation of the sample by the third harmonic (355nm) of a 5ns laser pulse generated by a Q-switched Nd: YAG laser (continuous laser II).

Elemental analysis was performed on a Fisons Instruments EA 1108CHNS-O elemental analyzer. The melting point was measured in an electrothermal capillary melting point measuring apparatus. Recording on 300MHzBrucker-Amx¹H-NMR、¹⁹F-NMR and¹³C-NMR spectrum. Using 2DCOSY and NOESY experiments¹H partitioning assay, performed using 2D HSQC and HMBC experiments¹³C, distribution measurement. MALDI-TOFMS data were obtained using an Applied Biosystems Voyager DE-STR apparatus (Framingham, MA, u.s.a.) equipped with a nitrogen laser (λ ═ 337 nm).

Electron Paramagnetic Resonance (EPR) spectroscopy of materials with at least one unpaired electron was performed using a Bruker ESP 300 spectrometer (IBM Instruments Inc.). A typical device is set up as: the microwave power is 10mW, the amplitude (amplitude) is adjusted to be 0.8G, and the scanning width is 60.0G. The EPR spectra were recorded under Hamamatsu diode laser in-situ illumination. The spectra were recorded using the following settings: high power (4mW), low modulation amplitude (0.2G) and narrow scan range (60G), and 20 scans are recorded per spectrum.

Irradiation of in vitro experiments was performed using halogen lamps or laser sources. In the first case, a 500W halogen lamp was placed 50cm from the illuminated plate to ensure uniform illumination. A cooled water filter (d ═ 35mm) and a 600nm cut-off filter were placed between the lamp and the sample. The fluence rate to the sample was 3mW/cm². In fig. 1, the emission spectrum of the halogen lamp was recorded using a spectroradiometer IL2000 (Spectrocube). In the second case, three Lyn lasers powered by a PilotPC 500 laser regulator (Sacher Lasertechnik, Marburg, Germany) were usedx external cavity diode laser TEC 500. The laser energy was stabilized at 40mW for the 748nm laser, 10mW for the 649nm laser, and 10mW for the 633nm laser. Laser energy was measured periodically using a Coherent laser check. In some in vitro experiments, the laser was focused on the optical fiber through a collimator of the purification stage and delivered to the cells. This system reduces the 748nm laser to 30 mW.

Irradiation of bacteriochlorin in photobleaching experiments was performed using a 748nm Lynx diode laser. For animal studies we used a costumer-madehamatsu diode laser model LA0873, S/N M070301, which delivers 140mW at 748 nm. The diode laser is controlled by a ThorLabs 500mA ACC/APC laser diode regulator and internal electronics. The laser energy of this laser and other higher energy lasers used in this operation were periodically measured using AN Ophir model AN/2E laser energy meter.

Time-dependent cellular uptake and cellular survival of the photosensitizer was confirmed by fluorescence microscopy using a Nikon ECLIPSE TS-100F instrument.

Fluorescent skin samples were analyzed by an Orlybach fluorescence microscope model BX51M using a U-MSWG2 fluorescence ruler set (mirrorunit) (excitation filter 480-. Confocal microscopy was performed with a Leica TCS SP5(Leica mycosystems CMS GmbH, Mannheim, Germany) inverted microscope (DMI6000) with a 63' water (1.2 optical port) apochromatic objective. Before the GUV suspension is converted into a confocal mode, a sodium lamp is used as a light source to directly observe GUV suspension, and a filter is used for selecting Rhod-DOPE fluorescence to evaluate the GUV forming yield. The excitation source in confocal fluorescence microscopy is from Ar⁺514nm of laser light, or 745nm of Ti: Sa laser light. The emitted light at 550-800nm was collected using acousto-optic tunable fibers and a beam splitter of the Leica TCSSPC5 system. Scattered light is minimized, which is consistent with an "offset value" that remains below 0.5% at all times (typically-0.1% to 0.1%), with a negligible number of photons outside the lipid structure. Obtaining small thickness by galvanometer motor stageIn 0.5mm confocal chips. The 3D projections were obtained using the Leica Application Suite-advanced fluorescence software.

II.D. Process

II.D.1. partition coefficient

N-octanol was determined by the shake flask method described in some of our references [35] with minor modifications: water distribution Coefficient (CP). The improvement is concerned with excitation with an absorption band of about 500nm and fluorescence collection in the red/IR region.

II.D.2. photochemistry and photophysics

Photobleaching experiments were performed in PBS, PBS: methanol (50: 50) and methanol solutions. These solutions were irradiated in a cuvette with a 1cm light path and a Lynx diode laser. The absorption of the solution at the beginning was about 0.8. The mechanism of photobleaching was evaluated using a Hamamatsu diode laser to irradiate the sensitizer at 80 mW. The sensitizers were irradiated in PBS and in the presence of ascorbic acid or azide.

Fluorescence quantum yield (Φ) determined in ethanol_F) As Cl₂Reference to fluorescence quantum yield of PhB in toluene [4]. The absorption of the reference and sample solutions at an excitation wavelength of 515.5nm matched at about 0.2, and these solutions were diluted 10-fold before fluorescence was collected. After correcting for the refractive index difference between ethanol and toluene, the ratio of the fluorescence bands of the sample and the reference was multiplied by the fluorescence quantum yield of the reference (according to [12 ]]0.012), a fluorescence quantum yield was obtained.

The triplet-triplet absorption spectrum and the lifetime of the triplet (. tau.) were determined by excitation at 355nm using the above-mentioned transient absorption spectroscopy apparatus_T) Wherein the solution has an absorbance of 0.25 to 0.30.

Time-resolved photoacoustic calorimetry (PAC) was performed using the above-described apparatus using the methods described in some of the documents [4] we provide. All measurements were performed in ethanol using 5, 10, 15, 20-tetraphenylporphyrinmanganese as a photoacoustic reference.

Using some of the documents we provide [37]]The method uses benzidine as a reference to obtain the singlet oxygen quantum yield. The literature value for the quantum yield of singlet oxygen from benzilketone in ethanol is Φ_Δ＝0.95[38]。

Electron Paramagnetic Resonance (EPR)

The reactive oxygen species generated by irradiation of the photosensitizer in PBS, i.e., superoxide ion and hydroxyl group, form adducts with various spin traps. These adducts can be identified by EPR. The PBS buffer used in these assays was pretreated with chelating resin Chelex 100 to remove any contaminating metal ions that would catalyze superoxide decomposition. Two spin traps were used: BMPO and DMPO. DMPO was first activated with charcoal/benzene and then used₂₂₆＝7200M^-1cm^-1To measure 1.0M stock concentrate with a spectrophotometer. EPR assays were performed at room temperature using the Bruker spectrometer described above, with in situ irradiation with a Hamamatsu diode laser.

II.D.4. skin penetration test

Considering that minipigs are similar in their skin properties to human skin, the best animal model to determine skin penetration is minipig [39 ]]. Different formulations of creams, ointments, gels, liquid solutions were used to enhance penetration of the photosensitizer through skin samples of mini-pigs. Penetration enhancer such as Azone in an amount of 0.1-10%And DMSO, as well as various excipients to incorporate the formulation into the photosensitizer. In vitro experiments used skin isolated from the back of mini-pigs. In vivo experiments were performed on the back of mini-pigs. In each test, the formulation was applied to an area of about 1cm with the aid of a blocking adjuvant²For a desired length of time. After this time, the formulation was removed with a spatula and washed with ethanol soaked cotton wool until no trace of sensitizer was visible on the cotton wool. In this in vivo test, a skin sample is taken by surgical operationThe animals were then sacrificed.

The first step of the procedure for tissue fixation of skin samples is to soak in paraformaldehyde (4% in water) for at least 24 hours. Next, the samples were transferred to a 25% sucrose solution for at least 48 hours. After this treatment, the skin sample became denser than the sucrose solution. Aliquots were extracted with a biopsy punch, frozen in dry ice, then mounted on scaffolds with Tissue-Tek o.c.t. compound (Sakura Finetek Europe b.v., Zoeterwoude, The Netherlands) and cut into sections at selected controlled thicknesses of 25-100mm in a cryostat. Skin sections were collected on microscope slides and kept cold before they were analyzed by fluorescence microscopy and confocal microscopy. Alternatively, the skin samples were directly frozen in dry ice without using paraformaldehyde as fixative.

II.D.5. in vitro experiments

The drugs described herein have been evaluated in vitro studies. One set of in vitro studies used halogen lamps equipped with several filters. Other groups use diode laser illumination to deliver light to individual cell cultures.

II.D.5.i) in vitro experiments with halogen lamp irradiation

The cell lines used in halogen lamp irradiation were MCF7 (human breast cancer), SKMEL188 (human melanoma) and S91/I3 (mouse melanoma) cells. They were used for cytotoxicity and photo-cytotoxicity experiments. MCF7 cells supplemented with 10% Fetal Bovine (FBS) serum, 25 units/ml penicillin and 25. mu.g/ml streptomycin were grown. Human melanoma cells SKMEL-188 were grown in F10 medium supplemented with 10% Fetal Calf Serum (FCS), 100 units/ml penicillin and 100. mu.g/ml streptomycin. Cloudman S91 melanoma cells of the I3 subline were grown in RPMI 1640 medium supplemented with 100 units/ml penicillin, 100. mu.g/ml streptomycin and 5% Fetal Calf Serum (FCS) (Gibco BRL). All cell lines were cultured as monolayers in 60mm diameter dishes and in 5% CO₂And incubated at 37 ℃.

Cytotoxicity. The metabolic efficiency and viability of cells were determined by uptake and reduction of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) to insoluble formazan (formazan) dye by cellular microsomal enzymes. The cell lines were grown in RPMI medium containing 10% fetal bovine serum, penicillin and streptomycin. Cells were maintained at 5% CO₂95% air and 100% humidity. To determine cytotoxicity, these cells were treated at 1 × 10⁴The density of cells/well was placed in complete medium in 96-well plates. After 24 hours, the cells were incubated with different concentrations (0.25-50. mu.M) of the photosensitizer at 37 ℃ for 18 hours. The cells were then washed 2 times with PBS and cultured in growth medium for 24 hours at 37 ℃. Next, the medium was replaced with 100. mu.l of fresh medium and 20. mu.l of MTT, and the cells were cultured with MTT for 3 hours at a final concentration of 0.5 mg/ml. The medium was then replaced with DMSO-methanol solution (1: 1) to dissolve the blue formazan crystals. The 96-well plate was shaken for 0.5 min at room temperature and immediately the optical density was read at 560nm with an ELISA reader (GENios Plus; Tecan tracing AG, Switzerland). Cell survival was expressed as the change in absorbance of formazan salt and survival was expressed as the percentage of cells that survived treatment relative to those that survived no treatment. The cell number was determined by linear regression of the calibration curve.

Time-dependent cellular uptake. At 1x10 per hole⁴Cells SKMEL188, S91 and MCF7 cells were seeded into 96-well plates and exposed to chlorins and bacteriochlorin photosensitizers at 20 μ M concentrations in PBS at various time intervals ranging from 10 minutes to 180 minutes to study time-dependent drug accumulation. At the end of the incubation interval, cells were washed 3 times with PBS and resuspended in 100. mu.L of 0.25% Triton X-100 in PBS. The retention of photosensitizer bound to the cells is detected by fluorescence measurement of the accumulated photosensitizer using an ELISA reader. In addition, uptake of the photosensitizer and survival of the cells were confirmed by fluorescence microscopy. In these experiments, SKMEL188, S91 and MCF7 cells were incubated with 20 μ M of photosensitizer for 2 hours, then washed 3 times with PBS, resuspended in PBS, and examined by fluorescence microscopy。

Photosensitization of cells. SKMEL188, S91 and MCF7 cells were prepared as above. On the basis of the cytotoxicity assay, a photosensitizer was selected for the photosensitizing assay at a concentration of 5 μ M. Cells were incubated at 37 ℃ for 12 hours, then at 0.53mW cm²The fluence rate of (2) and 0.1-0.64J cm²Dose range irradiation of (2). We recall: the photosensitizer uses only a filtered halogen lamp at an effective fluence rate of about 1/5. The MTT test was performed 24 hours after irradiation. Values were obtained from three independent experiments and expressed as a percentage of cell survival with reference to control cells treated in the same manner but incubated without the photosensitizer and light.

II.D.5.ii) in vitro experiments with laser irradiation

Cell lines used in laser irradiation were HT-29 (human colon carcinoma), PC-3 (human prostate carcinoma), SW2 (human small cell lung carcinoma), A-549 (human non-small cell lung carcinoma), S91/I3 (mouse melanoma), and CT26 (mouse colon carcinoma). They were used for cytotoxicity and photo-cytotoxicity experiments. PC-3, SW2 and S91/I3 cells were cultured in RPMI-1640 medium (Sigma-Aldrich, Steinheim, Germany) and HT-29, A-549 and CT26 cells in Dulbecco' S modified Eagle Medium (DMEM) (Cambrex Bioscience, Verviers, Belgium). Both cell culture media were supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Cambrex Bioscience, Verviers, Belgium) and 100IU/ml penicillin-100. mu.g/ml streptomycin (Cambrex Bioscience, Verviers, Belgium). DMEM medium for CT26 cells was also supplemented with HEPES 10 mM. At 37 ℃ and 5% CO₂The cell line was kept at 75cm in a humid atmosphere²In a flask (orange scientific, Braine-l' Alleud, Belgium). For in vitro studies, 85-90% of the fused cells were detached with Trypsin-Versene-EDTA solution (Cambrex Bioscience, Verviers, Belgium), counted, and seeded in flat bottom 96-well plates at the desired density.

Cell survival assay. After the experiment was completed, cell survival was assessed by resazurin reduction assay [40 ]. Briefly, stock solutions of Resazurin (Sigma-Aldrich, Steinhelm, Germany) diluted 10% (0.1mg/ml in Phosphate Buffered Saline (PBS) pH 7.4) in medium without FBS or antibiotics were added to 200. mu.l of the cells in each well. The plates were incubated at 37 ℃ for 3-4 hours. The absorbance values of each well were measured at 540nm and 630nm using a microplate reader MultiskaneEx (Thermo-Electron Corporation, Vartaa, Finland).

Cytotoxicity. Cells were seeded into flat bottom 96-well plates (Orange Scientific, brain-l' Alleud, Belgium) in 100. mu.l medium and allowed to attach overnight. The photosensitizer was added to each cell (diluted in 100. mu.l of medium) at a final concentration of 0.01-1 mM. Each concentration was determined in quadruplicate. The incubation time at 37 ℃ and in the dark is 2 times the doubling time of the test cell line. After culture, cell survival was evaluated. In a parallel control experiment, cells were not incubated with drug. Cytotoxicity was quantified by expressing cell survival (% of control cells) relative to untreated cells. The results are expressed as a dose response curve (% cell death as a function of drug concentration) that allows determination of the concentration that inhibits 50% of cell growth (IC)₅₀)。

Photosensitization of cells. Cells were seeded in 100. mu.l medium in DB Falcon Black 96-well plates (DB Biosciences-Labware, NJ, USA) with clear flat bottoms and allowed to attach overnight. Drugs were added to the cells (diluted in 100 μ l of medium) to obtain the desired concentration. Cells were incubated for a given time in the dark at 37 ℃. The incubation time is also referred to as drug-to-light interval. After incubation, cells were washed 1 time with 200 μ l PBS, unbound drug was removed, and 100 μ l of fresh medium was added. 748nm light with the above Lynx diode laser at 100.7mW/cm²The cells were irradiated (each well was irradiated). The irradiation time is chosen to obtain the desired light dose. Two parallel control conditions were determined: incubating the cells in the dark with the highest dose of the drug and without irradiation, and irradiating the cells with the highest light dose but without the drug. After irradiation, 100. mu.l of fresh medium was added. Cell survival was assessed approximately 24 hours after irradiation.

II.D.6. in vivo experiments

The mice used in this study were from two sources. Dark toxicity, biodistribution and pharmacokinetic studies were performed using DBA/2 mice weighing 20-30g from the animal culture laboratory of Faculty of biochemistry, Biophysics and Biotechnology, Jagilsonian University, Krakow. Mice were kept in a standard laboratory, without food, with free access to water. Jagiellonian University Committee for Ethics of experiments on Animals (precision No.384/99) approved these Animals for experimental purposes. These mice were also used for PDT.

Other mice were Balb/C weighing 20-25g from the animal culture laboratory of Charles river laboratories (Barcelona). Mice were kept in a standard laboratory, without food, with free access to water. The Board of Central de Neuroci E biologica cell (Coimbra) approved these animals for experimental purposes.

The 4 mini-pigs used in this study were from IMIDRA (Instituto)de Investigaci Lou n y Desarrolol Rural, Agrario y Alimentario) -Aranjuez (Madrid). They were all female, 6-8 months old, white with brown spots, and an average body weight of 56.8kg (66.2, 57.1, 43.5, 60.6 kg). They are as followsZoot nice Nacional, Vale de Santar, where they are received at 1.5m independently²The pigs were acclimatized with standard diets of pigs and with free access to water for an acclimatization period of 3 weeks. The study was based ondede Saúde eAnd the ethical guidelines of Portugal on the license granted by Animal, ref.0420/000/000/2007. Food intake was suspended 24 hours prior to treatment. The animal's back was shaved 24 hours before using the formulation. Topical administration of the photosensitizer is performed under anesthesia. Pretreatment-drugs pre-used 30 minutes in advance were: azaperone (Stressil)Veterinaria E STEVE-Spain), 2mg/kg intramuscular injection + atropine sulphate, 50mg SC. With ketamine (Clorketam)-vetoquinol, France), 20mg/kg, induced by intramuscular injection. 2-3 l/min oxygen + 3% isoflurane (Isoflo) was used-Veterin-ria ESTEVE, Spain) maintained under anesthesia by endotracheal intubation. Samples were collected from 3 mini-pigs under the above anesthesia. Skin aliquots of size 20x20x10 (length, width, thickness) were obtained by surgical excision. After collecting skin samples, animals were sacrificed with overdose of thiopentant sodium (25mg/kg) +20ml of 7.5% potassium chloride. The 4 th mini-pig was observed for 3 weeks while being fed on the standard diet of the pig and ad libitum. After 3 weeks, topical application of photosensitizer under anesthesia was performed. The animals were stunned by electric shock and sacrificed by jugular phlebotomy.

Properties of the Compounds

II.E.1. physical Properties

As mentioned in the background, the most important physical and chemical properties of PDT-related drugs are strong absorption in the 600-800nm range, high efficiency of singlet oxygen generation, chemical stability, controllable photobleaching and octanol: the water distribution coefficient is between-2 and 5. These properties meet the desired light absorbance for the light therapeutic window, efficient light production of cytotoxic substances, reduced side effects such as prolonged skin photosensitivity, and facilitate systemic or transdermal routes of administration.

The absorbance of the compound was measured at various concentrations from 1 to 20. mu.M, and in all cases it was observed whether it conformed to the Beer-Lambert law. Furthermore, the absorption maximum in the infrared region (λ) in the concentration range investigated_max) Is unchanged. This indicates that there is little aggregation between the molecules and that the molecules are present in the solvent under investigation at these concentrations almost exclusively as monomers. Table 1 lists typical red and infrared molar extinction coefficients (_max) And a wavelength maximum. The table also lists representative fluorescence quantum yields (. PHI.) for Luretins_F). The fluorescence quantum yield of these molecules is reduced in the presence of chlorin substituents, which is a fingerprint of the heavy atom effect expected for these molecules.

The triplet-triplet absorption spectra of Luzitins determined in this work are in good agreement with literature data for chlorins and bacteriochlorins. All triplet decays are clearly mono-exponential. In the presence of a catalyst composed of N₂Lifetime of triplet state (. tau.) in degassed solution prepared by rinsing for at least 30 min_T) In the millisecond range, this indicates ineffective photochemistry in the absence of oxygen. Representative values are given in table 1. In air-saturated ethanol, the lifetime of the triplet state was reduced to 200-300 nanoseconds, significantly shorter than that of the corresponding porphyrin. These values are consistent with diffusion-limited energy transfer of the triplet state of the sensitizer to molecular oxygen via charge transfer.

Survival with typical singlet oxygen (. tau.)_Δ) All singlet oxygen emissions determined in aerated ethanol solutions are described very well by single exponential decay. Phi of Table 1_ΔThe values are obtained by the above-mentioned methods and are representative of these photosensitizers.

Table 1. fluorescence quantum yield, triplet lifetime, singlet oxygen quantum yield, and singlet oxygen lifetime of representative photosensitizers in air saturated ethanol solution.

^a)[41]；^b)HpD monomer Unit in methanol_Δ0.64, but in water it is almost exclusively present as a dimer, Φ_Δ＝0.11[42]；^c)In an aqueous solution;^d)corrected for chlorin content^d)[43]；^e)In methanol, but down to half of this value in methanol with 65% water [44 ]]；^f)In methanol [45]。

Typically, the fluorescence and singlet oxygen quantum yields of Luzitins amount to 0.6-0.8, and absorb 20-40% of the light when used in other processes. These processes were studied using time-resolved photoacoustic calorimetry (PAC) and Electron Paramagnetic Resonance (EPR). PAC measures the amount of energy released by non-radiative processes (intrinsic conversion, intersystem crossing, chemical reactions) in the decay of an electronic excited state. EPR measures the amount of species with unpaired electrons (free radicals) present in a sample, here under direct laser irradiation for the measurement of photoinduced production of superoxide ions, hydroxyl radicals and other Reactive Oxygen Species (ROS). PAC-determined energy release was consistent with bacteriochlorin triplet formation with similar unit quantum yield and triplet energy of 105-125kJ/mol, as well as literature data for halogenated bacteriochlorins [12 ]]And (5) the consistency is achieved. EPR spectra collected during irradiation of Luzins in the presence of PBS and DMPO and BMPO indicated the presence of superoxide ions and hydroxyl groups. Taken together, the EPR and PAC data clearly indicate that: irradiation of these photosensitizers at 748nm results in the formation of superoxide ions, followed by the formation of hydroxyl groups, which render Luzitins phototoxic. Thus, the phototoxicity of these photosensitizers is higher than that expected from their singlet oxygen quantum yield, and they are almost greater than that of the photosensitizersThe phototoxicity of (2) is 5 times higher.

Chemical stability of compounds was studied by exposing them to light, air and pH changes. The most significant degradation of the aerated solution occurs under light irradiation at the appropriate wavelength. In these cases, the photobleaching of the compound follows a first order rate, proportional to the energy of the incident laser light. In general, it is also observed that the rate of photobleaching increases with the amount of water present in the solution. Bonnett reported Foscan(m-THPC) and similar bacteriochlorins (m-THPB) photoconversion in PBS: methanol (50: 50) [46]. Scherz reported TookadIn acetone and Triton-X: light conversion in PBS [3]. To compare Luzinins with FoscanAnd TookadFor the purpose of photostability, we present table 2, in which the half-lives (t) of representative drugs are depicted_1/2) Foscan and Tookad normalized with 100mW laser energy are also givenThe half-life of (c).

Table 2. half-life of bacteriochlorins and their n-octanol under 100mW laser irradiation at 748 nm: water distribution coefficient (K)_OW)。

^a)[3]；^b)[46]。

We combined our photobleaching of bacteriochlorins with m-THPB or TookadIs distinguished from the other. This distinction is based on the fact that: after a longer laser irradiation, most of our bacteriochlorins are converted to products with no detectable significant absorption, and can be properly described as photobleaching. In another aspect, m-THPB or TookadThe laser irradiation of (a) produces a large amount of the corresponding chlorins that do not absorb the same laser light, and this phenomenon is more properly described as light conversion to another dye. Our photo-bleaching of bacteriochlorins is advantageous because it produces less skin sensitivity than photo-conversion to another dye.

Biological Properties II.E.1

Photosensitive elementProvides an important benchmark for dark toxicity and in vitro PDT efficacy. Several photosensitizers were evaluatedConcentration of photosensitizerToxicity to the non-small cell lung cancer cell line H1299 in the dark, cell survival was reduced to 50% IC_{Dark 50}＝8.0μg/ml[47]. Photosensitive elementSimilar studies of dark toxicity on the murine colon carcinoma cell line Colo-26 led to IC_{Dark 50}20. mu.g/ml (30. mu.M, assumed to be monomeric) [48 ]]. The human adenocarcinoma (HT29) cell line is the most widely studied cell line in PDT. At 5J/cm²Filtering out halogen light, the photosensitive elementConcentration-dependent studies in HT29 cells resulted in a lethal dose IC of 50% death₅₀7.5. mu.g/ml. 90% of deathIncrease the concentration to IC₉₀＝40μg/ml[49]. For the same cell line, 10J/cm was delivered using a dye laser at 650nm²Hour FoscanIs a much more effective phototoxic agent, IC₅₀0.8 mug/ml; however, at this dose, the IC₉₀> 10. mu.g/ml and fall within the range of its dark toxicity, IC_{Dark 50}＝13μg/ml[50]. And a photosensitizerIn contrast, FoscanThe molecular composition of (a) is known, it is easier to express its ICs in molar units. In these units, FoscanThe dark cytotoxicity of is IC_{Dark 50}＝19μM，10J/cm²The toxicity of the light dose is IC₅₀1.2 μ M. Finally, it is interesting to use at 25J/cm²Under irradiation, the dose of Tookad was 48. mu.MIt results in a mortality rate of 50% in HT29 cells (patent US2003/6,569,846). Although IC₅₀And IC₉₀The values depend on the mode of administration, incubation time, light fluence and other details of the experiment, but the values given above represent the best mode of practice in the art.

Table 3 compares the photosensitizersAnd FoscanDark toxicity with Luzins. Using the above scheme and the examples described in more detail below, under filtered halogen lamp, the halo and sulfonic acids listed in Table 4In the presence of chlorins, light doses were required to kill 50% and 90% of multiple cell lines, and table 5 lists the corresponding values for halogenated and sulfonated bacteriochlorins. Table 6 shows the results at 6J/cm²The laser dose of (a) is the phototoxicity at the concentration of the various Luzitins required to kill 90% of the cells.

TABLE 3 dark toxicity of photosensitizers in human and murine cancer cell lines

^a)Cells were incubated in complete medium in the dark in the presence of different concentrations of photosensitizer 2-fold higher than the DT population.

TABLE 4 light dose required to kill 50% and 90% of the cells in the indicated cell lines under filtered halogen lamp irradiation, in J/cm²，[Luzitin-Cl-c]＝20μM(≈20μg/ml)。

Cell lines	S91	SKMEL 188	MCF7
				LD50	0.13	0.26	0.3
LD90	0.26	0.46	0.7

TABLE 5 light dose required to kill 50% and 90% of the cells in the indicated cell lines under filtered halogen lamp irradiation, in J/cm²，[Luzitin-Cl]＝20μM(≈20μg/ml)。

Cell lines	S91	SKMEL 188	MCF7
				LD50	0.15	0.1	0.08
LD90	0.3	0.32	0.25

TABLE 6J/cm at 28.5mW²Diode laser irradiation killed 90% of the cells human prostate cancer (PC-3), human adenocarcinoma (HT-29), human non-small cell lung carcinoma (A-549), mouse melanoma (S91I3) andphotosensitizer concentration required for murine colon cancer (CT26) cell line, in μ M.

Photosensitizers	PC-3	HT-29	A-549	S91I3	CT26
						Luzitin-Cl2	20	50		40
Luzitin-Cl2Et	5	10	10		5
						Luzitin-FMet	0.5	0.5	0.5		1.0
Luzitin-F2Met	0.5		0.5		1.0

Tables 4-6 show the low dark toxicity of Luzitins and their very high phototoxicity. Table 6 shows that the concentration of Luzitin required to kill 90% of the cells in various cell lines is a photosensitizerThe desired concentration is up to 1/100, FoscanUp to 1/20 for the desired concentration. At 6J/cm²At the laser dose of (60 seconds of irradiation under our experimental conditions), 100% of the cells can be killed in any cell line studied in the dark with a concentration of Luzitin with negligible cytotoxicity.

Methods of using compounds and compositions

Luzins may be administered in topical, oral, intravenous, subcutaneous, intraperitoneal, rectal, sublingual, nasal, ocular, otic or inhalant formulations depending on the clinical situation. The pharmaceutical formulation is adapted to the chosen route of administration. The formulation comprises, in addition to the Luzines, a pharmaceutically acceptable carrier (separately or in combination with each other). Luzitins may be derivatized to the corresponding salt, ester, enol ether or ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate, or prodrug prior to formulation.

After local or systemic administration or both, the treated area is exposed to light of a suitable wavelength, the preferred wavelength for chlorins is 630-690nm, the preferred wavelength for bacteriochlorins is 720-780nm, most preferably using a laser. Methods of irradiating various regions of the body are well known in the art. Depending on the mode of administration, the drug-light interval may range from minutes to days. The light dose depends on the route of administration, the light source and the target of treatment. For continuous laser irradiation, the light dose should be 10-250 joules/cm²The laser power is 20-200mW/cm². Pulsed laser irradiation may also be used, the energy per pulse being 0.001-10mJ/cm². The light dose may be used over one or more time periods. For diagnostic purposes, the light dose can be reduced. When a broadband light source is used in the illumination, the light dose is increased to maintain the source energy (at the level suggested by the laser illumination) in the spectral region absorbed by Luzitin.

Luzins may be administered immediately, or may be divided into a plurality of smaller doses to be administered at intervals. Luzinins may be administered with other drugs to achieve a synergistic effect. The light dose administered after the drug-light interval falls within the range of the single dose given above. The treatment may be repeated multiple times at different time intervals. It will be understood that the precise dose and duration of treatment is a function of the disease being treated and may be determined empirically using protocols known in the art.

Systemic administration (oral, injection, aerosol, rectal)

A common limitation associated with oral administration is the putative drug digestion under the acidic conditions found in the stomach. As shown in the examples below, Luzitins are relatively stable over 3 hours at pH 1. The spectral changes observed at lower pH are due to protonation of the pyrrole ring, but reversal occurs when acidity is neutralized. Another difficulty often encountered with oral administration is bioavailability. Luzitins can be made to approach 5 Lipinsky rules for intestinal absorption [51]。Luzitins may be log K_OW< 5, with 4 hydrogen bond donors, 12 hydrogen bond acceptors and a molecular weight of 1 kD. Only the last parameter clearly exceeds the scope of the above rule.

Solid, semi-solid and liquid dosage forms are contemplated in view of the physico-chemical properties of Luzitins. As such, the state of the art for pharmaceutical adjuvants for use in various pharmaceutical dosage forms and modes of administration should be considered.

Depending on the mode of administration, the preferred drug-light interval time in systemic administration may range from minutes to 3 days. The pharmaceutical composition should provide a dose of 0.01mg to 100mg of Luzitin (or combination of Luzitins) per kg of body weight per day. The preferred daily dosage of Luzitins is 0.1-10mg per kg body weight.

II.F.2. topical application

Topical application may be achieved with a suitable formulation comprising one or more surface penetration enhancers and other excipients in the form of a liquid, gel, hydrogel, cream, ointment, spray, or any other acceptable dermatological formulation. As described in the background section, the implementation of photosensitizersOr other macromolecular mass photosensitizers, are insufficient for effective PDT of skin diseases. Luzitins also have difficulty meeting most of the criteria for good transdermal delivery [32]. However, our ability to modulate the amphiphilicity and hydrogen bonding ability of these compounds, as well as to some extent their molecular mass, simplifies the task of finding formulations that promote their passive diffusion into the dermis. The gel formulations listed in example 21 were shown to produce rapid and effective local delivery to the dermis.

In topical application, the preferred drug-light interval is 15 minutes to 3 hours. The formulation should contain 0.01-10% Luzitin. In a preferred embodiment, the percentage of sensitizer in the formulation is 0.1-1%.

Topical application is also contemplated for application to the eye. Applications intended for ocular use may be formulated as 0.01% to 10% isotonic solutions, pH 5-7, containing appropriate salts.

The invention will now be described in more detail in the following non-limiting examples and with reference to the accompanying drawings, in which:

FIG. 1. spectral density of 500W halogen lamp using 600nm cut-off filter, and infrared absorption spectra of chlorins (dotted line) and bacteriochlorins (hatched line).

FIG. 2. absorption spectrum of Luzitin-FMet-c obtained from the reduction of the corresponding porphyrin in a synthetic route characterized by the absence of organic solvents and bases.

FIG. 3 Luzitin-Cl obtained by reduction of the corresponding porphyrin in a synthetic route characterized by the absence of organic solvents and bases₂Absorption spectrum of Et.

FIG. 4 shows Luzitin-Cl₂The more polar part of the atropisomer of Et (. beta.. alpha.)₃+α₄) Shows an increase in the absorption in the infrared region and is evaluated to reach 748nm_max＝150,000M^-1cm^-1。

FIG. 5 Luzitin-Cl in neutral aqueous solution (solid line), after 2 hours at pH1 (dotted line) and after neutralization (hatched line)₂The absorption spectrum of (1). The spectra were corrected for the dilution effect produced by dropping HCl and NaOH into the aqueous solution.

FIG. 6. absorption spectra of Luzitn-FMet in PBS: methanol (3: 2) before (left) and after (right) 65 minutes irradiation with a 748nm Lynx diode laser at 28.8mW (110J) show controlled photobleaching.

FIG. 7 Luzitin-Cl at 355nm in air-saturated ethanol₂And the singlet oxygen phosphorescence intensity collected at 1270nm after Hep or benzidine ketone excitation. The slope of phosphorescence versus laser intensity dependence was used to determine the singlet oxygen quantum yield of the sensitizer.

FIG. 8 EPR spectra obtained under irradiation in the presence of 80. mu.M Luzitin-Cl. Left side: using 40mM BMPO in PBS, the spectra correspond to the spin adduct of BMPO and hydroxyl groups. Right side: using 40mM DMPO in DMSO, the spectra correspond to the spin adduct of DMPO and superoxide ion.

Figure 9. fraction of S91 cell survival (percentage) for each light dose in the presence of [ luzitn-Cl-c ] ═ 20 μ M. Unincorporated (non-intercalated) photosensitizers were not removed prior to irradiation. Illustration is shown: Luzitin-Cl-c cytotoxicity on the S91 cell line in the dark at various concentrations of sensitizer.

FIG. 10 shows that [ Luretin-Cl ]]MCF7 cell survival fraction (percentage) for each light dose in the presence of 5 μ M after 12 hours incubation time. Unincorporated photosensitizer was removed prior to irradiation. Illustration is shown: Luzitin-Cl cytotoxicity on MCF cell lines in the dark at various concentrations of sensitizer. The effective optical power is 0.53mW/cm²。

FIG. 11 Luzitin-Cl after 24 hour drug-light interval₂Phototoxic effects in the prostate cancer cell line PC-3. Cells in the medium were irradiated with 748nm laser for 30 or 60 seconds, corresponding to 3 and 6J/cm, respectively²The light dose of (a).

FIG. 12 different Luretin-Cl in mouse colon carcinoma cell lines after a 18 hour drug-light interval₂Percentage of cell survival relative to control for Et concentration. Unincorporated photosensitizer was removed prior to irradiation. Cells in the medium were irradiated with a laser at 748nm for 60 seconds, corresponding to 6J/cm²The light dose of (a).

FIG. 13 percentage cell survival relative to control for different Luzitin-FMet concentrations in human adenocarcinoma cell lines after 18 hours incubation time. Unincorporated photosensitizer was removed prior to irradiation. Cells in the medium were irradiated with a laser at 748nm for 60 seconds, corresponding to 6J/cm²The light dose of (a).

FIG. 14 different Luretin-F in human non-small cell lung carcinoma lines after 18 hours incubation time₂Percentage of cell survival relative to control for Met concentration. Unincorporated photosensitizer was removed prior to irradiation. Cells in the medium were irradiated with a laser at 748nm for 60 seconds, corresponding to 6J/cm²The light dose of (a).

FIG. 15 Luzitin-Cl at human serum concentrations of 3.26nM (solid line) and 0.26nM (hatched line)₂The fluorescence of Et was used to determine its limit of detection. Shown are both a baseline (shaded-dashed line) and a gaussian curve (dashed line) used to model the lowest concentration for more accurate determination of low intensity fluorescence.

FIG. 16 fluorescence of Luzitin-Cl-c divided by the mass of liver, blood and brain (sorted by intensity, respectively) after 10mg/kg ip administration in DBA/2 mice.

FIG. 17 pharmacokinetics and biodistribution of Luzitin-Cl in DBA/2 mice after 10mg/kg ip administration. The fluorescence intensity of each tissue (blood, tumor, heart, lung, spleen, liver, kidney, intestine, muscle, skin) was normalized by their respective mass.

FIG. 18 Luzitin-Cl after 10mg/kg ip administration in DBA/2 mice₂Fluorescence of Et divided by mass of tumor and blood (sorted by intensity, respectively).

FIG. 19 tumor volumes in transplanted CT26 tumors and Balb/C mice treated with Luzitin-FMet. The denser line represents the average tumor size in 4 control animals to which Luzitin-FMet was administered but not irradiated. Error bars are standard deviations. The thinner lines represent the results obtained with Luzitn-FMet and 132J/cm at 748nm²Laser treated animals.

FIG. 20 is a schematic representation of the use of Luretin-Cl₂Size of S91 tumor transplanted in Et-treated DBA/2 mice. Two laser doses at 748nm were used.

FIG. 21 confocal fluorescence of Luzitin-FMet after 3 hours of topical application to the back of mini-pigs. Multiphoton excitation at 744 nm; completely eliminating noise; constructing a fluorescence plan by 6 pictures; an 800V detector; the pore size is 379 μm. The photosensitizer diffuses through the stratum corneum and epidermis, 50 microns.

FIG. 22 absorption spectra of Luzitin-FMet obtained by confocal spectroscopy after topical application to the back of mini-pigs for 3 hours. Each line corresponds to a measurement of a different portion of the sample.

Examples

Example 1 solid-state Synthesis of bacteriochlorin with Electron-withdrawing substituent

This example illustrates that a wide range of bacteriochlorins, which can be synthesized by a process characterized by the absence of solvents and bases, thus wherein only porphyrins and hydrazides (both in solid form) are used as starting materials.

In one preparation method, we will all be 60mg (6.8x 10) of fine powder^-5mol) of 5, 10, 15, 20-tetrakis (2-trifluoromethylphenyl) porphyrin and 500mg (2.7X 10)^-3mol) of p-toluenesulfonylhydrazide. They are introduced into a vacuum reactor under N₂Sealed and heated to a temperature above 100 c for several minutes. After cooling (room temperature), the solid was removed and 250mg (1.4X 10) of p-toluenesulfonylhydrazide was added in portions^-3mol) until the porphyrin sorel band completely disappears. Extracting bacteriochlorin with small amount of organic solvent. Excess hydrazide was removed by short filtration over silica gel (column height 8 cm; inner diameter of column 2.5cm) using dichloromethane/n-hexane as eluent. After evaporation of the solvent and recrystallization from diethyl ether/pentane, 90% CF is obtained₃PhB, containing less than 5% chlorin contaminants.

NMR of the isolated product was:

RMN¹H：(400，13MHz，CDCl₃)，ppm：7.47-7.45(m，4H)；7.26-7.20(m，8H)；7.15-7.12(m，4H)；2.42(s，4H)；2.37(s，4H)；-1.27(s，2H)。

example 2 solid-state Synthesis of halogenated chlorins

In one preparation of 5, 10, 15, 20-tetrakis (2-fluorophenyl-5-N-methylsulfamoylphenyl) chlorin, Luzitin-FMet-c, we will mix 50mg (4.72X 10)^-5mol) of 5, 10, 15, 20-tetrakis (2-fluorophenyl-5-N-methylsulfamoylphenyl) porphyrin and 18mg (9.44X 10)^-5mol) of p-toluenesulfonylhydrazide. The reactor is evacuated to a vacuum of N₂Sealed and heated to a temperature above 100 c for several minutes. After cooling (room temperature), the solid was removed with a small amount of organic solvent and the excess hydrazide was removed by short filtration over silica gel using ethyl acetate/hexane as eluent. A mixture of chlorins contaminated with about 10% bacteriochlorins was obtained. The mixture of chlorins and bacteriochlorins was dissolved in dichloromethane and oxidized to the corresponding chlorins by heating in the presence of air at 50 ℃.

After recrystallization from diethyl ether/pentane, Luzitin-FMet-c was obtained at 90% with less than 1% bacteriochlorin contaminants. Fig. 2 shows the absorption spectrum of the final product.

Example 3 solid-State Synthesis of Halobacterizin

This example describes a clean, simple, economical and environmentally benign synthesis involving a solvent-free one-pot synthesis of halogenated amphiphilic bacteriochlorins.

The appropriate porphyrin (solid) and p-toluenesulfonyl hydrazide (solid) were ground to a very fine powder and mixed well. They are then placed in a reactor and a high vacuum is drawn. The reactor is then sealed and maintained under vacuum or repeatedly washed with an inert gas. Finally, the reactor was heated (70-200 ℃) for 1-340 minutes while sealed. When the reaction was complete and the reactor was returned to room temperature, the corresponding bacteriochlorin was obtained in 90% yield. After short filtration through a silica gel column, bacteriochlorins were obtained containing less than 5% of the corresponding chlorin contaminants.

In 5, 10, 15, 20-tetrakis (2, 6-dichloro-3-N-ethylsulfamoylphenyl) bacteriochlorin, Luzitin-Cl₂In the preparation of Et, we used this method to mix 50mg (3.8X 10)^-5mol) of 5, 10, 15, 20-tetrakis (2, 6-dichloro-3-sulfoethylphenyl) porphyrin and 188mg (10)^-3mol) of p-toluenesulfonyl hydrazide, evacuating the reactor with an Edward pump, sealing, and then heating the reactor to above 70 ℃ for several minutes. After cooling (room temperature), the solid was removed with a small amount of diethyl ether and the excess hydrazide was removed by short filtration over silica gel (column height 4 cm; column internal diameter 2.5cm) using ethyl acetate/diethyl ether as eluent. After evaporation of the solvent, the resulting solid was recrystallized from diethyl ether/pentane to give Luzitin-Cl2Et in 90% yield. The absorption spectrum of fig. 3 shows that less than 5% of the corresponding chlorins are present. This and other bacteriochlorin molar extinction coefficients, corrected for the amount of chlorins sometimes present in the sample, are listed in table 1. Comparison of the spectra in figure 3 below with the spectra in figure 2 of patent PCT/EP2005/012212, which is the university of Coimbra, shows that chlorin impurities are reduced by at least a factor of 10 by this new synthetic method, which is also a more economical, less labour-intensive and environmentally benign process. NMR and MS of the isolated product were as follows:

RMN¹H：(300MHz，CDCl₃)，ppm：8,42(d，J＝8,55Hz，4H，p-H)；7,88(d，J＝8,55Hz，4H，m-H)；7,84-7,82(m，4H，β-H)；5,01(m，4H，N-H)；3,91(s，8H，β-H)；3,22(m，8H，CH₂)；1,24(t，12H，J＝6,73Hz，CH₃)；-1,29(s，2H，NH)。

MS：(MALDI-TOF)，m/z：1322，0[M]⁺。

example 4 atropisomers in Halobacteriaceae Green

This example shows the presence of stable atropisomers in halogenated and sulfonated bacteriochlorins, which are easily separated. It also indicates that the more polar atropisomers have a higher extinction coefficient in the infrared region.

Using the method described in example 3, but using a larger column (column height 8 cm; column internal diameter 2.5cm), with ethyl acetate/N-hexane (1: 1) as the first eluent and ethyl acetate/N-hexane (3: 1) as the last eluent, we observed 5, 10, 15, 20-tetrakis (2, 6-dichloro-3-N-ethylsulfamoylphenyl) bacteriochlorin, Luzitin-Cl₂Et was isolated in 2 portions. Each fraction showed two spots in TLC and was identified as the less polar atropisomer α β α β + α₂β₂Or atropisomers beta alpha with greater polarity₃+α₄A mixture of (a). Fig. 4 shows the absorption spectrum of a mixture of atropisomers with higher polarity. This fraction showed a 50% enhancement in the 750nm band, up to 150,000M^-1cm^-1。

Example 5 pH stability of halogenated and sulfonated bacteriochlorins

This example shows the stability of halogenated and sulphonated bacteriochlorins under the acidic conditions found in pH1 and 37 ℃, i.e. the stomach.

Mixing Luzitin-Cl₂Dissolved in a neutral aqueous solution and equilibrated to 37 ℃ to give the absorption spectrum shown in FIG. 5. Aqueous HCl was added dropwise to reduce the pH to 1, and after 2 hours Luzitn-Cl was recorded at a pH of 1₂The absorption spectrum of (1). Then, an aqueous NaOH solution was added to neutralize the solution and a new absorption spectrum was recorded after 3 hours. Fig. 5 shows the absorption spectrum corrected for dilution by addition of aqueous HCl and NaOH. The spectral change observed at lower pH is due to protonation of the pyrrole ring, but is reversed when acidity is neutralized. Under these conditions, almost half of Luzitn-Cl was present₂The original state is recovered, and the other half is converted into the corresponding chlorin.

Example 6 photostability of halogenated and sulfonated bacteriochlorins

This example demonstrates halogenated and sulfonated bacteriaThe increased stability of the chlorophyll under infrared light illumination solves the instability problems seen with other bacteriochlorins of synthetic origin, such as 5, 10, 15, 20-tetrakis (3-hydroxyphenyl) bacteriochlorins or from natural products, such as Tookad

Luzitin-FMet was dissolved in PBS: methanol (2: 3), transferred to a 1cm quartz cell and its absorption spectrum recorded, FIG. 6. The quartz cell was then placed under the light of a 748nm Lynx diode laser which had not been previously focused to have the same beam diameter as the window of the quartz cell. The laser power measured under these conditions was 28.8 mW. The irradiation was interrupted every 5 minutes and a new absorption spectrum was recorded. The process was carried out for 65 minutes. Within the time window of this experiment, the photobleaching action follows the kinetics of the first order reaction. Fig. 6 also shows the absorption spectrum after irradiation. Unlike the decrease in absorbance from 1.128 to 0.337 at the bacteriochlorin peak at 743nm, the absorbance of chlorins increases only from 0.114 to 0.151. Considering the molar absorption coefficient of the compound at these wavelengths, it is clear that only a small percentage of chlorins are formed, unlike 70% of bacteriochlorins destroyed during irradiation. The remaining product does not resolve a good spectrum in visible/IR and the dominant photo-degradation process can be described as photo-bleaching.

The photobleaching half-life was measured for different laser intensities and was shown to be proportional to the laser power. Table 1 lists the half-lives of other bacteriochlorins normalized to 748nm with 100mW laser irradiation, and Foscan normalized to the same light intensityAnd TookadLiterature data on photodegradation of (a). It was also observed that the half-life of Luzitin-Cl increased 30-fold when the oxygen concentration of the solution was reduced by saturating the PBS solution of the photosensitizer with argon. This indicates that ROS participates inAnd (4) photo-bleaching.

Example 7 efficient photogeneration of singlet oxygen

This example describes the efficient photogeneration of singlet oxygen in the presence of Luzinins, light of appropriate wavelength and molecular oxygen dissolved in solution.

Excitation of Luzitin-Cl with an absorbance of about 0.2 at 355nm by a pulsed Nd: YAG laser in a 1cm quartz cuvette₂Hep ethanol air-saturated solution, using the apparatus and method described previously to track singlet oxygen emission at 1270 nm. The intensity of singlet oxygen phosphorescence was studied as a function of laser intensity. Similar studies were performed with benzidine ketone in ethanol at a concentration similar to Luzitin-Cl₂The absorbance at 355nm of Hep was matched. FIG. 7 shows excitation of Luzitin-Cl at 355nm in air-saturated ethanol₂Laser energy dependence of singlet oxygen phosphorescence intensity measured at 1270nm after Hep or benzidine. The slope of the energy dependence of the emission from singlet oxygen and the phi of benzilketone in this ethanol using various laser energies_ΔBy 0.95 value we obtain luzitn-Cl₂Phi of Hep_Δ0.78. The values for the other bacteriochlorins are listed in table 1.

The photostability of these bacteriochlorins is related to their quantum efficiency of about 70% singlet oxygen production, suggesting that under continuous irradiation a given molecule of bacteriochlorin will locally produce a very large number of electronically excited oxygen molecules before it is photobleached. About 30% of the energy absorbed by the sensitizer is lost in other processes, some of which are identified in the examples below.

Example 8 photogeneration of Reactive Oxygen Species (ROS)

This example describes the generation of reactive oxygen species, i.e., superoxide ion and hydroxyl radical, using Luzitin-Cl as the sensitizer.

To evaluate the photoproduction of superoxide ions and hydroxyl groups by Luzitin-Cl, the EPR spectrum in the presence of 30-80. mu.M Luzitin-Cl and 40mM BMPO was determined under the following conditions:

a) air saturated aqueous solution in the dark.

b) The nitrogen-saturated aqueous solution was irradiated with a Hamamatsu diode laser for 8 minutes.

c) The air-saturated aqueous solution was irradiated with a Hamamatsu diode laser for 4 minutes.

d) The air-saturated aqueous solution was irradiated with a Hamamatsu diode laser for 8 minutes.

e) The air-saturated aqueous solution was irradiated with a Hamamatsu diode laser for 16 minutes.

f) A Hamamatsu diode laser was used to irradiate a 16 minute air-saturated aqueous solution in the presence of superoxide dismutase (50. mu.g/ml).

g) A Hamamatsu diode laser was used to irradiate an air-saturated aqueous solution for 16 minutes in the presence of catalase (30. mu.g/ml).

Under the experimental conditions (a) and (b), no ROS-BMPO adduct was detected. However, experimental conditions (c), (d) and (e) resulted in the detection of BMPO adduct formed between BMPO and hydroxyl group, fig. 8. The presence of light is necessary to form such adducts. The experimental condition (f) gave no signal, that is to say that superoxide dismutase, a known superoxide ion scavenger, inhibited the formation of the BMPO-hydroxyl adduct. Furthermore, no signal was obtained under experimental condition (g), demonstrating that catalase, which decomposes hydrogen peroxide into water and oxygen molecules, also inhibits the formation of BMPO-hydroxy adduct. These results indicate that the hydroxyl group is not formed directly from the photosensitizer and molecular oxygen, but is a secondary product. The inhibition observed by superoxide dismutase is clear evidence of superoxide ion formation. The inhibition observed by catalase suggests that hydrogen peroxide is also a precursor for hydroxyl groups.

Similar experiments were performed with DMPO in DMSO. We detected DMPO-superoxide adduct in the presence of air and light, fig. 8. However, in the absence of air or light, or in the presence of superoxide dismutase, such adducts are not observed.

Taken together, these data indicate that, following the formation of hydrogen peroxide, superoxide ions are formed followed by hydroxyl radicals, which are the active oxygen species that are frequently reported in the literature to be capable of causing cell damage. These ROS recruit approximately 70% of the singlet oxygen forming efficiency of these bacteriochlorins, indicating that the remaining 30% of the energy absorbed by the sensitizer is not lost, but is used to form other cytotoxic substances than singlet oxygen.

Example 9 in vitro phototoxicity of Luzitin-Cl-c to mouse melanoma cell lines under Standard Lamp irradiation

This example shows that Luzitins are very toxic to mouse melanoma cells under filtered halogen lamp illumination at concentrations where dark toxicity is negligible.

The aforementioned materials and methods were used to determine the cytotoxicity and photosensitizing activity of Luzitin-Cl-c against the S91 (mouse melanoma) cell line in the dark, except that after incubation, the cells were not washed to remove unincorporated photosensitizer prior to irradiation. FIG. 9 is an inset showing cytotoxicity of different concentrations of Luzitin-Cl-c in the dark over an incubation time of 120 minutes. FIG. 9 shows the compound when it is in [ Luzitin-Cl-c ]]S91 cell survival scores at different light doses when cells were irradiated in the presence of 20 μ M. Table 3 lists 90% kill (LD) in cultures₉₀) Or 50% (LD)₅₀) The light dose required by the cell.

Example 10 in vitro phototoxicity of Luzitin-Cl on mouse melanoma cell lines under standard Lamp irradiation

This example shows that Luzitins are very toxic to human melanoma cells under filtered halogen lamp illumination at concentrations where their dark toxicity is negligible.

The cytotoxicity and photosensitizing activity of Luzitin-Cl on MCF7 ((human breast cancer) cell line in the dark was determined using the foregoing materials and methods the inset in FIG. 10 shows the cytotoxicity of different concentrations of Luzitin-Cl in the dark over an incubation time of 12 hours FIG. 10 shows [ Luzitin-Cl ]]Survival score for MCF7 cells at 5 μ M and various light doses. It is clear that the exposure dose is 0.6J/cm²The filtered halogen lamp of (1) shows a killing effect of Luzitin-Cl at a concentration of 5. mu.M on 100% of cells. Table 4 lists 90% kill (LD) in culture medium₉₀) Or 50% (LD)₅₀) The light dose required by the cell.

Example 11 Luzitin-Cl under laser irradiation₂In vitro phototoxicity to human prostate cancer

This example shows that Luzitins are very toxic to human prostate cancer cells under laser irradiation at concentrations where dark toxicity is negligible.

Luzitin-Cl determination using the aforementioned materials and methods₂According to these experiments, 0.05mM is Luzitn-Cl which resulted in relatively no effect on cell viability in the dark in the experiments₂The highest concentration of (c). This will be the reference concentration for the following phototoxicity study.

Luzitin-Cl determination using the aforementioned materials and methods₂Photosensitizing activity on PC-3 cell line. FIG. 11 shows the light dose at 3 and 6J/cm for various concentrations of the photosensitizer²And incubation time is the survival score of 24 hours. According to FIG. 11, when exposed to 6J/cm²In the case of the laser dose of [ Luzitin-Cl ]₂]20 μ M produced a killing effect on 100% of the cells.

Example 12 Luzitin-Cl under laser irradiation₂In vitro phototoxicity of Et on mouse colon carcinoma cell lines

This example shows that Luzitins are very toxic to mouse colon cancer cells under laser irradiation at concentrations where dark toxicity is negligible.

In IC₅₀On the other hand, it is unnecessary to measure Luzitin-Cl₂Cytotoxicity of Et in CT26 (mouse colon cancer) cell line in the dark, since the compound precipitated in the medium before cytotoxic levels were reached. A concentration of 50. mu.M is Luzitin-Cl which results in relatively no effect on the survival of these cell lines in the dark₂Maximum concentration of Et.

Luzitin-Cl determination using the aforementioned materials and methods₂Photosensitizing activity of Et in CT26 cell line. FIG. 12 shows the light dose of 6J/cm for various concentrations of the photosensitizer²The incubation time was the survival score of 18 hours. The figure shows that the exposure to 6J/cm²Luzitin-Cl with a concentration of 5 mu M at laser dose₂Et exerts a killing effect on 90% of cells, LD₉₀5 μ M. According to FIG. 12, when exposed to 6J/cm²In the case of the laser dose of [ Luzitin-Cl ]₂]At 10 μ M, 100% of the cells were killed. Table 6 shows 6J/cm at 28.5mW²Achieving LD in various other cell lines under diode laser irradiation₉₀Required Luzitin-Cl₂The Et concentration.

Example 13 in vitro phototoxicity of Luzitin-FMet to human Colon cancer cells under laser irradiation

This example shows that Luzitins are very toxic to human colon cancer cells under laser irradiation at concentrations where their dark toxicity is negligible.

In IC₅₀In one aspect, Luzitin-FMet was not assayed for cytotoxicity in the dark on HT-29 (human colon adenocarcinoma) cell line, since the compound precipitated in the medium before cytotoxic levels were reached. The concentration of 50. mu.M is such thatThe highest concentration of Luzitin-FMet that had relatively no effect on the survival of these cell lines in the dark in the experiment.

The photosensitizing activity of Luzitin-FMet on HT-29 cell line was determined using the materials and methods described above. FIG. 13 shows the light dose of 6J/cm for various concentrations of the photosensitizer²The incubation time was the survival score of 18 hours. The figure shows that the exposure to 6J/cm²The Luzitin-FMet with the concentration of 0.5 mu M generates killing effect on 90 percent of cells under the laser dose, and LD₉₀1 μ M. According to FIG. 13, when exposed to 6J/cm²Laser dose of [ Luzitin-FMet ]]At 1 μ M, 100% of the cells were killed. Table 6 shows 6J/cm at 28.5mW²Achieving LD in various other cell lines under diode laser irradiation₉₀The desired concentration of Luzitin-FMet.

Example 14 Luzitin-F under laser irradiation₂In vitro phototoxicity of Met to human non-small cell lung carcinoma

This example shows that Luzitins are very toxic to human non-small cell lung cancer cells under laser irradiation at concentrations where their dark toxicity is negligible.

In IC₅₀On the other hand, it is not necessary to measure Luzitin-F₂Cytotoxicity of Met in A-549 (human non-small cell lung carcinoma) cell line in the dark, since the compound precipitates in the culture medium before cytotoxic levels are reached. A concentration of 50mM is Luzitn-F which results in relatively no effect on the survival of these cell lines in the dark in the experiments₂Maximum concentration of Met.

FIG. 14 shows the light dose of 6J/cm for various concentrations of the photosensitizer²The incubation time was the survival score of 18 hours. The figure shows that the exposure to 6J/cm²Luzitin-F with a concentration of 0.5. mu.M at a laser dose of₂Met exerts a killing effect on 90% of the cells, LD₉₀0.5 μ M. According to FIG. 14, when exposed to 6J/cm²Laser dose ofWhen it is not used (Luzitin-F)₂Met]0.5 μ M produced a killing effect on 100% of the cells. Table 6 shows 6J/cm at 28.5mW²Achieving LD in various other cell lines under diode laser irradiation₉₀Required Luzitin-F₂Met concentration.

Example 15 fluorescence detection Limit

This example shows that Luzitins are very sensitive and selective for detection in biological samples.

Solutions of different concentrations by dilution of 1% Luzitin-Cl in ethanol₂Human serum stock solution of Et. The concentration range is 0.2nM-10 nM. A4: 5: 3 slit was used for excitation and emission, and the sample was excited at 514nm in a fluorescence spectrometer as described above. As shown in fig. 15, the emitted light is collected in the infrared region.

The limit of detection was determined using the following formula:

wherein S_y/xIs the standard deviation of the calibration curve and b is its slope. A detection limit of 0.17nM (or 0.2ng/g) was obtained.

Example 16 in vivo toxicity in darkness

This example shows that Luzitins are not toxic to mice at much higher concentrations of PDT approved for use with commercially available photosensitizers.

Mice were divided into the following experimental groups:

a)10 animals received 2mg/kg of Luzitin-Cl

b)10 animals received 5mg/kg Luzitin-Cl

c)10 animals received 10mg/kg Luzitin-Cl

d)10 animals received 15mg/kg Luzitin-Cl

e)10 animals received 20mg/kg Luzitin-Cl

f)10 animals received no treatment and served as a control group.

A solution of Luzitin-Cl (0.5ml) was injected subcutaneously and the animals were closely observed for 30 days. On the first 6 days after injection, (e) group animals showed photosensitivity, (d) some of the groups also showed slight sensitivity. They show this sensitivity by being far from the light source directed towards them. Animals were weighed periodically, but none of them showed significant weight change. After 30 days, the animals were anesthetized with Morbital (Biowet, Poland), and organ and tissue samples were removed for hematology and histological testing of selected organs. No change was observed in either blood or organs.

Dose ratio photosensitizers used in these dark toxicity assaysThe recommended dosage is 10 times higher than FoscanThe dose of (a) was 100-fold higher, however, the threshold for measurable dark toxicity in mice was not yet reached. This provides evidence that specific photosensitizers can be used in PDTOr FoscanHigher doses of Luzitins.

Example 17 biodistribution of chlorins following intraperitoneal administration

This example shows that Luzitins can cross the blood-brain barrier 2 hours after intraperitoneal administration.

Luzitin-Cl-c was administered to DBA/2 mice at a dose of 10mg/kg by intraperitoneal injection. The distribution of chlorins in tissues was analyzed 2 hours after administration. The animals were anesthetized with Morbital (Biowet, Poland), some of their organs, including the brain, were removed, weighed, and then stored at-30 ℃ below until further analysis. The pigment content in the tissue samples was analyzed by fluorescence spectroscopy. To extract the pigments, tissue samples were homogenized in 7ml of ice-cold 90% aqueous acetone for 1 minute at 10000rpm using a tissue homogenizer MPW-120(Medical Instruments, Poland). The homogenate was centrifuged at 2000g for 10 min at 4 ℃, the supernatant collected and the precipitate re-extracted with 90% aqueous acetone to ensure complete recovery of the pigment. The extracts were combined and analyzed for pigment content. The sample was excited at 413nm and the fluorescence spectrum was recorded in the range of 600-800 nm. Fig. 16 shows fluorescence intensities of brain, blood and liver normalized by their respective masses.

The blood-brain distribution of the sensitizer was 4: 1 after 2 hours of intraabdominal administration in DBA/2 mice. This proves in principle: suitable Luzitins may cross the blood-brain barrier in significant amounts, becoming active photosensitizers in the brain. The latter examples show that such sensitizers will accumulate in the tumor, further increasing the amount of photosensitizer available for treating brain tumors.

Example 18 pharmacokinetics of bacteriochlorins after intraperitoneal administration

This example shows that upon intraperitoneal administration, Luzitins first accumulate in the tumor and then clear at a slower rate.

The tumor model was S91Cloudman melanoma cells cultured as monolayers in RPMI medium containing 5% fetal bovine serum supplemented with antibiotics. Cells were at 37 ℃ and contained 5% CO₂In a humid atmosphere. The melanoma cells (. about.1X 10)⁶) Taken in 0.1ml Phosphate Buffered Saline (PBS) and implanted subcutaneously in the right flank of the animal. Tumors grew significantly after 10 days of transplantation. Animals were treated 3 weeks after injection.

Luzitin-Cl was administered to DBA/2 mice at a dose of 10mg/kg by intraperitoneal injection. Tissue distribution of bacteriochlorins was analyzed at the following intervals: 2 hours, 6 hours, 12 hours, 24 hours and 48 hours after peritoneal administration. Animals were anesthetized with ketamine and xylazine, and their organ and tissue samples were removed, weighed, and then stored at-30 ℃ below until further analysis. The pigment content in the tissue samples was analyzed by fluorescence spectroscopy. To extract the pigments, tissue samples were homogenized in 7ml of ice cold ethanol/DMSO (75: 25) solution for 1 minute at 10000rpm using a tissue homogenizer MPW-120(Medical Instruments, Poland). The homogenate was centrifuged at 2000g for 10 min at 4 ℃, the supernatant collected and washed with ethanol: the DMSO solution re-extracts the precipitate to ensure complete recovery of the pigment. The extracts were combined and analyzed for pigment content. The sample was excited at 517nm and the fluorescence spectra were recorded in the range of 600-850 nm. Figure 17 shows the fluorescence intensity of the tumor and multiple organs normalized by their respective masses.

This photosensitizer significantly accumulates in the tumor 1 day after intraperitoneal administration, when it reaches 3 times the concentration in muscle. Furthermore, accumulation in the skin was negligible, which explains the lack of photosensitivity of mice in dark toxicity studies. Finally, the photosensitizer is cleared from most organs within 24 hours. These pharmacokinetics are highly advantageous for selecting the time window for PDT treatment, where side effects are reduced and treatment efficacy is enhanced.

Similar studies were performed with other Luzins, Luzinn-Cl₂Et shows very strong tumor to muscle selectivity18。

Example 19 PDT of mouse Colon cancer with Luzitin-FMet in Balb/C mice

This example shows that Luzitins produce tumor regression/necrosis when exposed to light of the appropriate wavelength.

The tumor model was CT26 mouse colon cancer cultured in RPMI medium containing 5% fetal bovine serum supplemented with antibiotics. Cells were at 37 ℃ and contained 5% CO₂In a humid atmosphere. The cancer cells (. about.1X 10)⁶) 0.1ml of Phosphate Buffered Saline (PBS) was taken up and subcutaneously transplanted into the right flank of Balb/C mice. Tumors grew approximately 1 week after transplantation and reached 5mm in diameter. No spontaneous necrosis was observed. Treatment was started when the tumor diameter of each animal reached 5 mm. On the day when the tumors reached therapeutic size, mice were injected intraperitoneally with a 10mg/kg dose of Luzitin-FMet in PEG400 solution. At 24 hours post-injection, 4 animals were anesthetized with ketamine and xylazine and dosed at a rate of 100mW/cm as described previously²Hamamatsu 748nm diode laser treatment for 22 minutes (total fluence 132J/cm)²). Another 4 mice were untreated and used as controls. Mice were examined daily and tumors were determined using two orthogonal measurements L and W (perpendicular to L) using the formula V-LxW²Volume was calculated and recorded,/2.

Fig. 19 shows the relative tumor volumes determined at different days after laser irradiation. The tumor size of the treated animals was smaller than the average size of the control animals, and in one case the tumor disappeared completely.

Example 20 use of Luretin-Cl in DBA/2 mice₂Et for PDT of melanoma cells

This example shows that Luzitins produce tumor regression/necrosis when exposed to light of the appropriate wavelength.

The tumor model is S91Cloudman melanoma cells, inMonolayers were cultured in RPMI medium containing 5% fetal bovine serum supplemented with antibiotics. Cells were at 37 ℃ and contained 5% CO₂In a humid atmosphere. The melanoma cells (. about.1X 10)⁶) Taken in 0.1ml Phosphate Buffered Saline (PBS) and transplanted subcutaneously into the right flank of DBA/2 mice. Tumors grew approximately 1 week after transplantation and reached 5mm in diameter. No spontaneous necrosis was observed. Treatment was started when the tumor diameter of each animal reached 5 mm. On the day when the tumors reached the therapeutic size, mice were injected intraperitoneally with Luretin-Cl at a dose of 10mg/kg₂PEG400 solution of Et. At 24 hours post-injection, mice were anesthetized with ketamine and xylazine and tethered to plastic racks and then treated with Hamamatsu 748nm diode laser at different light doses as described previously. At several minutes of irradiation, a significant signal of necrosis of the tumor area occurs and spreads throughout the irradiated area within hours. Mice were examined daily and tumor sizes were determined and recorded. Fig. 20 shows the tumor sizes measured at different days after laser irradiation. Apparently, a single treatment session produces long-lasting tumor regression.

Example 21 formulations for topical application

This example shows that Luzitins can be rapidly dispersed through the skin when using an appropriate transdermal formulation. 4 mini-pigs were used to determine the diffusion of the photosensitizer Luzitin-FMet into the skin and to evaluate the final side effects of the topically applied formulation.

The photosensitizer was first dissolved in absolute ethanol (5mg in 0.556ml), followed by 1.737ml of propylene glycol, then 0.22ml of azone and 0.3ml of water. The mixture was mixed thoroughly in a vortex and sonicated to promote dissolution, then a gel matrix consisting of water (76.65%), 96% ethanol (15%), glycerol (6%), triethanolamine (1.35%) and carbopol 940 (1%) was added. The mixture was mixed well to achieve good homogenization. In this formulation, the final concentration of photosensitizer is 0.1%, AzoneThe content was 4%.

Animals were treated as described above. Under sedation under anesthesia, the formulation was applied to the intended area by hand with surgical gloves. A circular area of about 3cm in diameter was covered with a gel several millimeters in thickness for each application. The applied drug was covered with a blocking patch. In some animals, the same procedure was repeated after 3 hours in different areas of the back. In one of the animals, the preparation was removed after 6 hours of application, the back of the animal was cleaned and kept alive within 10 days for subsequent evaluation. Skin samples were collected as described previously. Each sample was approximately square, 2cm on one side, and 1cm thick. No animals, in particular surviving animals, showed evidence of side effects caused by the formulation, with or without any sensitizers.

After fixation treatment, each sample was sectioned for evaluation by fluorescence microscopy and confocal microscopy. Fig. 21 shows a representative example of an image collected from a sample. These images show that Luzitin-FMet disperses throughout the epidermis within 3 hours of application of the gel. The fluorescence of Luzitin-FMet was further confirmed by its absorption spectrum in FIG. 22. Thus, formulations for topical application of Luzitins can be formulated which disperse throughout the stratum corneum and epidermis within hours, which is very convenient for PDT of skin disorders.

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Claims (2)

1. A process for preparing a compound having formula (I):

wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X¹，X²，X³，X⁴，X⁵，X⁶，X⁷，X⁸each independently selected from F, Cl, Br and hydrogen atoms, provided that all X' s²，X⁴，X⁶And X⁸Or all of X¹，X³，X⁵And X⁷Are both halogen, or X¹，X²，X³，X⁴，X⁵，X⁶，X⁷，X⁸Are all halogen;

R₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈independently selected from H, -OH and-SO₂R, wherein each R is independently selected from-Cl, -OH, -amino acid residue, -ORⁿ、-NHRⁿand-N (R)ⁿ)₂Wherein R isⁿIs an alkyl group of 1 to 12 carbon atoms;

y is fluorine or hydrogen;

comprises the following steps:

(i) solid state reduction of the corresponding substituted porphyrin to the compound of formula (I) using a hydrazide in the absence of a solvent and optionally in the absence of a base; wherein the corresponding substituted porphyrin has the formula:

2. a process for preparing a compound having formula (III):

wherein:

represents a carbon-carbon single bond or a carbon-carbon double bond;

X²selected from F, Cl, Br, X¹Selected from hydrogen, F, Cl, Br, and R' is-SO₂R；

Each R is independently selected from-Cl, -OH, -amino acid residue, -ORⁿ、-NHRⁿand-N (R)ⁿ)₂Wherein R isⁿIs an alkyl group of 1 to 12 carbon atoms;

the method comprises the following steps:

(i) solid state reduction of the corresponding substituted porphyrin to said compound of formula (III) using a hydrazide in the absence of a solvent and optionally in the absence of a base; wherein the corresponding substituted porphyrin has the formula: