WO1993012114A1 - NOVEL 10-HYDROXY PHEOPHYTIN $i(a) - Google Patents

Abstract

The present invention provides 10-hydroxy pheophytin a of formula (I) or pharmaceutically acceptable salts thereof. These compounds are useful as photosensitizers for photodynamic cancer therapeutics.

Description

NOVEL 10-HYDROXY PHEOPHYTIN a

TECHNICAL FIELD

The present invention relates to a novel 10-hydroxy pheophytin a (hereinafter, sometimes referred to as "CpD-1") and pharmaceutically acceptable salts thereof, and a process for preparing the same compound. The present invention also relates to a photosensitizer composition containing the same compound as an active ingredient, and a method for photodyna ically treating cancer using the same compound.

BACKGROUND ART

In recent years, the so-called "photodynamic therapy (PDT) " in which tumor cells are destroyed by applying actinic rays has attracted medical interest and attention. A representative of such photodynamic therapy has been reported by T. J. Dougherty. This photodynamic therapy involves the utilization of photosensitizers which are preferentially absorbed in a tissue portion where solid tumors grow rapidly. The photosensitizers once injected are concentrated around the tumor site. Light of maximum absorption wavelength intrinsic to the photosensitizers is then applied to the tumor to selectively destroy tumor cells without damaging healthy cells surrounding the tumor cells [See T.J. Dougherty et al . , Cancer Res. 38 , pp. 2628-2635, (1978)].

In treating cancer by means of the photodynamic therapy, the key point is the selection of photosensitizers to be used. Photosensitizers have specific properties in that they are^~~preferentially attached to tumor cells and fluores¬ cent. By the utilization of these properties, the cancer cells can be located. That is, when a photosensitizer is injected into a body to be examined and the body is then endoscoped, a fluorescent site(s) where the tumor cells are growing can easily be observed.

Upon irradiation, the photosensitizer in the ground state absorbs light. The light excited state of the photosensitizer transfers energy to molecular oxygen present in the body to generate singlet oxygen. The singlet oxygen oxidizes various biological compounds including structural components of cells such as steroids and phospholipids having unsaturated acyl chains, amino acids such as tryptophane, histidine, cysteine and methionine; and the components of nucleic acids such as guanosine and cytidine, and so forth. Thus, once singlet oxygen reacts with the tumor cells, it kills the tumor cells by oxidizing the cellular constituents of tumor tissue.

As explained above, the photodynamic therapy may be utilized in the field of the diagnosis and treatment of cancer. The photodynamic therapy is advantageous over other types of therapeutic methods. Therapy of this type has a high treatment effect and a broad spectrum of applica¬ tion since even tumors at any remote sites where light penetrates through an endoscope can be successively treated, regardless of the types of tumors. This therapeutic method has further advantages that it causes less side effects on the healthy cells and can thus be applied repeatedly.

So far, a number of photosensitizers have been developed which are of both natural and synthetic origins. Such photosensitizers include psoralens, flavins, porphyrins, acridines, phenothiazines, xanthenes, quinones, polyenes, haloaromatics, and inorganic ions [See J.D. Spikes and R. Livingston, Advances in Radiation Biology, Vol. II, pp. 29- 121 (1969)]. Many of these photosensitizers are now under clinical tests for use as photodynamic anti-tumor agents. A few photosensitizers are being commercially available for PDT. The representative example includes hematoporphyrin derivatives (HpD) which are being sold by The Quadra Logic Technology Inc. (Vancouver, Canada) under the tradenames "Phαtofrin-I," a mixture of ethers and esters of polyhe atoporphyrin, and "Photofrin-II," a product more purified.

DISCLOSURE OF THE INVENTION

We, inventors of the present invention, have intensively conducted a wide range of experiments in order to develop a potent photosensitizer having high treatment effects. As a result, the inventors have discovered that a novel pheophytin derivative isolated from silkworm excreta has significantly improved photosensitivity, and could accomplish the present invention.

Therefore, it is an object of the invention to provide a novel pheophytin derivative which is useful as a photosensitizer for the PDT treatment of cancer.

It is another object of the invention to provide a process for preparing the novel pheophytin derivative of the present invention.

It is still another object of the invention to provide a photosensitizer composition comprising, as an active ingredient, the novel pheophytin derivative of the present invention, in admixture and in association with conventional ingredients, such as carriers, excipients, extenders, and so forth.

It is still further object of the invention to provide a method for photodynamically treating cancer by using the novel pheophytin derivative of the present invention.

Any additional objects of the invention will become apparent through reading the remainder of the specification.

These and other objects of the invention can be achieved by providing a novel 10-hydroxy pheophytin a of the formula:

or pharmaceutically acceptable salts thereof.

The pheophytin derivative according to the invention is suitable to generate the photoactive singlet oxygen in higher yields, and it exhibits more preferential absorption in tumor tissues, compared to other conventional photosensitizers. Moreover, the photosensitizer of the present invention is superior to .other conventional ones in terms of selective destruction of cancer cells and efficacy in treating tumors in test animals.

The compound of the present invention is structurally similar to chlorophyll a, but has a structural feature in which Mg⁺⁺ and the hydrogen atom at C₁₀ position in chloro¬ phyll a are replaced by two hydrogen atoms and hydroxy group, respectively.

To the inventors* knowledge, the 10-hydroxy pheophytin a according to the invention is a novel compound which has not been structurally identified and has not been used as a photosensitizer in vivo and in vitro experiments.

The compound of the formula (I) may be converted to its non-toxic salt, i.e., its non-toxic pharmaceutically acceptable salts in accordance with any conventional methods. The non-toxic salts include its inorganic salts, for example, its metal salts, its inorganic acid salts, and its organic salts. Included among the metal salts are alkali metal salts such as sodium salt, potassium salt, etc. , and alkaline earth metal salts such as calcium salt, magnesium salt, etc. The inorganic acid salts may include hydrochloride, hydrobromide, sulfate, phosphate salts, and so forth. The organic salts may include organic a ine salts such as trimethylamine salt, triethylamine salt, pyridine salt, procaine salt, picoline salt, decyclohexylamine salt, N,N-dibenzylethylene-diamine salt, N-methyl glucamine salt, diethanolamine salt, triethanolamine salt, tris(hydroxy- methylamino) methane salt, phenylethylbenzylamine salt, dibenzylethylenediamine salt, and so forth. Also included among the non-toxic salts are organic carboxylic or sulfonic acid salts such as formate, acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc. , and a salt with a basic or acidic aminoacid such as arginine, aspartic acid, glutamic acid, lysine, etc. The present invention also provides a process for preparing the novel pheophytin derivative according to the invention which comprises the steps of:

(a) extracting chlorophyll metabolites from silkworm excreta with an appropriate solvent;

(b) dissolving the extracted substance in an appropriate solvent until supersaturated;

(c) subjecting the resulting solution to chromatography to carry out the separation of photosensitizer components; and

(d) collecting the fractions exhibiting a potent photo- sensitization function, followed by purification in a conventional manner.

The extraction of silkworm excreta (Step (a)) can be carried out by means of the conventional solvent extraction. As the solvent, any solvent known in the art such as ketones, ' alcohols, and a mixed solvent thereof may be used. The extracted substance is then dissolved in an appropriate solvent to give a supersaturated solution (Step (b) ) . Any solvent may be employed insofar as it dissolves the solid extract. However, a mixed solvent of t-butanol, acetone and pentane is preferred for effective solubilization. The resulting solution is then subjected to chromatography to separate the solid components contained in the extract (Step

(c) ) . Any preparative chromatography known in the art, such as liquid chromatography (LC) , thin layer chromatography

(TLC) , and the like may be used. After completing the separation, the fractions that have high photosensi ivity are collected, which is then purified by means of a conventional purification method (Step (d) ) . Preferably, high pressure liquid chromatography (HPLC) may be used for the purpose of the present invention.

The resulting product is analyzed to identify its chemical structure. To this end, any analytical methods known in the art, such as infrared spectrometry (IR) , Fourier transform infrared spectrometry (FT-IR) , ultraviolet spectrometry (UV) , optical rotatory dispersion and circular dichroic spectrometry, fluorimetry, flash spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, etc. , may be employed.

Alternatively, the compound of the formula (I) can be synthesized chemically by using chlorophyll a as a starting material. That is, the synthetic method for preparing the compound of the formula (I) comprises the steps of:

(a) reacting chlorophyll a with HC1 in ethanol or chloroform at room temperature to remove a Mg ion therefrom;

(b) gently bubbling air through the resulting solution under dim safelight or in a dark place for a day or longer at room temperature to replace the hydrogen atom at C₁₀ position by a hydroxy group; and (c) subjecting the resultant product to conventional purification.

The starting material, chlorophyll a , can be obtained from any appropriate source by a conventional manner. However, it is preferred to use chlorophyll-rich algae, Spirulina platensis as the chlorophyll a source. This synthetic method is advantageous in that it is suitable to the mass production of CpD on a large scale.

Although hereinabove the present invention has been described in respect of the single compound of the formula (I) , it should be noted that modification can be made to the same compound by replacing the substitutive groups with another ones. See, G.N. La Mar, "Model Compounds As Aids In Interpreting NMR Spectra Of Hemoproteins" in Biological Applications of Magnetic Resonance, R.G. Shul an Ed., pp.

305-343 (1979), Academic Press, New York; and J.D. Satterlee,

"NMR Spectroscopy Of Paramagnetic Haem Proteins" in Annual

Reports On NMR Spectroscopy, G.A. Webb Ed. , pp. 84-85 (1986) , Academic Press, New York. From these prior art teaching, it is apparent that a number of analogs having substitutive groups different from those of the compound of the formula

(I) may be designed and synthesized by those skilled in the art according to the conventional methods well-known in the art. Thus, the present invention encompasses such 10-OH- pheophytin a analogs having the following formula:

wherein, R_{1 f} R₃, R₄, R_g, R₆ and _g is a C,-^ alkyl aldehyde or a C_j-C₈ cyclo alkyl group ;

R₂ is a C,-^ alkenyl group ; and

R₇ is CH,

in which £ , m and n each denotes an integer of 1 to 3. In further aspect, the present invention provides a photosensitizer composition for use in treating tumor cells, which comprises, as an active ingredient, the compound of the formula (I) or pharmaceutically acceptable salts thereof, in admixture or in association with conventional ingredients such as carriers, excipients, extenders, and other additives.

In still another aspect, the present invention provides a method for photodynamically treating cancer which comprises administering into the body the novel pheophytin derivative of the formula (I) or pharmaceutically acceptable salts thereof.

The pheophytin derivatives of the formula (I) may be administered into the human body by means of various types of injection, for example, intravenous, intramuscular, subcuta¬ neous and intraperitoneal injections in a daily dose ranging from about 5 mg/kg to 10 mg/kg of body weight.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be described in greater detail with reference to the accompanying drawings, wherein;

Figs. 1A and IB represent a thin layer chromatogram (TLC) for each of silkworm excreta, spinach and mulberry;

Figs. 2A, 2B and 2C represent reverse phase high perfor¬ mance liquid chromatograms (HPLC) for Band 2 as depicted in Figs. 1A and IB; where Fig. 2A is a reverse phase HPLC for Band 2 which was exposed to fluorescent room light for a few hours, showing appearance of CpD-2 from photoconversion of CpD-1; Fig. 2B is a reverse phase HPLC for Band 2 which was exposed to light for 5 minutes; and Fig. 2C is a reverse phase HPLC for Band 2 which was not exposed to light; Fig. 3A represents a ^-N R spectrum for each of CpD-1 and Chlorophyll a, wherein the arrow indicates the location of C₁₀-OH and C₁₀-H;

Fig. 3B represents a ^TH-NMR spectrum for CpD-1, CpD-1 in admixture with heavy water (D₂0) , and chlorophyll a, heavy water being admixed with CpD-1 to determine ¹H resonance of C₁₀-OH;

Fig. 3C represents a ¹³C-NMR spectrum for CpD-1;

Fig. 3D represents a H two-dimensional COZY NMR spectrum for CpD-1;

Fig. 4 represents a FT-IR spectrum for CpD-1;

Fig. 5 is a graph showing the amount of singlet oxygen generated at the maximum initial photooxidation rate of 1,3- diphenylisobenzofuran (DPBF) at 295°K;

Fig. 6 is graphs showing the absorbability of CpD-1 and HpD in the tumor and normal cells; and

Figs. 7A and 7B are graphs showing the survival percentages of human T-4 lymphoma cell line (MOLT-4) and human peripheral blood lymphocyte (PBL) which were treated with CpD-1 and HpD, respectively, and then exposed to red colored light (Fig. 7A) and yellow colored light (Fig. 7B) .

PREFERRED EMBODIMENT OF THE INVENTION

The present invention will be illustrated in greater detail by way of the following examples. The examples are presented for illustration purpose only and should not be construed as limiting the invention which is properly delineated in the claims.

EXAMPLE 1 : Preparation of CpD-1 from Silkworm Excreta and Identification of Its Structure

Chlorophyll metabolites from silkworm excreta were extracted with acetone in accordance with a conventional solvent extraction method. Independently, the same extraction procedure was repeated with spinach and mulberry for comparison.

After drying, 0.1 g of the crude extract was dissolved in 1 mL of a mixed solvent of t-butanol, acetone and pentane (5:60:235 v/v/v) to give a supersaturated solution. The resulting solution was spotted on a plastic thin layer chromatographic (TLC) sheet of silica gel (20x20 cm) (TLC plastic silica gel, Art. 5735, EM Science) in a conventional manner. After spotting, the sample band was dried under a nitrogen stream. A filter paper (25 cm high) was placed in TLC chamber (30 x 28 x 10 cm) , and the same solvent was poured into the chamber for 20 minutes before running. Vacuum grease was used to seal the chamber covered with the cover glass. The running time was about 40 minutes. All manipulations were performed in the dark or under dim green light.

The results are shown in Figs. 1A and IB. In Figs. 1A and IB, the second band ("Band 2") does not appear in the developed chromatograms for spinach and mulberry. In vivo assay revealed that Band 2 has excellent photosensitivity.

Band 2 was carefully scraped off from the TLC sheet and extracted with acetone. The eluate was then evaporated under a nitrogen stream, and the residue was stored for further purification. Subsequently, purification was carried out by a reverse phase HPLC using an ISCO Model 2350 HPLC, model 2360 gradient programmer, UV-5 absorbance/fluorescence detector (ISCO, Inc.), and a C-18 Dynamax-300 (l x 25 cm) column (Rainin Instrument Company, Inc.) in accordance with a conventional method. HPLC conditions were as follows: Flow rate: 1.7 mL/ in;

Mobile phase: 35% methanol and 65% acetonitrile; Gradient: from 80% mobile phase and 20% water to 100% mobile phase for 25 min.

200 μL injection volumes of samples having an absorbance of about 7.0 at 660 nm (approximately 25 μg) were used. Each running time set for approximately 80 minutes, and the absorbance at 660 nm was monitored and stored -on a microcomputer. Each peak was automatically collected. This resulted in four sets of peaks, PI, P2, P3, and P4 as depicted in Fig. 2A-2C.

Referring now to Fig_* 2A-2C, Fig. 2A shows a reverse phase HPLC curve for Band 2 which was exposed to fluorescent room light for a few hours; Fig. 2B shows that for Band 2 which was exposed to light for 5 minutes; and Fig. 2C shows that for Band 2 which was not exposed to light. The main component was CpD-1 corresponding to the third peak, P3. When exposed to light, CpD-1 was oxidized to be converted into CpD-2, an isomer of CpD-1, corresponding to the second peak, P2.

Highly pure CpD-1 thus obtained was identified for its structure by employing FAB/MS, ¹H one- and two-dimensional COZY NMRs, ¹³C-NMR, and FT-IR.

Fig. 3A represents ^- MR spectra for CpD-1 and Chlorophyll a, in which the arrow indicates the locations of C₁₀-OH and C₁₀-H contained in CpD-1 and Chlorophyll a, respectively. Chemical shifts of CpD-1 and Chlorophyll a are listed in Table 1 below. Fig. 3B represents ¹H-NMR spectra for CpD-1, CpD-1 in admixture with heavy water (D₂0) and Chlorophyll a. Fig. 3C and Fig. 3D represent a ¹³C-NMR spectrum for CpD-1 and a ¹H two-dimensional COZY NMR spectrum for CpD-1, respectively. Fig. 4 represents a FT-IR spectrum for CpD-1.

From the above drawing Figures, it is confirmed that CpD-1 is of the structure, in which Mg⁺⁺ in chlorophyll a is replaced by two hydrogen atoms, and that the hydrogen atom at C₁₀ position is replaced by OH.

Table 1

Notes: * Solvent used : CDC1₃

** Locations designated in accordance with the Fischer's numbering system.

EXAMPLE 2 : Synthesis of CPD-1

About 40 g of dry algae, Spirulina platensiε , which is purchased from Mexico City, was submerged in 500 mL of methanol in a 1 L flask, and 5 mL of 0.01 M KOH was added thereto. Air was gently bubbled through the resulting solution in a dark place for two to six days, and then refluxed at approximately 50°C for 15 minutes. This solution was filtered through filter paper in a funnel and 100 mL of methanol was poured thereonto to rinse the remaining materials. The filtrate was then treated with 1 mL of concentrated HCl for 3 minutes. The resulting solution was evaporated to less than 30 mL in a rotavapor (Buchi, Switzerland) . 200 ML of ddH₂0 and 200 mL of CC1₄ were then added to the resulting materials. After separation, the organic phase was washed twice with 200 L of ddH₂0, and the aqueous phase was discarded. The organic solvents were further removed by evaporation and the resulting materials were dissolved in about 20 mL of CC1₄ to give a crude extract.

Approximately 60 g of 230 to 400 mesh silica-gel (Sigma Chemical Co.) was submerged in CC1₄ and then sonicated for 5 minutes. The column was packed in a 2.5_{I D}_ (cm) glass tube and pre-equilibrated with CC1₄. The same conditions were used to pack the second and third silica-gel columns. After loading the crude extracts, approximately 50 mL of CC1₄ was introduced to elute the pink and yellow materials. A 20% acetone solution in CC1₄ was then introduced to elute the fractions of.10-OH pheophytin a/pheophytin a containing CpD. The remaining material in the column was discarded. The solvents were evaporated out, and the residue was dissolved again in 15 mL of CC1 for a secondary silica-gel chromatog¬ raphy. After loading samples on a new silica-gel column, approximately 50 L of CC1₄ was used to elute the pink/yellow materials. A 10% acetone solution in CC1₄ was then used to elute the target CpD fractions. Similarly, the remaining materials, which could not have been eluted by 10% acetone in CC1₄, were discarded. Repeatedly, the solvent was evapo¬ rated out from the collected fractions, and the residues were then dissolved in 15 mL of CC1₄. After loading the samples taken from the second silica-gel column onto a third silica- gel column, approximately 150 mL (until enough) of a 2% acetone solution in CC1₄ was first introduced to elute non- target CpD (checked by taking absorption spectra) . Next, up to 5% acetone solution in CC1₄ was added stepwise to the mobile phase to elute fractions containing 10-OH pheophytin a. The collected fractions were dried and stored in the dark under nitrogen atmosphere for further purification by a reverse phase column chromatography. The above procedures were performed in a hood.

A preparation scale reverse phase column of C-18 (chromosorb LC-7, Sigma Chemical Co.) was packed in an adjustable glass tube (Amicon) of 2.2, _D X 12_length (cm). The elution program is as follows:

Mobile phase: 50% ethanol, 40% acetonitrile, and

10% CC1₄ Sample injection: 1 mL of approximately 250 μg CpD Flow rate: 2 mL/min Detection: 660 nm absorption

Elution: (1) gradient of 99% M.P. and 1% water to 100% M.P. for 15 min

(2) 100% M.P. for 13 min

(3) 20% CC1₄ in isopropanol for 7 min (4) 99% M.P. and 1% water for 20 min. The fraction of 10-OH pheophytin a was eluted at 28 minutes after the injection. The purity of this resulting product was greater than 95%, as determined by TLC assay. On TLC, the product migrated with the same Rf as the second band of the crude extract from silkworm metabolites. The conditions used for the TLC analysis were the same as described in Example 1.

The inventors also determined that Band 1 on the TLC chromatogram was identified as pheophytin a by assigning the NMR proton/carbon resonances in comparison with 10-OH pheophytin a and chlorophyll a. The retention time of pheophytin a in the C-18 column was longer than that of 10-OH pheophytin a, and they could be separated under the above elution conditions. The resulting product,- 10-OH pheophytin a, was dried and stored in the dark under nitrogen atmosphere. Yield: > 0.4% of dry weight algae powder, assuming 100% extraction of chlorophyll a (actual % extrac¬ tion of chlorophyll a varies from batch to batch) . This value is about 10 times higher than the CpD yield (0.04) from crude extract from silkworm excreta.

EXAMPLE 3: Productivity of Singlet Oxygen

One of the most important criteria for determining the ultimate efficiency of a PDT photosensitizer is its ability to generate singlet oxygen via energy transfer from the triplet photosensitizer to molecular oxygen. Two individual methods for the determination of the quantum yields of singlet oxygen as described in Methods I and II below were employed in this example. The photooxidation method

(Method I) uses 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen trap. The rate of photooxidation is followed in terms of decrease in DPBF fluorescence or absorbance to estimate the initial rates of photooxidation of DPBF in the presence of each sensitizer. The initial DPBF photo¬ oxidation rate inversely reflects the initial rate of singlet oxygen production.

Method I: Relative Photooxidation of DPBF

A 632.8 nm helium-neon laser (20 mW maximum output, Metrologic Instruments, Inc.) was used as an irradiation source to excite the sample solution. A solution of DPBF (recrystallized under nitrogen in the dark) in ethanol was prepared immediately before use, and the concentration was checked by using e = 22,500 cm^"1M^_1 at 420 nm. In a closed cuvette containing a total of 2 mL of solution, each sensi¬ tizer had almost the same absorbance at 632 nm. Oxygen gas presaturated with solvent was used to gently bubble the solution for 20 min, after which a small aliquot of different concentrations of DPBF was mixed with the solution. The decrease in the fluorescence intensity of DPBF at 455 nm in ethanol and 465 nm in CC1₄ was measured as a function of irradiation time to determine initial rates of photooxidation of DPBF.

Method II: Direct Luminescence Method

To detect singlet oxygen directly, the near-IR phospho¬ rescence was measured. A Q-switched Nd:YAG laser (Quantel, YG-571-C) was used operated at 355 nm. The silkworm CpD and chlorophyll a samples were dissolved in air-saturated ethanol-OD, and sample concentrations were kept almost the same by adjusting the absorbance to about 0.55 at 355 nm.

A double reciprocal plot of 1/rate vs. 1/[DPBF] yields a maximum initial rate from the reciprocal intercept

(Fig. 5) . The quantum yield of singlet oxygen of chlorophyll a in ethanol (φ& ,χ in C₂H₅OH) was then calculated from the maximum initial rate, relative to the known singlet oxygen yield of chlorophyll a in carbon tetrachloride as reference

in CC1₄) by Equation (1) below.

Equation (1) was also used for the quantum yield calculation for each sensitizer in ethanol. The results are shown in Table 2 below and Fig. 5. From the results, it is confirmed that CpD-1 is superior to chlorophyll a in terms of the productivity of singlet oxygen. CpD-1 exhibits higher productivity by over 20% relative to HpD.

Table 2

Singlet oxygen yields (φ.) determined by the relative photo¬ oxidation of DPBF (method I) and the direct luminescence method (method II)

Method Item Chlorophyll a CpD-1 CpD-2

Cellular absorption of CpD-1 and HpD was evaluated by using a human T-4 lymphoma cell line (MOLT-4) as a compara¬ tive standard tumor cell and a fleshly prepared human peripheral blood lymphocyte (PBL) as a comparative standard healthy cell. To 1 x 10⁶ test cells, 1 mL of photosensitizer solutions containing 10 μg/mL of CpD-1 and HpD, respectively,- was added and incubated in a roller drum for 30 to 210 minutes. The culture was centrifuged at 0°C, 2,000 rpm for 5 minutes. The precipitated cell pellet was washed with a cold physio¬ logical saline solution and then centrifuged again. 400 μL of DMF was added to the precipitates. The resulting mixture was shaken at room temperature and centrifuged at 4°C. Supernatant of 300 μL was taken and used as a cell extracted solution.

After 200 μL of distilled water and 500 μL of.1 N hydrochloric acid were added to 300 μL of the cell extracted solution to 1 mL. An absorbance of CpD-1 and HpD was measured at 660 and 550 nm, respectively. The observance .thus measured was compared with that of the standard solution the wavelength of which was predetermined to determine the amount of the test sensitizer uptaken in the cell.

The results are shown in Fig. 6. In Fig. 6, it can be seen that the amount of uptakes in cells increases with the passage of incubation time, regardless of types of porphyrins and test cells. The amount of uptakes in MOLT-4 is larger than that in human peripheral blood lymphocyte (PBL) as a comparative standard healthy cell. In the case of the same test cells, CpD shows larger absorbability than HpD. From these, it is confirmed that CpD-1 was absorbed preferentially at the site of tumor cells. The localization was found to be over two times higher than that of HpD.

EXAMPLE 5: Selective Destruction of Tumor Cells (Jn Vitro Assay)

Selective destruction ability of CpD-1 was evaluated by using the same tumor and healthy cells as in Example 2 above. CρD-1 was injected into each of 1 x 10⁶ tumor and healthy cells. The cells thus treated was then .exposed to red light for 10 minutes. The same procedure was repeated except that HpD was injected in place of CpD-1 as a compara- tive photosensitizer. Survival percentage of the cells was calculated at an interval of 1 hour during 8 hours immedi¬ ately after the light exposure. The results are shown in Fig. 7A.

MOLT-4 treated with CpD-1 and exposed to red light was found to be killed entirely within 2 hours after the light exposure, while PBL treated with CpD-1 and exposed to red light showed decrease in survival ratio with time and reached a survival ratio of 69.4% at 8 hours after the light exposure. MOLT-4 treated with HpD and exposed to red light showed a survival ratio of 83.4% at 8 hours after the exposure, while PBL treated with HpD and exposed to red light showed a survival ratio of 91.4% at 8 hours after the exposure.

The same procedure as the above was repeated except that MOLT-4 and PBL were exposed to yellow light in place of red light. The results are shown in Fig. 7B.

MOLT-4 treated with CpD-1 showed a decrease in survival ratio with time and reached a survival ratio of 2.7% at 8 hours after the yellow light exposure, while PBL treated with CpD-l showed a survival ratio of 81.5% at 8 hours after the yellow light exposure. MOLT-4 treated with HpD showed a survival ratio of 58.2% at 8 hours after the yellow light exposure, while PBL treated with HpD showed a survival ratio of 83.3% at 8 hours after the yellow light exposure.

These results indicates that CpD-1 exhibits a higher cytotoxicity at a longer wavelength (red light) than a shorter wavelength (yellow light) . In contrast, HpD showed a higher cytotoxicity in yellow light than in red light. Even in this case, the cytotoxicity of HpD was far less than that of CpD-1. In conclusion, HpD was not significantly effective relative to CpD-1.

From the above, it is confirmed that CpD-1 is superior to HpD in terms of the selective tumor destruction.

EXAMPLE 6: Treatment Effects on Mouse Skin Tumor (In Vivo Assay)

Female C3H/HeJ mice (whole body weight: about 20 g) and BA mammary carcinoma were used in this assay. Subcutaneous tumors were induced by injecting 1 μL pieces of BA mammary carcinoma into the hind flank of the recipient mice. PDT treatments were performed when the tumor grew to a diameter of 6-7 mm (about 10 days after the transplantation) .

In this PDT treatment, Group 1 consisting of 12 mice was injected in two different doses of CpD and exposed to light in two different light doses at 24 hours after the injection.

From Group 1 consisting of 12 mice, 6 mice were taken, treated with 0.2 L of stock solution 2 (5 mg/kg) and then exposed to 670 nm light at a time period of 24 hours. Among these 6 mice, 3 mice received a total light dose of 100

J/cm², and another 3 mice received 300 J/cm². Other 6 mice were injected in a higher doses of 0.4 L of stock solution 2 (10 mg/kg) , and exposed to light at 24 hours after the CpD injection. Similarly, 3 mice received 100 J/cm² light dose, and another 3 mice received 300 J/cm². Thus, the three mice constituted a sub-group receiving PDT treatment under the same conditions. In a separate set of PDT treatment on Group 2 consisting of 12 mice, the same PDT conditions were adopted except that the light treatment was started at 4 hours after the CpD injection. The results are shown in Tables 3 and 4, respectively.

Table 3 PDT Treatment at 24 Hours after Injection of CpD-1

CpD Dose Light Dose No. of (mg/kg) (J/cm²) Mice Toxicity Regrowth Cure

5 100 3 - 3 - 5 300 3 1 dead - ^• 2

10 100 3 - - 3

10 300 3 2 dead - 1

Table 4 PDT Treatment at 4 Hours after Injection of CpD-1

Mice Toxicity Regrowth Cure

3

3 1 dead, 2 2 lost foot 3 3 dead

3 3 dead - EXAMPLE 7: Treatment Effects on Solid Tumor (In Vivo Assay)

S-180 cells were administered to ICR mice by an abdomi¬ nal subcutaneous injection to induce solid tumor of 5-10 mm in size. S-180 cells was obtained by passaging a sarcoma- inducing cell line, Sarcoma 180, in ICR mice for a long time.

The test mice were divided into two groups, with each group being treated with CpD-1 and HpD, respectively. The two groups thus treated were divided again into two to give a total of four groups. One of the two among the four groups was exposed twice each for 10 minutes to light at the maximum porphyrin absorbance. Light was applied at 1 hour and then 24 hours after the treatment with the photosensitizer. Another of the two groups were exposed twice to the same light each for 10 minutes at 24 hours and then 48 hours on the same mice group after the treatment with the photosen¬ sitizer.

The group exposed to light twice at l hour and then 24 hours after the CpD-1 injection showed 100% of cure ratio (cured number/total number treated = 20/20) , while the group treated with HpD showed 80% of cure ratio (16/20) . These results indicate that CpD-1 exhibits superior photosensi- tizing capacity (P<0.025) as compared to HpD.

Also, the group exposed to light twice at 24 hours and then 48 hours after the CpD-1 injection showed 60% of cure ratio (12/20) , while the HpD treated group showed 80% of cure ratio (16/20). From these results (P>0.1), it is concluded that the retention time of CpD-1 in the tumor cells was very short and, thus, the secondary action of light could be minimized even in cases where the patient treated with CpD-1 was re-exposed to light within a relatively short time. As for HpD, the patient who had been subjected to photodynamic treatment had to be isolated from light for a period of a least 24 hours because of secondary cytotoxic effects of Hp such as flare, edema and pain of the skin [See Photobiolog of porphyrins, In Doiron DR, Gomer CJ eds., Porphyri localization and treatment of tumors, New York, Alan R Liss, Inc., 1984, pp. 19-39].

The experimental results mentioned above revealed that CpD-1 is superior to HpD in the various aspects and may be effectively used as a photosensitizer for treating tumor cells.

EXAMPLE 8: Treatment Effect on Abdominal Tumor (In Vivo Assay)

S-180 cells were administered to ICR mice by an abdominal subcutaneous injection to induce abdominal tumor and the mice were divided into five groups. Each group was comprised of 10 mice. The treatment effect of CpD-1 on abdominal tumor was evaluated on the basis of the increase in body weight of mice with treatment time.

Each group was treated with CpD-1 at 6 days (first group) , 13 days (second group) , and 20 days (third group) , respectively, after the injection of S-180, and then exposed to light three times each for 10 minutes at 24, 48 and 72 hours after the injection of CpD-1. The fourth group was treated with CpD-1, but was not exposed to light. The fifth group was not treated with CpD-1.

In the case where a mouse to be examined exhibited substantially the same increase in body weight as in the fifth group and survived during the period of longer than 50 days after the above treatment, the mouse was defined to be fully cured. In the first group, 8 mice were cured fully. In the second group, only 3 mice were cured fully and the remaining 7 mice were dead within 35 days owing to the continuing increase in body weight. In the third group, all mice were dead within 35 days owing to continuing increase in body weight. Also, in the fourth group, all mice were dead within 35 days owing to rapid increase in body weight, the weight of mice dead being about two times heavier than their initial weights.

The above studies also indicated that the smaller the tumor site, the higher the tumor clearance rate. It is concluded that CpD-1 can be used in the treatment of abdominal tumors by the PDT method.

EXAMPLE 9: Inhibitory Effects on Reverse Transcriptase from Retrovirus

A five fold diluted solution of infectious Gross Leukemia Virus (GLV) was first prepared. GLV was obtained by cultivating TGV cell line established from Leukemic C3HeB/Fe mouse in a medium (See Machala 0, Bodyd R, Youn JK, and Barski G: Long-term in vitro culture of pathogenic Gross leukemia virus in mouse thymus cells, Eur. J. Cancer 10, 467- 472, 1974). The resulting solution was treated with CpD-1 and then exposed to light. The production of reverse tran¬ scriptase was examined to evaluate the toxicity on virus.

GLV was inoculated on 1 x 10⁵ normal mouse fetal cells and cultivated in Eagle's minimal essential medium containing 10% of penicillin (100 units/mL) , streptomycin (100 μg/mL) and bovine fetal serum for 24 hours. After adding CpD-1 and subsequent exposure to light, the culture was treated with trypsin and dyed with trypan blue to count the survived cells. Virus pellets obtained by a ultrahigh speed centrifuge were triturated using a detergent and reacted with a reactive mixed solution capable of measuring the level of ³H dTTP to be incorporated with reverse transcriptase. The resulting mixture was spotted on a glass fiber filter, washed, and dried to determine a specific radioactivity by using a scintillation counter.

The virus treated with CpD-1 and exposed to light exhibited a radioactivity of 5,000 cpm or less, which is a remarkable effect on enzyme production (i.e., reduction) or photodynamic action on the enzyme (reverse transcriptase) , while the control group which was not subjected to the CpD-1 treatment but was exposed to light showed a radioactivity of more than 40,000 cpm. Also, the same virus which.was treated with CpD-1 and exposed to light showed a reduced infectivity to the mouse fetal cell.

From these results, it is concluded that CpD-1 has antivirus activity.

INDUSTRIAL APPLICABILITY

As discussed above, 10-hydroxy pheophytin a and pharmaceutically acceptable salts thereof according to the invention exhibit improved photosensitivity and superior absorbability by the tumor cells. Moreover, the compounds according to the invention are superior to other conventional photosensitizers, especially HpD, in terms of selective destruction of cancer cells and the productivity of singlet oxygen. Therefore, the compounds of the invention are expected to be suitable for use in the photodynamic therapeutic applications as photosensitizers.

Claims

1. 10-Hydroxy pheophytin a of the formula;

or pharmaceutically acceptable salts thereof,

2. A process for preparing the pheophytin a of Claim l which comprises the steps of:

(a) extracting chlorophyll metabolites from silkworm excreta with an appropriate solvent;

(b) dissolving the extracted substance in an appropriate solvent until supersaturated; (c) subjecting the resulting solution to chromatography to carry out the separation of photosensitizer components; and

(d) collecting the fractions exhibiting a potent photo- sensitization function, followed by purification in a conventional manner, or pharmaceutically acceptable salts thereof.

3. A process for preparing the pheophytin a of Claim 1 which comprises the steps of: (a) reacting chlorophyll a with HC1 in ethanol or chloroform at room temperature to remove a Mg ion therefrom;

(b) gently bubbling air through the resulting solution under dim safelight or in a dark place for a day or longer at room temperature to replace the hydrogen atom at C₁₀ position by a hydroxy group; and

(c) subjecting the resultant product to conventional purification, or pharmaceutically acceptable salts thereof.

4. A photosensitizer composition for use in photodynamic cancer therapeutics which comprises, as an active ingredient, 10-hydroxy pheophytin a or pharmaceu¬ tically acceptabl salts of Claim 1, in admixture or otherwise in association with conventional ingredients such as carriers, excipients, extenders, and other additives.

5. A method for photodynamic treatment of cancer by administering into the body 10-hydroxy pheophytin a or pharmaceutically acceptable salts of Claim 1.

6. Use of 10-hydroxy pheophytin a or pharmaceutically acceptable salts of Claim 1 as a photosensitizer for the photodynamic cancer therapeutics.